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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY


    ENVIRONMENTAL HEALTH CRITERIA 80




    PYRROLIZIDINE ALKALOIDS









    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of either the World Health Organization or the United Nations
    Environment Programme

    Published under the joint sponsorship of
    the United Nations Environment Programme
    and the World Health Organization

    World Health Organization
    Geneva 1988

    ISBN 92 4 154280 2

    (c) World Health Organization 1988

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    CONTENTS

    ENVIRONMENTAL HEALTH CRITERIA FOR PYRROLIZIDINE ALKALOIDS

    PREFACE

    INTRODUCTION - PYRROLIZIDINE ALKALOIDS AND HUMAN HEALTH

    1. SUMMARY AND RECOMMENDATIONS

        1.1. Summary
        1.2. Sources and chemical structure
        1.3. Mechanisms and features of toxicity
        1.4. Effects on man
             1.4.1. Nature and extent of health risks
        1.5. Methods for prevention
        1.6. Recommendations
             1.6.1. General recommendations
             1.6.2. Recommendations for research

    2. PROPERTIES AND ANALYTICAL METHODS

        2.1. Chemical structure and properties
        2.2. Analytical methods
             2.2.1. Extraction
                    2.2.1.1  Plant tissue
                    2.2.1.2  Biological fluids and tissues
             2.2.2. Analysis for pyrrolizidine alkaloids
                    2.2.2.1  Thin-layer chromatography (TLC)
                    2.2.2.2  High-performance liquid chromatography 
                             (HPLC)
                    2.2.2.3  Gas chromatography (GC) and mass 
                             spectrometry (MS)
                    2.2.2.4  Nuclear magnetic resonance (NMR) 
                             spectrometry
                    2.2.2.5  The Ehrlich reaction
                    2.2.2.6  Indicator dyes
                    2.2.2.7  Direct weighing
        2.3. Determination of metabolites in animal tissues

    3. SOURCES AND PATHWAYS OF EXPOSURE

        3.1. Hepatotoxic pyrrolizidine alkaloids and their sources
        3.2. Pneumotoxic and other toxic pyrrolizidine alkaloids 
        3.3. Pathways of exposure
             3.3.1. Contamination of staple food crops
             3.3.2. Herbal infusions
             3.3.3. Use of PA-containing plants as food
             3.3.4. Contaminated honey
             3.3.5. Milk
             3.3.6. Meat
             3.3.7. Use of PAs as chemotherapeutic agents for cancer

    4. METABOLISM

        4.1. Absorption, excretion, and tissue distribution
             4.1.1. Absorption
             4.1.2. Excretion and distribution
        4.2. Metabolic routes
             4.2.1. Hydrolysis
             4.2.2.  N-oxidation
             4.2.3. Conversion to pyrrolic metabolites
        4.3. Effects of treatments affecting metabolism
        4.4. Other factors affecting metabolism
        4.5. Other metabolic routes
        4.6. Metabolism of pyrrolizidine  N-oxides
        4.7. Metabolism in man

    5. MECHANISMS OF TOXICITY AND OTHER BIOLOGICAL ACTIONS

        5.1. Metabolites responsible for toxicity
             5.1.1. Metabolic basis of toxicity
             5.1.2. Isolation of pyrrolic metabolites
             5.1.3. Chemical aspects of pyrrolic metabolites
                    5.1.3.1  Preparation
                    5.1.3.2  Chemistry associated with toxic actions
             5.1.4. Possible further metabolites
        5.2. Toxic actions of pyrrolic metabolites
             5.2.1. Animals
                    5.2.1.1  Pyrrolic esters (dehydro-alkaloids)
                    5.2.1.2  Pyrrolic alcohols (dehydro-necines)
             5.2.2. Cell cultures
             5.2.3. Possible participation of membrane lipid 
                    peroxidation
        5.3. Chemical and metabolic factors affecting toxicity
             5.3.1. Structural features of a toxic alkaloid
             5.3.2. Activation and detoxication
             5.3.3. Factors affecting the toxicity of active 
                    metabolites
                    5.3.3.1  Reactivity of the metabolite
                    5.3.3.2  The number of reactive groups
        5.4. Metabolites associated with the biological actions of 
             pyrrolizidine alkaloids
             5.4.1. Acute hepatotoxicity
             5.4.2. Chronic hepatotoxicity
             5.4.3. Pneumotoxicity
             5.4.4. Toxicity in other tissues
             5.4.5. Carcinogenicity
             5.4.6. Antitumour activity
        5.5. Prevention and treatment of pyrrolizidine poisoning
             5.5.1. Modified diets
             5.5.2. Pre-treatment to enhance the detoxication of active 
                    metabolites
             5.5.3. Other treatments

    6. EFFECTS ON ANIMALS

        6.1. Patterns of disease caused by different plant genera and 
             of organ involvement in different species
        6.2. Field observations - outbreaks in farm animals
        6.3. Studies on farm animals
        6.4. Experimental animal studies
             6.4.1. Effects on the liver
                    6.4.1.1  Relative hepatotoxicity of different PAs 
                             and their  N-oxides
                    6.4.1.2  Factors affecting hepatotoxicity
                    6.4.1.3  Acute effects
                    6.4.1.4  Mechanism of toxic action
                    6.4.1.5  Chronic effects
             6.4.2. Effects on the lungs
                    6.4.2.1  Acute effects
                    6.4.2.2  Chronic effects
                    6.4.2.3  Mechanisms of toxic action
             6.4.3. Effects on the central nervous system
             6.4.4. Effects on other organs
             6.4.5. Teratogenicity
             6.4.6. Fetotoxicity
             6.4.7. Mutagenicity
                    6.4.7.1  Chromosome damage
             6.4.8. Carcinogenesis
                    6.4.8.1  Purified alkaloids
                    6.4.8.2  Plant materials
                    6.4.8.3  Pyrrolizidine alkaloid metabolites and 
                             analogous synthetic compounds
                    6.4.8.4  Molecular structure and carcinogenic 
                             activity
             6.4.9. Antimitotic activity
             6.4.10. Immunosuppression
             6.4.11. Effects on mineral metabolism
             6.4.12. Methods for the assessment of chronic 
                    hepatotoxicity and pneumotoxicity
        6.5. Effects on wild-life
             6.5.1. Deer
             6.5.2. Fish
             6.5.3. Insects

    7. EFFECTS ON MAN

        7.1. Clinical features of veno-occlusive disease (VOD)
        7.2. Salient pathological features of veno-occlusive disease
        7.3. Human case reports of veno-occlusive disease
        7.4. VOD and cirrhosis of the liver
        7.5. Differences between VOD and Indian childhood cirrhosis 
             (ICC)
        7.6. Chronic lung disease
        7.7.  Trichodesma poisoning
        7.8. Relationship between dose level and toxic effects
        7.9. Pyrrolizidine alkaloids as a chemotherapeutic agent for 
             cancer
        7.10. Prevention of poisoning in man

    8. BIOLOGICAL CONTROL

    9. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

        9.1. Human exposure conditions
             9.1.1. Reported sources of human exposure
             9.1.2. Plant species involved
             9.1.3. Modes and pathways of exposure
                    9.1.3.1  Contamination of grain crops
                    9.1.3.2  Herbal medicines
                    9.1.3.3  PA-containing plants used as food and 
                             beverages
                    9.1.3.4  Other food contaminated by PAs
             9.1.4. Levels of intake
        9.2. Acute effects of exposure
             9.2.1. Acute liver disease
        9.3. Chronic effects of exposure
             9.3.1. Cirrhosis of the liver
             9.3.2. Mutagenicity and teratogenicity
             9.3.3. Cancer of the liver
             9.3.4. Effects on other organs
        9.4. Effects on the environment
             9.4.1. Agriculture
             9.4.2. Wild-life
             9.4.3. Insects
             9.4.4. Soil and water

    REFERENCES

    APPENDIX I.  PYRROLIZIDINE ALKALOIDS AND THEIR PLANT SOURCES

    APPENDIX II.
    

    NOTE TO READERS OF THE CRITERIA DOCUMENTS

        Every effort has been made to present information in the 
    criteria documents as accurately as possible without unduly 
    delaying their publication.  In the interest of all users of the 
    environmental health criteria documents, readers are kindly 
    requested to communicate any errors that may have occurred to the 
    Manager of the International Programme on Chemical Safety, World 
    Health Organization, Geneva, Switzerland, in order that they may be 
    included in corrigenda, which will appear in subsequent volumes. 

                              *    *    *

    ENVIRONMENTAL HEALTH CRITERIA FOR PYRROLIZIDINE ALKALOIDS

        A WHO Task Group on Environmental Health Criteria for 
    Pyrrolizidine Alkaloids met in Tashkent, USSR, on 1 - 5 December 
    1986.  Dr M. Gounar opened the meeting on behalf of the three
    co-sponsoring organizations of the IPCS (UNEP/ILO/WHO).  The Task 
    Group reviewed and revised the draft criteria document and made an 
    evaluation of the health risks of exposure to pyrrolizidine 
    alkaloids. 

        Access to the original papers on the subject published in the 
    USSR was made possible by PROFESSOR M. ABDULLAHODJAEVA.  DR A.R. 
    MATTOCKS wrote the first drafts of the sections on Properties and 
    Analytical Methods, Metabolism, and Mechanisms of Toxicity and 
    Other Biological Actions.  DR C.C.J. CULVENOR, assisted PROFESSOR 
    H.D. TANDON in the finalization of the document after the Task 
    Group meeting.  Dr J. Parizek, who was originally the IPCS staff 
    member responsible for the preparation of the document, and was to 
    be Secretary of the Task Group, could not attend the meeting 
    because of sudden illness, and the Task Group  was assisted in his 
    place by Dr M. Gounar, former IPCS staff member.  Dr A. Prost was 
    responsible for the final version of the document. 

        The Secretariat acknowledge the help of both Professor H.D. 
    Tandon and Dr C.C.J. Culvenor.  The Task Group meeting in Tashkent 
    was organized by the Centre of International Projects, USSR State 
    Committee for Science and Technology. 

        The efforts of all who helped in the preparation and 
    finalization of the document are gratefully acknowledged. 

                                   * * *

        Partial financial support for the publication of this criteria 
    document was kindly provided by the United States Department of 
    Health and Human Services, through a contract from the National 
    Institute of Environmental Health Sciences, Research Triangle Park, 
    North Carolina, USA - a WHO Collaborating Centre for Environmental 
    Health Effects. 

                                   * * *

        A comprehensive data base on pyrrolizidine alkaloids has been 
    made available by CSIRO Division of Animal Health, Private Bag
    No. 1, Parkville, Vic. 3052, Australia.  The data base consists of 
    alkaloid occurrence tables and keyworded bibliography readable by 
    SCI-MATE software system (Bibliographic Manager, Institute for 
    Scientific Information), but adaptable to other systems.  It is 
    available from CSIRO on IBM - PC diskettes; price on application to 
    L.W. Smith. 

    PREFACE

        A disease caused by the consumption of plants containing 
    pyrrolizidine alkaloids (PAs) has been recognized independently as 
    an endemic disease in certain parts of the West Indies and in 
    Uzbekistan in the USSR.  Outbreaks of the disease have affected 
    significant segments of populations or large numbers of people in 
    geographically confined areas in Afghanistan, India, and 
    Uzbekistan.  The outbreaks have been caused through contamination 
    of the staple food crops with the seeds of plants containing PAs, 
    growing among the crops; such plants are likely to thrive following 
    periods of drought. 

        It is notable that the same family of plants that caused 
    endemic disease and large-scale outbreaks in Uzbekistan also caused 
    another outbreak of the disease in adjacent Afghanistan, long after 
    the chemical etiology of the disease (through consumption of toxic 
    seeds in the food) had been identified in the USSR.  This happened 
    because there was a lack of general awareness of the causal 
    relationship between the chemical present in the plant and the 
    disease.  Sporadic cases continue to occur in different parts of 
    the world through the consumption of seeds or plant parts 
    containing toxic PAs, as home remedies, beverages, or food. 

        The IPCS recognized that this was a health problem that might 
    be lethal, and that it was entirely preventable, provided that it 
    was recognized in time.  It was also recognized that the 
    dissemination of knowledge, about both the disease and the sources 
    of the chemicals involved, would be a critical step in its 
    prevention. 

        Accordingly, the IPCS invited Professor H.D. Tandon, who was 
    responsible for establishing such a causal relationship in the 
    outbreaks in Afghanistan and India, to prepare a draft criteria 
    document and to assist in its further development and finalization 
    after the Task Group meeting, which was held in Tashkent, USSR, on 
    1 - 5 December, 1986. 

        In most episodes of toxic human disease caused by PAs, the 
    liver has been the principal target organ, except for an outbreak 
    in the USSR caused by  Trichodesma alkaloids, in which the symptoms 
    were mostly extra-hepatic.  The Environmental Health Criteria 
    document provides comprehensive coverage of the hepatotoxic PAS, 
    but lack of relevant documentation prevented the Task Group from 
    analysing the role of  Trichodesma alkaloids in detail. 

    INTRODUCTION - PYRROLIZIDINE ALKALOIDS AND HUMAN HEALTH

        Pyrrolizidine alkaloids (PAs) are found in plants growing in 
    most environments and all parts of the world.  The main sources are 
    the families Boraginaceae (all genera), Compositae (tribes 
     Senecionae and  Eupatoriae), and Leguminosae (genus  Crotalaria), and 
    the potential number of alkaloid-containing species is as high as 
    6000, or 3% of the world's flowering plants (Culvenor, 1980).  They 
    have long been known to be a health hazard for livestock, at least 
    since 1902 (Schoental, 1963), and loss of livestock in various 
    parts of the world has been traced to their grazing on certain 
    plants growing in pastures, especially following periods of drought 
    or in arid climates.  They have been found to be toxic for all 
    species of animals tested (Schoental, 1963), though some species, 
    notably the guinea-pig, are resistant (Chesney & Allen, 1973a; 
    White et al., 1973).  Human disease caused by PA toxicity has been 
    known to be endemic in the central Asian republics of the USSR, at 
    least since the early thirties (Ismailov, 1948a,b; Mnushkin, 1949) 
    when several outbreaks occurred, and the cause was discovered to be 
    the seeds of plants of  Heliotropium species (Dubrovinskii, 1947, 
    1952; Khanin, 1948), which contaminated the staple food crops.  A 
    spate of reports followed, mostly from the West Indies, of acute 
    and chronic liver disease (Bras et al., 1954, 1961; Bras & Hill, 
    1956; Stirling et al., 1962), associated with the ingestion by 
    people of herbal infusions for the treatment of certain ailments.  
    Schoental (1961) and Davidson (1963) suggested that, in view of the 
    evidence of the hepatotoxicity of PAs, consumption of plants 
    containing them could be of etiological significance in human liver 
    disease, especially in developing countries where they are consumed 
    as food or herbal medicines. In spite of this, and the fact that 
    such an ubiquitous source of toxic material is capable of producing 
    animal and human disease and that there have been more recent 
    reports, the PAs have not attracted much attention in the world as 
    a health hazard.  In fact, a recent handbook on naturally occurring 
    toxic agents in food (Rechicigl, Jr, 1983) refers to them only in 
    passing and makes no mention of human disease caused by them.  
    Veno-occlusive disease (VOD) (Bras & Hill, 1956), which is  
    characterized by the dominant occlusive lesion of the centrilobular 
    veins of the liver lobule and is caused by these alkaloids, has 
    since been reported from all parts of the world, in both man and 
    animals (Hill, 1960; Bras, 1973).  It has been attributed to the 
    accidental contamination of food by toxic plant products or the 
    ingestion of herbal infusions. There have been reports of stray 
    cases and of small outbreaks from both developing and developed 
    countries.  However, in the most recent studies from Afghanistan 
    (Tandon & Tandon, 1975; Mohabbat et al., 1976; Tandon, B.N. et al., 
    1978; Tandon, H.D. et al., 1978) and India (Tandon, B.N. et al., 
    1976; Tandon, R.K. et al., 1976; Krishnamachari et al., 1977; 
    Tandon, H.D. et al., 1977; Tandon, B.N. et al., 1978), the disease 
    has been reported to affect large masses of the population, 
    resulting in high mortality, and has been attributed to the 
    accidental contamination of their staple food crops by PA-
    containing seeds of plants, following periods of drought. 

        There is conclusive evidence from studies on experimental 
    animals that the effects of a single exposure to PAs may progress 
    relentlessly to advanced chronic liver disease and cirrhosis 
    (Schoental & Magee, 1957, 1959; Nolan et al., 1966), following a 
    long interval of apparent well-being, and without any other latent 
    or provocative factor (Schoental & Magee, 1959).  The lowest levels 
    of such alkaloids administered thus far to experimental animals, 
    e.g., 1 - 4 mg/kg diet, have produced chronic liver disease and 
    tumours (Hooper & Scanlan, 1977; Culvenor & Jago, 1979).  
    Pyrrolizidine alkaloids have also been shown to act synergistically 
    with aflatoxin, another environmental toxin present in agricultural 
    products, in causing cirrhosis and hepatoma in primates (Lin et 
    al., 1974).  Though there is no conclusive evidence yet of a 
    carcinogenic role of PAs in man, such a possibility has been 
    suspected on the basis of experimental data (Hill, 1960; Williams 
    et al., 1967; IARC, 1976, 1983; Huxtable, 1980; Culvenor, 1983), 
    and experimental studies have demonstrated carcinogenicity in rats 
    given dosages equivalent to those reported to have been ingested in 
    human cases (Cook et al., 1950; Culvenor, 1983). 

        Alkaloids/toxic metabolites have been shown to be secreted in 
    the milk of lactating dairy cattle (Dickinson et al., 1976) and 
    rats, and the young of both sexes have been shown to suffer toxic 
    damage, even when suckled by mothers treated with retrosine, who 
    apparently are not affected themselves (Schoental, 1959).  Such 
    suckling animals may also be in apparent good health while the 
    livers show toxic effects. Protein-deficient and young suckling 
    animals are particularly vulnerable (Schoental, 1959). 

        Chromosomal aberrations have been demonstrated in rats and 
    humans with veno-occlusive disease (Martin et al., 1972). 

        Alkaloids have been found in the honey secreted by bees feeding 
    on the toxic plants (Deinzer et al., 1977).  According to Culvenor 
    and his co-workers, populations in some countries are exposed to 
    low levels of alkaloids in commonly used foodstuffs, e.g., honey in 
    Australia (Culvenor et al., 1981; Culvenor, 1983, 1985) and comfrey 
    in many countries (Culvenor et al., 1980a; Culvenor, 1985). 

        Human cases of acute disease following the brief ingestion of 
    the alkaloids have been known to progress to cirrhosis (Stuart & 
    Bras, 1957; Braginskii & Bobokhadzaev, 1965; Stillman et al., 1977; 
    Tandon, B.N. et al., 1977; Tandon, H.D. et al., 1977) in as short a 
    period as 3 months from the acute phase (Stuart & Bras, 1957).  The 
    initial disease may be cryptic (Braginskii & Bobokhadzaev, 1965) 
    and may not be ascribed to herbal consumption, and yet may progress 
    to cirrhosis (Huxtable, 1980).  Veno-occlusive disease was stated 
    to be the most common cause of cirrhosis in infants in Jamaica 
    (Bras et al., 1961) and has been believed to be a significant 
    etiological factor for adult cirrhosis, especially in developing 
    countries (Gupta et al., 1963). 

        Plants known or suspected to contain toxic alkaloids are widely 
    used for medicinal purposes as home remedies all over the world, 
    without systematic testing for safety (Schoental, 1963; Smith & 

    Culvenor, 1981) and some are even used as food (Schoental & Coady, 
    1968; Culvenor, 1980).  There are several reports of the continued 
    use of such herbs for medicinal purposes in technically advanced 
    countries (Culvenor, 1980).  Senecio jacobaea continues to be sold 
    at herbalists shops in the United Kingdom (Schoental, 1963; Burns, 
    1972), and  Symphytum spp. (comfrey) are still used as a vegetables, 
    beverages, or remedies (Mattocks, 1980).  Both these herbs are 
    known to be carcinogenic (IARC, 1976; Hirono et al., 1978).  Young 
    flower stalks of  Petasites japonicus Maxim, the pre-bloom flower 
    of coltsfoot,  Tussilago farfara, the leaf and root of comfrey, 
     Symphytum officinale, and the young leaves and stalks of  Farfugium 
     japonicum and  Senecio cannabifolius, which are all used in Japan 
    as human food or herbal remedies, are known to be carcinogenic for 
    rats (Hirono et al., 1983).   Symphytum x uplandicum Nyman (Russian 
    comfrey), which contains several toxic PAs (Culvenor et al., 1980b) 
    echimidine and 7 acetylycopsamine being the main constituents, is 
    used as a salad plant, green drink, and medicinal herb.  It has 
    been estimated that the rate of ingestion of alkaloids from this 
    herb may, over a period of time, exceed the levels reported to have 
    been taken during the Afghan outbreak.  There is a report of at 
    least one patient who developed toxic effects as a result of 
    consuming a comfrey preparation (Culvenor et al., 1980a; Ridker et 
    al., 1985).  Arseculeratne et al. (1981) found that 3 of the 50 
    medicinal herbs commonly used in Sri Lanka contained PAs that had 
    been proved to be hepatotoxic for animals.  They suggested that 
    consumption of such herbs might contribute to the high incidence of 
    chronic liver disease, including primary liver cancer, in Asian and 
    African countries, especially as they may act synergistically with 
    aflatoxin and hepatitis B virus.  The risk of toxic effects due to 
    these alkaloids may be particularly high in children (Schoental, 
    1959; Jago, 1970) and protein malnutrition, which exists in some 
    countries, may potentiate them (Schoental & Magee, 1957).  Recent 
    studies from Hong Kong (Kumana et al., 1985; Culvenor et al., 
    1986), the United Kingdom (McGee et. al, 1976; Ridker et al., 
    1985), and the USA (Stillman et al., 1977; Fox et al., 1978; Ridker 
    et al., 1985) report instances of human disease that have been 
    caused by the use of such herbs, resulting in fatality or the 
    development of cirrhosis, even in countries with well-developed 
    health services and among the higher economic and educated strata 
    of society. Indeed, Stillman et al. (1977), from the USA, called PA 
    toxicosis the "iceberg disease", implying that cases of this 
    disease might be more frequent than reported in the USA, especially 
    among populations of Mexican-American origin.  In general, the use 
    of herbal remedies is not elicited in the clinical history and 
    patients do not volunteer this information themselves.  
    Furthermore, the alkaloids are eliminated within 24 h (Huxtable, 
    1980) and, even though methods are available for their detection in 
    biological tissues and fluids, the suspicion cannot be confirmed, 
    as the symptoms may take several days or months to appear. 

        Contamination of food crops is particularly likely to occur in 
    parts of the world with arid climates, poor or uncertain rainfall, 
    poor irrigation facilities, and following periods of drought, all 
    of which promote the growth of the PA-containing plants that grow 
    as weeds among cultivated crops, as has been found in studies on 

    the outbreaks in Afghanistan, India, and the USSR (Terekhov, 1939; 
    Dubrovinskii, 1947; Ismailov, 1948a,b; Tandon & Tandon, 1975; 
    Mohabbat et al., 1976; Tandon, B.N. et al., 1976; Tandon, R.K. et 
    al., 1976; Tandon, H.D. et al., 1978) and in grazing pastures.  The 
    use of traditional medicines is common in these countries and there 
    is insufficient awareness of this hazard, the disease condition, 
    and its diagnostic pathological picture.  Furthermore, health 
    services are poorly developed. Thus, many of the cases or even 
    outbreaks may go unnoticed or unrecorded and may even be ascribed 
    to malnutrition (Lancet, 1984).  Also, many of the reported cases 
    of so-called "Budd-Chiari syndrome", a condition associated with 
    obstruction of major hepatic veins and/or inferior vena cava, may 
    actually be cases of veno-occlusive disease (Sherlock, 1968), in 
    which only the central veins of the liver lobule or sublobular 
    veins are occluded. 

        Another type of PAs,  Trichodesma alkaloids, has been known to 
    cause a human outbreak of disease in the USSR, through 
    contamination of the staple cereal with the seeds containing these 
    PAs; in this outbreak, the symptoms were principally extra-hepatic 
    (Ismailov et al., 1970). 

        This document is aimed at focusing on a health menace that is 
    insufficiently recognized, in order to evaluate the health risks on 
    the basis of published data, and to draft a set of recommendations 
    that would help in its recognition, prevention, and control. 

    1.  SUMMARY AND RECOMMENDATIONS

    1.1.  Summary

        The ingestion of pyrrolizidine alkaloids (PAs) in foods and 
    medicinal herbs results in acute and chronic effects in man, 
    affecting mainly the liver.  Data from experimental animal studies 
    indicate that PAs represent a potential cause of cancer in man. 

        The alkaloids are produced by numerous plant species and occur 
    throughout the world.  In the present document, the alkaloids and 
    their properties are described together with the sources of human 
    exposure and the diseases that they produce in man and animals.  
    The risks for human health are evaluated and recommendations are 
    made for reducing such risks. 

    1.2.  Sources and Chemical Structure

        The known pyrrolizidine alkaloids, most of which are 
    hepatotoxic, are produced by plant species within the following 
    families: Boraginaceae ( Heliotropium, Trichodesma, Symphytum, and 
    most other genera), Compositae ( Senecio, Eupatorium, and other 
    genera of the tribes  Senecioneae and  Eupatoriae), Leguminosae 
    (genus  Crotalaria), and Scrophul-ariaceae (genus  Castilleja).  
    These genera are mainly herbaceous and very widely distributed, 
    some species being found in most regions of the world.  The 
    majority of the species within these genera have not yet been 
    investigated, but are expected to contain pyrrolizidine alkaloids. 

        The hepatotoxic alkaloids have a 1,2-double bond in the 
    pyrrolizidine ring and branched chain acids, esterifying a
    9-hydroxyl and preferably also the 7-hydroxyl substituent. Modified 
    seco-pyrrolizidine alkaloids, in which the central bond between the 
    N and C8 atoms is broken, are also hepatotoxic.  Some  Senecio 
    species contain non-basic derivatives that are 5-oxopyrroles.  The 
    toxicity of these derivatives may be similar to that of the 
    alkaloids, but this aspect has not been investigated.  The 
    alkaloids occur as free bases and  N-oxides.  The latter are 
    reduced to the free bases in the gastrointestinal tract of animals 
    and have a similar toxicity when ingested orally. 

        Suitable analytical procedures are available for screening 
    plant species, including a simple field test for toxic alkaloids.  
    Thin-layer chromatography (TLC), high-performance liquid (HPLC), 
    gas chromatography (GC), and gas chromato-graphy-mass spectrometry 
    (GC-MS) have been applied for separating, characterizing, and 
    quantifying the alkaloids present.  Effective use of these 
    procedures requires authentic alkaloids for standards, few of which 
    are available.  Improved analytical methods are required for the 
    determination of very low levels of alkaloids in some foodstuffs. 

    1.3.  Mechanisms and Features of Toxicity

        The toxic effects of pyrrolizidine alkaloids are due to 
    activation in the liver.  Metabolism of the alkaloids by mixed-
    function oxidases leads to pyrrolic dehydro-alkaloids, which are 
    reactive alkylating agents.  Reaction of initial metabolites with 
    constituents of the liver cell in which they are formed are 
    probably the main cause of liver cell necrosis. Metabolites are 
    released into the circulation and are believed to pass beyond the 
    liver to the lung causing vascular lesions characteristic of 
    primary pulmonary hypertension, especially when alkaloids, such as 
    monocrotaline, are administered to animals. 

        In experimental animals, PAs are quickly metabolized and are 
    almost completely excreted in 24 h, so that no residual products 
    are detectable in the biological fluids or body tissues after this 
    period. 

        The rate of formation of pyrrolic metabolites is influenced by 
    the induction or inhibition of the mixed-function oxidases in the 
    liver, but the relationship between the rate of metabolism and 
    expression of toxicity is uncertain. 

        Several pyrrolizidine alkaloid-derivatives and related 
    compounds are known to cause chromosome aberrations in plants, 
    leukocyte cell cultures of the marsupial  (Potorus tridactylus),  
    and in hamster cell lines.  Some pyrrolizidine alkaloids induce 
    micronuclei formation in erythrocytes in the bone marrow and fetal 
    liver in mice, sister chromatid exchanges in a Chinese hamster cell 
    line and human lymphocytes  in vitro, and repair DNA synthesis in 
    rodent hepatocyte cell cultures.  Chromosome aberrations have been 
    reported in the blood cells of children suffering from veno-
    occlusive disease VOD, presumably caused by fulvine. 

        A number of pyrrolizidine alkaloids have been shown to be 
    mutagenic in the  Salmonella typhimurium assay, after metabolic 
    activation.  The carcinogenic activity of pyrrolizidine alkaloids 
    appears to parallel their mutagenic behaviour, but not their 
    hepatotoxicity. 

        Heliotrine at doses of 50 mg/kg body weight or more, 
    administered to rats during the second week of gestation, has been 
    shown to induce several abnormalities in the fetus. Doses of
    200 mg/kg body weight resulted in intrauterine deaths or resorption
    of fetuses.  Dehydroheliotridine, the metabolic pyrrole derivative of 
    heliotrine, was 2.5 times more effective on a molar basis than its 
    parent PA in inducing teratogenic effects. 

        The ability of PAs to cross the placental barrier in the rat 
    and to induce premature delivery or death of litters has been 
    demonstrated.  The embryo  in utero appears to be more resistant to 
    the toxic effects of pyrrolizidine alkaloids than the neonate.  PAs 
    are known to have passed through the mother's milk to the 
    sucklings. 

        Megalocytosis, the presence of enlarged hepatocytes containing 
    large, hyper-chromatic nuclei, is a characteristic feature of 
    pyrrolizidine alkaloid-induced chronic hepatotoxicity in 
    experimental animals.  The enlarged hepatocytes arise through the 
    powerful antimitotic action of the pyrrole metabolites of 
    pyrrolizidine alkaloids.  This change has not been observed in the 
    human liver, though human fetal liver cells  in vitro culture 
    become enlarged when exposed to PAs, indicating susceptibility to 
    the antimitotic effect of the alkaloids. 

        In experimental animals, protein-rich and sucrose-only diets 
    have given some measure of protection against the effects of the 
    alkaloids, as has pre-treatment of animals with thiols, anti-
    oxidants, or zinc chloride. 

        PAs are noted mainly for the poisoning of livestock due to the 
    animals grazing on PA-containing toxic weeds, and large-scale 
    outbreaks have been recorded.  Such episodes have been reported 
    from most parts of the world, including those with temperate or 
    cold climates.  Studies carried out on a wide variety of farm and 
    laboratory animals have revealed generally common features of 
    toxicity with some species variations.  The liver is the principal 
    target organ.  In small laboratory animals, doses approaching a 
    lethal dose produce a confluent, strictly zonal haemorrhagic 
    necrosis in the liver lobule, within 12 - 48 h of administration of 
    PAs. Simultaneously in non-human primates, or after a short time in 
    the rat, chicken, and swine, changes begin to occur, and later 
    become organized, in the subintima of the central or sublobular 
    veins in the liver resulting in their occlusion. The reticulin 
    framework in the central zone of the lobule collapses following 
    necrosis leading to scarring.  Repeated administration of suitable 
    doses leads to chronic liver lesion characterized by megalocytosis, 
    and increasing fibrosis, which may result in cirrhosis.  Chronic 
    liver disease including cirrhosis has been shown to develop in the 
    rat following administration of a single dose of a PA.  In a number 
    of animal species, the lungs develop vascular lesions 
    characteristic of primary pulmonary hypertension with secondary 
    hypertrophy of the right ventricle of the heart.  In rats, 
    appropriately low repeated doses of several alkaloids have been 
    shown to induce tumours, mainly in the liver.  In some studies, a 
    single dose has been carcinogenic. 

        The central nervous system is the target organ of the toxic PAs 
    contained in Trichodesma, which produce spongy degeneration of the 
    brain. 

    1.4.  Effects on Man

        In man, PA poisoning is usually manifested as acute veno-
    occlusive disease characterized by a dull dragging ache in the 
    right upper abdomen, rapidly filling ascites resulting in marked 
    distension of the abdomen, and sometimes associated with oliguria, 
    and massive pleural effusion.  It can also manifest as subacute 
    disease with vague symptoms and persistent hepatomegaly.  Children 
    are particularly vulnerable.  Many cases progress to cirrhosis and, 

    in some cases, a single episode of acute disease has been 
    demonstrated to progress to cirrhosis, in spite of the fact that 
    the patient has been removed from the source of toxic exposure and 
    has been given symptomatic treatment.  Mortality can be high with 
    death due to hepatic failure in the acute phase or due to 
    hematemesis resulting from ruptured oesophageal varices caused by 
    cirrhosis.  Less severely affected cases may show clinical, or even 
    apparently complete, recovery.  The Task Group was not aware of any 
    substantiated report of primary pulmonary hypertension resulting 
    from PA toxicity.  However, in view of the evidence in experimental 
    animals and circumstantial evidence in one case report, the 
    possibility of the development of toxic pulmonary disease in man 
    cannot be ruled out.  There is a report of an outbreak of 
     Trichodesma poisoning in the USSR in which the symptoms were mainly 
    neurological. 

    1.4.1.  Nature and extent of health risks

        The two main sources of pyrrolizidine alkaloid poisoning 
    reported in human beings are the consumption of cereal grain 
    contaminated by weeds containing the alkaloids and the use of 
    alkaloid-containing herbs for medicinal and dietary purposes. A 
    third form of exposure, with the potential to affect large 
    populations is the possible low-level contamination of some 
    foodstuffs, such as honey and milk, but the Task Group was not 
    aware of any cases of human toxicity having been caused through the 
    contamination of these foods. 

        Liver disease caused by the contamination of cereal grains has 
    been reported in rural populations in Afghanistan, India, South 
    Africa, and the USSR.  A contributing factor appears to be 
    abnormally dry weather, resulting in the growth of an exceptionally 
    high proportion of the alkaloid-containing weeds in the crops, the 
    seeds of which contaminate the cereal grain on harvesting.  The 
    weeds responsible for known outbreaks have been  Heliotropium, 
     Trichodesma, Senecio, and  Crotalaria species.  Mortality in such 
    outbreaks has been reported to be high.  In the largest reported 
    outbreak in northwestern Afghanistan, an estimated 8000 people were 
    affected in a total population of 35 000 with 1600 - 2000 deaths. 

        Human poisoning through the medicinal use of herbs containing 
    pyrrolizidine alkaloids has been reported from all parts of the 
    world.  PAs were responsible for a common liver disease in children 
    in Jamaica, and individual cases in Ecuador, Hong Kong, India, the 
    United Kingdom, and the USA. The plants involved were species of 
    Crotalaria, Heliotropium,  Senecio, Symphytum, and  Gynura.  
     Symphytum-containing preparations present a particular hazard 
    because of their widespread use and the generally high levels of 
    individual exposures.  The use of herbs is almost universal in 
    traditional folk medicine and is increasing in developed countries. 
    Some of the herbs used contain pyrrolizidine alkaloids and have a 
    long-term toxicity that is unsuspected by the people taking them.  
    Knowledge of the species used in herbal medicine and the frequency 
    of such use is very limited in the scientific literature.  About 40 
    such species are listed in this report, about one-third of which 

    are in use in developed countries.  They are often prescribed by 
    herbalists, naturopaths, and other non-orthodox practitioners.  The 
    extent of the contribution to acute and chronic liver disease 
    cannot be accurately assessed.  It may also constitute an 
    etiological factor in cirrhosis of the liver and, once this stage 
    is reached, it may not be possible to identify the cause as a PA. 

        PAs are known to be transmitted from the feed of dairy animals 
    into milk and to cause toxic damage in the suckling young.  One 
    instance of large-scale contamination of honey is known to have 
    been caused by a common weed rich in PAs, which was the source of 
    nectar and pollen for the honey-secreting bees.  No reports of 
    cases of acute toxicity caused by consumption of contaminated dairy 
    products or honey were available to the Task Group.  Furthermore, 
    no information is available on the possible presence of PAs or 
    their metabolites in the meat of animals fed toxic weeds before 
    slaughter; however, the possibility of toxic disease being caused 
    through this medium is considered to be low. 

        There are no substantial, long-term follow-up data to assess 
    whether exposure to PAs results in increased incidence of chronic 
    liver disease or cancer in man.  Available clinical and 
    experimental data suggest that a single episode of PA toxicity and 
    possibly also a long-term low level exposure may lead to cirrhosis 
    of the liver.  PAs could also be possible carcinogens in man, since 
    a number of them have been demonstrated to induce cancer in 
    experimental animals, the main target organ being the liver.  These 
    include some which have caused episodes of human toxicity, and some 
    others which are found in herbs traditionally used as items of 
    food.  Also, in several instances of human toxicity, the reported 
    daily rates of intake of such PAs were in close range of those 
    known to induce tumours in rats.  However, these risks cannot be 
    adequately assessed on a quantitative basis.  There are indications 
    that PA intoxications leading to liver disease are more prevalent 
    than the reported frequency of cases would seem to indicate. 

        Because of their known involvement in human poisoning and their 
    possible carcinogenicity, exposure to pyrrolizidine alkaloids 
    should be kept as low as practically achievable. The setting of 
    regulatory tolerance levels for certain food products may be 
    required in some situations. 

    1.5.  Methods for Prevention

        The only known method of prevention is to avoid consumption of 
    the alkaloids.  In the USSR, a set of agricultural (or 
    agrotechnical) legislative, phyto-sanitary and educational measures 
    has prevented new outbreaks of poisoning due to  Heliotropium and 
     Trichodesma, since 1947. 

    1.6.  Recommendations

    1.6.1.  General recommendations

     1. Cereal crops should be assessed throughout the world for 
        possible contamination by weeds likely to contain pyrrolizidine 
        alkaloids.  Appropriate grain inspection systems are desirable 
        in order to achieve near-zero levels of contamination by such 
        weeds. 

    2.  There is a need to create awareness, among the general 
        population  and those responsible for the delivery of health 
        services, with regard to the hazards of consuming such plants 
        as contaminants in food or as food, or for medicinal purposes. 
        Advice on hazards should include mention of possible increased 
        risks, if the alkaloid intake is associated with drug 
        treatment, (e.g. phenobarbitone) or foods which increase the 
        level of liver metabolizing enzymes. 

    3.  Ethnobotanical and taxonomic studies are required in many 
        countries to provide specific information on the use of plant 
        species containing pyrrolizidine alkaloids for medicinal and 
        dietary purposes.  There may be a need to control the sale of 
        some species, and their prescription by herbalists and other 
        practitioners of traditional systems of medicine. 

    4.  Honey and dairy products, both local and bulk supplies, should 
        be assayed for pyrrolizidine alkaloids in all regions where a 
        risk of contamination of these foodstuffs has been identified. 

    1.6.2  Recommendations for research

    1.  Long-term follow-up studies of the survivors of both alkaloid 
        poisoning in human beings and animal outbreaks are required, in 
        order to determine the possible development of chronic liver 
        disease or cancer.  Similar studies are also desirable on 
        individuals who regularly consume comfrey or other PA-
        containing herbs over a substantial period of time. 

    2.  Epidemiological studies should be carried out in countries with 
        a high incidence of primary liver cancer, in order to determine 
        whether there is an association with the intake of herbs 
        containing pyrrolizidine alkaloids. 


    3.  A network of reference laboratories is needed to assist member 
        states in identifying plants and their seeds suspected of 
        producing toxic effects and for the assay and identification of 
        PAs.  Provisions may be made for the easy availability of pure 
        alkaloids for use as reference standards for assays. 

    4.  It is necessary to develop improved assay procedures, suitable 
        for the purposes of recommendation (4) in section 1.6.1, 
        particularly using fluorescence and immunochemical methods.

    5.  There is a need for further toxicological studies, such as 
        studies on the carcinogenicity of echimidine and the toxicity 
        of the 5-oxopyrrole constituents of  Senecio species, and for 
        studies that would provide more quantitative information on the 
        various adverse biological effects of PAs.  A study of the 
        carcinogenicity of the alkaloids in the pig is also indicated, 
        since the pig exhibits a high sensitivity to acute and subacute 
        toxicity similar to that seen in man. 

    6.  Study is required of the possible alkaloid content of the meat, 
        organs, and fat of animals that have recently consumed plants 
        containing pyrrolizidine alkaloids. 

    7.  Experimental studies are needed on the influence of nutritional 
        status on the metabolism, and acute and chronic effects of PAs.

    8.  Further metabolic studies are required to define more 
        specifically the enzymes involved in the microsomal activation 
        and detoxification of PAs, to determine whether organelles 
        other than microsomes are involved, and to explore further, 
        quantitative relationships between different routes of 
        metabolism. 

    9.  The maximum no-observed-adverse-effect dose levels for repeated 
        long-term administration in the rat and the pig need to be 
        determined. 

    10. Experimental studies should be conducted to determine:

        (a)  whether pyrrolizidine alkaloid  N-oxides may be
             metabolized directly into the pyrrolic dehydroalkaloid
             in mitochondria, especially in tumour cells; and

        (b)  which P450 enzymes are involved in the activation and
              N-oxidation of PAs and thence in the selective
             induction of  N-oxidation enzymes.

    11. A study might be conducted of human variability and its genetic 
        aspects in relation to factors that influence susceptibility to 
        PAs;  for example, the study of mixed-function oxidase levels 
        in the liver by metabolism of appropriate test substances 
        recognized as harmless. 

    2.  PROPERTIES AND ANALYTICAL METHODS

    2.1  Chemical Structure and Properties

        The chemical structure of PAs in relation to their toxic 
    effects has been reviewed recently by Mattocks (1986).  The 
    pyrrolizidine alkaloids with which this document is concerned are 
    those that have previously been called "hepatotoxic" or 
    "nucleotoxic".  Here it is proposed to refer to them as "toxic" 
    PAs, because of the weight of evidence now available that they 
    produce damage in other organs as well as the liver, and the need 
    to avoid a restrictive term.  There are other types of 
    pyrrolizidine alkaloids, such as those that occur in the plant 
    family Orchidaceae, which are not toxic and are not discussed here. 

        The toxic PAs are esters of the amino-alcohols derived from the 
    heterocyclic nucleus.  The pyrrolizidine molecule is made up of two 
    5-membered rings inclined to each other as shown in Fig. 1 so that 
    geometric isomerism is possible, and which share a common nitrogen 
    at position 4. 

    FIGURE 1

        Most hepatotoxic alkaloids are esters of molecules similar to 
    that shown in Fig. 1(b) (1-hydroxymethyl-1:2-dehydro-
    pyrrolizidine).  However, a few hepatotoxic alkaloids are esters of 
    the amino-alcohol otonecine, e.g., petasitenine (Fig. 2, No.7).  
    The unsaturated pyrrolizidine nucleus itself is not toxic, but 
    esters of branched-chain acids are.  Ester linkages may be at 
    positions 9, 7, or (rarely) 6.  Some esters have an "open" 
    molecule, e.g., heliotrine, whereas others are macrocyclic 
    diesters, e.g., monocrotaline and retrosine. Examples of some 
    pyrrolizidine alkaloid structures are shown in Fig. 2. 

        The ring nucleus contains a double bond at the 1:2 position, 
    which is essential for the toxic effects of the alkaloid, but not 
    for unrelated effects. 

    1.   Echimidine

    Chemical structure:

    FIGURE 2

    Chemical formula:   C20H31NO7

    Relative molecular mass:    397

    CAS registry number:    520-68-3

    2.   Heliotrine

    Chemical structure:

    FIGURE 2

    Chemical formula:   C16H27NO5

    Relative molecular mass:    313

    CAS registry number:    303-33-3

    3.   Indicine- N -oxide

    Chemical structure:

    FIGURE 2

    Chemical formula:   C15H25NO6

    Relative molecular mass:    315

    CAS registry number:    41708-76-3

    4.   Jacobine

    Chemical structure:

    FIGURE 2

    Chemical formula:   C18H25NO6

    Relative molecular mass:    351

    CAS registry number:    6870-67-3

    5.   Lasiocarpine

    FIGURE 2

    Chemical structure:

    Chemical formula:   C21H33NO7

    Relative molecular mass:    411

    CAS registry number:    303-34-4

    6.   Monocrotaline

    FIGURE 2

    Chemical structure:

    Chemical formula:   C16H23NO6

    Relative molecular mass:    325

    CAS registry number:    315-22-0

    7.   Petasitenine

    FIGURE 2

    Chemical structure:

    Chemical formula:   C19H27NO7

    Relative molecular mass:    381

    CAS registry number:    60132-19-6

    8.   Retrorsine  (retrosine  N -oxide = isatidine)

    FIGURE 2

    Chemical structure:

    Chemical formula:   C18H25NO6

    Relative molecular mass:    351

    CAS registry number:    480-54-6

    9.  Senecionine

    FIGURE 2

    Chemical structure:

    Chemical formula:   C18H25NO5

    Relative molecular mass:    335

    CAS registry number:    130-01-8

    10.  Symphytine

    FIGURE 2

    Chemical structure:

    hemical formula:   C20H31NO6

    Relative molecular mass:    381

    CAS registry number:    22571-95-5

    11.  Trichodesmine

    FIGURE 2

    Chemical structure:

    Chemical formula:   C18H27NO6

    Relative molecular mass:    353

    CAS registry number:    548-90-3

    12.  Incanine

    FIGURE 2

    Chemical structure:

    Chemical formula:   C18H27NO5

    Relative molecular mass:    337

    CAS registry number:    480-77-3

        As the Task Group met in Tashkent, it is of historical interest 
    to recall that the structures of heliotrine and lasiocarpine, the 
    main alkaloids of  Heliotropium lasiocarpum, were worked out by 
    Dr G.P. Men'shikov and associates in Moscow in the 1930s.  This 
    work included determining the structure of heliotridine, the parent 
    compound of the amino-alcohol, heliotridane.  Dr Men'shikov's 
    studies were carried out at essentially the same time, but 
    independently of studies by English and American authors on 
    retronecine-based alkaloids. 

        The alkaloids in plants are often found together with their 
     N-oxides, which are also toxic, when ingested orally.  The 
    pyrrolizidine alkaloids acquire their toxic properties only through 
    the toxic pyrrolic intermediates (the general structure of which is 
    shown in Fig. 3) formed by the mixed-function oxidases of the 
    hepatocytes.  To form these pyrrolic derivatives, the alkaloid 
    molecule should have: 

        (a)  a double bond at the 1:2 position of the ring nucleus;

        (b)  esterified hydroxyl groups in the nucleus at the C 9
             and/or C 7 positions; and

        (c)  a branched carbon chain in at least one of the ester side-
             chains (McLean, 1974).

    FIGURE 3

    Substitution at the a position of the acid and esterification of 
    the C-7 hydroxy group both enhance the toxicity of the alkaloid 
    (Robins, 1982). 

        A group of related alkaloids, isolated from  Senecio species by 
    Bohlmann et al. (1979), have non-basic pyrrolic structures similar 
    to those of toxic pyrrolizidine alkaloid metabolites, but they are 
    chemically deactivated by the presence of a carbonyl group at 
    position 3 of the pyrrolizidine nucleus, e.g., senaetnine (Fig. 4).  
    Senaetnine does not possess the acute hepatotoxic characteristics 
    of basic pyrrolizidine alkaloids.  However, it had a direct 
    irritant action on tissues near the site of intraperitoneal 
    administration and caused damage to pulmonary vascular tissue when 
    given intraveinous to rats (Mattocks & Driver, 1987). 

    FIGURE 4

        The alkaloids are fairly stable chemically, but the ester 
    groups may undergo hydrolysis under alkaline conditions.  Some 
    alkaloids in plant material may decompose during drying (Bull et 
    al., 1968), but others appear to be stable under similar conditions 
    (Pedersen, 1975; Birecka et al., 1980).  The  N-oxides of 
    unsaturated pyrrolizidines are more readily decomposed by heat than 
    the basic alkaloids, especially when dry.  However, the stability 
    of the alkaloids and  N-oxides in hot water as, for example, in 
    cooking, is not known. 

        Some pyrrolizidine alkaloids have a limited water solubility, 
    unless neutralized with acid; but others (e.g., indicine), and all 
    the  N-oxides, are readily soluble. 

    2.2  Analytical Methods

        When analysing for PAs, it is important to recognize that this 
    group consists of many different compounds (section 2.1) and that 
    these often occur as mixtures in plants or in materials of plant 
    origin.  They may vary in structure, relative molecular mass, 
    response to analytical procedures, and toxicity.  Both basic 
    alkaloids and corresponding  N-oxides may be present at the same 
    time.  Thus, where such mixtures are present, analyses will 
    inevitably be approximate, unless the individual components are 
    separated and identified. 

        Nevertheless, such estimates can be useful.  In particular, all 
    hepatotoxic PAs are unsaturated in the sense that they possess a 
    1:2-double bond in the pyrrolizidine nucleus, and analytical 
    methods that are specific for this structure can be of value in 
    screening for potential toxicity.  A simple qualitative field test 
    for screening plant materials for the presence of such alkaloids 
    and their  N-oxides, without the need of high technology equipment, 
    is described in section 2.2.2.5. 

    2.2.1  Extraction

    2.2.1.1  Plant tissue

        Pyrrolizidine alkaloids are usually extracted from dried, 
    milled plant material with hot or cold alcohol.  The alcohol is 
    evaporated, the bases taken up in dilute acid, and fats extracted 
    with ether or petroleum.  It is usual, at this stage, to reduce any 
     N-oxides present to the corresponding basic alkaloids with zinc, 
    before making the solution alkaline and extracting the alkaloids 
    with chloroform (Koekemoer & Warren, 1951).  Alternatively, alcohol 
    can be continuously circulated through the plant material and then 
    cation exchange resin, and the alkaloids subsequently eluted from 
    the resin (Mattocks, 1961; Deagen & Deinzer, 1977).  PAs can also 
    be extracted by soaking plant material in dilute aqueous acid 
    (Briggs et al., 1965; Craig et al., 1984). 

    2.2.1.2  Biological fluids and tissues

        Pyrrolizidine alkaloids have been extracted for analytical 
    purposes from honey (Deinzer et al., 1977), milk (Dickinson et al., 
    1976), blood-plasma (Ames & Powis, 1978; McComish et al., 1980), 
    urine (Mattocks, 1967a; Jago et al., 1969; Evans et al., 1979), and 
    bile (Jago et al., 1969; Lafranconi et al., 1985). 

        When attempting to isolate PAs from animal tissues, it must be 
    appreciated that the toxic alkaloids are often metabolized very 
    rapidly in animals, so that the amounts that are recoverable 
    (except from urine), only a few hours after alkaloid ingestion, may 
    be extremely small.  Various methods have been used to separate 
    PAs, but some mixtures are extremely difficult to separate.  On the 
    analytical scale, the most useful methods are thin-layer 
    chromatography (TLC), high-performance liquid chromatography 
    (HPLC), and gas chromatography (GC) (section 2.2.2). 

    2.2.2  Analysis for pyrrolizidine alkaloids

    2.2.2.1  Thin-layer chromatography (TLC)

        For TLC, silica plates are usually used, eluted with chloroform:
    methanol:aqueous ammonia mixtures (Sharma et al., 1965; Chalmers
    et al., 1965); solvents suitable for the  N-oxides, which 
    are more water-soluble, have been described by Mattocks (1967b)
    and Wagner et al. (1981).  The most sensitive methods for 
    detecting PAs on TLC are those using Ehrlich reagent
    (4-dimethylaminobenzaldehyde) (Mattocks, 1967b).  The unsaturated
    alkaloids are best visualized by spraying the plates first with a 
    solution of orthochloranil, then with Ehrlich reagent, heating 
    after each spray (Molyneux & Roitman, 1980).  The  N-oxides of 
    unsaturated pyrrolizidines are detected by spraying a solution of 
    acetic anhydride, heating the plate, and then spraying Ehrlich 
    reagent (Mattocks, 1967b). 

        Pyrrolizidine alkaloids with a saturated base moiety must be 
    detected in other ways (which are not specific for pyrrolizidines), 
    e.g., by exposing the dried plates to iodine vapour, or by spraying 
    with an iodobismuth (Dragendorff) reagent (Munier, 1953). 

    2.2.2.2  High-performance liquid chromatography (HPLC)

        Analytical or preparative scale HPLC separation of 
    pyrrolizidine alkaloids has been described by Segall (1979a,b) and 
    Dimenna et al. (1980), and an improved method has been reported by 
    Ramsdell & Buhler (1981).  Alkaloids from  Symphytum officinale  
    (comfrey) have been separated on an analytical scale by Tittel et 
    al. (1979), and partially separated on a preparative scale by 
    Huizing et al. (1981).  UV detectors are usually used for the HPLC 
    of pyrrolizidine compounds (Mattocks, 1986). 

    2.2.2.3  Gas chromatography (GC) and mass spectrometry (MS)

        The GC characterization of PAs using packed columns has been 
    described by Chalmers et al. (1965) and Wiedenfeld et al. (1981).  
    Mixtures of alkaloids from comfrey ( Symphytum sp.), normally hard 
    to separate, were resolved by Culvenor et al. (1980a) and Frahn et 
    al. (1980) by GC of the methylboronate derivatives. 

        Gas chromatography combined with mass spectrometry (GC-MS) has 
    become a valuable and highly sensitive means for both the 
    identification and the quantitative determination of pyrrolizidine 
    alkaloids.  Thus, alkaloids extracted from honey were separated and 
    identified by Deinzer et al. (1977) and (as butylboronate 
    derivatives) by Culvenor et al. (1981).  Deinzer et al. (1978) 
    described a method for the recognition (but not the individual 
    identification) of retronecine-based pyrrolizidine alkaloids, by 
    hydrolysing them to retronecine (the amino alcohol moiety) followed 
    by GC-MS of its bis-trifluoroacetate.  The use of capillary GC has 
    greatly improved the sensitivity of pyrrolizidine alkaloid 
    analysis, especially when used with MS (Luthy et al., 1981).  The 
    MS of pyrrolizidine compounds has been reviewed (Bull et al., 1968; 
    Mattocks, 1986). 

        Pyrrolizidine  N-oxides generally undergo thermal decomposition, 
    when subjected to GC, but they can first be reduced to the 
    corresponding basic alkaloids (Koekemoer & Warren, 1951).  
    Alternatively they may be derivatised.  Thus, trimethylsilylation 
    of indicine  N-oxide or heliotrine  N-oxide can lead either to the 
    trimethylsilyl (TMS) derivative of the parent alkaloid or to the 
    TMS derivative of the dehydro-alkaloid (pyrrolic derivative), 
    depending on the reagents used, and these products will run 
    successfully on GC-MS (Evans et al., 1979, 1980). 

    2.2.2.4  Nuclear magnetic resonance (NMR) spectrometry

        A convenient, but relatively insensitive, method, specifically 
    for the determination of unsaturated PAs, has been described by 
    Molyneux et al. (1979).  The basic alkaloids are extracted, then 
    subjected to NMR spectrometry along with an internal standard 

    ( p-dinitrobenzene).  This enables quantitative measurements to be 
    made of the signal(s) representing the H2 proton(s) in unsaturated 
    pyrrolizidines, and thus the alkaloid(s) can be determined.  
    Quantitative NMR analysis of pyrrolizidine alkaloid mixtures from 
     Senecio vulgaris has been described by Pieters & Vlietinck (1985) 
    and compared with an HPLC method by the same authors (1986). 
    Qualitative aspects of the NMR spectrometry of pyrrolizidine 
    alkaloids have been reviewed by Bull et al. (1968) and Mattocks 
    (1986). 

    2.2.2.5  The Ehrlich reaction

        This method (Mattocks, 1967a, 1968b) is specific for 
    unsaturated pyrrolizidine alkaloids and is not suitable for other 
    alkaloids.  Thus, it is the most useful colorimetric method for 
    potentially hepatotoxic pyrrolizidine compounds.  The procedure 
    converts the alkaloid into its  N-oxide, using hydrogen peroxide.  
    The product reacts with acetic anhydride to form a pyrrolic 
    derivative (dehydro-alkaloid) that gives a magenta colour with a 
    specially modified Ehrlich reagent.  The latter contains boron 
    trifluoride to give maximum sensitivity.  As little as 5 µg of most 
    unsaturated pyrrolizidines can be measured by this method.  If the 
    oxidation stage is omitted, only the unsaturated pyrrolizidine 
     N-oxides can be determined.  The determination of pyrrolizidine 
     N-oxides has also been discussed by Mattocks (1971b). 

        A simplification of the above colorimetric procedure was 
    described by Mattocks (1971d) to provide a qualitative test that 
    could be used to screen large numbers of plant samples for the 
    presence of unsaturated pyrrolizidine alkaloid  N-oxides.  An 
    improved version of this field test is now available (Mattocks & 
    Jukes, 1987).  It is suitable for any plant parts, such as leaves, 
    stems, flowers, seeds, or roots, or materials of plant origin, such 
    as cereals or herbal teas, but has not yet been applied to cooked 
    food. 

        The plant material (0.2 - 1 g) is extracted by grinding it with 
    aqueous ascorbic acid (5%) and a small amount of sand.  The 
    solution is filtered and divided into two equal portions ("test" 
    and "blank").  An aqueous solution (0.2 ml) of sodium nitroprusside 
    (5%) containing sodium hydroxide (10-3 mol) is added to the "test" 
    sample.  Both portions are heated for approximately 1 min at 70 - 
    80 °C; then Ehrlich reagent is added and heating is continued for 
    1 min.  The Ehrlich reagent contains 4-dimethylaminobenzaldehyde
    (5 g) dissolved in a mixture of acetic acid (60 ml), water (30 ml), 
    and 60% perchloric acid (10 ml).  A magenta colour in the "test" 
    compared with the "blank" indicates the presence of an unsaturated 
    PA  N-oxide.  The "blank" may show a colour if the plant contains 
    compounds, such as indoles or pyrroles, which can themselves give a 
    colour with Ehrlich reagent.  The intensity of colour in the 
    "sample" compared with the "blank" can give a rough idea of the 
    amount of alkaloids present, and indicate whether further chemical 
    or toxicological testing of the plant material is adviseable. 

        In practice, the majority of PA-containing plants contain 
    enough alkaloid in the  N-oxide form (often a large proportion) to 
    react positively in this test.  The main exceptions are some seeds 
     (Crotalaria), which may contain much alkaloid base, but little or 
    no  N-oxide.  These (and any other sample not containing 
    chlorophyll) can be tested for basic PAs by grinding them with 
    chloroform, heating the filtered extract with a solution (0.1 ml) 
    of orthochloranil (0.5%) in acetonitrile, and then heating it with 
    Ehrlich reagent.  A magenta colour indicates the presence of an 
    unsaturated PA.  Non-toxic pyrrolizidine alkaloids having a 
    saturated pyrrolizidine nucleus, and pyrrolizidine alkaloids that 
    are otonecine esters, such as petasitenine, will not respond to 
    this test. 

    2.2.2.6  Indicator dyes

        A method generally applicable to tertiary bases has been 
    adapted for pyrrolizidine alkaloids by Birecka et al. (1981).  It 
    is sensitive, but is not specific for this group of alkaloids, and 
    it does not distinguish between the saturated and unsaturated 
    alkaloids.  A chloroform solution of the alkaloid is shaken with 
    acidified aqueous methyl orange.  The yellow alkaloid:dye complex 
    is subsequently released from the chloroform phase, using ethanolic 
    sulfuric acid, and measured spectrophotometrically. 

    2.2.2.7  Direct weighing

        An insensitive way to determine the alkaloids in, for example, 
    a plant sample, providing enough is available, is to extract the 
    alkaloids (section 2.2.1) and weigh them.  This will provide a 
    rough measure of the total bases present in the sample; however, 
    these may not necessarily be PAs.  Nevertheless, the sample can 
    then be subjected to further tests, e.g., GC-MC, nuclear magnetic 
    resonance (NMR), or colorimetric analysis.  Furthermore, 
    pyrrolizidine  N-oxides are generally too water soluble to be 
    appreciably extractable from aqueous solution by chloroform.  Thus, 
    if two portions of the sample are extracted, and one of them is 
    reduced to convert  N-oxides to bases, the weight difference between 
    the two products will represent the alkaloid existing in the form 
    of  N-oxide in the original sample. 

    2.3  Determination of Metabolites in Animal Tissues

        Important metabolites of toxic pyrrolizidine alkaloids in 
    animals include "pyrrolic" derivatives (dehydro-alkaloids) and 
     N-oxides.  A procedure for measuring pyrrolic metabolites in tissue 
    samples (such as liver or lung) has been described by Mattocks & 
    White (1970).  The sample (usually 0.5 g) is homogenized in an 
    ethanolic solution of mercuric chloride; the solids are separated 
    by centrifugation and heated with Ehrlich reagent to give a soluble 
    colour that can be measured spectrophotometrically. 

        The measurement of pyrrolic and  N-oxide metabolites, formed by 
    the action of hepatic microsomal preparations on PAs  in vitro, is 
    an improvement described by Mattocks & Bird (1983). 

    3.  SOURCES AND PATHWAYS OF EXPOSURE

    3.1  Hepatotoxic Pyrrolizidine Alkaloids and Their Sources

        Plants constitute the only natural source of pyrrolizidine 
    alkaloids (PAs) that cause toxic reactions in man and animals.  PAs 
    occur in a number of species in the families Boraginaceae, 
    Compositae, Leguminosae (genus  Crotalaria), Ranunculaceae (genus 
     Caltha), and Scrophulariaceae (genus  Castilleja) (Table 1).  The 
    most important genera of PA-containing toxic plants are  Crotalaria  
    (Leguminosae),  Senecio (Compositae),  Heliotropium, Trichodesma, 
     Amsinckia, Echium, and  Symphytum (Boraginaceae) (Hooper, 1978).  
    The recorded cases of human toxicity have mainly been caused by at 
    least 12 different pyrrolizidine alkaloids, mostly derived from 
     Heliotropium, Senecio, and  Crotalaria genera.  The  Senecio spp. 
    grow throughout the world; the  Crotalaria spp. are mainly found in 
    the tropics and subtropics (Culvenor, 1980). 
        Table 1.  List of plant genera containing toxic pyrrolizidine alkaloids 
              (with number of species investigated)
    -------------------------------------------------------------------------------------
    Family            Genera
    -------------------------------------------------------------------------------------
    Apocynaceae        Fernaldia (1),  Parsonsia (4),                       

    Boraginaceae       Alkanna (1),  Amsinckia (4),  Anchusa (2),  Asperugo (1),  Borago (1), 
                       Caccinia (1),  Cynoglossum (9),  Echium (3),  Hackelia (1), 
                       Heliotropium (25),  Lappula (2),  Lindelofia (7),  Lithosperum (1),  
                       Macrotomia (1),  Messerschmidtia (1),  Myosotis (2),  Paracaryum (1), 
                       Paracynoglossum (1),  Rindera (5),  Solenanthus (4),  Symphytum (7), 
                       Tournefortia (2),  Trachelanthus (2),  Trichodesma (2),  Ulugbekia (1)

    Compositae         Adenostyles (3),  Brachyglottis (1),  Cacalia (4),  Conoclinium (1), 
                       Crassocephalum (1),  Doronicum (2),  Echinacea (2),  Emilia (2), 
                       Erechtites (1),  Eupatorium (8),  Farfugium (1),  Gynura (2), 
                       Ligularia (5),  Petasites (4),  Senecio (142),  Syneilesis (1), 
                       Tussilago (1)                       

    Leguminosae        Crotalaria (60)                                     

    Ranunculaceae      Caltha (2)                                          

    Scrophulariaceae  Castilleja (1)                                      
    -------------------------------------------------------------------------------------
            An alphabetical list of pyrrolizidine alkaloids with their 
    plant sources has been published by Smith & Culvenor (1981) and 
    Mattocks (1986).  An updated version is attached as Appendix I.  
    The plant genera containing toxic PAs are listed in Table 1 
    indicating the number of species investigated for PAs.  A 
    comprehensive list of species of plants belonging to each of these 
    genera, the alkaloids isolated from each, and the part of the plant 
    containing the alkaloid are presented in Appendix II.  Table 1 in 
    Appendix II includes species known to contain alkaloids of proved 
    hepatotoxicity, or of a molecular structure that would make them 
    very probably hepatotoxic.  Table 2 in Appendix II includes species

    containing pyrrolizidine amino-alcohols or esters, which, while not 
    having all the features of hepatotoxicity, would need only minor 
    structural modifications to render them hepatotoxic.  Plants of the 
    same taxonomic groups as the plants of proven hepatotoxicity are 
    listed in part (a) of the table.  There is a possibility that, on 
    further examination, hepatotoxic alkaloids may be found, as minor 
    constituents, in strains or parts of these plants not yet 
    investigated or under specific conditions of growth.  It should be 
    noted that the species that have been investigated and are listed 
    are only few compared with the total number of species in each 
    genera.  It has been recommended by Smith & Culvenor (1981) that it 
    would be prudent to regard all species in the family Boraginaceae 
    and the genera  Crotalaria, Senecio, and  Eupatorium as potentially 
    hepatotoxic. 

        It is pertinent to note that the alkaloid content in different 
    parts of the plant (e.g., roots, leaves, stalks, flowers, and buds) 
    varies and is subject to fluctuations according to the climate, 
    soil conditions, and time of harvesting (Danninger et al., 1983; 
    Hartmann & Zimmer, 1986).  Mattocks (1980) demonstrated that the 
    alkaloid content of the leaves of  Symphytum spp. (Russian 
    comfrey), which are used as an item of food, varies with their 
    maturity.  The toxic PA content is highest at the beginning of the 
    vegetative period and declines as the leaves mature.  The PA 
    content of the roots is much higher than that of the leaves, and 
    dried leaves contain a higher concentration than fresh leaves 
    (Mattocks, 1986).  According to Danninger et al. (1983), in some 
    species  (Symphytum asperum), relatively long storage may lead to a 
    reduction in the alkaloid content, presumably because enzymes are 
    released during drying.  Candrian et al. (1984b) studied the 
    stability of PAs in hay and silage containing various amounts of 
     Senecio alpinus.  The PA content of hay remained constant for 
    several months, but the PAs in silage were mainly degraded.  
    However, the degradation of PAs was much less complete in the lower 
    concentration range.  A quantitatively significant PA-degradation 
    product in silage was identified as retronecine.  Silage with an
     S. alpinus percentage of 3.5 - 23 still contained macrocyclic PAs at 
    a concentration of about 20 mg/kg wet weight.  Such silage was not 
    considered safe for cattle bearing in mind that a 600-kg calf eats 
    about 30 kg silage/day, amounting approximately to a daily intake 
    of about 1 mg PAs/kg body weight.  In feeding trials with  Senecio 
     jacobaea, Johnson (1979) found that the minimum lethal dose for 
    cattle was between 1 and 2 mg PAs/kg body weight per day. 

        PAs known to have been associated with instances of human toxic 
    liver disease in different parts of the world are listed in Table 
    2.  Two groups of alkaloids that, according to Culvenor (1983), are 
    consumed in significant amounts by people in different parts of the 
    world include: 

        (a)   Echimidine, acetyllycopsamine, and related alkaloids
              (many countries)

        Leaves of plants of the  Symphytum sp. ( Symphytum officinale  
    (comfrey) and  Symphytum x uplandicum) are used traditionally as a 
    salad and as a medicinal herb in Australia, many countries of 

    Europe, and the USA.   S. officinale has been shown to be 
    carcinogenic for rats (Hirono et al., 1978).  Leaves of Russian 
    comfrey contain a concentration of alkaloids (mainly echimidine) of 
    0.1 - 1.5 g/kg.  The highest level of daily consumption of the 
    alkaloids has been estimated to be 5 - 6 mg (Culvenor, 1983). 

        (b)   Echimidine and related alkaloids (Australia)

        PAs derived from  Echium plantagineum, with echimidine as the 
    major component, have been found in honey secreted by bees feeding 
    on the plant (Culvenor et al., 1981).  The plant is a major source 
    of honey (section 3.3.4). 

    3.2  Pneumotoxic and Other Toxic Pyrrolizidine Alkaloids

        Not all hepatotoxic alkaloids are pneumotoxic.  The commonest 
    ones used to produce experimental lung injury are fulvine (Barnes 
    et al., 1964; Kay et al., 1971a; Wagenvoort et al., 1974a,b) and 
    monocrotaline (Lalich & Ehrhart, 1962; Chesney & Allen, 1973b; 
    Huxtable et al., 1977).  These are also the most active (Mattocks, 
    1986).  The seeds of  Crotalaria spectabilis, which contain 
    monocrotaline, have also been used to study pneumotoxic effects on 
    experimental animals (Turner & Lalich, 1965; Kay & Heath, 1966; Kay 
    et al., 1967a) and  C. spectabilis has been called the pulmonary 
    hypertension plant (Kay & Heath, 1969), because of the pulmonary 
    hypertensionogenic properties of the PAs it contains.  Culvenor et 
    al. (1976a) screened 62 PAs for hepatotoxicity and pneumotoxicity.  
    Chronic lung lesions were produced by most compounds that induced 
    chronic liver lesions, though high doses were required in some 
    instances.  It is possible that chronic lung lesions may not occur 
    in experimental animals because of early death due to acute 
    toxicity.  However, the authors identified a number of PAs that 
    were particularly prone to produce chronic lung damage in rats 
    including crispatine, senecionine, seneciphylline, and usaramine 
    (12-membered macrocyclic, retronecine diesters), anacrotine and 
    madurensine (crotonecine esters), and the heliotridine esters, 
    heliosupine, lasiocarpine, and rinderine. 

        The molecular structure-activity requirements for 
    pneumotoxicity are the same as those for hepatotoxicity.  This is 
    consistent with their both being caused by the same toxic 
    metabolites and by the metabolic activation of the alkaloids in the 
    liver cells to form a reactive pyrrolic dehydro-alkaloid (Culvenor 
    et al., 1976a). 

        Trichodesmine and incanine, found in the seeds of  Trichodesma 
     incanum (Yunusov & Plekhanova, 1959), are believed to have been 
    the causative factors of the "Ozhalangar encephalitis" that was 
    endemic in Uzbekistan, USSR (1942 - 51), in which the symptoms and 
    signs were related primarily to the central nervous system 
    (Shtenberg & Orlova, 1955) (section 7.7). 

        Table 2.  Instances of human toxicity caused by pyrrolizidine alkaloidsa

                                                                                                          
    Principal              Plant               Country/         Cause of intake     Reference
    alkaloid                                   Region
                                                                                                          

    Heliotrine and         Heliotropium        Afghanistan      contamination       Tandon & Tandon
     other alkaloids       popovii                                                  (1975); Tandon,
     similar to                                                                     B.N. et al.
     lasiocarpine                                                                   (1978); Tandon,
                                                                                    H.D. et al.
                                                                                    (1978);
                                                                                    Mohabbat et al.
                                                                                    (1976)

    Senecionine            Senecio             South            contamination       Wilmot &
                           illiciformis;       Africa                               Robertson
                           Senecio-burchelli                                        (1920)

                           Senecio spp.        South            contamination       Selzer &
                                               Africa                               Parker (1951)

    Alkaloids of           Crotalaria          Ecuador          medicine            Lyford et al.
     trichodesmine         juncea                                                   (1976)
     and senecionine
     type

    Heliotrine and         Heliotropium        Hong Kong        medicine            Kumana et al.
    lasiocarpine           lasiocarpum                                              (1985);
                                                                                    Culvenor et al.
                                                                                    (1986)
                                                                                                          
    Table 2.  (cont'd)

                                                                                                          
    Principal              Plant               Country/         Cause of intake     Reference
    alkaloid                                   Region
                                                                                                          

    Crotananine and        Crotalaria          India            contamination       Tandon, R.K.
     cronaburmine          nana                                                     et al. (1976);
                                                                                    Krishnamachari
                                                                                    et al. (1977);
                                                                                    Siddiqui et al.
                                                                                    (1978a,b)

    Heliotrine             Heliotropium        India            medicine            Datta et al.
     N-oxide               eichwaldii                                               (1978a,b)

    Monocrotaline          Crotalaria          West Indies      medicine            Bras et al.
     fulvine               retusa;                                                  (1954, 1957)
                           Crotalaria
                           fulva                                                    Stuart & Bras
                                                                                    (1957)

                           Ilex sp.            United           medicine            McGee et al.
                                               Kingdom                              (1976)

    Riddelline             Senecio             USA              medicine            Stillman et al.
     retrorsine            longilobus                                               (1977); Fox et
     N-oxide                                                                        al. (1978);
     (with others)                                                                  Huxtable (1980)

    Indicine N-oxide       purified            USA              medicine            Letendre et al.
                           chemical                                                 (1984)

    Symphytine,            Symphytum sp.       USA              medicine            Ridker et al.
     symglandine, and                                                               (1985);
     other symphytum                                                                Huxtable
     alkaloids                                                                      et al (1986)
                                                                                                          
    Table 2.  (cont'd)

                                                                                                          
    Principal              Plant               Country/         Cause of intake     Reference
    alkaloid                                   Region
                                                                                                          

    Lasiocarpine and       Heliotropium        USSR             contamination       Dubrovinskii
     heliotrine            lasiocarpum                                              (1952);
                                                                                    Mnushkin
                                                                                    (1952)

    Trichodesmine and      Trichodesma         USSR             contamination       Shtenberg &
     incanine              incanum                                                  Orlova (1955);
                                                                                    Yunosov &
                                                                                    Plekhanova
                                                                                    (1959)
                                                                                                          

    a  Adapted from: Culvenor (1983) and Mattocks (1986). Refer also to Table 15 for
       details and section 7.
    
    3.3  Pathways of Exposure

        Naturally-occurring animal disease is caused by the alkaloid-
    containing plants growing in fields and pastures or being fed 
    accidentally as fodder.  They are mostly herbaceous or small shrubs 
    and many thrive in dry and arid climates.  One such plant 
    containing toxic PA alkaloids has been reported to grow in the 
    western desert of Egypt (Hammouda et al., 1984).  The growth of 
    this group of plants is particularly prolific during, and 
    following, periods of drought, as has been reported in association 
    with the outbreaks of human disease in Afghanistan (Tandon & 
    Tandon, 1975; Mohabbat et al., 1976) and India (Tandon, B.N. et 
    al., 1976).  Alkaloid-containing plants are widespread in the 
    tropics, especially  Crotalaria, of which there are over 300 
    species in Africa.  Ordinarily, the alkaloid-containing plants have 
    a bitter taste and grazing animals will reject them, unless their 
    normal fodder is scarce.  However, PAs often occur largely as 
     N-oxides, which are said not to be bitter, and plants containing 
    PAs are readily eaten by some animal species. 

        Human intoxication may result from the ingestion of the toxic 
    substance in either contamined food or herbal infusion. 

    3.3.1  Contamination of staple food crops

        The products of pyrrolizidine alkaloid-containing plants, 
    generally seeds, may contaminate the staple food and may be eaten 
    over long periods of time.  The fact that these plants may cause 
    disease is generally not recognized by the people and such 
    contamination is known to have resulted in large-scale outbreaks of 
    poisoning (Dubrovinskii, 1952; Mnushkin, 1952; Shtenberg & Orlova, 
    1955; Tandon & Tandon, 1975; Mohabbat et al., 1976; Tandon, B.N. et 
    al., 1976, 1977; Tandon, R.K. et al., 1976; Krishnamachari et al., 
    1977; Tandon, H.D. et al., 1977) (Table 2, section 3.1). 

    3.3.2  Herbal infusions

        Plants have been used traditionally for medicinal purposes all 
    over the world.  Herbs have been the mainstay of the indigenous 
    systems of medicine, especially in China, Greece, and India, since 
    ancient times.  Table 3 includes a list of some plants that are 
    suspected, or known, to contain PAs and have been used as herbal 
    medicines in different countries (Mattocks, 1986). 

        Several PA-containing plants are included among the list of 
    plants used in indigenous systems of medicine in India (Chopra, 
    1933).  As a part of a research study on the etiological factors of 
    chronic liver disease in Sri Lanka, Arseculeratne et al. (1981) 
    chemically screened the first 50 plants used in Sri Lanka's 
    traditional medicine pharmacopoaea, and found that 3 of them 

    contained PAs.  All 3 were hepatotoxic in rats.  Of the 3, the 
    presence of alkaloids in  Cassia auriculata  and that of PAs in 
     Hollarhena antidysenterica  had not previously been recorded.  It 
    should be noted that the amount of experimental plant material used 
    in this study was approximately 6.5 g/kg body weight per day, in 
    contrast to the approximate intake by a human being estimated to be 
    in the range of 0.3 - 0.6 g/kg body weight per day.  Some, but not 
    all, of the plants reported to be etiological agents in human cases 
    of veno-occlusive disease can be found in an inventory of medicinal 
    plants used in different countries (WHO, 1980), which also 
    indicates the countries that they are used in.  The above lists may 
    not be complete as many such plants may be used in folk medicine 
    but have not been mentioned in the scientific literature.  However, 
    the lists do indicate the wide and varied use of such toxic herbs 
    in all parts of the world. 

        Lately, there has been a growing interest in the developed 
    countries in organically grown products for food, as well as home 
    remedies (Table 3), and some of the PA-containing herbs have been 
    freely available in herbal shops (Schoental, 1968; Burns, 1972).  
    Danninger et al. (1983) listed plants containing PAs that are 
    commonly used in the Federal Republic of Germany as medicaments 
    (Table 4).  He also listed 9 plants in which alkaloids have only 
    been identified qualitatively, the toxicity of which has not been, 
    or has been insufficiently, investigated (Table 5).  Similarly, 
    Roitman (1983) listed 10 plants, in which the presence of PAs is 
    suspected or has been proved and which are used as herbal teas in 
    the USA.  The lists include 10 plants containing PAs, most of which 
    have been proved hepatotoxic experimentally, some having highly 
    carcinogenic promoter activity.  Some of these alkaloids have been 
    associated with human case reports of PA toxicity.  The more recent 
    reports (Table 2) of instances of PA poisoning through the use of 
    herbal medicines are from developed countries (Lyford et al., 1976; 
    Stillman et al., 1977; Fox et al., 1978; Kumana et al., 1985; 
    Ridker et al., 1985).  Such use of the herbs is the reason that 
    veno-occlusive disease is endemic in Jamaica (Bras et al., 1954; 
    Jellife et al., 1954a,b; Bras & Watler, 1955; Stuart & Bras, 1955, 
    1957).  There are obvious difficulties in exercising any kind of 
    control to restrict this use only to plants that have been tested 
    and certified as safe for human use.  It is impossible to identify 
    many such herbs, as they are sold as plants or their amorphous 
    products in the herbal shops. 

        Table 3.  Some plants containing (or suspected of containing) PAs, which have been used
              by people either as herbal medicines (M) or foods (F)
                                                                                                  

    Plant                    Mode      Country             Referencea
                             of use    or region
                                                                                                  

    BORAGINACEAE

    Anchusa officinalis      M         Europe              Broch-Due & Aasen (1980)           B

    Borago officinalis       M         USA                 Delorme et al. (1977)              A

    Cynoglossum              M         East Africa         Schoental & Coady (1968)           A
      geometricum

    Cynoglossum              M         Iran                Coady (1973)                       B
      officinale

    Heliotropium             M         India               Gandhi et al. (1966a);             B
      eichwaldii                                           Datta et al. (1978a,b)             A

    H. europaeum             M         India, Greece       IARC (1976)                        A

    H. lasiocarpum           M         Hong Kong           Kumana et al. (1985);              A
                                                           Culvenor et al. (1986)             A

    H. indicum               M         India, Africa,      Schoental (1968a);                 B
                                       South America,      Hoque et al. (1976)                B
                                       and elsewhere

    H. ramossissimum         M         Arabia              Macksad et al. (1970);             B
      (ramram)                                             Coady (1973)                       B

    H. supinum               M         Tanzania            Schoental & Coady (1968)           A
                                                                                                  

    Table 3 (contd.)
                                                                                                  

    Plant                    Mode      Country             Referencea
                             of use    or region
                                                                                                  

    Pulmonaria spp.          M         USA                 Delorme et al. (1977)              A

    Symphytum officinale     F, M      Japan and           Hirono et al. (1978, 1979b)        A
                             M         USA                 Furuya & Hikichi (1971);           A
                                                           Delorme et al. (1977)              A

    S. x uplandicum          F, M      General             Hills (1976)                       B

                                       USA                 Culvenor et al. (1980a,b)          A

    S. asperum               M         USA                 Pedersen (1975)                    A

    COMPOSITAE

    Cacalia decomposita      M         USA                 Sullivan (1981)                    B
      (matarique)

    C. yatabei               F         Japan               Hikichi & Furuya (1978)            B

    Farfugium japonicum      M         Japan               Furuya et al. (1971)               B

    Ligularia dentata        F         Japan               Asada & Furuya (1984)              B

    Petasites japonicus      F, M      Japan               Hirono et al. (1973, 1979b)        A

    Senecio abyssinicus      M         Nigeria             Williams & Schoental (1970)        B

    S. aureus                M         USA                 Wade (1977)                        B
                                                                                                  
    Table 3 (contd.)
                                                                                                  

    Plant                    Mode      Country             Referencea
                             of use    or region
                                                                                                  

    S. bupleuroides          M         Africa              Watt & Breyer-Brandwijk (1962)     A

    S. burchelli             F, M      South Africa        Rose (1972)                        A

    S. coronatus             M         South Africa        Rose (1972)                        A

    S. discolor              M         Jamaica             Asprey & Thornton (1955)           B

    S. doronicum             M         Germany             Roeder et al. (1980a)              B

    S. inaequidens           F         South Africa        Rose (1972)                        B

    S. jacobaea              M         Europe              Schoental & Pullinger (1972);      B
      (ragwort)                                            Wade (1977)                        B

    S. longilobus            M         USA                 Stillman et al. (1977);            A
      (S. douglassi)                                       Huxtable (1979a)                   B

    S. monoensis             M         USA                 Huxtable (1980)                    A

    S. nemorensis            M         Germany             Habs et al. (1982)                 A
      spp. fuchsii

    S. pierotti              F         Japan               Asada & Furuya (1982)              B

    S. retrorsus             M         South Africa        Rose (1972)                        A
      (S. latifolius)
                                                                                                  
    Table 3 (contd.)
                                                                                                  

    Plant                    Mode      Country             Referencea
                             of use    or region
                                                                                                  

    S. vulgaris              M         Europe              Watt & Breyer-Brandwijk (1962)     A
      (common groundsel)

                                       Netherlands         Wade (1977)                        B

                             M         Iran                Coady (1973)                       B

    Syneilesis palmata       F         Japan               Hikichi & Furuya (1976)            B

    Trichodesma africana     M         Asia                Omar et al. (1983)                 B

    Tussilago farfara        M         Japan               Culvenor et al. (1976a)            A
      (coltsfoot)

                             M         China               Hirono et al. (1976b)              A

                             M         Norway              Borka & Onshuus (1979)             B

                             M         USA                 Borka & Onshuus (1979);            B
                                                           Culvenor et al. (1976b);           B

    LEGUMINOSAE

    Crotalaria brevidens     F         East Africa         Coady (1973)                       B

    C. fulva                 M         Jamaica             Barnes et al. (1964);              A
                                                           McLean (1970, 1974)                A
                                                                                                  
    Table 3 (contd.)
                                                                                                  

    Plant                    Mode      Country             Referencea
                             of use    or region
                                                                                                  

    C. incana                M         East Africa         Schoental & Coady (1968)           A
                                                           Watt & Breyer-Brandwijk            A
                                                           (1962)

    C. laburnifolia          M         Tanzania            Schoental & Coady (1968)           A

                             F         Asia                Coady (1973)                       B

    C. mucronata             M         Tanzania            Coady (1973)                       B

    C. recta                 M, F      Tanzania            Schoental & Coady (1968);          A
                                                           Coady (1973)                       B

    C. retusa                M, F      Africa              IARC (1976)                        A

                                       India               Watt & Breyer-Brandwijk (1962)     A

    C. verrucosa             M         Sri Lanka           Arseculeratne et al. (1981)        A
                                                                                                  

    a  A = Reference in the reference list of this document.
       B = Reference in Mattocks (1986).
    
        Manufactured preparations may also contain PA-containing herbs, 
    e.g., comfrey-pepsin capsules sold as a digestive aid (Huxtable et 
    al., 1986). 

    3.3.3  Use of PA-containing plants as food

        Several PA-containing plants are used as food as can be seen in 
    Table 3 (Mattocks, 1986).   Petasites japonicus Maxim,  Tussilago 
     farfara L. (coltsfoot), and  Symphytum officinale L. (comfrey or 
    Russian comfrey) are known as edible plants in Japan, and have been 
    proved to contain carcinogenic pyrrolizidine alkaloids (Hirono et 
    al., 1973, 1979a,b).  The young flower-stalks of  P. japonicus and 
    the buds of coltsfoot have been used in Japan as human food or 
    herbal remedies.  The leaf and root of comfrey are also used as an 
    edible vegetable or tonic (Hirono et al., 1978) in Japan and other 
    countries (Culvenor, 1985).  The carcinogenic PAs in these plants 
    are petasitenine  (P. japonicus), senkirkine (coltsfoot), and the 
    group including symphytine (comfrey). They were also mutagenic in 
    the Ames system of  Salmonella typhimurium and V79 hamster cell line 
    and induced transformation in cryo-preserved hamster embryonic 
    cells (Hirono et al., 1979b).  Other such PA-containing plants, 
    used as food in Japan, include young leaves of  Syneilesis palmata, 
    various  Cacalia species, and young  Senecio pierotti (Mattocks, 
    1986).  According to Culvenor (1985), consumers of comfrey could be 
    ingesting up to 5 mg PAs per day.  Rose (1972) listed a number of 
    plants of the genus  Senecio that are used as spinach in South 
    Africa.  These include  S. burchelli, which is known to have caused 
    an episode of PA poisoning through the ingestion of contaminated 
    bread (Wilmot & Robertson, 1920). 

    3.3.4  Contaminated honey

        In the USA, Deinzer et al. (1977) reported the presence of all 
    PAs contained in  Senecio jacobaea (ragwort) and proved to be 
    hepatotoxic, in the honey secreted by bees feeding on the plant.  
    The total alkaloid content ranged from 0.3 to 3.9 mg/kg.  It has 
    been estimated that an average annual human intake of honey (600 g) 
    at the highest alkaloid level quoted would contain less than 3 mg 
    of PAs (Mattocks, 1986).  Culvenor et al. (1981) and Culvenor 
    (1983, 1985) drew attention to the same potential hazard in honey 
    from  Echium plantagineum, a weed that grows widely in Southern 
    Australia and is a major source of honey, yielding an estimated 
    2000 - 3000 tonnes per annum for human consumption.  Echimidine is 
    the major component of the alkaloids of  Echium, which are present 
    in concentrations of up to 1 mg/kg.  Culvenor (1983) estimated that 
    individuals may consume up to 80 g honey/day with a corresponding 
    alkaloid intake of 80 µg/day, if only the  Echium honey were used.  
    No reports of acute human toxicity through this source are 
    available. 


    
        Table 4.  Medicinal plants containing PAs of known hepatotoxicity, reported as commonly
              used in the Federal Republic of Germany, and the PAs contained in thema
                                                                                             

    Family         Genus                    Species                  Pyrrolizidine
                                                                     alkaloids
                                                                                             

    Compositae     Eupatorium               E. cannabinum            amabiline±
                                            (hemp agrimony)          supinineb

                   Petasites                P. hybirdus              senecionineb,c
                                                                     integerrimineb
                                                                     senkirkineb

                   Senecio                  S. nemorensis            fuchsisenecionine
                   (groundsel)              sp. fuchsii              senecionineb,c
                                            (Fuch's groundsel)

                                            S. vulgaris              senecionineb,c
                                            (groundsel)              seneciophyllineb
                                                                     retrorsineb
                                                                     riddellineb,c

                                            S. Jacobaea              jacobineb
                                            (ragwort)                senecionineb,c
                                                                     seneciphyllineb
                                                                     jacoline, jaconine
                                                                     chlorinated PAsd

                                            S. aureus                senecionineb,c
                                            (American golden
                                             ragwort)

                   Tussilago                T. farfara               senkirkineb
                   (coltsfoot)              (coltsfoot)              senecionineb,c
                                                                     tussilagine

                                                                                             

    Table 4 (contd.)
                                                                                             

    Family         Genus                    Species                  Pyrrolizidine
                                                                     alkaloids
                                                                                             

                   Alkanna                  A. tinctoria             7-angelylretronecine
                                                                       triangularine
                                                                     dihydroxytriangularine

                   Anchusa                  A. officinalis           lycopsamine

    Boraginaceae   Borago                   B. officinalis           lycopsamine/intermedine±
                                            (borage)                 acetyllycopsamine/
                                                                       acetylintermedine
                                                                     amabiline
                                                                     supinine

                   Symphytum                S. officinale            symphytineb
                   (comfrey)                (comfrey)                echimidine(?)
                                                                     lycopsamine
                                                                     acetyllycopsamineb
                                                                     lasiocarpineb,c
                                                                     heliosupine N-oxide

                                            S. peregrinum            lycopsamineb
                                            S. x uplandicum          intermedineb
                                                                     symphytineb
                                                                     echimidineb
                                                                     7-acetyllycopsamine
                                                                     7-acetylintermedine
                                                                     symlandine
                                                                     uplandicine

                                            S. asperum               asperumine
                                            (prickly comfrey)        heliosupine N-oxide
                                                                     echimidineb
                                                                     echinatine
                                                                                             

    Table 4 (contd.)
                                                                                             

    Family         Genus                    Species                  Pyrrolizidine
                                                                     alkaloids
                                                                                             

                   Cynoglossum              C. officinale            heliosupine N-oxide
                   (hound's                 (hound's tongue)           echinatine
                    tongue)                                          acetyl heliosupineb
                                                                     O-7-angelylhelio-
                                                                       tridineb

                   Heliotropium             H. europaeum             heliotrineb,c,e
                   (Heliotrope)             (common heliotrope)      lasiocarpineb,c,e
                                                                     supinine
                                                                     heleurine
                                                                     europine
                                                                     acetyllasiocarpineb
                                                                                             

    a  Modified from: Danninger et al. (1983).
    b  Toxic alkaloids.
    c  Alkaloids known to have caused human toxicity.
    d  Alkaloids with highly carcinogenic promoter activity.
    e  Used only in homeopathy.
    
    Table 5.  Medicinal plants containing PAs, reported as 
              commonly used in the Federal Republic of Germany,
              the toxicity of which has not been, or has been
              insufficiently, investigateda
    -----------------------------------------------------------
    Family        Genus          Species
    -----------------------------------------------------------
    Compositae     Eupatorium     E. perforatum                     

                   Brachyglottis  B. repens                         

                   Arnica         A. montana (mountain arnica)      

    Boraginaceae   Lappula        L. intermedia (stickseed)         

                   Pulmonaria     P. officinalis (lungwort)         
    -----------------------------------------------------------
    a Modified from:  Danninger et al. (1983).

    3.3.5  Milk

        PAs have been shown to produce toxic effects via transference 
    into the milk of dams (Schoental, 1959).  Retrorsine was 
    administered orally to 17, and intraperitoneally to 6, lactating 
    rats weighing 185 - 350 g in 5 - 10 mg doses daily, the first dose 
    being given during the first 24 h after parturition.  The rats 
    received from 1 to 14 doses, the total intake amounting to
    21 - 335  mg/kg body weight.  The litters were separated from the
    mothers for ´ h following the administration of PA to avoid direct 
    contamination of the former by licking.  Apparently the milk 
    production was not affected as the stomachs of many of the young, 
    examined postmortem, were distended with milk.  All animals whose 
    mothers had received a total dose of 138 mg PA or more died within 
    30 days.  Many of the young whose mothers had received smaller 
    doses survived until they were killed at 6 months.  Biopsy of the 
    liver of the young at various intervals or at autopsy showed marked 
    changes, even in cases where the mothers did not appear to be 
    affected.  Animals dying at 18 - 30 days showed hydropic or fatty 
    vacuolation of liver cells.  In the liver of animals dying or 
    killed later, various degrees of haemorrhagic necrosis and increase 
    in the centrilobular reticulin of the liver, and some thickening of 
    centrilobular veins were seen.  In animals that survived 6 months, 
    the appearance was less abnormal, but some hyperplastic nodules and 
    bile-duct proliferation were seen.  The lactating rats dosed with 
    the PAs generally survived longer than the suckling animals and 
    usually did not show any ill effects, suggesting that the 
    susceptibility of the suckling rats was greater than that of the 
    mothers. 

        Dickinson et al. (1976) demonstrated the presence of PAs in the 
    milk of dairy cattle fed or dosed with ragwort  (Senecio jacobaea).
    When 4 cows were administered the dried plant material at levels of 
    up to 10 g/kg body weight per day through rumen cannula, PA levels 
    of up to 0.84 mg/kg were observed in the milk.  However, only one 

    (jacoline) of the several PAs contained in the plant was secreted.  
    Calves, bucket fed on the milk did not show any signs of PA 
    toxicity. 

        Dickinson (1980) repeated the study on goats.  Four milk goats 
    were freshly prepared with rumen cannulae.  The kids were separated 
    from their dams and were fed milk twice a day.  Dried tansy ragwort 
    plant material with a PA content of 0.16% (dry weight) was 
    administered through the cannulae to each goat at a dosage rate of 
    10 g/kg body weight per day over 125 days.  During this period, 
    each of the 4 kids received milk from their dams at approximately 
    125 ml/kg per day in addition to  ad lib feeding on alfalfa grass 
    hay.  Six PAs were isolated from the plant material:  jacobine, 
    jaconine, jaconline, jacozine, senecionine, and seneciphylline.  
    Milk samples collected twice daily showed PA contents of
    225 - 530 µg/litre with a mean of 381 µg/litre.  No apparent health
    effects were noted in the kids, and only mild hepatic damage was
    suspected in the dams, on the basis of liver function tests.  Fifty
    percent of the kids were killed after 10 weeks.  No lesions of PA
    toxicity were seen.  The dams were rebred and appeared normal
    throughout the gestation period.  However, three dams aborted at
    almost full term, and the fetuses were born dead. One of the dams
    died shortly after parturition and showed evidence of severe liver
    damage characteristic of PA toxicity.  Another, which delivered
    normally, also showed a lesser degree of liver damage at biopsy. 

        Data relating to PA secretion were compared with similar 
    earlier data on cows.  Mean secretion of PAs in cows appeared much 
    higher, e.g., 684 µg/litre.  The authors concluded that the amount 
    of PAs secreted in the goat's milk did not cause any serious 
    deleterious effects in the kids. 

        Johnson (1976) fed long-term lethal doses of  Senecio jacobaea, 
    by stomach tube, to 6 cows.  Feeding started at term or within 30 
    days post-partum, and continued until what was considered to be a 
    lethal dose had been fed.  The daily dose of the plant ranged from 
    1 to 4.4 g/kg body weight, the total amount fed representing 5 - 
    15% of body weight over a period of 54 - 126 days.  Five cows died 
    within 98 days; one, in a moribund state, was killed on day 126.  
    The calves suckled for 30 - 126 days.  Suckling started immediately 
    after birth in the case of 4 calves and 10 and 30 days later, 
    respectively, in the 2 remaining calves.  Three calves were killed 
    with their dams or soon after, and 3 were retained for 1 year for 
    observation.  Milk samples from 2 cows were collected and pooled in 
    14- to 16-day lots during 64 days of feeding of the  Senecio plant.  
    Each pooled sample was administered intragastrically to a group of 
    rats in daily doses of 12 ml for 15 - 30 days.  A control group of 
    rats were fed raw milk from cows not fed  Senecio.  Blood samples of 
    the dams and the calves were analysed for glutamic oxaloacetic 
    transaminase (GOT), lactic dehydrogenase (LDH), and gamma-glutamyl 
    transpeptidase (GGTP).  Serum-enzyme levels in all cows indicated 
    statistically significant deviations suggesting liver dysfunction, 
    and the livers at autopsy had characteristic features of PA 
    toxicosis.  The LDH and GOT levels in calves were generally 
    abnormal after 20 - 45 days of suckling.  The abnormalities ranged 

    from mild to a 15- to 170-fold increase.  One calf was autopsied at 
    the peak increase of serum-enzymes and was found to have mild focal 
    hepatitis.  No significant pathological features were seen in the 
    livers of other animals, nor of the rats, some of which were 
    retained for up to 150 days. 

        Goeger et al. (1982) fed dried  Senecio jacobaea (tansy ragwort) 
    to lactating goats in a proportion of 25% of the feed.  The milk 
    contained 7.5 µg PA/kg dry weight.  The milk produced by the goats 
    was pooled and then bottle fed to appetite to 2 Jersey bull calves 
    (1 day old) that also had access to tansy ragwort-free hay for 109 
    and 124 days, respectively.  They were then weaned and given normal 
    feed and observed for 6 months, after which they were killed and 
    autopsied.  In another study, rats were fed a diet containing the 
    freeze-dried milk at 80% level for 180 days with a calculated total 
    PA intake of 0.96 mg/rat.  Other groups of rats were fed tansy 
    ragwort at dietary levels of 0.01 - 10 g/kg (corresponding to PA 
    intakes of 39.77, 5.04, 0.52, and 0.05 mg/rat).  The calf livers 
    only showed very mild non-specific changes, but the livers of rats 
    fed tansy ragwort or the milk from tansy ragwort-fed goats 
    showed definite, but mild, changes including swollen 
    hepatocytes, megalocytosis, biliary hyperplasia, and fibrosis.  
    Histopathological changes in milk-fed rats were similar to those in 
    the group fed tansy ragwort in the diet at 0.01 g/kg.  The authors 
    concluded that there was evidence of PA transfer into milk, which 
    proved hepatotoxic for rats.  It was also noted that the goats had 
    been fed high levels of tansy ragwort at the upper limit of their 
    acceptance, and that the hepatic changes observed in rats fed high 
    levels of milk, for extensive periods, were slight. 

        Luthy et al. (1983) produced direct evidence of excretion of 
    macrocyclic esters of retronecine of the senecionine and 
    seneciphylline-type into rat milk.  3H-retronecine, an 3H-necic 
    acid-labelled senecionine, and seneciphylline were prepared 
    biosynthetically with seedlings of  Senecio vulgaris L.  Two 
    lactating rats (Ivanovas, Sprague Dawley), weighing 300 - 400 g, 
    were fed the first of the second compound by stomach tube, in doses 
    of 2.7 mg/kg and 5.5 mg/kg body weight, respectively.  Samples of 
    blood were examined 1, 3, and 6 h after treatment, and those of 
    milk 1 and 3 h after.  Animals were killed after 6 h.  They were 
    found to have excreted approximately 0.08% of the applied 
    radioactivity in the milk within 3 h, mainly as unidentified 
    retronecine-derived metabolites, and approximately 0.02% as 
    unchanged PAs.  The highest levels of PAs and metabolites in 
    tissues were found in the liver and lungs, 6 h after 
    administration. 

        Candrian et al. (1984a) also demonstrated that  Drosophila 
     melanogaster flies fed on milk from lactating rats that had been 
    administered an oral dose of seneciphylline showed 1.2% sex-linked 
    recessive lethals, compared with 0.3% in controls, indicating the 
    transfer of the mutagenic properties of the PA via milk (section 
    6.4.7). 

        The implications of the above studies on the possibility of 
    carry-over of PAs into foodstuffs of animal origin are obvious.  
    However, no reports of human cases of acute PA toxicity, ascribed 
    to the consumption of contaminated milk, are available. 

    3.3.6  Meat

        There have not been any reports of the detection of PAs in meat 
    products from livestock exposed to them. 

    3.3.7  Use of PAs as chemotherapeutic agents for cancer

        An alkaloid of  Heliotropium indicum L. (indicine  N-oxide) has 
    been found to have antitumour activity and has been used in 
    experimental clinical chemotherapy for cancer (section 7.9). 

    4.  METABOLISM

    4.1  Absorption, Excretion, and Tissue Distribution

    4.1.1  Absorption

        There have been few studies on the absorption of PAs in man, 
    but absorption has been inferred from studies on tissue 
    distribution and the amounts of alkaloids and their metabolites 
    excreted in the urine, faeces, and bile of animals (section 4.1.2). 

        Swick et al. (1982c) measured the transfer of a mixture of 
    pyrrolizidine alkaloids extracted from  Senecio jacobaea, across 
    isolated intestine and stomach segments from rabbits.  The alkaloid 
    mixture contained seneciphylline, jacobine, jacozine, jacoline, and 
    senecionine.  The alkaloids were transferred across the ileum and 
    jejunum, but not the stomach.  Brauchli et al. (1982) compared the 
    oral and percutaneous absorption in rats of a crude alkaloid 
    mixture obtained from comfrey ( Symphytum officinale L.).  The 
    mixture consisted of  N-oxides of 7 alkaloids, principally 7-acetyl-
    intermedine and 7-acetyl-lycopsamine.  A dose of 194 mg/kg was 
    either given by gavage, or was applied to the shaved skin and left 
    for 44 h.  After the dermal application, the excreted  N-oxides in 
    urine (up to 48 h) amounted to 0.1 - 0.4% of the dose.  After oral 
    dosage the excreted level of  N-oxides and alkaloid bases was quoted 
    as being 20 - 50 times greater. 

    4.1.2  Excretion and distribution

        The excretion and distribution of heliotrine in rats has been 
    reported in Bull et al., 1968.  Young rats (150 g), given the LD50 
    of heliotrine by ip injection, were killed at intervals, bled 
    quickly, and the organs and tissues analysed.  Heliotrine was 
    present in the liver after 2 min (3.7% of total dose), the level 
    peaking at 5 min (6.3%), and dropping to 2.2% at 1 h and 0.5% at 
    2.5 h.  In adult rats, the level in the liver at 5 h was 0.07% of 
    the total dose.  Five min after dosing, 30 - 40% of the initial 
    dose remained in the peritoneal cavity, and the blood level of 
    heliotrine was 60 mg/litre, dropping to 3 mg/litre at 1 h.  The 
    urinary excretion of base and metabolites other than pyrrolic 
    metabolites, collected and measured 16 h after administration of 
    several alkaloids by ip injection, is shown in Table 6.  The 
    proportion of base excreted unchanged increased with the 
    hydrophilicity of the alkaloid, being 62% for heliotrine  N-oxide, 
    30% for heliotrine, and only 1 - 1.5% for lasiocarpine.  
    Heliotridine, the hydrolysis product from heliotrine and 
    lasiocarpine, was excreted in the form of the  N-oxide in larger 
    quantities after the administration of each of these alkaloids. 

        The distribution and excretion of monocrotaline was studied in 
    rats by Hayashi (1966) who found that 50 - 70% was excreted in the 
    urine within the first day.  However, the analysis was by a non-
    specific chemical method that did not distinguish between the 
    unchanged alkaloid and its metabolites.  Mattocks (1968a) gave 
    toxic pyrrolizidine alkaloids intraperitoneally to male rats and 

    measured the urinary excretion of the unchanged alkaloid, and of 
     N-oxide and pyrrolic metabolites.  The excretion of  N-oxide and 
    unchanged alkaloid was rapid and almost complete in the first 24 h.  
    Excretion of pyrroles was also rapid but continued for a little 
    longer.  For example, in rats given retrosine (60 mg/kg body 
    weight), the urine in the first 24 h contained 10.6% unchanged 
    alkaloid, 13.3%  N-oxide, and 13.4% pyrrolic metabolites.  During 
    the second day, only 0.1% alkaloid, 0.2%  N-oxide, and 1.8% pyrroles 
    were excreted.  Biliary excretion also occurred.  About one-quarter 
    of an iv dose of retrosine in rats was excreted in the bile as 
    pyrrolic metabolites, and 4% as unchanged alkaloid; most of this 
    excretion occurred during the first hour after the injection 
    (White, 1977). 

        Jago et al. (1969) gave heliotrine iv to sheep; urinary 
    excretion of the unchanged alkaloid together with metabolites 
    ( N-oxide, and demethylation and hydrolysis products) occurred 
    rapidly and continued for up to 8 h.  Excretion in the bile was 
    only 2% of that in the urine. 

        The tissue distribution of radioactivity from a tritiated toxic 
    pyrrolizidine alkaloid analogue was studied by Mattocks & White 
    (1976) using synthanecine A bis- N-ethylcarbamate (40 mg/kg body 
    weight).  The highest concentrations of radioactivity were seen in 
    the liver (where metabolism occurs), lungs, kidneys, and spleen 
    (respectively, 3.9%, 0.19%, 0.18%, and 0.27% of the dose given), 
    and about 69% of the dose was eliminated in the urine during the 
    first day.  Radioactivity in the expired air was negligible.  The 
    binding of radioactivity in the liver, and especially the lungs, 
    was more persistent than in other organs.  Similar results were 
    given by the semisynthetic pyrrolizidine alkaloid analogue, 
    retronecine bis- N-ethylcarbamate (Mattocks, 1977). 


        Table 6.  Urinary metabolites of pyrrolizidine bases in the rat (16-h urine)a

                                                                                                             
                                  Urine constituent (amount in percentage of dose injected)

    Base                Unchanged   Base       Heliotridine      Heliotridine     Heliotridine   Heliotridine
    administered        base        N-oxide    trachelanthate    trachelanthate                  N-oxide
    (ip injection)                                               N-oxide
                                                                                                             

    Heliotrine          30          Trace      10                5                3              15
    Heliotrine          62          (62)       2.7               ca. 6            ca. 1          ca. 10
      N-oxide
    Lasiocarpine        1-1.5                                                     1.5-3          6
    Heliotridine        35          ca. 1      (35)              (ca. 1)          5              20
      trachelanthate
    Heliotridine        40          20                                            (40)           (20)
                                                                                                             

    a  From: Bull et al. (1968).
            The distribution of the uniformly 14C-labelled natural 
    pyrrolizidine alkaloid senecionine in lactating mice was studied by 
    Eastman et al. (1982).  After 16 h, 75% of the radioactivity had 
    been recovered in the urine, 14% in the faeces, but only 0.04% was 
    in the milk; the liver contained 1.92%.  The mice were milked using 
    teat cups.  Candrian et al. (1985) studied the distribution of 
    radioactivity in rats given small doses of senecionine or 
    seneciphylline (0.3 - 3.3 mg/kg), tritiated in the pyrrolizidine 
    (retronecine) moiety.  Most radioactivity was eliminated in the 
    urine and faeces within 4 days.  Using mass spectrometry, Dickinson 
    et al. (1976) found a concentration of up to 0.84 mg PAs/litre in 
    the milk of cows fed  Senecio jacobaea.  Blood levels of senecionine 
    in rats given 0.1 LD50 ip were determined by Culvenor (1978).  The 
    levels were 0.38, 0.32, and 0.14 mg/litre at 0.5, 1, and 2 h after 
    injection, respectively. 

        To summarize, the available evidence suggests that ingested 
    toxic pyrrolizidine alkaloids are rapidly metabolized and that the 
    excretion of unchanged alkaloid and of most metabolites is also 
    rapid.  Thus, within a few hours, only a relatively small 
    proportion of the dose remains in the body, much of this in the 
    form of metabolites bound to tissue constituents.  It appears 
    improbable that a significant amount of unchanged alkaloid will 
    remain in the body after the first day. 

        Pyrrolizidine  N-oxides are much more water soluble than their 
    parent alkaloids.  Indicine  N-oxide (which is exceptionally water 
    soluble) is very rapidly excreted, either unchanged or conjugated.  
    Thus, indicine  N-oxide given iv to mice, monkeys, or rabbits 
    disappeared from the serum with initial half-lives ranging from 3 
    to 20 min (Powis et al., 1979; El Dareer et al., 1982).  Over 80% 
    of tritium-labelled indicine  N-oxide given iv was excreted in the 
    urine of mice or monkeys within 24 h (El Dareer et al., 1982); at 
    2 h, the highest concentrations of radioactivity were in the 
    kidneys, liver, and intestines.  Urinary excretion of indicine 
     N-oxide was also rapid in rabbits, but somewhat slower in human 
    beings (Powis et al., 1979). 

    4.2  Metabolic Routes

        The major metabolic routes of unsaturated pyrrolizidine 
    alkaloids in animals are:  (a) hydrolysis (of the ester groups); 
    (b)  N-oxidation; and (c) dehydrogenation (of the pyrrolizidine 
    nucleus) to dehydro-alkaloids (pyrrolic derivatives).  Other minor 
    routes of metabolism are known, but the three pathways account for 
    the major known toxic effects of these alkaloids (Fig. 5).  Routes 
    (a) and (b) are believed to be detoxification mechanisms.  Route 
    (c) leads to toxic metabolites and appears to be the major 
    activation mechanism.  Route (a) may occur in various tissues, 
    including the liver and blood.  Routes (b) and (c) are brought 
    about in the liver by the microsomal mixed-function oxidase system. 

    FIGURE 5

    4.2.1  Hydrolysis

        The hydrolysis of a PA leads to the formation of the amino-
    alcohol moiety (necine base) and the acid moiety.  Neither of these 
    is hepatotoxic (Schoental & Mattocks, 1960; Culvenor et al., 
    1976a).  The highly water-soluble necine base is readily excreted, 
    is not accessible to the microsomal system, and is not activated to 
    a toxic metabolite.  Thus, pyrrolizidine alkaloids that are very 
    susceptible to (enzymic) hydrolysis have low toxicity (Mattocks, 
    1982).  A major factor contributing to resistance to esterase is 
    the steric hindrance in the acid moiety.  Thus, the chain branching 
    near the carbonyl groups slows hydrolysis allowing the formation of 
    relatively high levels of pyrrolic metabolites; a conformation of 
    the basic moiety, which brings the two ester groups close together, 
    thus leading to mutual steric hindrance, can also prevent 
    hydrolysis (Mattocks, 1981a). 

        The influence of hydrolysis  in vivo on alternative metabolic 
    pathways is demonstrated by the fact that treatment of rats with an 
    esterase inhibitor, before giving pyrrolizidine alkaloids (or 
    synthetic analogues), can lead to greatly increased production of 
    pyrrolic metabolites from alkaloids that are normally susceptible 
    to hydrolysis, but little increase in those from alkaloids normally 
    resistant to hydrolysis (Mattocks, 1981a). 

    4.2.2  N-oxidation

        The  N-oxidation of pyrrolizidine alkaloids is induced by the 
    hepatic microsomal enzymes.  The  N-oxide metabolites are highly 
    water soluble and are rapidly excreted in the urine (Mattocks, 
    1968a).  Pyrrolizidine  N-oxides are not converted to any 
    significant extent to pyrrolic metabolites by microsomal enzymes 
    (Jago et al., 1970; Mattocks & White, 1971a), and there is no 
    evidence that they are toxic, unless first reduced to the 
    corresponding basic alkaloids, which can then be activated by the 
    microsomal system (Mattocks, 1971c).  Thus, it appears that the 
    formation of  N-oxides represents a detoxification pathway. 

    4.2.3  Conversion to pyrrolic metabolites

        In laboratory animals, toxic pyrrolizidine alkaloids are 
    metabolized to pyrrolic derivatives, so-called because the 
    unsaturated ring of the pyrrolizidine system loses 2 hydrogen atoms 
    to form what is in effect a pyrrole ring (though the structure is 
    more correctly a dihydropyrrolizidine).  Pyrrolic metabolites are 
    easily detectable in the tissues shortly after giving a toxic 
    pyrrolizidine alkaloid to an animal, by treating the tissue with an 
    Ehrlich reagent containing boron trifluoride, when a red colour is 
    produced; this reaction also occurs with the urine (Mattocks, 
    1968a; Mattocks & White, 1970).  In rats given retrosine, pyrrolic 
    metabolites were found principally in the liver, with highest 
    levels associated with the microsomal and solid debris fractions 
    and less in the mitochondrial fraction; low levels were found in 
    the lungs, heart, spleen, and kidneys, within 4 h of giving 
    retrosine.  Rats given 60 mg retrosine/kg body weight excreted 14% 
    of the dose in the urine, within 48 h. 

        Pyrrolic metabolites are formed by the hepatic mixed-function 
    oxidase system, with a requirement for cytochrome P450, oxygen, and 
    NADPH, as has been demonstrated  in vitro (Jago et al., 1970; 
    Mattocks & White, 1971a).  Conversion of pyrrolizidine alkaloids to 
    pyrrolic metabolites by the lung tissue of the human embryo 
    (Armstrong & Zuckerman, 1970), rat (Mattocks & White, 1971a; 
    Hilliker et al., 1983), or rabbit (Guengerich, 1977) was 
    negligible.  The formation of pyrrolic metabolites does not proceed 
    via  N-oxide intermediates, but appears to result from an initial 
    hydroxylation of the unsaturated pyrrolizidine ring adjacent to the 
    nitrogen atom (Mattocks & White, 1971a; Mattocks & Bird, 1983).  
    This would lead to a chemically unstable intermediate that would be 
    expected to decompose spontaneously to the pyrrolic product.  The 
    primary pyrrolic metabolites (or dehydro-alkaloid) formed by 
    dehydrogenation of pyrrolizidine alkaloids are chemically 
    dehydropyrrolizidine esters (Fig. 5).  These are highly reactive 
    compounds that can rapidly react with tissue constituents or 
    hydrolyse to the corresponding pyrrolic alcohols, or dehydro-
    necines, which can thus be regarded as secondary metabolites.  The 
    latter can also react with tissue constituents, but more slowly.  
    Because of their high chemical reactivity, the primary metabolites 
    would be expected to have a short life in the liver cell (minutes 
    or seconds) before they are hydrolysed or react with nucleophilic 
    tissue constituents.  Some might escape into the blood stream and 
    reach other organs, especially the lungs.  Dehydro-necines are more 
    stable and also more water soluble, and can become more widely 
    distributed throughout the body.  However, they are also capable of 
    reacting with tissue constituents.  Thus, measurements of pyrroles 
    formed from pyrrolizidine alkaloids in tissue samples, using a 
    colour reaction (Mattocks & White, 1970), will not represent a 
    single metabolite, but mixtures of the metabolites together with 
    various reaction products of these with tissue constituents.  It 
    will be seen (section 5) that pyrrolic metabolites are believed to 
    be responsible for major toxic actions of pyrrolizidine alkaloids 
    (Mattocks, 1972a). 

        A pyrrolic metabolite with reactivity midway between that of 
    dehydromonocrotaline and dehydroretronecine has been reported to be 
    formed from monocrotaline in isolated, perfused rat liver 
    (Lafranconi et al., 1985).  Studies on this metabolite (isolated 
    from bile) indicated that it is a monoester, and that it is toxic 
    in perfused rat lung.  This suggests that monoester pyrrolic 
    metabolites may play a part in the toxic actions of PAs in extra-
    hepatic tissues. 

        When rats or other laboratory animals are given a toxic 
    pyrrolizidine alkaloid, pyrrolic metabolites accumulate rapidly in 
    the liver (Mattocks, 1973; White et al., 1973), reaching a peak 
    within 1 - 2 h, then falling slowly during the next 24 h; the 
    metabolites may still be detectable after 2 days.  Accumulation is 
    especially rapid after intraperitoneal injection, a very high level 

    of pyrrolic metabolites being attained within 20 min; this 
    indicates how rapid the metabolism of pyrrolizidine alkaloids can 
    be. 

        The level of pyrrolic metabolites in rat liver is generally 
    directly related to the amount of alkaloid given 2 h previously, at 
    least up to an acute LD50 dose.  The pyrrole level depends on the 
    alkaloid used, and is related to the acute hepatotoxicity of the 
    alkaloid (Mattocks, 1972a). 

        To be converted to the type of chemically reactive, toxic 
    pyrrolic metabolites described above, an alkaloid must possess a 
    1-hydroxymethyl pyrrolizidine system, unsaturated in the 
    1,2-position (this makes the ring susceptible to dehydrogenation), 
    and at least one hydroxyl group must be esterified, usually by a 
    branched-chain acid.  Otonecine esters are converted to similar 
    pyrrol metabolites by a different media involving  N-demethylation.  
    Pyrrolizidine amino-alcohols (e.g., retronecine) are not 
    metabolized to more than small amounts of pyrroles (Jago et al., 
    1970; Mattocks, 1981a), possibly because they are too water soluble 
    to reach the microsomal enzymes. 

        The metabolic formation of pyrroles is catalysed by cytochrome 
    P450 and specificity exists in the various isozymes (Guengerich, 
    1977; Juneja et al., 1984). 

        A few non-toxic pyrrolizidine alkaloids (e.g., rosmarinine and 
    hygrophylline) are converted to pyrrolic metabolites  in vivo  
    (Mattocks, 1973).  Such metabolites are chemically different from 
    the pyrroles of toxic alkaloids, and they are neither reactive nor 
    toxic (Mattocks & White, 1971b).  The balance of structural 
    features necessary for a pyrrolizidine alkaloid to be converted to 
    give high concentrations of toxic pyrrolic metabolite has been 
    discussed by Mattocks (1981a); the optimum conditions appear to be 
    met in some alkaloids that are macrocyclic diesters, such as 
    retrosine. 

    4.3  Effects of Treatments Affecting Metabolism

        The formation of pyrrolic metabolites (and of  N-oxides) is 
    altered by treatments that affect the hepatic microsomal enzymes.  
    Such effects have been studied by measuring rates of metabolism of 
    pyrrolizidine alkaloids  in vitro using microsomal preparations 
    from animals pre-treated in various ways (Table 7).  For example, 
    microsomes from rats given the microsomal enzyme inducers 
    phenobarbitone or DDT (but not those from rats given 
    3-methylcholanthrene) induce greatly increased pyrrole formation 
    and smaller increases in  N-oxide formation, from the alkaloid 
    retrorsine (Mattocks & White, 1971a).  Enzyme preparations from 
    rats treated with inhibitors of microsomal enzymes, including SKF 
    525A and chloramphenicol, are much less active in converting 
    monocrotaline to pyrroles (Chesney et al., 1974).  The ability to 
    metabolize retrorsine is diminished in microsomes from rats fed a 
    protein-free diet, or from rats acutely poisoned with retrorsine 
    (Mattocks & White, 1971a). 


        Table 7.  Effect of pre-treatment of male rats on the conversion of PAs to 
              pyrrolic derivatives and to  N-oxides by liver microsomes
               in vitro
    ----------------------------------------------------------------------------
    Alkaloid       Pre-treatment, and       Enzyme activity as   Reference
                   time before enzyme       % of control values
                   measurements             for formation of:  
                                            Pyrroles    N-oxides
    ----------------------------------------------------------------------------
    retrorsine     phenobarbitone, ip,      311        232       Mattocks &
                   3 x 100 mg/kg,                                White (1971a)
                   1 - 3 days

    retrorsine     DDT, ip, 75 mg/kg,       407        203       Mattocks &
                   3 days                                        White (1971a)

    retrorsine     3-methylcholanthrene,    95 (ns)    116 (ns)  Mattocks &
                   ip, 3 x 20 mg/kg,                             White (1971a)
                   1 - 3 days

    retrorsine     retrorsine, ip,          63         -         Mattocks &
                   35 mg/kg, 20 h                                White (1971a)

    retrorsine     protein-free diet,       39         -         Mattocks &
                   3 days                                        White (1971a)

    monocrotaline  phenobarbitone, sc,      448        165       Chesney et al.
                   4 x 75 mg/kg,                                 (1974)
                   1 - 4 days

    monocrotaline  chloramphenicol, sc,     10         109 (ns)  Chesney et al.
                   200 mg/kg, 1 h                                (1974)

    monocrotaline  SKF 525A, ip, 75 mg/kg,  10         87 (ns)   Chesney et al.
                   1 h                                           (1974)
    ----------------------------------------------------------------------------
    ns = not significantly different from controls.
            The effects of  in vivo treatment with several types of enzyme 
    inducers on the toxicity of lasiocarpine and senecionine for 
    primary rat hepatocyte cultures was investigated by Hayes et al. 
    (1985).  Pre-treatment with phenobarbitone potentiated the 
    cytotoxicity of senecionine towards the cultured cells, whereas 
    pre-treatment with 3-methylcholanthrene diminished the toxic action 
    of senecionine, but had little effect on lasiocarpine cytotoxicity.  
    The cytocidal effects of both alkaloids were substantially 
    inhibited in the presence of SKF 525A. 

    4.4  Other Factors Affecting Metabolism

        Variations between animal species have been investigated by 
    White et al. (1973) and Shull et al. (1976).  For instance, 
    metabolism to form pyrroles is high in rats and very low in guinea-
    pigs, which, however, have higher rates of  N-oxidation.  For 
    example, 2 h after an ip dose of retrosine (100 mg/kg body weight), 

    the liver-pyrrole level in male rats was 13 times higher than that
    in male guinea-pigs (White et al., 1973).  Liver microsome 
    preparations from male rats were 28 times more active than 
    microsomes from male guinea-pigs in the dehydrogenation of 
    monocrotaline (Chesney & Allen, 1973a). 

        The development with age of the ability of Wistar rats to 
    metabolize retrosine was studied by Mattocks & White (1973).  The 
    ability to form pyrroles is very low in new-born rats, but, by 5 
    days of age, it is nearly as high as in adult males.  This activity 
    continues at a similar level in male rats, but, in females, it 
    falls after the age of about 20 days until, by 60 days, it is about 
    one-eighth that in males.  Such a sex difference was not observed 
    in mice (White et al., 1973). 

    4.5  Other Metabolic Routes

        The actions of hepatic microsomal enzymes on pyrrolizidine 
    alkaloids can produce other metabolites as well as pyrroles and 
     N-oxides, but there are few reports of these.  Eastman & Segall 
    (1982) demonstrated hydroxylation of the acid moiety of senecionine 
    by liver microsomes from female mice.  Such metabolism should not 
    prevent the subsequent conversion of the product to pyrrolic or 
     N-oxide metabolites.  The formation of other microsomal metabolites 
    of senecionine has been reported by Segall et al. (1984). 

        The  O-demethylation of the acid moiety of heliotrine has been 
    demonstrated by Jago et al. (1969) and represents a partial 
    detoxification mechanism, since the product is about half as toxic 
    as heliotrine.  Other detoxification mechanisms exist in the rumen 
    of sheep (Dick et al., 1963; Lanigan & Smith, 1970a,b), which are, 
    thus, particularly resistant to the effects of pyrrolizidine 
    alkaloids. 

    4.6  Metabolism of Pyrrolizidine  N-Oxides

        As mentioned in section 4.2, the  N-oxides of pyrrolizidine 
    alkaloids are not converted to pyrrolic metabolites by liver 
    microsomes.  It appears that their main route of metabolism in 
    animals is reduction to the corresponding basic alkaloids, which 
    may then be further metabolized as already described.  This 
    reduction has been shown to occur in the rat or rabbit gut 
    (Mattocks, 1971c; Powis et al., 1979), and may be brought about by 
    intestinal bacteria or possibly by gut enzymes.  Such reduction can 
    also be brought about by hepatic microsomal fractions (Powis et 
    al., 1979) in the presence of NADH or of NADPH, and by sheep rumen 
    fluid (Lanigan et al., 1970a, b). 

        The reduction of pyrrolizidine  N-oxides  in vivo is of great 
    importance as a step in the bioactivation of these compounds 
    (Mattocks, 1971c), as shown in section 4.2.2. 

    4.7  Metabolism in Man

        Powis et al. (1979) found that indicine  N-oxide given iv to 
    3 human patients as an antitumour drug was partially reduced to 
    indicine base, detectable in the urine and plasma.  Armstrong & 
    Zuckerman (1970) showed that human embryo liver slices, but not 
    lung slices, converted the pyrrolizidine alkaloids lasiocarpine, 
    retrorsine, and fulvine to pyrrolic metabolites  in vitro. 

    5.  MECHANISMS OF TOXICITY AND OTHER BIOLOGICAL ACTIONS

    5.1  Metabolites Responsible for Toxicity

    5.1.1  Metabolic basis of toxicity

        The toxic effects of pyrrolizidine alkaloids are mediated 
    through their toxic metabolites and not by the alkaloids 
    themselves.  The following observations are evidence for the above 
    statement (Mattocks, 1972a): 

        (a)  The alkaloids are chemically rather unreactive and it is 
        hard to envisage reactions with cell constituents that they 
        could undergo readily under physiological conditions.  On the 
        other hand, chemically prepared derivatives, similar or 
        identical to known metabolites of these alkaloids, are highly 
        reactive and are capable of causing toxic effects similar to 
        those of PAs, often at dose levels much lower than those 
        required by the alkaloids themselves.

        (b)  The liver is usually the main organ affected, whatever the 
        route of administration of the alkaloid.  The alkaloids are 
        known to be metabolized in the liver.

        (c)  Direct application of these alkaloids to the skin does not 
        cause local toxic effects (Schoental et al., 1954), nor do 
        cytotoxic effects occur at sites of injection.

        (d)  The susceptibility of animals to the toxic actions of PAs 
        is related to the ability of the animal to metabolize the 
        alkaloids.  For example, the hepatic microsomal enzymes of rats 
        less than 1 h old have very low activity towards retrorsine and 
        these rats are relatively resistant to it, whereas rats aged 
        several days have a high enzyme activity and are highly 
        susceptible to the alkaloid (Mattocks & White, 1973).  Guinea-
        pigs are very resistant to retrorsine, unless they have been 
        given phenobarbitone, which potentiates the enzymes that 
        metabolize it (White et al., 1973).  Rats pre-treated with 
        microsomal enzyme inhibitors, such as SKF 525A or 
        chloramphenicol, have increased resistance to retrorsine or 
        monocrotaline (Allen et al., 1972; Mattocks, 1973).  In 
        general, there is a good relationship between the rate of 
        hepatic metabolism of PAs to pyrrole  in vitro (Shull et al., 
        1976) and chronic toxicity.  Highly resistant species, e.g., 
        guinea-pigs, Japanese quail, and sheep, have a low rate of 
        pyrrole formation, while susceptible species, such as the 
        horse, cattle, and rat, have a high rate.  Notable exceptions 
        are the rabbit and hamster, which have high rates of pyrrole
        formation, but are resistant.  It is possible that this may be 
        due to changes in the balance between activation and the 
        involvement of other factors, such as activity of 
        detoxification.  For example, sheep have a high epoxide
        hydrolase activity in the liver (Swick et al., 1983), which may 
        affect PA detoxification (Cheeke & Pierson-Goeger, 1983).

    5.1.2  Isolation of pyrrolic metabolites

        There is plenty of evidence that many unsaturated PAs are 
    converted into pyrrolic esters (dehydro-alkaloids) in the mammalian 
    liver (section 4.2.3).  These primary pyrrolic metabolites cannot 
    be isolated, because of their high reactivity and rapid rate of 
    hydrolysis.  However, their more stable hydrolysis products 
    (pyrrolic alcohols; dehydronecines) have been isolated and 
    identified.  Thus dehydroheliotridine has been obtained from the 
     in vitro incubation of both the heliotridine-based alkaloids, 
    lasiocarpine and heliotrine, with rat liver microsomes (Jago et 
    al., 1970) and dehydro-retronecine was found to be the main 
    detectable pyrrolic metabolite in the liver, blood, and urine of 
    rats injected with the retronecine-based alkaloid, monocrotaline 
    (Hsu et al., 1973).  There is evidence that these materials are 
    identical, i.e., the (±)-form resulting from racemization during 
    hydrolysis of the parent pyrrolic esters (Kedzierski & Buhler, 
    1985). 

        The results of these studies confirm that rat liver enzymes 
    convert PAs into metabolites with known cytotoxic activity (section 
    5.2), and imply that these metabolites are formed via the yet more 
    toxic and short-lived dehydro-alkaloids (Jago et al., 1970). 

    5.1.3  Chemical aspects of pyrrolic metabolites

    5.1.3.1  Preparation

        Chemical methods are available for converting unsaturated PAs 
    into pyrrolic esters (dehydro-alkaloids), the putative primary 
    toxic metabolites, enabling the physical, chemical, and 
    toxicological properties of the latter to be studied. 

        Small amounts of dehydro-pyrrolizidine alkaloids are usually 
    prepared by the reaction of the corresponding alkaloid  N-oxides 
    with either acetic anhydride (Mattocks, 1969; Culvenor et al., 
    1970a) or methanolic ferrous sulfate (Mattocks, 1969).  The 
    products must be protected from moisture and from acids, which can 
    cause their immediate decomposition. 

        A variety of reagents can dehydrogenate the alkaloid bases to 
    pyrrolic derivatives, these include manganese dioxide (Culvenor et 
    al., 1970a,b; Mattocks, 1969), potassium permanganate (Culvenor et 
    al., 1970a), chloranil (Culvenor et al., 1970a), 2,3-dichloro-5,6-
    dicyanobenzoquinone (Mattocks, 1969), iodine (Culvenor et al., 
    1970b), and aryl thiols (Juneja et al., 1984).  Some PAs are slowly 
    oxidized to pyrroles by molecular oxygen (Bick et al., 1975). 

        The more stable pyrrolic alcohol, dehydroretronecine (Fig. 6), 
    is prepared from retronecine using chloranil (Culvenor et al., 
    1970a) or aqueous potassium nitro-sodisulfonate (Mattocks, 1981c) 
    or from retronecine  N-oxide (isatinecine) using ferrous sulfate 
    (Mattocks, 1969).  The enantiomeric dehydroheliotridine can be 
    prepared from heliotridine in similar ways.  Racemic dehydro-
    heliotridine has been synthesized (Viscontini & Gilhof-
    Schaufelberger, 1971; Bohlmann et al., 1979). 

    FIGURE 6

    5.1.3.2  Chemistry associated with toxic actions

        Dehydro-pyrrolizidine alkaloids and dehydronecines (pyrrolic 
    esters and alcohols) act chemically as alkylating (electrophilic) 
    agents, i.e., they can react with compounds possessing electron-
    rich (nucleophilic) groups, such as amines, thiols, and some 
    hydroxyl compounds.  The products of alkylation consist of the 
    "pyrrole" moiety covalently bonded to the substrate molecule.  The 
    mechanism of alkylation is illustrated in Fig. 7 (Mattocks, 1972a).  
    An ester (R = COR) or hydroxyl group (R = H) attached to the 
    pyrrole ring via one carbon atom (i.e., at C7 or C9) is highly 
    reactive, being easily cleaved, leaving a positively charged 
    pyrrole moiety with a high affinity for electron-rich substrates.  
    Pyrrolic esters are the most reactive, RCOO being a better "leaving 
    group" than HO.  When 2 oxygen functions are present (as 
    illustrated), either (in turn) can act as an alkylating centre.  
    Such bifunctional alkylation could lead to cross linking of 
    macromolecules (Mattocks, 1969; White & Mattocks, 1972; Petry et 
    al., 1984, 1986).  When the groups (R) are the same or similar, C7 
    is the more reactive site.  Examples of such alkylations using pure 
    chemicals (amines or alcohol) have been given by Mattocks (1969) 
    and Culvenor et al. (1970a).  Mattocks & Bird (1983) showed that a 
    variety of nucleophiles of biological interest could be alkylated 
    by dehydroretronecine.  Black & Jago (1970) demonstrated the 
     in vitro alkylation of DNA by dehydroheliotridine, and Robertson 
    (1982) and Wickramanayake et al. (1985) the alkylation of 
    deoxyguanosine by dehydroretronecine.  The alkylation of mouse or 
    rat liver DNA by pyrrolizidine alkaloids has been shown  in vivo by 
    Eastman et al. (1982) and Candrian et al. (1985). 

    FIGURE 7

    5.1.4  Possible further metabolites

        The possibility that pyrrolic metabolites of PAs might 
    themselves be metabolized by microsomal enzymes to further 
    cytotoxic derivatives was suggested by Guengerich & Mitchell 
    (1980).  These authors showed that the tritium-labelled model 
    compounds 1,2,3-trimethylpyrrole and 1-methyl-3-4 bishydroxy-
    methylpyrrole could be metabolized in rats or by rat liver 
    microsomes to unidentified derivatives able to bind covalently to 
    proteins and nucleic acids.  It is possible that liver damage, seen 
    in some rats given iv injections of pneumotoxic pyrrolic esters, 
    might have been due to metabolites of the latter formed in the 
    liver (Mattocks & Driver, 1983).  Segall et al. (1985) have 
    identified trans-4-hydroxy-2-hexenal in an  in vitro mouse liver 
    microsomal system metabolizing the PA senecionine and suggested 
    that it might have been formed from the alkaloid via a pyrrolic 
    intermediate.  The compound is capable of causing liver damage and 
    might contribute to the acute hepatotoxicity of senecionine and 
    other alkaloids.  However, this has not been proved, and the highly 
    reactive and toxic primary pyrrolic metabolites from PAs are 
    themselves capable of causing the known hepatotoxic effects of 
    these alkaloids. 

    5.2  Toxic Actions of Pyrrolic Metabolites

        Pyrrolic derivatives prepared chemically from PAs, as well as 
    some analogous compounds, have been tested in experimental animals 
    and  in vitro systems, and shown to have a variety of toxic actions. 

    5.2.1  Animals

    5.2.1.1  Pyrrolic esters (dehydro-alkaloids)

        Dehydro-pyrrolizidine alkaloids are very reactive and their 
    effects  in vivo are largely confined to the first tissues they 
    encounter.  When given orally to rats, they are destroyed almost 
    immediately in the aqueous acid of the stomach and show no toxic 
    action.  When given ip, they cause severe local irritation and 
    peritonitis (Mattocks, 1968a; Butler et al., 1970); subcutaneous 
    injection leads to skin lesions (Hooson & Grasso, 1976).  After iv 
    injection of pyrroles, such as dehydromonocrotaline (monocrotaline 
    pyrrole), into the tail veins of rats, the toxic injuries appear 
    principally in the lungs.  Depending on the dose, these include 
    vascular lesions and pulmonary oedema (Plestina & Stoner, 1972); a 
    progressive alveolar proliferation similar to that produced by 
    very much larger doses of the parent alkaloid (Butler et al., 
    1970) and pulmonary hypertension (Hilliker et al., 1983).  
    Dehydromonocrotaline does not require further metabolism to express 
    its pneumotoxicity, and it is rapidly rendered inactive after 
    exposure to aqueous media (Bruner et al., 1986).  Similar 
    pneumotoxicity is produced by totally synthetic pyrrolic esters 
    having a simpler structure but the same type of chemical reactivity 
    as the alkaloid derivatives (Mattocks & Driver, 1983), thus 
    confirming the chemical mechanism of this action. 

        Injections of dehydro-pyrrolizidine alkaloids or synthetic 
    analogues into mesenteric veins of rats lead to liver damage after 
    smaller doses than the alkaloids themselves (Butler et al., 1970; 
    Shumaker et al., 1976).  The liver damage differs somewhat from the 
    alkaloid damage, consistent with the toxin being introduced via the 
    hepatic vascular system rather than being produced within the 
    hepatocytes, as is the case with the alkaloids.  Nevertheless, the 
    progressive liver lesions are very similar to those produced by PAs 
    (Butler et al., 1970).  The lung damage after tail vein injections 
    bears a closer resemblance to pyrrolizidine damage, since the 
    latter is also believed to be caused by metabolites entering the 
    lungs via the bloodstream (Barnes et al., 1964). 

    5.2.1.2  Pyrrolic alcohols (dehydro-necines)

        Dehydroheliotridine (Fig. 6), a secondary pyrrolic metabolite 
    from heliotridine-based PAs, such as heliotrine and lasiocarpine, 
    is less acutely toxic than its parent alkaloids; it has an LD50 (7 
    days) of about 250 mg/kg body weight in mice (Percy & Pierce, 
    1971).  Its effects on 14-day-old rats were studied by Peterson et 
    al. (1972).  All rats given ip doses of 0.4 mmol/kg body weight 
    survived, but a dose of 0.6 mmol/kg killed most animals within 10 
    days.  Toxic effects were mainly found in rapidly developing 
    tissues.  In young rats, it caused fur loss, tooth defects, and 
    atrophy of hair follicles, gut mucosa, spleen, thymus, testis, and 
    bone marrow.  The lungs were not affected.  Pathological effects in 
    the liver were confined to necrosis of isolated cells and 
    antimitotic action, which was manifested as a mild megalocytosis 
    (development of giant hepatocytes) in rats surviving 4 weeks or 
    more.  The persistent antimitotic action of dehydroheliotridine and 
    of its parent alkaloid lasiocarpine in the liver of rats was 
    investigated by Samuel & Jago (1975), who located the mitotic block 
    as being either late in the DNA synthetic (S) phase or early in the 
    post synthetic (G2) phase of the cell cycle. 

        Dehydroheliotridine is also carcinogenic.  Peterson et al. 
    (1983) showed that rats given 9 ip injections of this compound
    (60 - 76.5 mg/kg body weight) over 23 weeks had a shorter life span
    and suffered a significantly higher incidence of tumours than control
    rats.  The authors concluded that dehydroheliotridine is responsible
    for some, or possibly all, of the carcinogenicity of its parent
    alkaloids. 

        Dehydroheliotridine was found to be teratogenic when given ip 
    to female hooded rats on the 14th day of pregnancy.  A dose of
    40 mg/kg body weight produced effects similar to those produced by the
    alkaloid heliotrine at a dose of 200 mg/kg (Peterson & Jago, 1980).  
    For the immunosuppressant activity of this compound, see section 
    6.4.10. 

        The toxic actions of dehydroretronecine (DHR) (Fig. 7) when 
    given sc to rats are similar to those of dehydroheliotridine (Hsu 
    et al., 1973; Shumaker et al., 1976).  Repeated large doses also 
    caused ulceration of the glandular stomach.  Daily sc doses
    (4 mg/kg body weight), administered to rats for 1 week, caused lung

    damage leading to right ventricular hypertrophy (Huxtable et al., 
    1978).  DHR was carcinogenic when applied repeatedly to mouse skin 
    (Johnson et al., 1978; Mattocks & Cabral, 1982). 

    5.2.2  Cell cultures

        Dehydroheliotridine and dehydrosupinidine both have an 
    inhibitory action in cultures of KB cells (human epidermoid 
    carcinoma of the nasopharynx) with ED50 concentrations of 10-4 mol 
    and 10-5 mol, respectively (Culvernor et al., 1969). 

        Bick & Culvenor (1971) found dehydroheliotridine (DHR) to be 
    considerably more effective than the alkaloid heliotrine in 
    suppressing cell division and causing chromosome breaks, in 
    cultures of leukocytes from the marsupial  Potorus tridactylus; at a 
    concentration of 6 x 10-5 mol, the mitotic index was zero, and more 
    than half the cells had disintegrated.  In a study by Mattocks & 
    Legg (1980), dehydroretronecine and several synthetic analogues 
    completely inhibited cell division in a cultured rat liver cell 
    line at a concentration of 10-4 mol.  Ord et al., (1985) found that 
    DHR induced sister chromatid exchange in human lymphocytes without 
    the need for metabolic activation.  Analogous pyrroles with only 
    one functional (reactive) group were much less effective.  DHR was 
    also weakly active in inducing mutations in the  Salmonella 
     typhimurium base substitution strain, TA92, and gave positive 
    results in an  in vitro cell transformation test using a culture 
    derived from hamster kidney cells (Styles et al., 1980). 

        The toxicity of the pyrrolic ester, dehydromonocrotaline, for 
    cultures of mouse fibroblasts was studied  in vitro by Johnson 
    (1981).  The level of exposure was approximately 1 ng per cell.  
    Cell death was preceded, first by the swelling and disruption of 
    organelles, including mitochondria, and then by the rupture of 
    plasma membranes with the release of cell components. 

        Bick et al., (1975) investigated whether the effects of PAs on 
    leukocyte cultures of  Potorus tridactylus were due to pyrrolic 
    metabolites.  Levels of dihydropyrrolizines, which could be 
    demonstrated in the culture media, were insufficient to account for 
    the observed effects of heliotrine, lasiocarpine, and monocrotaline 
    on the cells, but the actual amounts formed within the cells may 
    have been higher than those observed. 

    5.2.3  Possible participation of membrane lipid peroxidation

        Distinct increases in NADPH- and ascorbate-dependent 
    peroxidation of microsomal membrane lipids were found in rats given 
    heliotrine subcutaneously (300 mg/kg body weight) (Savin 1983).  
    The primary biochemical interactions and cellular macromolecular 
    targets for the pathogenesis of PA-induced toxicity remain 
    unidentified. 

    5.3  Chemical and Metabolic Factors Affecting Toxicity

        The toxicity of an alkaloid depends on the extent to which it 
    is converted into active metabolites and on the disposition and 
    reactivity of these metabolites, once formed. 

    5.3.1  Structural features of a toxic alkaloid

        The essential structural features of a hepatotoxic PA (Fig. 8) 
    are: 

        (a)  a 1-hydroxymethylpyrrolizidine ring system unsaturated in 
             the 1:2-position, with preferably a second hydroxyl group 
             in the 7-position;

        (b)  esterification of at least one of the hydroxyls, though 
             toxicity is much greater when both hydroxyls are 
             esterified; and

        (c)  ester groups that are resistant to enzymic hydrolysis, 
             which usually means that there is a high degree of chain 
             branching in the acid moiety.

        The above requirements apply to natural PAs but, strictly 
    speaking, only the right hand (pyrroline) ring is essential, being 
    the ring that is metabolized to a pyrrole derivative.  Thus, esters 
    of 2,3-bis-hydroxymethyl-1-methyl-3-pyrroline (synthanecine A) 
    (Fig. 9) have pyrrolizidine-like hepatotoxicity (Mattocks, 1971a; 
    Driver & Mattocks, 1984). 

    FIGURE 8

        Structural requirements for  N -oxides are the same as those for 
    the hepatotoxic alkaloids.  However, it is important to note that a 
    PA  N -oxide is not hepatotoxic itself; toxicity depends on it being 
    reduced to the corresponding basic alkaloid, chiefly in the gut 
    (Mattocks 1971c), but possibly in other organs, such as the liver 
    (Powis et al., 1979). 

    FIGURE 9

    5.3.2  Activation and detoxication

        Factors affecting the proportion of an ingested alkaloid that 
    is converted into toxic metabolites in an animal include the 
    following: 

        (a)   Lipid solubility

        Highly water-soluble alkaloids (such as indicine) are easily 
    excreted and have low toxicity.  Alkaloids that are more lipophilic 
    are more open to activation by liver microsomes (Mattocks, 1981a). 

        (b)   Subceptibility to hydrolysis

        This is determined by the molecular structure and conformation 
    of the alkaloid (Mattocks, 1981a,b).  If the alkaloid is open to 
    esterase attack, it may be largely detoxified by hydrolysis. 

        (c)   Susceptibility to N-oxidation

        The relative amounts of an alkaloid converted by hepatic 
    microsomal enzymes to  N-oxide and to pyrrolic metabolites depends 
    on its molecular structure and conformation (Mattocks & Bird, 
    1983).   N-oxidation represents a detoxication pathway (Mattocks, 
    1972b). 

    5.3.3  Factors affecting the toxicity of active metabolites

    5.3.3.1  Reactivity of the metabolite

        Toxic metabolites are formed in liver cells.  Primary pyrrolic 
    metabolites (dehydro-alkaloids) are very reactive and, thus, are 
    quickly hydrolysed or deactivated by reaction with cell constituents.
    To damage tissues other than the cells in which they are formed,
    active metabolites must cross the cell membrane and survive while
    being transported in the bloodstream.  The more stable pyrrolic
    metabolites, such as dehydromonocrotaline from the alkaloids
    monocrotaline, are able to reach, and become bound to, lung tissue
    (Mattocks, 1973).  Thus, monocrotaline frequently damages the lungs,
    whereas retrorsine, which yields a more reactive pyrrolic metabolite,
    normally does not. 

        Secondary metabolites (pyrrolic alcohols, e.g., dehydroretronecine),
    formed by the hydrolysis of primary pyrrolic metabolites, are water
    soluble, relatively stable compounds that can become more widely
    distributed throughout the body or excreted; these are not acutely
    toxic. 

    5.3.3.2  The number of reactive groups

        The toxicity of a pyrrolic alkylating agent is affected by the 
    number of reactive ester or hydroxyl groups (1 or 2) present as the 
    following examples show: 

        (a)  Many pyrrolic esters can cause acute lung damage when
        given iv to rats, but only bifunctional ones also cause
        delayed effects on the lungs (Mattocks & Driver, 1983).

        (b)  Bifunctional pyrrolic alcohols are more effective
        inhibitors of mitosis in cultured cells than monofunctional 
        pyrroles (Mattocks & Legg, 1980).

        (c)  Bifunctional pyrrolic alcohols are much better inducers 
        of sister chromatid exchange (SCE) in lymphocytes than 
        monoalcohols (Ord et al., 1985).

        Reasons for these differences might be that the bifunctional 
    pyrroles are able to crosslink macromolecules or simply that they 
    can bind more strongly to target molecules. 

    5.4  Metabolites Associated with the Biological Actions of
         Pyrrolizidine Alkaloids

    5.4.1  Acute hepatotoxicity

        The following is good evidence that acute liver necrosis is 
    caused by primary pyrrolic ester metabolites (dehydro-alkaloids): 

        (a)  The liver, in which these metabolites are formed, is the 
        only organ exposed to them in relatively high concentrations.

        (b)  There are good correlations between amounts of pyrroles 
        bound to liver tissue and acute hepatotoxicity (Mattocks, 
        1973).

        (c)  Pyrrolic alcohols are not acutely hepatotoxic, even when 
        given to animals in very large amounts.

        (d)  Pyrrolic esters injected iv into the liver are much more 
        acutely hepatotoxic than the parent alkaloids (Butler et al., 
        1970).

        It is possible that other metabolites, such as 4-hydroxy 
    2,3-unsaturated aldehydes, might also contribute to the acute 
    hepatotoxicity of some PAs (Segall et al., 1985).  However, this 
    has still to be confirmed. 

    5.4.2  Chronic hepatotoxicity

        The persistent antimitotic action on the liver that leads to 
    the formation of giant hepatocytes can be produced both by pyrrolic 
    ester metabolites, such as dehydromonocrotaline (Hsu et al., 1973), 
    and by pyrrolic alcohols, such as dehydroheliotridine (Peterson et 
    al., 1972).  Both kinds of metabolites can lead to similar 
    alkylation products and both are likely to be present in the liver 
    when the alkaloids are metabolized.  Thus, either could be 
    responsible for chronic hepatotoxic effects.  However, the 
    antimitotic action alone is not sufficient.  It must be accompanied 
    or followed by a stimulus of cell division.  This may be provided 
    by the acute necrotic effect of primary pyrrolic metabolites or by 
    any other cause of acute liver injury that leads to tissue 
    regeneration.  In very young animals, the stimulus can be the 
    enhanced rate of replication that already exists in them. 

    5.4.3  Pneumotoxicity

        Characteristic pyrrolizidine lung damage is produced by iv 
    injections of pyrrolic ester metabolites, which are effective at 
    much lower doses than the parent alkaloids.  The latter are not 
    metabolized in lung tissue; thus, lung damage from PAs is believed 
    to be due to pyrrolic esters reaching the lungs from the liver 
    (Butler et al., 1970).  Chronic lung damage appears to be caused by 
    bifunctional rather than by monofunctional pyrrolic alkylating 
    agents (Mattocks & Driver, 1983) (section 5.3.3.2). 

        There is some evidence that pyrrolic alcohol metabolites might 
    also be able to contribute to chronic (but not acute) 
    pneumotoxicity (Huxtable et al., 1978). 

    5.4.4  Toxicity in other tissues

        Chronic heart damage including right ventricular hypertrophy is 
    a consequence of pyrrolizidine lung damage (pulmonary hypertension) 
    (Hayashi et al., 1967).  Brain damage is attributed to ammonia 
    intoxication secondary to severe pyrrolizidine liver injury 
    (Hooper, 1972).  This view has been contested and some PAs are 
    known to have direct effects on the central nervous system (section 
    6.4.3).  There is no evidence that PAs are appreciably metabolized 
    in tissues other than the liver.  Thus, damage to other organs is 
    probably due to metabolites transported from the liver.  For 
    example, in the relatively uncommon cases of chronic kidney damage 
    after pyrrolizidine intoxication (Hooper, 1974; Hooper & Scanlan, 
    1977) megalocytosis in this organ suggests that pyrrolic 
    metabolites (either ester or alcohol) are involved.  Overall, 
    patterns of disease, as observed in extrahepatic sites, are 

    consistent with a "spillover" effect of the pyrroles produced in 
    the liver (Hooper, 1978).  Toxicity of an alkaloid reflects its 
    rate of metabolism to a pyrrole (Tuchweber et al., 1974) and so the 
    spillover effect is likely to be more evident at higher doses.  
    Studies of Culvenor et al. (1976a) suggest that the PAs that are 
    hepatotoxic for rats should also be pneumotoxic when administered 
    at higher doses.  In acute poisoning, the hepatotoxic effects could 
    outweigh the pneumotoxic effects or those on other organs, to such 
    a degree that the latter are not manifested.  Variation in 
    expression of disease (primarily hepatic or extrahepatic) also 
    depends on the reactions of host tissues in different species of 
    animals, in addition to the quantities of the pyrroles (Hooper, 
    1978).  The sensitivity of the blood vessels might explain severe 
    interstitial pneumonias in some animals, or severe nephroses in 
    pigs (McGrath et al., 1975). 

    5.4.5  Carcinogenicity

        The pyrrolic alcohols dehydroretronecine and dehydroheliotridine
    are known carcinogens (Johnson et al., 1978; Peterson et al., 1983),
    whereas the pyrrolic esters dehydromonocrotaline and dehydroretrorsine
    are only carcinogenic in conjunction with a tumour promotor (Mattocks
    & Cabral, 1979, 1982).  This suggests that the more persistent
    secondary metabolites (pyrrolic alcohols) might account for the rather
    weak carcinogenicity of some PAs. 

    5.4.6  Antitumour activity

        Some PAs and their  N-oxides are active as tumour inhibitors in 
    test systems (Culvenor, 1968; Suffness & Cordell, 1985).  Indicine 
     N-oxide, in particular, showed high activity against B16 melanoma, 
    mammary xenograft, M5076 sarcoma, P388 leukaemia, and Walker 256 
    carcinoma.  In clinical studies, indicine  N-oxide has shown 
    significant activity against some forms of leukaemia, with dosage 
    limited mainly by myelosuppression and sometimes by hepatotoxicity. 
    It is tempting to suppose that this action is related to the 
    powerful antimitotic action of their pyrrolic metabolites, even 
    though some of these alkaloids and derived pyrroles are themselves 
    carcinogenic.  On the other hand, there is evidence suggesting that 
    indicine  N-oxide owes it activity to a property of the compound 
    itself rather than to the pyrrolic metabolites, which could be 
    formed through reduction to indicine (Powis et al., 1979).  The 
    evidence, that indicine is less effective than indicine  N-oxide, is 
    not conclusive and other structure-activity data (Milkowsky, 1985) 
    point to a need for a structural capability to form a pyrrolic 
    metabolite.  It is also possible that indicine  N-oxide is converted 
    directly to dehydroindicine by mitochondrial enzymes in liver or 
    tumour cells, since the type of reaction required has been observed 
    in the mitochondrial metabolism of the  N-oxides of tryptamine 
    alkaloids and certain methylated amino acids (Fish et al., 1956; 
    Smith et al., 1962). 

    5.5  Prevention and Treatment of Pyrrolizidine Poisoning

        There is no known way to prevent pyrrolizidine liver damage, 
    once a hepatotoxic dose of the alkaloid has been ingested.  A 
    number of dietary regimes have been found to partially protect 
    animals (chiefly rodents) from the acute effects of subsequent 
    alkaloids ingestion.  None of these are of any practical use for 
    preventing pyrrolizidine intoxication in livestock.  Furthermore, 
    chronic toxic effects in the liver or in other organs are sometimes 
    more severe in animals receiving higher doses of alkaloids after 
    being protected against acute hepatotoxicty. 

    5.5.1  Modified diets

        The mechanism of action of modified diets is not clear, but 
    they may be associated with the decreased metabolic activation of 
    the alkaloids.  Some examples follow: 

        (a)  A protein-rich diet can give some protection to rats
        against  Senecio jacobaea alkaloids (Cheeke & Gorman, 1974).  
        Rats fed a high casein diet survived longer than rats given a 
        normal diet, when poisoned with retrorsine or riddelline, but 
        the survivors were more liable to develop liver tumours  
        (Schoental & Head, 1957).  However, whether this was simply due 
        to a prolongation of life of the animals by the diet is open to 
        question.

        (b)  Male rats previously fed a sucrose-only diet for 4 days 
        were considerably protected against the acute hepatotoxicity of 
        retrorsine (LD50 120 mg/kg body weight compared with 34 mg/kg 
        in normal rats).  However, lung damage, rare in control rats, 
        was frequently seen in "protected" rats given high doses of 
        retrorsine (Mattocks, 1973).

        (c)  Restriction of feed intake to 40% of normal attenuated the 
        increase in lung weight and lavage protein concentration in 
        cell-free bronchopulmonary lavage fluid and abolished the right 
        ventricular hypertrophy in monocrotaline-treated rats.  
        Furthermore, the percentage of diet-restricted animals that 
        survived was significantly higher than that in animals that had 
        eaten ad libitum up to day 28, but, from this time onwards, 
        there was no difference.  Alterations of dietary sodium intake 
        alone did not affect the results of monocrotaline-induced
        toxicity (Ganey et al., 1985).

    5.5.2  Pretreatment to enhance the detoxication of active metabolites

        Treatments that have afforded some protection against 
    pyrrolizidine hepatotoxicity (probably by increasing the liver 
    level of sulfydryl compounds, which are known to react with 
    pyrrolic metabolites) (White, 1976) include the following: 

        (a)  Pre-treatment of rats with mercaptoethylamine (150 mg/kg 
        body weight ip) partially protected rats against the acute 
        hepatotoxicity of monocrotaline given 15 min later (Hayashi & 
        Lalich, 1968); it gave no protection when administered 2 h 
        after the alkaloid.  Mercaptoethylamine, when given orally 
        (300 mg/kg body weight) at the same time as the lasiocarpine, 
        also increased the resistance of rats to the alkaloid (Rogers & 
        Newberne, 1971).

        (b)  Cysteine (1% in the diet) partially protected rats against 
         Senecio jacobaea alkaloids (Buckmaster et al., 1977) and mice 
        against monocrotaline (Miranda et al., 1981c).

        (c)  The antioxidant ethoxyquin fed at a level of 2.5 g/kg diet 
        to female mice for 38 days, increased the liver thiol 
        concentration and raised the acute LD50 of monocrotaline, given 
        ip on the 10th day, to 364 mg/kg compared with 243 mg/kg in 
        control mice (Miranda et al., 1981a).

        (d)  Rats or mice also had increased resistance to acute
        pyrrolizidine hepatotoxicity when fed the antioxidant butylated 
        hydroxyanisole (BHA) (up to 7.5 g/kg diet) (Miranda et al., 
        1981c, 1982a,b; Kim & Jones, 1982).

        (e)  Heliotrine-induced toxicity can be modified by the
        co-administration of cupir (a copper-containing complex) at a 
        level of 1 mg/kg per day for 20 days.  It prevented the exit of 
        hepatic cytosolic enzymes into the blood and improved all the 
        energy reactions studied in the mitochondria of heliotrine-
        intoxicated rats (Yuldasheva & Sultanova, 1983).  Inhibition of 
        lipid peroxidation by cytoplasmic copper was shown later 
        (Wittig & Stephen, 1964).  Savin (1983) found that lethality to 
        rats of heliotrine (300 mg/kg sc) was completely prevented by
        co-administration of alpha-tocopherol (6 ml/kg ip).

        (f)  Rats pre-treated with ip doses of zinc chloride (72 µmol/kg
        body weight) had increased resistance to the hepatotoxicity of
         Senecio jacobaea alkaloids, as assessed by histology and enzyme
        measurements (Miranda et al., 1982c).  The zinc treatment increased
        the liver level of metallothionein, a sulfhydryl-rich protein that
        might react with pyrrolic metabolites.

        Metabolic inhibitors of the microsomal P450 mixed-function 
    oxidase system, SKF 525A, metyrapone, and allylisopropyl acetamide, 
    which inhibit the formation of toxic pyrroles in the liver, have 
    been tried successfully in the prevention of the toxic effects of 
    monocrotaline in rats (Eisenstein & Huxtable, 1979).  The use of 
    P450 inhibitors was stated to show "potential therapeutic promise".  
    However, this would seem impracticable considering that, at least 
    in the rat, PAs undergo a high rate of metabolism commencing a few 
    minutes after ingestion (Mattocks, 1972b).  In some instances, they 
    have been known to lead to an increase in toxicity, e.g., with 
    lasiocarpine as reported by Tuchweber et al. (1974). 

    5.5.3  Other treatments

        Lanigan & Whittem (1970) attempted, unsuccessfully, to protect 
    sheep against  Heliotropium europaeum poisoning by treating them 
    with cobalt, in the hope that this would enhance the vitamin 
    B12-mediated detoxication of the alkaloids in the rumen (Dick et 
    al., 1963). 

        Lanigan et al. (1978) found that the resistance of sheep to 
    dietary  Heliotropium europaeum was increased by giving them large 
    daily doses of the antimethanogenic drug, iodoform.  However, Swick 
    et al., (1983) found that  Senecio jacobaea alkaloids were not 
    detoxified by incubation for 48 h with sheep rumen fluid  in vitro. 

    6.  EFFECTS ON ANIMALS

    6.1  Patterns of Disease Caused by Different Plant Genera and of 
         Organ Involvement in Different Species

        The most important genera of PA-containing plants listed in 
    section 3.1 are all hepatotoxic.  Among these,  Crotalaria spp.  
    cause damage in the broadest range of tissues in most domestic 
    species.  In pigs, they are known to be severely nephrotoxic 
    (Peckham et al., 1974; McGrath et al., 1975; Hooper & Scanlan, 
    1977).  Some species are known to be pneumotoxic for horses (Watt & 
    Breyer-Brandwijk, 1962; Gardiner et al., 1965), cattle (Sanders et 
    al., 1936; Berry & Bras, 1957), sheep (Laws, 1968), and pigs 
    (Peckham et al., 1974; Hooper & Scanlan, 1977), as well as 
    hepatotoxic. 

        Although several  Crotalaria spp. are known to be pneumotoxic 
    for horses (Gardiner et al., 1965),  C. retusa is an exception.  It 
    is an important cause of disease in horses in northern Australia 
    (Hooper, 1978) and has been shown to be pneumotoxic for pigs in the 
    same area (Hooper & Scanlan, 1977); yet it produces only hepatic 
    disease in horses (Rose et al., 1957a,b). 

        Similarly,  Senecio spp. are primarily hepatotoxic, but 
     S. jacobaea has been demonstrated to be pneumotoxic for pigs 
    (Harding et al., 1964), though it could probably be an inconsistent 
    change (Bull et al., 1968).  This plant is also known to cause 
    pulmonary disease in rats and mice (Hooper, 1974).  However, there 
    are no reports of its affecting the lungs in cattle, sheep, horses, 
    or chicken.  Renal megalocytosis and mild nephrosis are reported in 
    most species poisoned with  S. jacobaea (Harding et al., 1964; Bull 
    et al., 1968).   Heliotropium spp., Amsinckia spp., and  Echium spp. 
    are all mainly hepatotoxic. 

        Roitman (1983) summarized the pattern of organ involvement 
    observed in man and different species of farm and experimental 
    animals affected by pyrrolizidine alkaloids (Table 8).  Even within 
    a single species, the nature of a toxic effect, as well as the 
    organ affected, can be altered by changing the dose rate and 
    duration. 

    6.2  Field Observations - Outbreaks in Farm Animals

        The veterinary problem of PA toxicity has been reviewed by Bull 
    et al. (1968) and McLean (1970).  Mattocks (1986) listed the cases 
    of livestock poisoning and feeding trials since 1968, and cited 
    relevant literature.  Peterson & Culvenor (1983) produced a useful 
    and comprehensive table of the plant species known or suspected of 
    causing natural outbreaks of poisoning in each animal species.  The 
    influence of factors such as species, age, sex, and diet, on 
    toxicity is also reviewed in the same paper. 

    Table 8.  Animal species and organs affected by pyrrolizidine 
              alkaloidsa
    ---------------------------------------------------------------
    Species  Liver  Lung  Kidney  Heart  Pancreas  Gastric  Muscle
                                                   mucosa
    ---------------------------------------------------------------                
    Man      +
    Monkey   +      +     +       +
    Horse    +      +     +
    Pig      +      +     +              +         +
    Sheep    +      +     +
    Goat     +      +
    Cattle   +      +
    Dog      +
    Mouse    +            +
    Rat      +      +             +                 +
    Chicken  +      +     +                         +       +
    Turkey   +      +                                       +
    ---------------------------------------------------------------
    a From:  Roitman (1983).

        The first cases of pyrrolizidine poisoning were described in 
    cattle as early as 1903 (Gilruth, 1903).  Since then, there have 
    been numerous reports from most parts of the world, of poisoning 
    among farm animals caused by grazing or feeding on PA-containing 
    weeds (Bull et al., 1968; Mattocks, 1986).  One of the first clues 
    to the etiology of the human disease in Jamaica, came from a study 
    in which calves fed with  Crotalaria fulva (Bras et al., 1957) 
    developed characteristic veno-occlusive disease in the liver. 

        Laws (1968) described a field outbreak in sheep in a herd of 
    100 adult merino ewes, which developed within 2 weeks of moving 
    into a coastal farm in Australia, where they grazed  Crotalaria 
     mucronata.  The etiology was confirmed by feeding the plant to 6 
    sheep, 4 of which died within 24 h of feeding, with severe 
    pulmonary oedema.  However, the rapidity of poisoning and the 
    atypical lung lesions suggest that possibly a toxin other than 
    pyrrolizidine alklaoids was also present. 

        An outbreak of  Crotalaria retusa poisoning was observed in a 
    piggery near Darwin, Northern territory, Australia (Hooper & 
    Scanlan, 1977) containing approximately 350 sows.  It was caused by 
    feeding sorghum contaminated by  Crotalaria seeds at the rate of 
    about 0.1% by weight for about 3 weeks, and at a rate of about 
    0.05% for a further week.  The disease was indicated by reduced 
    body weight gain and inappetence.  The dominant pathological 
    features at autopsy were severe nephrosis with chronic uraemia, and 
    to a lesser degree, severe diffuse interstitial pneumonia.  Both 
    were accompanied by microscopic disease in the liver, and both the 
    liver and kidney showed megalocytosis. 

        Walker & Kirkland (1981) reported outbreaks in the Hunter river 
    valley of New South Wales in Australia, in cattle that had been 
    grazing a pasture in which  Senecio lautus was growing.  There were 
    sporadic deaths among the cattle as well as two protracted 

    outbreaks affecting calves 3.5 months of age and older animals, in 
    which groups of 3 - 16 head of cattle died in addition to sporadic 
    deaths of animals over periods of 1 and 6 months.  Clinical signs 
    were weakness, emaciation with recumbency, aimless wandering and 
    ataxia, which suggested neurological involvement.  At autopsy, the 
    liver showed characteristic megalocytosis, periportal fibrosis, and 
    focal necroses.  An aged animal had cirrhosis.  The etiology of 
     S. lautus was proved in feeding studies on 3 calves. 

        Knight et al. (1984) reported the deaths of 10 horses during a 
    3-year period after being fed hay from the same pasture.  The 
    animals became sick in the spring after being fed only the suspect 
    hay throughout the winter.  The hay was found to be contaminated 
    with  Cynoglossum officinale (hound's tongue), which contained two 
    PAs (heliosupine and echinatine) in much higher quantities than is 
    generally reported in  Senecio species.  The animals developed 
    weight loss, icterus, ataxia, and symptoms of hepatic failure.  At 
    autopsy, there was diffuse severe megalocytosis, biliary 
    hyperplasia, and fibrosis.  The  C. officinale was proved as the 
    etiological factor in a feeding trial on a 14-year-old pony, that 
    developed clinical features and pathological changes in the liver 
    suggesting PA poisoning.  The signs of PA toxicity in horses are 
    mostly neurological, though non-specific gastric and oesophageal 
    lesions have also been reported (McLean, 1970) (section 6.4.3).  
    Rose et al. (1957a,b) described a disease in which the dominant 
    symptom was "compulsive" walking in a straight line.  It occurred 
    in areas where  Crotalaria retusa was growing, and was ascribed to a 
    steep rise in blood-ammonia levels, which accompanied chronic liver 
    failure. 

        Farm animals differ widely in their sensitivity to PAs, sheep 
    and goats being fairly resistant, cattle and horses, less so, and 
    poultry and pigs, rather sensitive (section 6.4.1.2).  Sheep are 
    not immediately affected and generally survive one season, after 
    feeding on heliotrope and  Senecio (Bull & Dick, 1959).  During the 
    second season of feeding, they die of neurological symptoms caused 
    presumably by rising blood-ammonia levels associated with chronic 
    liver disease, or with haemoglobinuria and very high copper levels 
    in the blood (Bull et al., 1956, 1958) (section 6.4.1.2).  With 
     Crotalaria, the lung seems to be the target organ (Hooper, 1978).  
    Similar acute responses to a single feed of the plant  Crotalaria 
     spectabilis were described in cattle by Emmel (1948) and in the 
    chicken by Piercy & Rusoff (1946). 

        Poisoning of cattle in northwestern USA has reached such 
    proportions that it has become a considerable economic problem 
    (Johnson, 1982).  Culvenor (1985) has reviewed the problem of 
    livestock losses due to PA toxicosis in Australia, where it has 
    been estimated that about 10 million sheep are exposed to 
    heliotrope and  Echium plantagineum (Paterson's curse) to a greater 
    or lesser extent and may suffer a shortening of their productive 
    life by as much as two years.  Most of the PA-containing plants are 
    reported to grow in fallow fields and pastures and thrive 
    particularly in a dry climate or following periods of drought.  
    However, instances of cattle poisoning have been reported from most 
    parts of the world, including countries with temperate or cold 

    climates, which do not ordinarily suffer drought.  Three herds of 
    cattle have been reported to have been affected in the 
    alpine/subalpine region of Switzerland, after they grazed pastures 
    that had  Senecio alpinus growing on them.  Nine different 
    hepatotoxic PAs were found in the weed, the main one being 
    seneciphylline.  Analyses of urine samples from one cow confirmed 
    the presence of PA metabolites.  Several cows had to be 
    slaughtered, because of cirrhosis of the liver (Luthy et al., 
    1981). 

    6.3  Studies on Farm Animals

        There are several reports of the production of disease 
    characteristic of PA toxicity in farm animals, by feeding them 
    PA-containing plants. 

         Senecio jacobaea (tansy ragwort) is a weed that commonly grows 
    in pastures and has been the cause of extensive livestock losses in 
    the United Kingdom (Forsyth, 1968) and the USA (Johnson, 1982).  
    Extensive studies on this plant have been carried out using a 
    variety of farm animals.  Dickinson et al. (1976) fed tansy ragwort 
    to cows through a rumen canula at the rate of 10 mg/kg body weight 
    per day, for 2 weeks.  Liver biopsies showed characteristic 
    megalocytosis and fibroplasia, and autopsy also showed 
    centrilobular necrosis.  A PA, jacoline, was found in the milk, but 
    when the calves were bucket fed the milk, there were no detectable 
    effects on them.  Thorpe & Ford (1968) made similar observations in 
    5 calves fed ragwort in their diet.  Animals eventually dying of 
    toxicity showed characteristic necrosis, megalocytosis, and veno-
    occlusive lesions.  In a study by Goeger et al. (1982), goats were 
    fed dried ragwort mixed in the diet at 250 g/kg.  Four of the 11 
    goat kids and lactating dairy goats died.  Characteristic 
    megalocytosis was seen in the liver.  Goats are more resistant to 
    tansy ragwort toxicosis than cattle and horses, the chronic lethal 
    dose for cattle or horses being 0.05 - 0.2 kg ragwort/kg body 
    weight and, for goats, 1.25 - 4.04 kg/kg body weight.  The alkaloid 
    levels in the plant, and thus its toxicity, varies with season and 
    locality. 

        Hooper & Scanlan (1977) studied the long-term effects of 
    feeding very low levels of ground  C. retusa seeds, mixed with the 
    feed, to pigs and chickens.  Seven groups of 4 pigs each (sex not 
    mentioned) bred from Saddleback-large white cross sows and Large 
    White or Landrace boars, were maintained on diets containing 0 
    (control), 0.004%, 0.01%, 0.02%, 0.05%, 0.1%, or 0.5% body weight 
    ground seeds.  Another 8 pigs were fed a diet containing 0.1% 
     C. retusa for 21 days followed by 0.05% for 7 days and then kept on 
    a  C. retusa-free diet.  Pigs either died or were killed when 
    moribund, or at the end of 136 days of feeding. 

        In a second study, groups each of 4 2-week-old chickens were 
    fed diets containing 0 (control), 0.005%, 0.01%, 0.05%, 0.1%, and 
    0.5% ground seeds of  C. retusa.  Chickens fed 0.5% started dying 12 
    days after the commencement of feeding and were all dead by day 45.  
    Five out of 8 birds fed 0.1% or more died between days 22 and 56.  
    No deaths occurred in animals fed 0.01% - 0.005%. 

        In the study on pigs, all animals died between days 63 and 107 
    except for 2 that survived 136 days.  In these animals, pulmonary 
    disease was the main cause of death.  Hepatic and renal 
    megalocytosis was seen in almost all animals in both the field 
    outbreak and study group.  The lungs showed extensive consolidation 
    and oedema.  Besides megalocytosis in the glomeruli and tubules, 
    the kidneys showed glomerular atrophy and tubular necrosis.  In the 
    study on poultry, the major disease was hepatic necrosis of 
    irregular distribution.  The kidneys showed mild megalocytosis. 

        In the above study, the low levels of contamination that 
    produced serious disease are worthy of note. 

        Johnson & Molyneux (1984) fed 55 cattle, by gastric lavage, 
    with hay mixed with threadleaf groundsel ( Senecio douglasii var. 
     longilobus), which grows commonly in the pastures of southwestern 
    USA.  The PA dosage in different groups ranged from 5 to 40 mg/kg, 
    daily, and the total intake ranged from 80 to 284 mg/kg body 
    weight.  The groups were fed for periods ranging from 2 to 20 days.  
    One hundred percent mortality occurred in 3 out of 9 groups, each 
    consisting of 2 - 8 calves, receiving doses of 13 mg/kg or more.  
    Mean survival time was generally inversely proportional to the 
    dosage received.  All sick calves had typical clinical signs of 
    seneciosis.  At autopsy, the principal lesion was seen in the liver 
    and consisted of swelling of the hepatocytes, necrosis, biliary 
    hyperplasia, and marked fibrosis.  The estimated minimum lethal 
    dose of the PA was 13 mg/kg body weight for 15 days, or a total 
    intake of approximately 200 mg PA/kg, over a 15-day period.  Cattle 
    that consumed up to 600 mg PA/kg, in hay, in a 20- to 100-day 
    period, were unaffected or only slightly affected.  The authors 
    concluded that the time-dose relationship for PA toxicosis in 
    cattle is important and that there is a threshold level that must 
    be exceeded for the toxicosis to develop. 

        A similar study was conducted by Johnson et al. (1985) in which 
    the dry whole or ground leaves of  Senecio riddelli, mixed with the 
    feed, were fed to calves in gelatin capsules or by gavage.  Forty-two
    female Hereford calves were divided into 3 groups.  One group
    of 12 was fed the leaves mixed with alfalfa hay feed estimated to 
    have 20 - 40 mg PA/kg body weight per day over a 20-day period in 
    different regimes, receiving a total of 400 - 800 mg PAs per 
    animal.  The second group of 12 animals received the plant packed 
    into gelatin capsules, receiving an estimated PA content of
    10 - 20 mg/kg body weight, daily, over 20 days, with a total of
    200 - 400 mg per animal.  The third group of 18 animals was
    administered (by gavage) finely ground leaves in a water slurry at
    various PA dosages ranging from 10 to 60 mg/kg body weight per day
    and a total of 200 - 500 mg over 20 days. 

        Calves that received 10 mg PA/kg body weight per day for 20 
    days did not develop clinical signs of disease or show any changes 
    in serum-enzyme.  However, feed containing the plant that provided 
    15 - 20 mg PA/kg per day or more, administered by gavage or fed in 
    capsules, resulted in high mortality.  Malaise, depression, erratic 
    or unprecedented behaviour, aimless walking, and ataxia, were 

    observed in the affected calves; diarrhoea with tenesmus and rectal 
    collapse were frequently observed.  The feed intake decreased 
    progressively and was negligible terminally.  The animals that died 
    and those that were moribund or in a state of irreversible wasting, 
    were autopsied.  Hepatobiliary lesions were present in all such 
    animals.  The most consistent change was portal biliary hyperplasia 
    and periportal fibrosis.  Centrilobular or zonal haemorrhage and 
    necrosis were observed in some lobules.  Fibrosis of some central 
    veins was common, often encroaching on the lumen, resulting in 
    complete occlusion.  Hepatocytes also showed nonspecific changes.  
    Central nervous system changes were present in all animals with 
    clinical signs of seneciosis, consisting mainly of spongy 
    degeneration of the brain. 

        The plant mixed in the hay ration was eaten slowly and 
    reluctantly and was tolerated at dosages > 20 mg/kg per day, 
    emphasizing that the toxicity depended on the rate at which the 
    dosage was consumed and that mortality was not necessarily 
    dependent on the cumulative dosage. 

        Burguera et al. (1983) produced the disease in turkey poults by 
    feeding them seeds of  C. spectabilis.  Simultaneous addition of 
    sodium selenite at doses of 0.1, 5, or 10 mg selenium/kg diet did 
    not provide any protection. 

    6.4  Experimental Animal Studies

    6.4.1  Effects on liver

    6.4.1.1  Relative hepatotoxicity of different PAs and their  N-oxides

        The LD50 values for rats, listed in Table 9, are for some of 
    the most commonly used hepatotoxic alkaloids, calculated from data 
    on animals dying from acute haemorrhagic necrosis of the liver, 3 - 7
    days after intraperitoneal administration of a single dose.  It 
    is evident that the toxicity varies widely between the alkaloids.  
    The most toxic are certain macrocyclic diesters of retrorsine and 
    the least toxic are the monoesters of heliotridine, retrorsine, and 
    supinine (Mattocks, 1986). 

        The relative toxicity of  N-oxides compared with that of their 
    basic alkaloids depends on the route of administration.  The 
     N-oxides of lasiocarpine, monocrotaline, and fulvine were reported 
    to be as toxic as their basic alkaloids (Schoental & Magee, 1959) 
    when administered orally; however, when given by the ip or 
    intravenous (iv) routes to rats, they were much less toxic 
    (Mattocks, 1971c).  Similarly, the LD50 of retrorsine  N-oxide when 
    administered ip to male rats was 250 mg/kg (Table 9) but when given 
    orally, it was 48 mg/kg.  This has been explained by the 
    observations on the metabolic pathways of the basic alkaloids and 
    their  N-oxides.  The PAs or their  N-oxides exert toxic effects only 
    after being metabolized to pyrroles by the hepatic microsomal 
    enzymes (section 5.1.1).  Hepatic microsomes act directly on the 
     N-oxides (Mattocks & White, 1971b) only after they have been 
    converted to the basic alkaloids (Mattocks, 1986); this mainly 

    occurs in the gut (Mattocks, 1971c; Powis et al., 1979).  This 
    matter is of practical importance as the alkaloids are often 
    present as their  N-oxides in weeds grazed by farm animals. 

    Table 9.  LD50s in male rats after a single intraperitoneal dose of 
              some hepatotoxic alkaloids
    -------------------------------------------------------------------
    Alkaloid              LD50     Time range  Reference
                          (mg/kg)  (days)
    -------------------------------------------------------------------
    heliotrine            296      3           Bull et al. (1958)

    lasiocarpine          77       3           Bull et al. (1958)

    lasiocarpine N-oxide  547      3           Bull et al. (1958)

    monocrotaline         175      3           Bull et al. (1968)

    retrorsine            34       4 or 7      Mattocks (1972a)

    retrorsine N-oxide    250      7           Mattocks (1972a)

    senecionine           50       7           Mattocks (1972a)

    seneciphylline        77       3           Bull et al. (1968)

    senkirkine            220      -a          Hirono et al. (1979a)

    symphytine            130      -a          Hirono et al. (1979a)
    -------------------------------------------------------------------
    a Not stated.

    6.4.1.2  Factors affecting hepatotoxicity

        These factors have been reviewed by Mattocks (1986). 

        (a)   Route of administration

        Most studies on LD50 values have been carried out using the ip 
    route, and very few experimental data are available on toxicity 
    using the oral route.  There is a close similarity between the iv 
    data and the ip data.  Furthermore, toxicity data on rats 
    administered PAs by the oral route (Schoental & Magee, 1959), 
    including retrorsine, lasiocarpine, heliotrine, and monocrotaline, 
    closely resemble those relating to the LD50 values for the same 
    strain administered PAs intraperitoneally (Mattocks, 1972b).  Thus, 
    the hepatotoxicity of PAs in rats does not differ very much, 
    irrespective of the route of administration.  However, rabbits 
    appear to be less susceptible to PAs in the plant  Senecio jacobaea  
    when administered orally than when administered intravenously 
    (Pierson et al., 1977). 

        (b)   Species

        Wide differences have been observed in the hepatotoxic effects 
    of PAs and alkaloid-containing plants between different species of 
    both farm animals and laboratory animals, and in the same animal 
    exposed to PAs derived from different plants.  Sheep are resistant 
    to PA-containing plants (section 6.2) and when fed  Echium 
     plantagineum pellets containing 1.3 g alkaloid/kg as the sole diet 
    for 12 months, over a period of 2 years, showed almost no liver 
    damage (Culvenor et al., 1984).  However, adult rats fed the same 
    pellets as only 50% of the diet for 14 days died 4 - 13 weeks later 
    (Peterson & Jago, 1984).  Pigs were found to be 5 - 10 times as 
    susceptible to PAs in  Crotalaria retusa as chickens (Hooper & 
    Scanlan, 1977).  Overall, the approximate ratios of quantities of 
    plant material required to prove toxic in the various species 
    listed are about 200 for the sheep (approximately the same for the 
    goat), 150 for the mouse, 50 for the rat, 14 for cattle 
    (approximately the same for the  horse), 5 for the chicken, and 1 
    for the pig (Hooper, 1978). 

        Cheeke & Pierson-Goeger (1983) studied the chronic toxicity of 
     Senecio jacobaea for several laboratory animals by feeding the 
    dried plant as a component of a mixed diet.  The degree of 
    susceptibility to PA poisoning was compared in terms of the chronic 
    lethal dose of the dried plant as a percentage of the initial body 
    weight among the animals themselves, and with similar data on 
    livestock in other studies.  Gerbils, hamsters, and guinea-pigs 
    were resistant to chronic toxicity, gerbils being the most 
    resistant, consuming over 35 times their body weight of the dried 
    plant.  Comparison with similar data in other studies indicated 
    that the rabbit (Pierson et al., 1977), Japanese quail (Buckmaster 
    et al., 1977), and goat (Goeger et al., 1982) were resistant, 
    requiring a long-term lethal dose of the plant of 113% or more of 
    the initial body weight, whereas the rat was highly sensitive 
    requiring only 21% (Goeger et al., 1983).  Chicks and turkey poults 
    were also susceptible with severe inhibition of growth occurring 
    when there was 5% and 10% contamination of the feed with the plant; 
    survival time was short (Cheeke & Pierson-Goeger, 1983). 

        In a study by Fushimi et al. (1978), on the carcinogenicity of 
    the flower stalks of  Petasites japonicus Maxim in mice and Syrian 
    golden hamsters, species and strain differences were observed, not 
    only with regard to hepatotoxicity, but also with regard to the 
    carcinogenicity of PAs.  Mice of ddN, Swiss, and C57BL/6 strains 
    and Syrian golden hamsters were fed on a diet containing young 
    flower stalks of the plant for 480 days.  High incidences of lung 
    adenoma and adenocarcinoma were observed in ddN mice, but no 
    significant differences in tumour incidence were observed between 
    the experimental groups of Swiss mice and hamsters and the 
    corresponding control group.  No tumours were induced in an 
    experimental group of C57BL/6 strain mice. 

        These differences have been explained by the differences in the 
    rate of metabolic conversion of PAs to toxic metabolites (pyrroles) 
    by the hepatocyte microsomes in the different animal species (White 
    et al., 1973; Shull et al., 1976; Peterson & Jago, 1984). 

        The resistance of sheep has been ascribed to destruction of the 
    alkaloids in the rumen by a reductive conversion into non-toxic 
    1-methylenepyrrolizidine derivatives (Bull et al., 1968; Lanigan, 
    1971, 1972).  It has also been suggested that the resistance of 
    sheep is due to a low level of activation in liver cells (Shull et 
    al., 1976), but this factor was not prominent in some Australian 
    sheep, which were as sensitive as rats to PAs injected 
    intraperitoneally (Hooper, 1974). 

        Thus, it is possible for ruminants to graze plants containing 
    PAs for a period of months without evident harm, e.g., cattle 
    eating  Crotalaria juncea in Africa (Srungboonmee & Maskasame, 
    1981), but long-term effects may arise in animals exposed over 
    several years. 

        Considerable differences in LD50 values have been reported for 
    the same alkaloids in different species.  For example, the LD50 for 
    retrorsine varies from 34 mg/kg for male rats to 279 mg/kg for 
    quail and over 800 mg/kg body weight for guinea-pigs (White et al., 
    1973).  Guinea-pigs are also resistant to monocrotaline (Chesney & 
    Allen, 1973a), but not to jacobine or to mixed alkaloids of  Senecio 
     jacobaea, which are highly toxic (Swick et al., 1982a). 

        (c)   Sex

        Significant differences in the hepatotoxicity of the same 
    alkaloid have been observed between sexes in some species.  Male 
    rats are much more susceptible to the acute toxicity of retrorsine 
    or monocrotaline than females (Mattocks, 1972b).  Mattocks & White 
    (1973) reported a higher level of metabolic transformation in young 
    male rats to form pyrroles from retrorsine, compared with females 
    (section 4.4).  Jago (1971) reported a higher susceptibility in 
    male rats to the chronic hepatotoxic effects of heliotrine, while 
    female rats were more susceptible to lasiocarpine.  It is possible 
    that this may be due to the potentiating effect of male sex 
    hormones.  Campbell (1957a,b) reported that diethylstilboesterol 
    protects against the effects of seneciphylline and promotes repair 
    of damaged liver in poultry.  Protein-deficient rats of both sexes, 
    or female animals pre-treated with testosterone, were more 
    susceptible to monocrotaline (Ratnoff & Mirick, 1949). 

        (d)   Age

        Available data on the effects of age are highly conflicting.  
    It has been stated that young rats, particularly suckling animals 
    (Schoental, 1959), are more susceptible than adults to the 
    hepatotoxic effects of some alkaloids (Jago, 1970), such as 
    monocrotaline (Schoental & Head, 1955), and retrorsine and 
    lasiocarpine (Schoental, 1959).  Rats, 1 - 4 days old, were far 
    more susceptible to retrorsine and senkirkine than rats aged 25 - 30
    days (Schoental, 1970); yet new-born rats (within 1 h of birth) 
    were relatively more resistant to the hepatotoxic effects of 
    retrorsine than 1- to 4-day-old rats (Mattocks & White, 1973).  
    McLean (1970) has critically reviewed the data.  In comparing the 
    data on small animals from several studies, new-born and 4-week-old 
    animals appear to have about the same susceptibility as adults.  
    Data for the intervening period obtained by Harris et al. (1957), 
    Schoental (1959), and Hayashi & Lalich (1968) are conflicting, 
    suggesting decreased susceptibility in some studies (Harris et al., 
    1957 and one series of Schoental's studies, 1959), and increased 
    susceptibility in others (Hayashi & Lalich, 1968 and the second 
    series of Schoental's studies, 1959).  Furthermore, Jago (1971) 
    demonstrated that, while rats aged 1 - 2 weeks were more 
    susceptible to the acute effects of heliotrine and lasiocarpine 
    than older rats, sensitivity to the effects of long-term 
    administration of these alkaloids increased with age, after 2 - 3 
    months. 

        (e)   Diet

        Effects of both qualitative and quantitative changes in diet on 
    the hepatotoxicity of PAs have been investigated in several 
    studies.  Restriction of protein levels in the diet enhanced the 
    acute hepatotoxic effects of retrorsine in rats (Selzer & Parker, 
    1951) and the chronic effects of a single dose of orally 
    administered lasiocarpine (Schoental & Magee, 1957) (section 
    6.4.5.1) as well as the toxicity of PAs in  Senecio jacobaea, 
    whereas a high protein diet had a protective effect (Cheeke & 

    Gorman, 1974).  Likewise, low lipotrope diet enhanced the toxic 
    effects of orally administered lasiocarpine in pregnant rats and 
    also in the fetal livers (Newberne, 1968).  On the other hand, it 
    protected young male rats against the acute toxicity of 
    monocrotaline, because of the reduced metabolic conversion of the 
    alkaloid into pyrrolic metabolites (Newberne et al., 1971, 1974). 

        Caloric restriction reduced the cardiopulmonary toxicity of a 
    single dose of monocrotaline in rats (Hayashi et al., 1967).  This 
    was ascribed to the reduced growth rate in animals on a restricted 
    diet rather than to a reduction in the rate of metabolic conversion 
    of the alkaloid, since dietary restriction started only after 
    administration of the alkaloid.  When the animals were put back on 
    the  ad libitum feeding regimen, they developed signs of increased 
    toxicity. 

        A high copper content in the diet has been shown to enhance the 
    toxic effects of PAs (Miranda et al., 1981b).  Incorporation of 
    copper sulfate at 50 mg/kg in the diet containing the plant  Senecio 
     jacobaea increased the hepatotoxicity in rats, as judged by enzyme 
    measurements.  The implications of this observation are obvious if 
    some PA-containing plants being grazed by farm animals also have a 
    high copper content. 

        Mattocks (1972b) demonstrated the protective effects of sucrose 
    against the acute hepatotoxic effects of retrorsine in rats, if 
    administered for 3 days prior to alkaloid administration (section 
    5.5.1). 

    6.4.1.3  Acute effects

        Experimental animal studies on the pathological effects of PAs 
    on the liver have been reviewed by Bull et al. (1968) and McLean 
    (1970).  Most studies have been carried out on the rat (Schoental & 
    Magee, 1957, 1959; Bull & Dick, 1959; Schoental, 1963; Barnes et 
    al., 1964; McLean et al., 1964; Nolan et al., 1966; Jago, 1969; 
    Butler et al., 1970; Peterson & Jago, 1980), but several other 
    species of animals have been studied including the monkey (Wakim et 
    al., 1946; Rose et al., 1959; Allen & Carstens, 1968, 1971; Allen 
    et al., 1969), turkey (Allen et al., 1963), chicken (Allen et al., 
    1960), hamster (Harris et al., 1957), mouse, guinea-pig (Chen et 
    al., 1940), quail, cat, rabbit, and pig (Emmel et al., 1935; Hooper 
    & Scanlan, 1977).  All animals tested, except the guinea-pig (Chen, 
    1945), have been found to be susceptible in studies using purified 
    alkaloids and their  N-oxides and crude extracts of PA-containing 
    plants. 

        Typically, the most common lesion produced in small laboratory 
    animals by doses close to the LD50 is a confluent haemorrhagic 
    necrosis in the liver, which appears within about 12 h of exposure 
    and peaks at 24 - 48 h.  It is strictly zonal in distribution in 
    different species of animals but may vary within the same animal, 
    depending on the alkaloid used, species, nutritional status, and 
    pretreatment with other chemicals. 

        Retrorsine produces centrilobular necrosis in the rat, mouse, 
    and guinea-pig, periportal necrosis in the hamster, and focal 
    necrosis in the fowl and in the monkey (White et al., 1973).  In 
    the monkey, monocrotaline produces centrilobular necrosis (Allen & 
    Carstens, 1968), but senecionine produces necrosis in the 
    periportal and midzonal areas of the liver lobule (Wakim et al., 
    1946).  Almost simultaneously, or shortly after the development of 
    acute haemorrhagic necrosis of the liver cells, various levels of 
    change appear in the central and sublobular veins of the liver 
    lobules, consisting of subintimal oedema or even necrosis, deposits 
    of fibrin, thrombosis, and occlusion of the lumen, which later 
    becomes organized.  While haemorrhagic necrosis is a constant 
    feature, attempts to produce occlusive lesions in the veins of 
    experimental animals have produced variable results (Allen et al., 
    1967).  In man and non-human primates, hepatocellular necrosis and 
    venous occlusion occur simultaneously but, in the rat (McLean et 
    al., 1964), chicken (Allen et al., 1960), and swine (Emmel et al., 
    1935), the vascular changes follow hepatic necrosis. 

        Selzer & Parker (1951) produced a lesion comparable to human 
    veno-occlusive disease in albino rats by administrating retrorsine 
    hydrochloride, the active alkaloid of  Senecio ilicifolius, as well 
    as the crude plant extract.  Four batches of rats were administered 
    alkaloids orally in a single dose of 1 - 1.5 mg/10 g body weight or 
    repeated doses of 5 - 50 mg/kg body weight for 31 days or as single 
    subcutaneous injection of 100 mg/kg body weight.  One batch was fed 
    on a diet of  Senecio ilicifolius constituting 10% of the ration as 
    crude plant or its extract; the animals lived for 21 - 84 days.  
    Some groups were kept on a normal diet, and others on a diet that 
    was protein-deficient.  Animals, administered a single dose orally, 
    developed the earliest degenerative changes in the centrilobular 
    hepatocytes and sinusoidal dilatation, and the vascular lesion 
    appeared after 36 h.  Protein deficiency enhanced the toxic effect.  
    Only 5 out of 9 animals administered repeated doses orally showed 
    early centrilobular fibrosis and none showed the vascular lesion, 
    possibly due to scarring. 

        Bull et al. (1958) studied the effects of a single ip LD70 dose 
    of heliotrine (320 mg/kg body weight), lasiocarpine (80 mg/kg), or 
    lasiocarpine  N-oxide (629 mg/kg) in rats of a hooded strain of both 
    sexes.  Eighty-one rats were used and 3 rats from each treatment 
    were killed at intervals of 4 - 36 h.  Heliotrine produced marked 
    centrilobular necrosis at 24 h, but venous changes were not 
    evident, except for some aggregation of mononuclear macrophages on 
    the endothelium.  With lasiocarpine, the hepatic changes were 
    similar, but the necrosis was not clearly centrilobular and, with 
    its  N-oxide, it was midzonal at 34 - 49 h.  The earliest toxic 
    effect of the PAs was manifested as a temporary loss of 
    mitochondria at 8 h.  The authors concluded that PAs have an early 
    toxic effect on the hepatocytes and that this does not follow 
    vascular injury, as suggested by Davidson's earlier studies (1935). 

        McLean et al. (1964) administered an aqueous extract of 
     Crotalaria fulva to Wistar rats in a single intragastric dose of 
    0.8 - 1.5 g/kg body weight.  Lesions identical to those of human 
    veno-occlusive disease were produced in the animals by adjusting 
    the dose to permit survival for 8 - 12 days.  Loss of cytoplasmic 
    glycogen in the centrilobular cells occurred 3 h following 
    administration.  Centrilobular necrosis, which occurred after 24 h, 
    increased with time.  The central veins gradually filled up with 
    thickened endothelial cells at about 7 days, later progressing to 
    collagenization.  Evidence was presented that the histological 
    occlusion of the central veins was preceded by several days by a 
    functional blocking of the blood flow. 

        Barnes et al. (1964) observed similar results in rats 
    administered a single oral dose of fulvine  N-oxide at 50 mg/kg 
    body weight and studied at intervals of 1 - 4 days after the 
    administration.  One hundred and thirty-five rats of both sexes 
    were used.  Acute lesions resembling human disease were observed 
    during days 1 - 8.  During days 19 - 21, 3 out of 25 animals showed 
    liver damage consisting of some centrilobular haemorrhage and 
    fibrous thickening of the central veins.  Of the 78 animals studied 
    at 22 - 44 days, 50% still had centrilobular congestion and some 
    had fibrous thickening of the central veins. 

        The effects of pyrrolic derivatives of PAs on rats were studied 
    by Butler et al. (1970).  Male albino rats of Porton strain were 
    administered solutions of pyrrole derivatives of monocrotaline
    and retrorsine in dimethyl formamide as a single injection of
    0.05 - 0.1 ml solution.  When injected in the tail vein at a
    concentration of 5 mg/kg body weight, it produced progressive
    proliferation of alveolar epithelium of the lungs and the animals
    developed signs of respiratory distress in 2 - 3 weeks.  When injected
    in the mesenteric vein at a concentration of 15 mg/kg body weight, as
    a rule, the animals remained well in the postoperative period and
    only 1/26 animals died of mesenteric vein thrombosis; the livers 
    developed multiple infarcts in the left lobes that developed into 
    multiple coarse nodules at 6 - 12 weeks.  The above studies 
    substantiated the view that PAs act only when converted in 
    hepatocytes to pyrroles.  When pyrroles were injected, they 
    affected the smaller vessels at the portal of entry; in animals 
    injected through the mesenteric vein, the main target was the 
    portal vein with only secondary damage to the parenchymal cells, 
    thus sparing the animals from the effects of hepatocellular injury.  
    On the other hand, pyrroles injected through the tail vein went 
    directly to the pulmonary arteries through the heart and damaged 
    the alveolar capillaries. 

        Acute veno-occlusive disease was produced in monkeys 
    administered monocrotaline (Allen et al., 1967, 1969) and ground 
     Crotalaria spectabilis seed (Allen & Carstens, 1968).  In a study 
    published in 1967, these authors used 14 monkeys  (Macaca speciosa)  
    of both sexes, each weighing approximately 4 kg.  Seven of the 
    animals were administered 1 mg monocrotaline in distilled water by 
    gastric intubation on days 1 and 14.  The remaining 7 were used as 
    controls and received distilled water only.  Wedge biopsies of 

    liver were examined weekly.  The survival time ranged from 14 to 38 
    days, the mean being 21 days.  The livers of treated animals were 
    small and firm and showed changes characteristic of human veno-
    occlusive disease including centrilobular necrosis, and vascular 
    changes in the central veins of liver lobules ranging from 
    subintimal oedema to progressive collagenization and extension of 
    collagen fibres into the sinusoids.  Similar observations were made 
    in studies on  Macaca mulatta monkeys (Allen & Carstens, 1968), 
    administered ground  Crotalaria spectabilis seed.  Sixty-four 
    animals, averaging 6.2 kg in weight, were divided into 3 groups.  
    Group I, comprising 10 experimental animals (4 control animals), 
    received seeds in the diet containing the equivalent of 0.074 mg 
    monocrotaline/kg body weight, daily, up to death.  Group II, 
    consisting of 14 treated animals (4 control animals), received a 
    single dose of seeds containing 1.3 g monocrotaline/kg body weight, 
    and Group III, consisting of 26 experimental animals (6 controls), 
    received 3 weekly doses containing the equivalent of 0.116 g 
    monocrotaline/kg body weight, by gastric intubation.  Liver 
    biopsies were carried out each month in Group I and each week in 
    Groups II and III.  Animals of the last 2 groups were killed when 
     in extremis.  The mean survival times for the groups were:  
    Group I, 269 days (176 - 425 days); Group II, 28 days (6 - 43 
    days); and Group III, 41 days (23 - 91 days).  In Group I animals, 
    occlusive lesions of the central and sublobular veins of the liver 
    were seen in 7/10 animals at autopsy.  These consisted of oedema, 
    haemorrhages, and fragmentation of the vessel walls, the lumina 
    being filled with fibrin, and degenerating liver cells.  The 
    lobular pattern was distorted because of connective tissue bands 
    encircling small groups of liver cells, especially in the central 
    zones of the lobules.  In Groups II and III, various levels of 
    focal or centrilobular necrosis were observed and the liver cells 
    were replaced by stromal tissue.  Vascular lesions, as described 
    above, were seen in 25 monkeys, but no collagen was demonstrated. 

        In the studies of Wakim et al. (1946) on the rhesus monkey, 
    senecionine administered iv as a 2% solution at doses of 10 - 30 mg/kg
    body weight to 4 animals, daily, until they appeared to be sick,
    produced periportal, or midzonal necrosis in 3 animals accompanied
    by haemorrhage.  No mention was made of any vascular changes. 

         Electron microscopic studies

        Svoboda & Soga (1966) studied the effects of lasiocarpine and 
     Crotalaria fulva on the livers of male Sprague Dawley rats weighing 
    110 - 150 g each.  One group of 22 rats was given an ip injection 
    of lasiocarpine at 80 mg/kg body weight and pairs of animals were 
    killed at various intervals ranging from 15 min to 6 days.  A 
    second group of 22 animals was administered a single dose of an 
    aqueous extract of  Crotalaria fulva at 0.5 mg/g body weight, by 
    gastric tube, and killed at the same intervals.  A third group of 8 
    rats was administered a total of 3.2 times the LD50 dose of 
    lasiocarpine in small doses, 3 times a week, and killed at 9 - 20 
    weeks.  The changes primarily involved the nucleus and 
    interchromatin granules.  The first change, seen after 30 min in 

    the nueleoli of the hepatocytes and Kupffer cells of animals 
    receiving lasiocarpine or crotalaria extract, consisted of a 
    separation of the fibrillar and granular components.  The 
    hepatocyte nuclei had returned to normal after 72 h and remained so 
    throughout the rest of the study.  In animals receiving a single 
    dose of lasiocarpine or crotalaria, round periodic acid schiff 
    (PAS) positive eosinophilic bodies appeared in the cytoplasm after 
    12 h, consisting of dense masses of cytoplasmic material.  Five 
    days after treatment with crotalaria, large cells lined the luminal 
    surface of the central veins; the centrilobular cells had undergone 
    necrosis by this stage.  Animals receiving 3.2 times the LD50 of 
    lasiocarpine developed megalocytosis after 9 weeks (section 
    6.4.1.5).  The cytoplasm showed vesicles of smooth endoplasmic 
    reticulum with mitochondria of various shapes and sizes.  The 
    appearance resembled an exaggerated regenerative response. 

        Allen et al. (1967, 1969) studied ultrastructural changes in 
    the liver tissue, in general, including the hepatic veins in  Macaca 
     speciosa monkeys treated with PAs.  In the study on hepatic veins 
    (Allen et al., 1969), 18 treated and 6 control adult animals were 
    used with an average weight of 5.8 kg.  Animals were divided into 3 
    groups of 6 treated and 2 control animals each.  The experimental 
    animals received 0.125 g monocrotaline/kg body weight by ip 
    injection.  Liver wedge biopsies were examined at various intervals 
    in Group I during hours 1 - 48, in Group II at 4 - 12 days, and in 
    Group III at 16 - 32 days.  The earliest changes, observed by light 
    microscopy, were seen at 24 h and consisted of progressive loss of 
    endothelial cells and other associated changes in the lumen and 
    wall leading to occlusion by collagenization by the third week.  
    Under the electron microscope, within 24 h of administration, 
    marked changes were observed in the endothelial cells resulting in 
    their rupture and release of organelles in the lumen.  This was 
    followed by penetration of fluid though the vessel walls in the 
    first week and changes in the fibroblasts.  By the third to fourth 
    week, hepatic veins showed various levels of occlusion and the 
    vessel was scattered with cell debris, free organelles, etc.  The 
    authors concluded that, in this species, hepatocellular necrosis 
    was not the primary factor causing veno-occlusive disease, as the 
    association of cellular necrosis and venous occlusion occurred only 
    in the central area of liver lobules, and the hepatocytes 
    surrounding the sublobular and medium sized hepatic veins were 
    unaffected. 

        In their study of 1967, Allen et al. also investigated the 
    ultrastructural changes in the liver of  M. speciosa monkeys after 
    administering 2 doses of 1 g monocrotaline each, on days 1 and 14.  
    At autopsy, after a mean survival time of 21 days, a wide spectrum 
    of changes was observed in the hepatocytic organelles, many of 
    which were lying, discharged into sinusoids, and also phagocytosed 
    by the Kupffer cells.  By the third week, proliferation of 
    connective tissues had started in the sinusoids near the central 
    veins and also in the walls of central veins.  The authors 
    concluded that the vascular and parenchymal cell changes were 
    simultaneous and appeared to be equally instrumental in the 
    development of the occlusive lesion. 

    6.4.1.4  Mechanism of toxic action

        The mechanism of toxic action in acute pyrrolizidine 
    hepatotoxicity and the sequence of events, judged from the 
    collective experimental studies, appears to be as follows. 

        The PA, which is inactive as a cell poison by itself, becomes 
    cytotoxic through its metabolism in the hepatic parenchymal cells 
    to pyrroles, which act preferentially on the hepatocytes and the 
    endothelium of blood vessels in the liver or lungs.  In the 
    hepatocytes, the immediate action is a rapid fall in cytoplasmic 
    protein synthesis reaching 30% of control levels at 15 min and 6% 
    at 1 h (Harris et al., 1969).  This is manifested as disaggregation 
    of polyribosomes and is followed by failure of pyruvate oxidation, 
    loss of glycogen, structural damage to the mitochondria, lysosomal 
    activity, failure of mitochondrial nicotine-adenine-dinucleotide 
    (NAD) systems and nuclear NAD synthesis, and necrosis (McLean, 
    1970).  The necrosis is zonal in the liver lobule, the particular 
    zone affected depending on the metabolic enzymic geography of the 
    lobule in the particular animal species, and also in man and monkey 
    on the vascular endothelium of the central and sublobular veins. 

        The sequence of events of the vascular lesion has been studied 
    by McLean et al (1964).  After a single dose of  Crotalaria fulva  
    extract in the rat, centrilobular necrosis is present after the 
    first day, but collagenous veno-occlusion of the central veins of 
    the liver lobule only appears between 7 and 10 days later.  
    Evidently, the necrosis of the liver cells is not secondary to 
    venous occlusion.  Centrilobular haemorrhage is seen from day 2 
    onwards and signs of hepatic venous outflow tract obstruction 
    appear after 2 - 5 days (McLean & Hill, 1969). 

        Rappaport et al. (1967) and McLean (1969) demonstrated, through 
    transillumination studies on rats, that the outlet end of the 
    sinusoids is blocked by stationary columns of red cells, 16 - 24 h 
    following administration of PAs.  The reaction is typically patchy 
    and results in stasis and extravasation of red cells spreading 
    backwards from the centre of the lobule.  For at least 3 days, no 
    circulatory detail can be seen with transillumination.  Portal 
    pressure is significantly raised 3 days after administration of 
    fulvine (Rappaport et al., 1967), notably before the first 
    appearance of collagenous venous occlusion at 7 days.  McLean 
    (1969) observed that 6 - 10 days after PA administration, a new 
    irregular pattern of vascular flow, contrasting with the uniform 
    radial pattern of flow in the normal liver lobule, develops, which 
    corresponds to the bypass channels represented by dilated 
    paraseptal sinusoids, as observed in human liver biopsies (section 
    7.3).  Segments of central vein into which the blocked sinusoids 
    open, are gradually abandoned in favour of such by-pass routes and 
    undergo occlusion first by oedematous connective tissue and then by 
    fibrosis.  The mechanism of closure of the sinusoids is not clear.  
    A toxic action on the sinusoidal or venous endothelium, which 
    swells and occludes the lumen, seems possible, as suggested by 
    Allen & Carsten's (1968) electron microscopic studies on the 
    monkey, and studies on children (Brooks et al., 1970).  The 

    endothelial lining of the vessels is denuded and replaced by a 
    fibrinous and proteinaceous precipitate, which, together with the 
    oedematous wall of the vessel, becomes organized and slowly 
    replaced by fibrous connective tissues.  The occlusion of sinusoids 
    is further contributed by the discharge of cellular debris into the 
    space of Disse.  The lumen of the sinusoids becomes occluded 
    simultaneously with the fibrosis occurring in the central vein.  
    Collagen fibres extend into the space of Disse and sinusoids 
    leading to a creeping fibrosis. 

        The proximate toxin that escapes from the liver is returned to 
    the heart, after which it damages the first portal of entry into 
    the alveolar capillaries of the lung and pulmonary arteries. 

    6.4.1.5  Chronic effects

        The chronic hepatotoxic effects of PAs have been described in a 
    number of studies on a variety of animals and have been reviewed by 
    Bull et al. (1968) and McLean (1970).  A notable feature is that an 
    appropriate single dose of PA has been demonstrated by Schoental 
    and Magee (1957, 1959) to lead to a relentlessly progressive course 
    and eventually kill the animal, more than 18 months after 
    administration.  Schoental & Bensted (1963) demonstrated that rats 
    receiving a single dose of PA may develop chronic liver disease and 
    finally hepatocellular carcinoma more than 13 months after 
    administration. The morphological changes in the liver are similar 
    in a given species of animal, whether a single sublethal dose is 
    administered or multiple small doses. 

        Schoental & Magee (1957) studied the long-term effects of a 
    single dose of lasiocarpine on rats receiving normal and protein-
    deficient diets.  Albino rats of Porton strain were used.  In the 
    first study, 66 rats fed a normal diet were administered a single 
    oral dose of lasiocarpine at one of 3 dose levels (50 - 74 mg/kg, 
    75 - 100 mg/kg, or 101 - 150 mg/kg body weight); 24 animals served 
    as controls.  In another study, 46 young female rats were 
    administered a protein-deficient diet.  Of these, 13 and 10 animals 
    received a single oral dose of lasiocarpine at 50 - 100 mg/kg and 
    50 - 75 mg/kg body weight, respectively.  Each of these groups was 
    pair-fed with an identical number of animals that did not receive 
    any PA.  Of the 66 rats fed a normal diet, very few died in the 
    first 10 days.  Thirty-one animals survived longer than 3 months.  
    They continued to be in good health until shortly before death.  
    The numbers of animals that survived for 13 months after 
    administration of lasiocarpine were 8/10 males and 7/7 females 
    (lowest dose) and 5/25 males and 11/18 females (intermediate dose).  
    In the group that received the highest dose, neither of the 2 male 
    animals survived longer than 35 days, and only 1/4 female animals 
    survived longer than 3 months.  In the animals that died or were 
    killed when moribund, parenchymal damage was invariably present 
    with prominent megalocytes, ductular proliferation, and invasion of 
    lobules by oval or elongated cells, thought to be derived from the 
    bile-duct epithelium or the reticuloendothelial cells.  Animals 
    that survived showed various degrees of fibrosis and nodular 
    hyperplasia and, in some, a mild thickening of the central veins.  

    No obliterative lesions of the veins were seen.  The 31 animals 
    that survived 13 months showed similar changes, but to a much 
    lesser extent.  In the livers of animals that had repeat liver 
    biopsies, there was no tendency to regression of the lesions. 

        The above data indicate that very few animals died of acute 
    disease.  In most animals, there was a latent period of 3 - 4 
    weeks, during which they remained well and showed little evidence 
    of liver cell injury, followed by a progressive course often 
    leading to fibrosis and nodular hyperplasia. 

        The low-protein diet adversely affected the growth of all the 
    rats in the control as well as the treated group.  Only 3 out of 23 
    treated animals remained alive and in apparent good health, 8 - 11 
    months after the treatment.  Liver biopsies taken at various 
    intervals between 4 and 10 months showed very severe fatty changes 
    in the liver cells.  There was little fibrosis and no bile-duct 
    proliferation.  In areas where the fatty changes were less severe, 
    characteristic megalocytes were seen.  Control animals had either 
    normal livers or showed only slight fatty changes that were not 
    comparable in severity with those in the livers of PA-treated 
    animals.  Thus protein deficiency in the diet was shown to enhance 
    the toxic effects of the PA. 

        Schoental & Magee (1959) extended these studies on young Wistar 
    rats using several other PAs including heliotrine, retrorsine, 
    riddelliine, seneciphylline, monocrotaline, and its  N-oxide in 
    various dosages ranging from 25 to 300 mg/kg body weight; the 
    animals were studied at death from 1 - 10 days to 18 - 30 months.  
    Pathological changes were similar to those observed with 
    lasiocarpine in the previous study.  Notable observations were that 
    necrosis did not necessarily precede subacute or chronic changes.  
    The livers of some animals became severely damaged and showed 
    nodular hyperplasia.  Liver biopsy, 2 - 3 days after PA treatment, 
    did not show pathological changes in some animals, but a repeat 
    biopsy at 41 days showed characteristic changes.  Fibrous 
    thickening of the central veins was observed in some animals, more 
    often with monocrotaline  N-oxide, but no occlusion of the hepatic 
    veins was seen. 

        The studies of Nolan et al. (1966) confirmed the observations 
    of Schoental & Magee (1957, 1959).  They gave a single dose of 
    lasiocarpine at 120 mg/kg body weight, by stomach tube, to 108 
    (equal numbers of both sexes) Sprague Dawley weanling rats (60 - 
    135 g body weight).  Thirty animals of both sexes served as 
    controls.  Groups of 10 animals, each consisting of 8 treated and 2 
    control animals, were killed at various intervals from 1 to 123 
    days.  Of the 80 treated animals, 28 died within 26 days.  No 
    delayed hepatic lesions were found in 59 rats between days 1 and 
    18.  Between 19 and 123 days, delayed lesions were found in 34/49 
    rats.  These 34 rats showed megalocytosis, but no ductular 
    proliferation or fibrosis. 

        In a second study, 127 twenty-one-day-old male Wistar rats were 
    given a single oral dose of lasiocarpine at 80 mg/kg body weight; 
    65 animals served as controls.  Liver biopsy was carried out on day 
    3 in 108 and on day 9 in 15 of the treated animals.  In 47 animals, 
    additional biopsies were carried out at intervals.  Of the 127 
    PA-treated rats, 98 died during the first 9 days, and 29 after 
    10 - 50 days.  In contrast to the first study, 32/58 survivors 
    exhibited delayed subacute and chronic lesions, as described by 
    Schoental & Magee (1957).  Of these, 8 animals developed cirrhosis.  
    The observations indicated that the lesions of acute zonal 
    necrosis, which appeared on, or before, the third day, healed 
    without residual lesions.  However, 55% of the 58 survivors 
    developed subacute/chronic lesions that tended to be progressive 
    after a latent period of 2 - 3 weeks.  There appeared to be an 
    intimate relationship between chronic lesions and megalocytosis, 
    which was seen in 52/58 surviving animals.  No obliterative 
    vascular changes were observed and so the lesions could not be 
    ascribed to impaired circulation. 

        Schoental (1959) demonstrated the toxic effects on the newborn 
    of PAs administered to lactating mother rats.  Wistar rats
    (200 - 300 g) were administered lasiocarpine, orally or by ip
    injection, at 25 - 40 mg, in 5 - 10 doses of 5 - 10 mg, twice weekly
    or more (24 rats), or retrorsine at 4 - 10 mg per dose, in 1 - 14
    approximately daily doses (23 rats).  The litters were left with 
    the mothers for 24 days or more (except for 1/2 hour separation 
    during the PA treatment).  The litters were examined by biopsy at 
    frequent intervals or at autopsy when they died or were killed in a 
    moribund state.  Litters of the lasiocarpine-treated rats showed 
    only insignificant fibrosis or some megalocytosis.  In the 
    retrorsine-treated group, the majority of the young rats survived 
    for about 18 days, but all rats died before reaching the age of 30 
    days. The milk secretion of the mothers was apparently not affected 
    by the PA treatment.  Of the 98 animals in 9 litters, 45 died by 
    the 20th day and 45 survived 30 days.  Animals dying in the first 
    fortnight did not show gross liver lesions, but those that died at 
    weaning time or later, all showed signs of liver disease.  Animals 
    dying at 18 - 30 days showed hydropic or fatty vacuolation of 
    hepatocytes and haemorrhage into distended sinusoids.  The change 
    was severe in animals dying at 1 - 2 months, and some central veins 
    showed a narrowed lumen.  The lactating rats that received the 
    alkaloids survived longer than their young, and most showed no ill 
    effects from the treatment.  This evidently indicates either a high 
    susceptibility of suckling rats or a high concentration of PAs in 
    milk. 

        In studies by Allen et al. (1963), 2 groups of 4-week-old 
    turkeys, each consisting of 12 birds, were fed diets containing 
    ground  Crotalaria spectabilis seed at 2.5 g/kg and 5 g/kg, 
    respectively, for 120 days.  Twelve animals served as controls.  At 
    the end of the study, 11/12 birds receiving 5 g/kg seed and 6/12 
    receiving 2.5 g/kg seed in the diet developed cirrhosis.  The 
    minimum period of feeding required to produce cirrhosis was 75 
    days, provided the diet was reduced to a level that was not lethal. 

        Allen & Carstens (1971) induced the Budd-Chiari syndrome in 
    monkeys by monocrotaline.  Six adult female and 9 adult male  Macaca 
     speciosa monkeys, weighing 5.2 - 6.5 kg each, were divided into 2 
    groups, each comprising 5 control (3 males and 2 females) and 10 
    treated animals (6 males and 4 females).  The treated group was 
    given a subcutaneous (sc) injection of monocrotaline at 60 mg/kg 
    body weight at monthly intervals for 3 months.  Needle biopsy of 
    the liver was carried out every month for 5 months and laparotomy, 
    6 months after PA treatment. 

        The treated animals showed marked vascular changes and various 
    degrees of occlusion in the centrilobular and sublobular veins as 
    well as the larger vessels.  There was also characteristic 
    haemorrhagic necrosis in the centrilobular zones and megalocytes 
    were seen.  The portal venous pressures were raised.  The animals 
    were autopsied at 6 months.  The livers were markedly shrunken 
    weighing an average of 68 g in contrast to those of control 
    animals, which weighed 130 g.  There were severe occlusive vascular 
    changes and irregular fibrosis in the lobules.  The adjacent 
    sinusoids were dilated as a compensatory mechanism. 

        Swick et al. (1982a) studied the effects on guinea-pigs of 
    long-term dietary administration of  Senecio jacobaea and compared 
    them with the toxic effects of single doses of injected  Senecio  
    alkaloids and monocrotaline.  The possible protective effect of 
    cysteine was also examined.  Fifteen guinea-pigs of 250 - 300 g 
    initial body weight were divided into 2 treated groups, being fed 
    10%  Senecio jacobaea, or 10%  Senecio jacobaea plus 1% cysteine in 
    the diet, and a control group.  The whole plant of  Senecio jacobaea  
    was air dried and powdered for incorporation into the diets.  The 
    animals were fed for 365 days.  They were autopsied at death or at 
    the termination of the study.  In a second study, 7 guinea-pigs of 
    500 g body weight were injected intraperitoneally with either 
    monocrotaline, jacobine, or mixed  Senecio jacobaea PAs.  The 
    chronic lethal dose LD100 of  Senecio jacobaea was 1264 g/kg initial 
    body weight or 526% of the initial body weight with an average 
    survival time of 279 days.  No mortality was observed in control 
    animals.  This contrasts with the chronic LD100 of  Senecio jacobaea  
    for rats of 58% of initial body weight (Swick et al., 1979) and 
    that of cattle equivalent to 5 - 20% body weight (Bull et al., 
    1968).  Addition of cysteine to the diet was only slightly, but not 
    significantly, protective.  Pathological examination of the livers 
    of the guinea-pigs fed  Senecio jacobaea revealed extensive 
    megalocytosis and severe cytoplasmic vacuolation with biliary 
    hyperplasia and fibrosis, primarily in periportal areas.  The 
    centrilobular and midzonal areas were spared. 

        Monocrotaline was non-toxic at doses up to 1000 mg/kg body 
    weight, whereas jacobine and mixed alkaloids from  Senecio jacobaea  
    were lethal at much lower levels.  Similar results showing 
    resistance to monocrotaline in guinea-pigs were also reported by 
    Chesney & Allen (1973a).  In  in vitro studies, they related this 
    resistance to lack of conversion of PA to pyrroles by guinea-pig 
    microsomes. 

        A morphological peculiarity of chronic hepatotoxicity in a 
    large variety of laboratory and farm animals is megalocytosis 
    (Bull, 1955; McLean, 1970), i.e., the appearance of exceptionally 
    large hepatocytes, 10 - 30 times the volume of normal cells with 
    proportionately large nuclei.  Relevant literature has been 
    reviewed by Jago (1969), McLean (1970), and Mattocks (1986).  
    Advanced megalocytosis was produced by Jago (1969) within 4 weeks 
    in 2-week-old rats by administering a single dose of lasiocarpine 
    at 76 µmol/kg body weight.  Megalocytes tend to appear in the 
    periportal and midzones of the liver lobules with normal sized 
    cells around the central veins.  The nuclear chromatin is 
    proportionately increased, but the cells appear incapable of 
    entering into mitosis, as only abnormal mitoses are seen.  Jago 
    (1969) demonstrated a fall in the mitotic index (from 1.61 to 0.04) 
    in liver cells of 2-week-old rats, one day after injection of 50 µmol
    lasiocarpine/kg.  The electron microscopic appearance also 
    supports the above observations (Afzelius & Schoental, 1967).  A 
    striking proliferation of rough endoplasmic reticulum and multiple 
    centrioles is seen in the cytoplasm, and the cytoplasmic organelles 
    are disorganized, suggesting increased metabolic activity but 
    inability of the cells to divide.  Such cells may persist for the 
    life-time of the animal (up to 2 years in the rat) and the liver 
    never returns to normal (Mattocks, 1986).  Megalocytes have also 
    been described in the kidney (Bull et al., 1968), the lung (Barnes 
    et al., 1964; Butler et al., 1970; Hooper, 1974), and the duodenum 
    (Hooper, 1975c). 

        Data on the total chronic lethal dose of heliotrine in rats 
    were discussed by Bull & Dick (1959) and Bull et al. (1968).  For a 
    variety of dosing rates, and with withholding periods of 10 - 20 
    weeks interposed, the total doses ranged from 2.2 to 7.8 LD50.  In 
    Table 10, these data are extended with results for other alkaloids.  
    The overall range of the total lethal dose is 1.2 - 10.3 LD50. 

    6.4.2  Effects on lungs

        Current literature has been extensively reviewed by Kay & Heath 
    (1969), and Mattocks (1986).  PAs have been shown to produce 
    pulmonary hypertension with associated vascular changes in the 
    pulmonary circulation in a number of experimental animal species 
    including the rat, mouse, frog, turkey, pig, sheep, rabbit, and 
    horse (McLean, 1970) as well as in non-human primates (Allen & 
    Chesney, 1972; Chesney & Allen, 1973b) and the dog (Miller et al., 
    1978).  The alkaloids have been administered by feeding the animals 
    with:  PA-containing seeds of plants (notably  Crotalaria 
     spectabilis) (Turner & Lalich, 1965; Kay & Heath, 1966; Kay et al., 
    1967a) or the dried plant itself (e.g.,  Senecio jacobaea) (Burns, 
    1972), aqueous solutions of fulvine (Barnes et al., 1964; 
    Wagenvoort et al., 1974a,b) or monocrotaline (Lalich & Ehrhart, 
    1962; Huxtable et al., 1977), subcutaneous injections of 
    monocrotaline (Allen & Chesney, 1972; Chesney & Allen, 1973b) and 
    seneciphylline (Ohtsubo et al., 1977), or intravenous injections of 
    some pyrrolic esters and analogues of pyrrolizidine alkaloids and 
    their metabolites (Mattocks & Driver, 1983).  Lafranconi & Huxtable 
    (1984) studied the hepatic metabolism and pulmonary toxicity of 

    monocrotaline in  in vitro perfusion studies.  Some of the 
    representative studies on the morphological effects of toxic lung 
    injury are listed in chronological order in Table 11. 

        Chronic lung lesions have been produced by most compounds that 
    produce chronic liver lesions, though higher doses were required in 
    some instances (Culvenor et al., 1976a).  However, not all PAs that 
    are hepatotoxic are also pneumotoxic.  Among the pneumotoxic 
    alkaloids, fulvine (Barnes et al., 1964) and monocrotaline are 
    particularly active (Mattocks, 1986).  Molecular structure activity 
    requirements are the same as for hepatotoxicity, since both are 
    caused by the same toxic metabolites produced in the hepatocytes. 

    6.4.2.1  Acute effects

        Pulmonary lesions produced by PAs have been extensively 
    investigated, mostly in rats, but also in non-human primates. 
    Monocrotaline has been the alkaloid most frequently used, but lung 
    lesions have also been seen in rats following fulvine and 
    seneciphylline administration.  Besides pure alkaloids, 
    PA-containing seeds of some plants, most notably  Crotalaria 
     spectabilis, have also been used. 

        Miller et al. (1978) gave a single iv injection of 
    monocrotaline at 60 mg/kg body weight to 10 mongrel dogs.  Toxic 
    effects, recorded within 2 h, included ultrastructural changes in 
    the endothelial cells of the alveolar capillaries, prominent 
    accumulation of platelets, and the appearance of interstitial 
    oedema (Table 11).  Valdivia et al. (1967a,b) used 25 Sprague 
    Dawley rats in their study and made similar observations on the rat 
    lung within 4 h of a single subcutaneous injection of monocrotaline 
    at a dose of 60 mg/kg body weight (Table 11).  Interstitial oedema 
    and elastolysis of the alveolar wall, increase in number of mast 
    cells, and other associated changes were observed within 4 h of the 
    injection, followed by alterations in endothelial and interstitial 
    cells.  All of the changes progressed steadily for up to 3 weeks.  
    It was concluded that the initial changes of destruction of 
    pulmonary capillaries and the other components of the alveolar wall 
    preceded the arteriolar hypertrophy and arteritis observed by other 
    investigators following monocrotaline administration, and alone was 
    sufficient to cause right ventricular hypertrophy.  Sugita et al. 
    (1983a,b) administered a single dose of monocrotaline at 40 mg/kg 
    body weight to 5 Sprague Dawley rats and adduced further evidence, 
    by biochemical and radioisotopic studies, of microvascular leak in 
    the alveolar wall within the first 3 days of injury, which preceded 
    right ventricular hypertrophy observed 2 weeks following 
    administration (Table 11). 


        Table 10.  Total chronic lethal doses in rats (ip administration, 2 or 3 
               times per week)
    --------------------------------------------------------------------------
    Dose (x LD50)               Time to   Total lethal  Reference
                                death     dose
                                (days)    (x LD50)
    --------------------------------------------------------------------------
     Heliotrine (male rats, unless otherwise stated)

       0.2                      58        5             Bull & Dick (1959)
       0.1                      123       5.1           
       0.04                     303       4.1
       0.02                     508       4.1
       0.01                               5.2 - 5.3     Bull & Dick (1960)
    (with interval of 10 -                              
    20 weeks after 21 days)
       0.11                               2.2 - 4.3     Bull & Dick (1959)
       0.1                                7.8           Jago (1971)
    (35-day-old male rats)
       0.1                                4.7           Jago (1971)
    (337-day-old male rats)
       0.1                                5.8           Jago (1971)
    (35-day-old female rats)
       0.1                                4.5           Jago (1971)
    (337-day-old female rats)

     Lasiocarpine (male rats)

       0.1                      210       9             Culvenor & Jago (1979)
       0.05                     482       10.3          
       0.02                     676       5.7         
       0.01                     595       2.6
       (0.005)                  (638)     (1.4)
       0.1                      81 - 175  2.4 - 5       Bull & Dick (1959)
       0.1                      -         6.3 - 10.9    Jago (1971)

     Lasiocarpine (female rats)

       0.1                      108       4.6           Culvenor & Jago (1979)
       0.05                     274       5.8           
       0.02                     471       4
       0.01                     487       2.1
       (0.005)                  (692)     (1.5)
       0.1                                2.4 - 7       Jago (1971)

     Monocrotaline
       0.1                                1.2 - 2.4     Bull et al. (1968)
       0.05                               2.5 - 4.4      

     Senecioninea
       0.2                                2 - 7.4       Bull et al. (1968)
       0.1                                1.7 - 5.7
       0.04                               (3 survived)
    --------------------------------------------------------------------------
    a Assuming LD50 mg/kg (c.f., Mattocks, 1986).
    
        Table 11.  Summary of experimental data on the morphological effects of toxic lung injury due to pyrrolizidine alkaloids (in 
               chronological order)
    ---------------------------------------------------------------------------------------------------------------------------------------
    Animal/     Number     Toxic agent     Dose     Administration   Single/   Killed after  Pathological            References
    strain/     of                         (mg/kg)  dose             multiple  or survival   effects
    sex         animals/                                                       for
                controls
    ---------------------------------------------------------------------------------------------------------------------------------------
    Acute effects

    Male        25/5       monocrotaline   60       subcutaneous     single    2 - 48 h      interstitial oedema,    Valdivia et al.
    Sprague                                (body                               1 - 3 weeks   endothelial cell        (1967a,b)
    Dawley rat                             weight)                                           alterations,            
                                                                                             elastolysis, etc.

    Mongrel     10/0       monocrotaline   60       intravenous      single    2 h           interstitial oedema,    Miller et al. (1978)
    dog                                    (body                                             changes in endothelial  
                                           weight)                                           cells                   

    Male        5/3        monocrotaline   40       subcutaneous     single    0 - 21 days   microvascular leak,     Sugita et al. (1983a)
    Sprague                                (body                                             right ventricular     
    Dawley rat                             weight)                                           hypertrophy after 2   
                                                                                             weeks

    Chronic effects

    Female      35/12      monocrotaline   10 - 30   ad libitum                               pulmonary arteritis     Lalich & Ehrhart
    Sprague                                (diet)   feeding                                  (with 20 - 30 mg dose   (1962)
    Dawley rat                                                                               only)            

                6          alcohol-        2500      ad libitum                 51 days       none
                           extracted       (diet)   feeding
                           seeds of
                            Crotalaria
                            spectabilis

                19/4       monocrotaline   10 - 75   ad libitum                 26 - 232                              Turner & Lalich (1965)
                                           (diet)   feeding                    days                              
    ---------------------------------------------------------------------------------------------------------------------------------------                                                                

    Table 11.  (contd.)
    ---------------------------------------------------------------------------------------------------------------------------------------
    Animal/     Number     Toxic agent     Dose     Administration   Single/   Killed after  Pathological            References
    strain/     of                         (mg/kg)  dose             multiple  or survival   effects
    sex         animals/                                                       for
                controls
    ---------------------------------------------------------------------------------------------------------------------------------------
     Chronic effects (contd.)

    Female      24/6        Crotalaria      200 -     ad libitum                 105 - 172      right ventricular       Allen & Chesney (1972)
    Wistar                  spectabilis     1600     feeding                    days           hypertrophy and       
    Furth rat              seeds           (diet)                                            dilatation, pulmonary     
                                                                                             arterial hypertrophy,
                                                                                             pulmonary arteriolar
                                                                                             hypertrophy, 
                                                                                             endocardial fibrosis

    Female      10/34       Crotalaria      1000      ad libitum                 30 - 60 days   right ventricular       Kay & Heath (1966)
    weanling                spectabilis     (diet)   feeding                                   hypertrophy, pulmonary  
    Wistar rat             seeds                                                             arterial hypertrophy,      
                                                                                             pulmonary arteritis

    Male        22/12      monocrotaline   120      subcutaneous     single    20 - 47 days  right ventricular       Hayashi & Lalich 
    suckling                               (body                                             hypertrophy, pulmonary  (1967)
    Sprague                                weight)                                           arterial hypertrophy,     
    Dawley rat                                                                               pulmonary arteritis,    
                                                                                             fibrin thrombi

    Female      30/10      fulvine         50       intraperitoneal  single    3 - 37 days   right ventricular       Kay et al. (1971a)
    Wistar rat                             (body                                             hypertrophy, pulmonary  
                                           weight)                                           arterial hypertrophy,   
                                                                                             pulmonary arteriolar
                                                                                             hypertrophy, pulmonary
                                                                                             arteritis, arteriolar
                                                                                             thrombi

                                           80       intragastric     single    7 - 35 days
                                           (body    
                                           weight)
    ---------------------------------------------------------------------------------------------------------------------------------------

    Table 11.  (contd.)
    ---------------------------------------------------------------------------------------------------------------------------------------
    Animal/     Number     Toxic agent     Dose     Administration   Single/   Killed after  Pathological            References
    strain/     of                         (mg/kg)  dose             multiple  or survival   effects
    sex         animals/                                                       for
                controls
    ---------------------------------------------------------------------------------------------------------------------------------------
     Chronic effects (contd.) 

    Monkey      12 (30     monocrotaline   30       subcutaneous     one       199 - 325    right ventricular        Allen & Chesney (1972)
    (Macaca     days old)                  (body                     followed  days         hypertrophy and        
    arctoides)                             weight)                   by 3      (average,    dilatation, left          
    (both                                                            (2, 4,    241)         ventricular 
    sexes)                                                           and 6                  hypertrophy, pulmonary 
                                                                     months                 arterial hypertrophy, 
                                                                     after                  pulmonary arteriolar 
                                                                     first                  hypertrophy, pulmonary 
                                                                     injection              hypertension 
                12 (15                                                         163 - 334    pulmonary arterial
                months                                                         days         hypertension (isolated,
                old)                                                           (average,    veno-occlusive disease
                                                                               217)         (liver)

    Monkey      20/6       monocrotaline   30       subcutaneous     as above  165 - 325    right ventricular        Chesney & Allen
     (Macaca                                (body                               days          hypertrophy and          (1973b)
     arctoides)                             weight)                             (average,     dilatation, pulmonary     
    (both                                                                      326)         arterial hypertrophy,
    sexes)                                                                                  pulmonary arteriolar
                                                                                            hypertrophy, pulmonary
                                                                                            arteritis, endocardial
                                                                                            fibrosis

    Female      50/12      fulvine         80       intragastric     single    1 - 6 weeks  vasoconstriction,        Wagenvoort et al.
    Wistar rat                             (body                                            pulmonary arterial       (1974a,b)
                                           weight)                                          hypertrophy, pulmonary 
                                           or 50                                            arteriolar hypertrophy, 
                                           (body    intraperitoneal                         right ventricular
                                           weight)                                          hypertrophy, thickening
                                                                                            of veins and venules
    ---------------------------------------------------------------------------------------------------------------------------------------

    Table 11.  (contd.)
    ---------------------------------------------------------------------------------------------------------------------------------------
    Animal/     Number     Toxic agent     Dose     Administration   Single/   Killed after  Pathological            References
    strain/     of                         (mg/kg)  dose             multiple  or survival   effects
    sex         animals/                                                       for
                controls
    ---------------------------------------------------------------------------------------------------------------------------------------
     Chronic effects (contd.)

    Male        16/0       seneciphylline  50 - 80  subcutaneous     single    1 - 3 weeks  pulmonary arteriolar     Ohtsubo et al. (1977)
    Wistar                                 (body                                            and right ventricular 
    rat (4                                 weight)                                          hypertrophy (after 3   
    weeks old)                                                                              weeks), right 
                                                                                            ventricular dilatation
                                                                                            in 2 animals

    Male        21/14       Crotalaria      1000      ad libitum                 3 - 35 days   pulmonary arterial       Meyrick & Reid (1979,
    Sprague                 spectabilis     (diet)   feeding                                  hypertrophy and          1982)
    Dawley rat                                                                              pulmonary arteritis      
                                                                                            (2/21); right           
                                                                                            ventricular hypertrophy
    ---------------------------------------------------------------------------------------------------------------------------------------
            A histological and electron microscopic study was made by 
    Hurley & Jago (1975) of the lungs of rats administered 
    dehydromonocrotaline.  Female black and white hooded rats weighing 
    80 - 100 g were used.  Dehydromonocrotaline dissolved in 
    dimethylformamide (DMF) was administered iv as a single dose at
    30 mg/kg body weight to 12 rats and at 15 mg/kg body weight to 7 rats.  
    Four control rats were administered DMF alone.  A colloidal 
    suspension of carbon black was injected iv, 6 - 18 h after 
    injection of dehydromonocrotaline, and the animals killed 19 - 44 h 
    after treatment. 

        After an interval of 6 - 8 h, there was a direct toxic effect 
    on the endothelial cells of pulmonary capillaries and small 
    venules.  Many endothelial cells had prominent nuclei and thickened 
    cytoplasm, which contained more RNA granules than usual.  There was 
    also an increase in the number of mitochondria.  The endothelial 
    damage did not seem to have caused permanent disruption of the 
    small blood vessels, and, 2 days after injury, all vessels were 
    patent.  Large numbers of mononuclear cells, which appeared in the 
    interstitial tissues of the lung 44 h after injury, seemed to be 
    altered emigrated blood monocytes. 

    6.4.2.2  Chronic effects

        Lalich & Erhart (1962), fed 35 Sprague Dawley rats a diet 
    containing monocrotaline at 10 - 30 mg/kg (Table 11).  Animals 
    receiving a daily dose of monocrotaline of 20 mg/kg diet or more, 
    showed progressive changes in the lungs after 24 days of feeding.  
    Of the 23 animals receiving 20 - 30 mg/kg, 12 showed pulmonary 
    arteritis, 4 of these even at the dose level of 20 mg/kg diet.  
    Pulmonary haemorrhages were observed in 16 animals.  No changes 
    were observed in animals fed alcohol-extracted seeds or other 
    derivatives of monocrotaline. 

        Identical changes were observed in similar studies by Turner & 
    Lalich (1965) on two strains of rats, Sprague Dawley (19) and 
    Wistar Furth (24).  The first group of 19 female Sprague Dawley 
    rats was fed a diet containing monocrotaline at an initial level of 
    10 mg/kg diet.  Depending on the response of each individual 
    animal, the monocrotaline level was raised to a maximum of 75 mg/kg 
    diet (Table 11).  Fourteen rats survived for more than 100 days and 
    8 reached the maximum dietary level of monocrotaline, the last 
    animal dying after 232 days.  The second group of 24 female Wistar 
    Furth rats was fed a diet contaminated with  Crotalaria spectabilis  
    seeds, initially at 0.2 mg/kg diet, gradually rising to 1.6 mg/kg 
    by increasing the levels by 0.2 mg/kg every week.  All test animals 
    survived 100 days and 15 reached the maximum levels of  Crotalaria  
    fed.  The last animal died after 172 days of feeding.  Animals 
    developed signs of toxicity and right ventricular strain, e.g., 
    cyanosis, etc.  Progressive thickening of media in the muscular 
    pulmonary arteries, progressive muscularization of arterioles, and 
    changes characteristic of pulmonary hypertension, were seen.  Some 
    pulmonary arteries showed medial necrosis.  No changes were 
    observed in the pulmonary veins.  Significant hypertrophy of the 
    heart, as judged by the heart weight in relation to body weight, 

    was seen in almost all animals that survived 100 days or more 
    (32/38), and the right ventricles were dilated.  The hypertrophy 
    affected the right side of the heart only, and generally 
    corresponded with the vascular changes.  There was a marked 
    hyperplasia of the mast cells in the mediastinal lymph nodes and 
    around bronchi and pulmonary arteries.  Similar observations were 
    made by Barnes et al. (1964) and Valdivia et al. (1967a,b). 

        Kay and his group studied cardiac and pulmonary vascular 
    changes in rats fed  Crotalaria spectabilis seeds (Kay & Heath, 
    1966; Kay et al., 1967a,b) or administered fulvine (Kay et al., 
    1971a). 

        A group of 10 female weanling Wistar albino rats were fed a 
    diet containing 1 g powdered seeds of  Crotalaria spectabilis/kg 
    until they died of cardiorespiratory distress, after 36 - 60 days 
    of feeding.  Thirty-four control rats were fed a normal diet.  At 
    autopsy, the atria of the heart, the right ventricle, and the left 
    ventricle with the interventricular septum were weighed separately.  
    The medial thickness of the muscular pulmonary arteries was 
    measured, and expressed as percentage of external diameter.  The 
    medial thickness of the muscular pulmonary arteries increased in 
    all test rats; acute or healing pulmonary arteritis was seen in 3 
    animals. Statistically significant cardiomegaly was present in all 
    rats fed the seeds, contributed chiefly by the right ventricle. The 
    readings from all the test rats were well outside the upper 95% 
    confidence limit.  The increase in the medial thickness of the 
    pulmonary arteries correlated well with the weight of the right 
    ventricle (Fig. 10).  Three rats showed pulmonary arteritis 
    (indicated by a solid triangle).  It was presumed that the organic 
    basis for increased pulmonary resistance was the abnormal 
    muscularization of the radicles of the pulmonary arterial system.  
    Essentially similar results were obtained in an identical study 
    repeated on 8 test rats and 5 controls (Kay et al., 1967a).  The 
    test rats developed pulmonary hypertension in 37 days, levels of 
    which were correlated with the medial thickness of the muscular 
    pulmonary arteries and that of the pulmonary trunk, as well as with 
    the weight of the right ventricle. 

    FIGURE 10

        Ghodsi & Will (1981) made similar observations in Sprague 
    Dawley rats given a single subcutaneous injection of monocrotaline 
    at 60 mg/kg body weight.  Forty rats weighing 180 - 200 g were 
    used; of these, 20 constituted the control group.  The control 
    animals received the same volume of saline.  Each week, 3 rats from 
    each group were catheterized and pulmonary artery pressures were 
    measured.  In the treated group, 2 out of 5 animals showed a mild 
    increase in pulmonary artery pressure at the end of 8 days.  A 
    further 4 out of 5 animals showed a mild to moderate rise in 
    pulmonary artery pressure after 2 weeks.  The highest value 
    recorded in test rats was 56 mmHg compared with a normal upper 
    limit of 22 mmHg.  The medial thickness of pulmonary arteries was 
    correlated with pulmonary artery pressures ( P < 0.02) as was the 
    thickness of the right ventricle.  The correlation between the 
    pulmonary artery pressures and right ventricular hypertrophy was 
    statistically significant ( P < 0.05). 

        Kay et al. (1971a) administered a single dose of fulvine to 
    rats, intraperitoneally at 50 mg/kg body weight, or through a 
    stomach tube at 80 mg/kg.  Of the 30 treated rats, 17 survived 23 
    days.  All of the animals showed changes characteristic of 
    hypertensive pulmonary vascular disease with right ventricular 
    hypertrophy and muscular hypertrophy of the pulmonary trunk and the 
    muscular pulmonary arteries.  Pulmonary arterioles were also 
    muscularized and contained fibrin thrombi.  Four animals showed 
    pulmonary arteritis. 

        Essentially similar changes in the pulmonary arterial system 
    were produced within 20 - 28 days by Hayashi & Lalich (1967) in 22 
    male suckling rats administered a single injection of monocrotaline 
    at 120 mg/kg body weight, and within 28 days by Ohtsubo et al. 
    (1977) in 16 male, 4-week-old rats given a single injection of 
    seneciphylline at 80 mg/kg body weight.  Hooper (1974) did not find 
    any such effect on feeding powdered  Senecio jacobaea at
    100 - 200 mg/kg diet to 9 male white mice for up to 193 days. 

        In studies by Allen & Chesney (1972), non-human primates 
     (Macaca arctoides) were administered 4 doses of monocrotaline at 
    30 - 60 mg/kg body weight by subcutaneous injection (Table 11).  
    Twelve infant monkeys (30 days old) and 12 adults (15 months old) 
    were studied with different results.  Vascular changes, 
    characteristic of pulmonary hypertension and resultant cor 
    pulmonale, were observed in the infant monkeys, as described by 
    earlier workers in the rat.  Only isolated small hepatic veins were 
    occluded.  On the other hand, the adult animals showed a more 
    severe involvement of the liver with changes characteristic of 
    veno-occlusive disease and only an occasional pulmonary blood 
    vessel was involved.  The authors postulated that the different 
    responses in infant and adult animals were due to the different 
    stages of maturation of the enzyme systems of the hepatocyte in the 
    two age groups.  It is possible that the different reactions in the 
    liver and lung in the 2 groups may be due to the fact that the 
    enzymatic pathways responsible for producing metabolites that cause 
    hepatic damage are poorly developed in the infant, but those 
    responsible for causing pulmonary lesions are better developed. 

        Chesney & Allen (1973b) made observations similar to those of 
    Allen & Chesney (1972) in twenty, 30-day-old monkeys in a similar 
    study using monocrotaline injections and recorded, in addition, 
    endocardial fibrosis of the right heart.  The treated animals 
    developed classical clinical features of cardiopulmonary distress, 
    which was also evidenced by changes in the blood-gas parameters.  
    The raised right heart pressures were confirmed by actual 
    measurements of the blood pressure in the right ventricle, 
    pulmonary artery, and descending aorta.  The authors considered 
    this study to be a good experimental model to investigate 
    hypertensive pulmonary vascular disease, or pulmonary and 
    endocardial fibrosis.  The type of vascular changes seen in the 
    animals were comparable with those associated with pulmonary 
    hypertension in man in cardiopulmonary disease (Barnes et al., 
    1964; Kay & Heath, 1966; Kay et al., 1967a; Chesney & Allen, 
    1973b). 

        Wagenvoort et al. (1974a,b) made light microscopic and ultra-
    structural studies on 50 female Wistar rats, 1 - 6 weeks following 
    a single oral dose of fulvine at 80 mg/kg body weight or an ip dose 
    at 60 mg/kg body weight.  Twelve animals served as controls.  
    Vasoconstriction of muscular pulmonary arteries and arterioles was 
    seen initially, one week following administration.  This was 
    evident by the coiled appearance of the muscular nuclei and 
    excessive crenation of the internal elastic lamina.  The nuclei of 
    smooth muscle cells as well as those of endothelial cells were 
    partly squeezed between the folds of the lamina.  After 3 - 4 
    weeks, these blood vessels began to thicken, with muscular 
    hypertrophy and fibrinoid necrosis of the arterial muscle.  Animals 
    surviving administration of fulvine developed right ventricular 
    hypertrophy, proliferation of endothelial cells in the arteries and 
    even thickening of the veins. 

        In a study by Meyrick and Reid (1979), 21 Sprague Dawley rats 
    were fed a diet containing 1 g powdered seeds of  Crotalaria 
     spectabilis/kg for various periods ranging from 3 to 35 days.  The 
    earliest demonstrable change in the pulmonary arterial system of 
    the animals was seen on day 3 and consisted of the appearance of 
    muscle in normally non-muscular arteries of the lung.  The muscular 
    pulmonary arteries began to show hypertrophy of the media from day 
    7, which reached statistically significant levels on day 10 in 
    smaller arteries and on day 14 in the larger arteries.  Significant 
    right ventricular hypertrophy was seen on day 21.  These changes 
    were confirmed by 3H-thymidine uptake studies (Meyrick & Reid, 
    1982). 

    6.4.2.3  Mechanisms of toxic action

        Considerable progress has been made recently in the 
    understanding of biochemical and pharmacological changes that occur 
    in PA-induced lung disease. 

        Turner & Lalich (1965) and Takeoka et al. (1962) postulated 
    that pulmonary hypertension was mediated by the release of 
    5-hydroxytryptamine from the mast cells, which became hyperplastic 

    in the mediastinal lymph nodes and around bronchi and pulmonary 
    arteries (Turner & Lalich, 1965) (section 6.4.2.2) following 
    administration of monocrotaline, causing vasoconstriction.  On the 
    other hand, Kay et al. (1967b) found that the number of mast cells 
    corresponded with the severity of exudative changes in the lung and 
    were not related to the genesis of pulmonary hyperplasia. 

        Besides the medial muscular hypertrophy of pulmonary arteries 
    reported in several studies cited above, swelling and lysis of the 
    endothelial cells, contributing to luminal narrowing and thickening 
    of the wall with fibrosis, have been described (Allen & Carstens, 
    1970). 

        Weanling rats are more susceptible to these changes than older 
    animals, and the changes follow a strict temporal sequence.  Oral 
    administration of monocrotaline to rats at 20 mg/litre in drinking-
    water produced a sequence of changes over 3 weeks, that included an 
    increase in lung mass, which was significant by day 9, stimulation 
    of pulmonary RNA and protein synthesis (maximal on day 10), 
    increased pulmonary arterial blood pressure (significant by day 
    10), and right ventricular hypertrophy by day 14 (Huxtable et al., 
    1978; Lafranconi et al., 1984).  The increase in the lung mass was 
    not accompanied by change in the total collagen content and was 
    contributed possibly by hypertrophy of endothelial cells, but the 
    increased mass of the right ventricle was associated with a 4-fold 
    increase in collagen content (Lafranconi et al., 1985). 

        An early event is inhibition of serotonin removal by pulmonary 
    endothelium (Huxtable et al., 1978).  This phenomemon, combined 
    with the increased release of serotonin by mast cells that has been 
    observed, may be involved in the development of pulmonary 
    hypertension (Carillo & Aviado, 1969).  Right ventricular 
    hypertrophy is blocked by propanolol, whereas the development of 
    pulmonary hypertension is unaffected (Huxtable et al., 1977).  
    Novel metabolites have been found to be released by livers perfused 
    with monocrotaline  in vitro, and these metabolites block serotonin 
    transport  in vitro, when perfused through isolated lungs 
    (Lafranconi & Huxtable, 1984).  These data suggest that the slow 
    release of metabolites from the liver into the circulation 
    following low-level exposure to monocrotaline results in specific 
    inhibition of endothelial cell function (Huxtable et al., 1978). 

        The effect of monocrotaline treatment on pulmonary angiotensin 
    converting enzyme (ACE) activity in the rat is disputed.  Hayashi 
    et al. (1984) observed a reduction in the ACE activity of pulmonary 
    tissue in pyrrolizidine-exposed rats in parallel with the 
    development of pulmonary alterations, while the ACE activity of the 
    plasma remained unchanged.  However, other authors have reported 
    that, though the specific activity of ACE falls in the isolated 
    perfused lungs of monocrotaline-treated rats, or in lung 
    homogenates from such animals, when activity is expressed as total 
    activity per lung, there is no significant alteration in the lungs 
    of treated animals compared with those of untreated animals 
    (Huxtable et al., 1978; Lafranconi & Huxtable, 1983).  Therefore, 
    the significance of changes in ACE activity is open to question. 

        Molteni et al. (1984) also found evidence of endothelial cell 
    damage by monocrotaline in their ultrastructural and biochemical 
    studies on rats.  Eighty male Sprague Dawley rats were used; half 
    were administered monocrotaline at 20 mg/litre in the drinking-
    water and half were given plain water.  The average daily water 
    consumption was 35 ml/rat.  Thus, the treated rats were estimated 
    to have received 2 mg/kg per day.  Five animals each from the 
    treated and control groups were killed at intervals of 1 - 12 weeks 
    after the start of the study.  The endothelial damage was measured 
    by ACE activity, plasminogen-activator (PLA) activity, and 
    prostacyclin (PGI2) production.  These were correlated with 
    pulmonary arterial perfusion and ultrastructural changes in the 
    lung.  In the treated groups, after an initial rise at 1 week, the 
    ACE activity showed a steady decline from 1 to 6 weeks, after which 
    it plateaued at 55% of normal.  PLA activity did not change for 2 
    weeks, but decreased by 59 and 79% of the control value after 6 and 
    12 weeks, respectively.  On the other hand, the PGI2 production 
    increased progressively reaching 140 and 270% of the control level 
    after 6 and 12 weeks, respectively.  These endothelial functional 
    changes were not accompanied by significant changes in pulmonary 
    arterial perfusion as visualized by 99mTc-labelled macroaggregated 
    albumin perfusion studies.  The activities of ACE and PLA and the 
    production of PGI2 are considered sensitive indices of endothelial 
    function in rats.  The above results indicated endothelial cell 
    dysfunction.  The ultrastructural studies also revealed oedema of 
    capillary subendothelial, perivenous and periarterial tissues at 1 
    week, and interstitial inflammatory infiltrates at 2 weeks.  At 
    6 - 12 weeks, there was thickening of the pulmonary arteries and 
    enlargement of right side of the heart. 

        Stenmark et al. (1985) studied the role of alveolar 
    inflammation and arachidonate metabolism in monocrotaline-induced 
    pulmonary hypertension in rats.  Five groups of male Sprague Dawley 
    rats were treated as follows:  (a) 20 rats received 40 mg 
    monocrotaline/kg body weight, sc; (b) 20 rats received 
    monocrotaline, 40 mg/kg sc plus diethylcarbamazine (DEC) 100 mg/kg 
    sc, every 12 h; in addition, 250 mg DEC was added to 100 ml of 
    drinking-water.  This treatment started 2 days prior to the start 
    of the study and was continued daily for 3 weeks; (c) 12 control 
    rats received normal saline plus monocrotaline at 40 mg/kg sc; (d) 
    12 rats received indomethacin at 2 mg/kg sc for 2 days, prior to 
    receiving monocrotaline at 40 mg/kg and then daily for 3 weeks; (e) 
    6 animals each received a single sc injection of normal saline and 
    served as additional controls. 

        One, 2, and 3 weeks after monocrotaline or saline injection, 
    lung lavage was carried out for cell counts and assay for enzyme 
    activity and cyclooxygenase metabolites, the degradation products 
    of prostacyclin (PGI2) and thromboxane A2(TXA2), as 6-keto-
    prostaglandin (PGF1alpha) and TXB2, respectively.  At 3 weeks, the 
    animals were anaesthetized, right ventricular pressures measured by 
    catheterization and the heart removed.  The 2 ventricles were 
    separated and weighed for the determination of heart-weight ratio 
    (right ventricle/left ventricle + septums RV/LV + S) an indicator 
    of right ventricular hypertrophy. 

        The right ventricle showed hypertrophy at 2 weeks and the right 
    ventricular pressure was increased at 3 weeks following 
    monocrotaline administration (Fig. 11).  The leukocyte count in the 
    lavage fluid increased at 3 weeks, with a rise in the percentage of 
    polymorphonuclear leukocytes and large, abnormal alveolar 
    macrophages in the test animals.  B- N-acetyl-D-glucoseaminidose 
    activity was also elevated at 3 weeks, indicating activation of 
    leukocytes.  There was also a rise in the concentration of 6-keto-
    PGF1alpha at 1 and 3 weeks, as well as in TXB2 at 3 weeks, compared 
    with those in control animals. 

        The administration of DEC inhibited both the increase in heart-
    weight ratio (RV/LV + S) and the increase in pulmonary artery 
    pressure (Fig. 11) that occurred 3 weeks after monocrotaline 
    administration, and reduced the percentage of polymorphonuclear 
    cells, abnormal alveolar macrophages, and hexoseaminidase activity 
    in the lavage fluid, compared with that from animals that had 
    received monocrotaline only.  The rise in the levels of 6-keto-
    PGF1alpha was inhibited ( P < 0.05) by 73% and that of TXB2 by 74% 
    in the lung lavage. 

    FIGURE 11

        The administration of indomethacin did not have any effects on 
    either the heart-weight ratio or the pulmonary arterial pressure 3 
    weeks after monocrotaline administration (Fig. 11), but it 
    inhibited ( P < 0.05) the rises in 6-keto-PGF1alpha (by 90%) and 
    TXB2 (by 91%) that occurred in the lung lavage of monocrotaline-
    treated animals at 3 weeks. 

        The above studies indicate that both the cyclo-oxygenase and 
    the lipo-oxygenase pathways of arachidonate metabolism are 
    activated by monocrotaline as early events in its toxic effect. 
    Activation of the cyclo-oxyenase pathway, demonstrated by increased 
    concentrations of the prostaglandin metabolites 6-keto-PGF1alpha 
    and TXB2 in lavage fluid, was inhibited by indomethacin, but this 
    inhibition did not prevent the monocrotaline-induced injury.  DEC 
    attenuated both the inflammatory response and pulmonary 
    hypertension and inhibited the formation of slow reacting 
    substances including leukotriene D4.  Since DEC produces a 
    pharmacological blockade of the lipo-oxygenase pathway, it seems 
    that the latter, rather than the cyclo-oxygenase pathway, is 
    responsible for perpetuating the pathophysiological mechanism 
    leading to monocrotaline-induced pulmonary hypertension. 

        Hilliker et al. (1984) demonstrated that antibody-induced 
    thrombocytopaenia attenuates right ventricular hypertrophy induced 
    by monocrotaline in rats.  In another study, Hilliker & Roth (1984) 
    also produced evidence that hydrallazine, a vasodilator and 
    inhibitor of platelet prostaglandin synthesis, dexamethason, an 
    antiinflammatory agent and inhibitor of phospholipase, and 
    sulfinopyrazone, an inhibitor of platelet prostaglandin synthesis 
    inhibited monocrotaline-induced right ventricular hypertrophy in 
    rats, supporting the hypothesis that platelets and vasoconstrictor 
    agents play a role in monocrotaline-induced pulmonary hypertension.  
    Likewise, prior chemical sympathectomy with 6 hydroxydopamine 
    (100 mg/kg) or inhibition of serotonin synthesis with 
     p-chlorophenylalanine (500 mg/kg) reduced the degree of 
    monocrotaline-induced right ventricular hypertrophy in rats, but 
    did not prevent or reduce pulmonary vascular muscularization 
    (Tucker et al., 1983).  Thus, the sympathetic nervous system and 
    serotogenic mechanisms seemed to be involved in the development of 
    right ventricular hypertrophy, but not in the development of the 
    pulmonary vascular lesion induced by monocrotaline.  Kay et 
    al. (1985) also demonstrated that pretreatment with 
     p-chlorophenylalanine, which inhibits 5-hydroxytryptamine (5HP) 
    synthesis, also significantly ( P < 0.05) reduced right ventricular 
    systolic pressure, right ventricular hypertrophy, and medial 
    thickness of muscular pulmonary arteries in monocrotaline-treated 
    rats.  Similar observations were made in rats exposed to hypoxia.  
    It was therefore suggested that 5HP might play a role in 
    monocrotaline-induced or chronic hypoxic pulmonary hypertension. 

        The biosynthesis of rat lung polyamines, putrescine, 
    spermidine, and spermine, generally considered to be important 
    regulators of cell growth and differentiation, is increased prior 
    to the evolution of monocrotaline-induced pulmonary hypertension in 
    rats.  Continuous administration of alpha-difluoromethylornithine 

    (DFMO), which is a highly specific irreversible inhibitor of 
    ornithine decarboxylase (DDC), a rate-limiting enzyme in polyamine 
    biosynthesis, attenuated the development of monocrotaline-induced 
    pulmonary hypertension in rats (Olson et al., 1984).  This effect 
    was mediated by the DFMO, by inhibiting the synthesis of putrescine 
    and spermidine, and not by blocking the hepatic metabolism of 
    monocrotaline to pyrroles (Olson et al., 1985).  Thus, it was 
    suggested that lung polyamine biosynthesis might be essential for 
    the expression of monocrotaline-induced perivascular oedema as well 
    as medial thickening in the development of monocrotaline-induced 
    pulmonary hypertension vascular disease. 

        On the basis of the preceding studies, mostly on the rat, the 
    mechanism of chronic long-term injury to the lung by monocrotaline 
    seems to be as follows.  Within hours of PA administration, there 
    is damage to the pulmonary endothelial cells accompanied by 
    vascular leak leading to pulmonary oedema.  Platelet aggregation 
    also occurs.  The endothelial damage indicated by ultrastructural 
    and biochemical studies activates the production of prostacyclin 
    and lipogenic products, which mediate increases in vascular 
    permeability and inflammatory reaction.  There is simultaneous 
    production of 5 hydroxytryptamine and several polyamines.  The 
    injected monocrotaline is completely metabolized within hours, and 
    no significant quantity is found in the body at 24 h (Hayashi, 
    1966) and, though some active metabolites may still be detectable 
    by isotope studies, even at 14 days (Hsu et al., 1974), the rats do 
    not have any lung lesions.  The slow evolution of vascular changes 
    suggests that it is not caused by monocrotaline but through 
    biological pathways activated by the initial injury. 

        Methylprednisolone (MP), which reduces acute lung oedema caused 
    by monocrotaline (MCT), has been shown to reduce MCT-induced 
    pulmonary hypertensive vascular changes in rats and the resultant 
    right ventricular hypertrophy (Langleben & Reid, 1985).  Daily ip 
    administration of MP at 5 mg/kg body weight, was found to be more 
    effective than 2 large doses of MP at 30 mg/kg, 2 h before and 2 h 
    after a single sc injection of MCT at 60 mg/kg.  It was suggested 
    that secondary changes, though triggered by the acute MCT injury, 
    become self sustaining and are more significant for vascular 
    structural remodelling. 

        Structural arterial remodelling with vasoconstriction, medial 
    hypertrophy of the muscular pulmonary arteries, and muscularization 
    of the pulmonary arterioles follow as late effects, resulting in 
    pulmonary hypertension and right ventricular hypertrophy of the 
    heart. 

        The results of the above studies suggest a direct toxic effect 
    of the alkaloid on the endothelial cells of the alveolar 
    capillaries and on the pulmonary arteries, as well as a pulmonary 
    hypertensive effect on the heart. 

    6.4.3  Effects on the central nervous system

        The dominant signs of pyrrolizidine poisoning in horses are 
    neurological (Rose et al., 1957a,b; McLean, 1970).  Similar signs 
    can also occur in cattle and sheep.  It has been claimed that such 
    signs are probably non-specific secondary effects following primary 
    liver disease resulting in hyperammonaemia (Rose et al., 1957a).  
    However, neurological abnormalities in which animals walk in a 
    straight line until they come to an object, and then stand with 
    their heads pressed against the object, indicate specific lesions 
    in the central nervous system.  Spongy degeneration of the central 
    nervous system occurs in cattle, sheep, and pigs (Hooper et al., 
    1974; Hooper, 1975a,b). 

         Trichodesma alkaloids, in particular, appear to be neurotoxic.  
    There is a considerable body of literature in the USSR on 
     Trichodesma intoxication of mice, rabbits, and dogs, which has 
    been reviewed (Ismailov et al., 1970).  Mice given  Trichodesma  
    alkaloids subcutaneously at 0.5 mg/kg develop paresis of the hind 
    limbs within 12 - 17 days.  Opisthotonus and clonic convulsions are 
    also seen.  Doses of 10 - 15 mg/kg of alkaloids produces death in 
    all animals within 2 - 6 h, as the result of respiratory 
    depression.  Higher doses produce immediate death. 

    6.4.4  Effects on other organs

        Right ventricular hypertrophy, secondary to the primary effects 
    on the pulmonary arteries, and the resultant pulmonary hypertension 
    in animals treated with PAs or PA-containing plants have been dealt 
    with in section 6.4.2.  Lalich & Merkow (1961) reported myocarditis 
    consisting of focal oedema and infiltration with a minimal number 
    of lymphocytes and mononuclear cells in some rats fed  Crotalaria 
     spectabilis seeds mixed with the diet at a concentration of 
    0.13 - 2 g/kg.  Treated groups consisted of 11 - 24 animals each; 
    there were 12 controls.  The changes were seen in all groups of 
    animals, but the maximum number of rats (10 out of an unspecified 
    number of the group) showing these changes was in the group that 
    received 0.5 g/kg diet for 20 - 31 days.  Generally, there was a 
    close correlation with the presence of pulmonary arteritis. 

        Renal changes have been described by a number of investigators.  
    Hayashi & Lalich (1967) observed mild to moderate changes in renal 
    glomeruli consisting of necrosis, capillary thrombosis, and 
    degenerative changes in the epithelial and mesangial cells, 
    thickening of interlobular arteries, and arterial thrombosis in 
    suckling, male Sprague Dawley rats given a single sc dose of 
    monocrotaline at 120 mg/kg body weight.  Renal changes were seen, 
    to some extent, in all animals surviving for 41 - 47 days. 

        Carstens & Allen (1970) studied the effects of feeding 
     Crotalaria spectabilis seed on the rat kidney.  Fifty male Sprague 
    Dawley rats were fed a diet containing ground  Crotalaria 
     spectabilis seed at 0.2 - 0.8 g/kg for 8 months.  The seeds were 
    estimated to contain approximately 3.5 g monocrotaline/kg; 10 
    animals served as controls.  Renal changes were seen in 33/50 

    PA-treated rats.  In 22 rats, over 75% of the glomeruli were 
    hyalinized and capsules thickened.  In the less severely affected 
    kidneys, the glomerular basement membrane was thickened and 
    homogeneous deposits were seen in mesangial areas.  Afferent 
    arterioles and interlobular arteries were markedly thickened.  In 
    the most severely affected vessels, the internal elastic lamina was 
    necrosed and the larger arteries showed fibrinoid necrosis. 

        Renal tubular megalocytosis was the dominant lesion described 
    by Hooper (1974) in mice.  Nine male white mice, 10 weeks of age, 
    were fed  Senecio jacobaea, which contained a concentration of 
    alkaloids (jaconine, jacobine, and seneciphylline) of 2.7 g/kg and 
    a concentration of  N-oxide of 0.9 g/kg, mixed with the diet.  The 
     S. jacobaea was given at 100 g/kg diet for 9 weeks, before being 
    raised to 200 g/kg diet.  Five animals served as controls.  The 
    animals were killed from 63 to 193 days after the start of the 
    study.  All treated animals, except 2 killed on day 63, showed 
    changes.  The large cells occurred in both the proximal tubules and 
    the loop of Henle.  Similar cells were seen in the alveolar and 
    bronchiolar epithelium.  No glomerular lesions were described.  The 
    author mentioned having seen the above changes in rats given 
    repeated sublethal injections of fulvine and spectabiline.  On the 
    other hand, Kurozumi et al. (1983) observed glomerular lesions in 
    rats given a single injection of monocrotaline. 

        A variety of renal lesions has been observed in pigs, a common 
    pathological feature being renal megalocytosis, which was observed 
    in pigs poisoned by at least 4 different plant genera containing a 
    variety of toxic alkaloids (Harding et al., 1964; Peckham et al., 
    1974) and has also been observed in wild pigs grazing in areas rich 
    in PA-containing plants in northern Australia (Hooper, 1978).  
    McGrath et al. (1975) described glomerular lesions in pigs given 
     Crotalaria spectabilis seed daily for 43 days.  Severe renal 
    lesions comprising tubular dilatation, megalocytosis, and necrosis 
    of tubular epithelial cells with casts in the lumen, interstitial 
    and periglomerular fibrosis, and glomerular hyalinization were 
    reported by Hooper & Scanlan (1977) in pigs fed  Crotalaria retusa  
    seeds containing monocrotaline.  Renal megalocytosis has also been 
    reported in  C. retusa poisoning in horses, sheep, and mice poisoned 
    by  S. jacobaea but not by  H. europaeum, and in vervet monkeys with 
    chronic retrorsine poisoning (Van der Watt et al., 1972). 

        Lesions have been reported in the stomach and intestines in 
    field and experimental animals after poisoning with pyrrolizidine 
    alkaloids, but are difficult to identify as specific PA injury.  
    Hooper (1975c) conducted studies on sheep, rats, and mice.  In the 
    study on sheep, 12 male cross-bred lambs, 7 - 8 weeks of age, were 
    newly weaned on to a standard commercial calf grower diet.  
    Lasiocarpine was administered at the rate of 15 - 20 mg/kg body 
    weight every 2 - 4 days.  Each animal was killed when in terminal 
    coma.  Survival time ranged from 4 to 17 days.  In the rat study, 
    young Wistar-Furth rats (sex not stated) weighing 150 - 200 g were 
    used.  In one group of 11 rats, each animal received an ip 
    injection of lasiocarpine at the rate of 40 mg/kg body weight.  
    Three animals received isotonic saline.  Animals were killed or 

    died 2 - 6 days after the injection.  A second group of 13 rats 
    received a dose of 35 mg lasiocarpine/kg body weight; 4 control 
    animals received saline.  All rats received a second injection
    48 h later.  They were killed 3 - 60 days after the second 
    administration of lasiocarpine.  In the mouse study, 3 mature male 
    white mice received 6 injections each of lasiocarpine at the rate 
    of 45 mg/kg body weight followed by 4 injections of 90 mg/kg body 
    weight at 48-h intervals.  There was one control animal. 

        All animals showed characteristic hepatic lesions.  Sheep also 
    showed severe oedema, haemorrhage, and epithelial necrosis in the 
    gall bladder; lesions were also found in the central nervous system 
    and occasionally in the kidney.  All animals showed severe 
    intestinal atrophy. There was inhibition of crypt cell mitosis 
    leading to mitotic irregularities, abnormal large cells and 
    syncitial cells, especially in the duodenum of sheep, and severe 
    villous atrophy with ulceration.  Lesions in the intestines were 
    similar to those caused by radiation and radiomimetic agents.  It 
    was suggested that the local intestinal radiomimetic effect was due 
    to local exposure to the pyrrole metabolite of lasiocarpine after 
    excretion through the bile duct.  It was proposed that a more 
    conspicuous and rapid development of duodenal megalocytosis was due 
    to very rapid turnover of cells in the duodenum. 

        Other probably secondary effects included haemolysis in sheep 
    in association with advanced liver disease and high liver-copper 
    levels (Bull et al., 1956), anaemias and disturbance in iron 
    metabolism and haematopoiesis (Schoental & Magee, 1959; Schoental, 
    1963; Peckham et al., 1974; Hooper & Scanlan, 1977) (section 
    6.4.11), pancreatic oedema and fibrosis (Bras & Hill, 1956; 
    Schoental & Magee, 1959), cerebral oedema, haemorrhage, and 
    congestion in the rat brain (Davidson, 1935; Rosenfield & Beath, 
    1945). 

        Tumours in the different organs have been dealt with separately 
    under carcinogenesis (section 6.4.8). 

    6.4.5  Teratogenicity

        The teratogenic potential of PAs was demonstrated by Green & 
    Christie (1961) who produced a variety of dose-related fetal 
    abnormalities in the rat, with a single intraperitoneal injection 
    of heliotrine administered during the second week of gestation.  
    The dosages ranged from 15 to 300 mg/kg maternal body weight.  
    Litters exposed to a dose of less than 50 mg did not show any 
    abnormalities, but abnormalities were observed in litters exposed 
    to higher doses, and increased in frequency and severity with 
    increasing dose.  The abnormalities included retardation of 
    development, musculoskeletal defects, especially hypoplasia of the 
    lower jaw, cleft palate, and other abnormalities.  Doses above 200 mg
    resulted in the intrauterine death or resorption of many fetuses. 

        Similar studies were performed by Peterson & Jago (1980) who 
    compared the effects of heliotrine with its metabolic pyrrole 
    derivative dehydroheliotridine (DHH), when administered in a single 
    ip injection to rats on the 14th day of gestation.  Heliotrine was 
    administered at 200 mg/kg body weight and DHH at 30 - 90 mg/kg, 14 
    days after conception.  Effects on embryos, evaluated on the 20th 
    day, showed that both heliotrine and DHH retarded growth and were 
    teratogenic, but that the effects of a 40 mg/kg dose of DHH were 
    equivalent to those of 200 mg/kg heliotrine, i.e., the metabolite 
    was 2.5 times as effective on a molar basis.  DHH produced a number 
    of skeletal abnormalities including retarded ossification, 
    distorted ribs, long bones, cleft palate, and feet defects.  At 
    higher doses, growth almost ceased in many tissues and the fetuses 
    were very immature.  However, the embryonic liver parenchyma did 
    not show the antimitotic effects of DHH. 

        The teratogenic properties of heliotrine were also demonstrated 
    in  Drosophila larvae fed low levels of the alkaloid (Brink, 1982). 

    6.4.6  Fetotoxicity

        The subject of fetotoxicity has been reviewed by Mattocks 
    (1986).  Sundareson (1942) demonstrated the ability of 
    pyrrolizidine alkaloids to cross the rat placenta.  Twice weekly 
    injections of the PA, starting at, or after, the 12th day of 
    gestation, resulted in premature delivery of some litters and many 
    were born dead.  The same author showed that the alkaloids 
    themselves and not just the pyrroles formed in the dams' livers, 
    could pass the placental barrier by injecting senecionine into 
    19-day-old rat fetuses  in utero, which produced the characteristic 
    toxic lesions in the dams.  The fetuses were also found to be more 
    resistant to the lethal effects of the PA than the mother rats.  
    When 4 fetuses were each administered 1.25 mg of PA, representing 
    about 200 - 400 mg/kg body weight, which is much higher than the 
    LD50 for an adult rat, 3 of them were still alive after 2 days.  
    Green & Christie (1961) did not find any liver damage in fetuses 
    from pregnant rats given teratogenic doses of heliotrine.  Only 
    mild liver damage was found in the embryo rats whose mothers had 
    been injected with PAs (heliotrine, lasiocarpine, retrorsine, or 
    monocrotaline) (Bhattacharya, 1965).  In contrast, Schoental (1959) 
    demonstrated that lasiocarpine and retrorsine, when administered to 
    lactating rats produced little effect on the mothers, but produced 
    acute liver lesions in the suckling infants.  The lesions were most 
    severe in 3- to 7-week-old animals.  It was suggested that the 
    infants were affected by the milk from the lactating mothers, which 
    possibly contained the metabolic products of the PAs. 

        It would seem from the above studies that the embryo is 
    relatively more resistant to the toxic effects of PAs  in utero than 
    it is after birth.  Mattocks & White (1973) postulated that this 
    could be due to the low capacity for the metabolic activation of 
    PAs of the embryo liver, as they had shown that the ability of 
    liver enzymes to convert retrorsine to toxic metabolites was low in 
    rats, immediately after birth, but picked up rapidly afterwards.  
    The susceptibilities of rats of various ages to the hepatotoxic 

    effects of the PAs was proportional to their capacity to form and 
    retain the pyrrolic metabolites.  Twenty-day-old rats were found to 
    be more sensitive than older animals. 

        The effects of fulvine administration on pregnant rats between 
    9 and 12 days of gestation were studied by Persaud & Hoyte (1974).  
    Dose-related fetal resorptions were observed, but no hepatic 
    lesions were seen in the fetuses.  On the other hand, Newberne 
    (1968) observed damage, in both the maternal and fetal livers, when 
    lasiocarpine was administered to pregnant rats.  Acute liver 
    necrosis was observed in the livers of mothers as well as fetuses 
    in animals that had received 100 mg lasiocarpine/kg body weight on 
    day 13 of gestation.  However, in animals that received 2 doses of 
    35 mg/kg body weight on days 13 and 17 of pregnancy, liver necrosis 
    was seen in the fetal liver but not in that of the mother.  It is 
    not known why lasiocarpine acts differently from other alkaloids 
    and has a greater effect on the fetal liver.  Mattocks (1986) has 
    postulated the possibility that fetotoxicity was caused chiefly by 
    toxic metabolites formed in the maternal liver, and that a greater 
    proportion of such metabolites reached the fetus from lasiocarpine 
    than from other PAs. 

    6.4.7  Mutagenicity

        A number of PAs that have been shown to be powerful dose 
    dependent mutagens in  Drosophila melanogaster have been listed by 
    Mattocks (1986).  All the compounds are hepatotoxic though the 
    degree of mutagenicity is not necessarily proportional.  Table 12 
    provides a summary of the mutagenicity tests on different PAs, 
    related compounds and plant extracts.  Clark (1959) demonstrated 
    the mutagenic effect of heliotrine in  Drosophila, in which a 
    considerable increase in sex-linked recessive lethals was produced, 
    apparently by interfering with the maturation of germ cells, so 
    that as soon as the available spermatozoa were used, the males were 
    no longer capable of breeding.  The cell damage was irreversible.  
    The mutagenic effect of feeding  Drosophila males for 24 h with a 
    medium containing 10-3 mol monocrotaline was comparable to about 
    1000 R of X rays (Clark, 1976).  The Basc test with  Drosophila 
     melanogaster is considered a highly sensitive mutagenicity test 
    for PAs (Candrian et al., 1984a). 

        Seneciphylline and senkirkine, known to occur in animal feeds 
    and medicinal herbs, respectively, were tested for their ability to 
    produce sex-linked recessive lethals in males of  Drosophila 
     melanogaster using the Basc (3-day feeding method) by Candrian et 
    al. (1984a).  Seneciphylline was found to be mutagenic at 
    concentrations of 10-5, 10-4, and 10-3 mol, which produced 3.8% 
    (983 chromosomes tested), 9% (708 chromosomes tested), and 15.3% 
    (327 chromosomes tested) sex-linked recessive lethals, 
    respectively.  Senkirkine (10-5 mol) was found to produce 4.4% sex-
    linked recessive lethals (2541 chromosomes tested) against 0.17% 
    maximum sensitivity in the late spermatid stage of spermatogenesis 
    indicating that PAs act as indirect mutagens.  Flies fed with milk 
    from lactating rats given an oral dose of 25 mg seneciphylline/kg 
    showed 1.2% sex-linked recessive lethals (1477 chromosomes tested) 
    compared with 0.3% (1533 chromosomes tested) in controls. 

    Table 12.  Mutagenicity tests on pyrrolizidine alkaloids, 
               related compounds, and source plants
    ---------------------------------------------------------
    Compound or material          Type of testa  Responseb

    ---------------------------------------------------------
    Clivorine                     A              +
                                  HPC            +

    Echimidine                    D              +

    Echinatine                    D              +

    Fulvine                       D              +

    Heliotrine                    D              +
                                  P              +
                                  F              +
                                  CC             +
                                  A              +
                                  B              +
                                  TM             +
                                  CM/CC          +

    Integerrimine                 D              +

    Jacobine                      D              ±
                                  P              +

    Lasiocarpine                  D              +
                                  P              +
                                  F              +
                                  A              +
                                  HPC            +
                                  CM/CC          +
                                  TM             +

    Ligularidine                  A              +

    Lindelofine                   A              0

    Lycopsamine                   A              0

    Monocrotaline                 D              +
                                  P              +
                                  CC             +
                                  B              +
                                  A              0
                                  HPC            +
                                  CT             +

    Petasitenine (fukinotoxin)    A              +
                                  HPC            +
                                  CM             +
    ---------------------------------------------------------

    Table 12.  (contd.)
    ---------------------------------------------------------
    Compound or material          Type of testa  Responseb

    ---------------------------------------------------------
    Platyphylline                 D              0

    Retrorsine                    A              +                 
                                  D              +                 
                                  CT             +                 

    Rosmarinine                   CT             0                 

    Senecionine                   D              +                 
                                  A              0                 

    Seneciphylline                A              0                 
                                  P              +                 
                                  D              +                 

    Senkirkine                    A              +                 
                                  HPC            +                 
                                  CM/CC          +                 
                                  D              +                 

    Supinine                      D              ±                 
                                  P              +                 

    Mixed alkaloids from
    Senecio jacobaea              A              0                 

    Senecio numorensis
    spp. fuchsii (extract)        CM             +                 
                                  A              0                 
                                  A              +                 

    Senecio jacobaea (extract)    A              +                 

    Senecio longilobus (extract)  A              0                 

    Symphytum officinale
    (comfrey extract)             A              0                 

    Retronecine bis-p-chloro-     P              +                 
    benzoate                                       

    Synthanecine A bis-N-ethyl-   CT             +                 
    carbamate

    Retronecine                   A              0

    Heliotridine                  D              0

    Viridofloric acid             A              0
    ---------------------------------------------------------

    Table 12.  (contd.)
    ---------------------------------------------------------
    Compound or material          Type of testa  Responseb

    ---------------------------------------------------------
    Heliotric (heliotrinic) acid  D              ±
                                  HPC            +

    Dehydroretronecine            CT             +
                                  A              ±
                                  SCE            +

    Dehydroheliotridine           CM             +

    Pyrrole                       HPC            0

    2,3-Bishydroxymethyl-1-       CT             +
    methylpyrrole                 A              ±
                                  SCE            +

    2-Hydroxylmethyl-1-methyl-    A              0
    pyrrole                       SCE            ±

    3-Hydroxylmethyl-1-methyl-    A              +
    pyrrole                       SCE            ±
    ---------------------------------------------------------
    a A =  Salmonella ("Ames") test.
      B = Other bacterial tests.
      CC = Clastogenic activity in cultured cells.
      CM = Mutagenicity in cultered mammalian cells.
      CT = Cell transformation test.
      D = Mutagenicity in  Drosophila.
      F = Tests in fungus  (Aspergillus nidulans).
      HPC = Hepatocyte primary culture/DNA repair test.
      P = Chromosomal aberrations in plant cells.
      SCE = Sister chromatid exchange.
      TM = Transplacental micronucleus test.
    b + = active.
      ± = marginally active.
      0 = inactive.

        Mutagenic properties of 7 PAs extracted from plants to 
     Salmonella typhimurium TA100 have been demonstrated by a modified 
    Ames method by Yamanaka et al. (1979).  The PAs were clivorine, 
    fukinotoxin, heliotrine, lasiocarpine, ligularidine, LXC201, and 
    senkirkine.  Pre-incubation of these alkaloids with liver S9 mix 
    and bacteria in liquid medium was essential for demonstration of 
    the property.  PAs in the heliotridine and otonecine family were 
    mutagens, while retronecine bases were inactive.  Monocrotaline and 
    heliotrine were not active mutagens to  Escherichia coli WP2, even 
    though they were quite cytotoxic (Green & Muriel, 1975).  They were 
    active in repair deficient strains.  Retrorsine was active in 
    inducing mutations on the Ames  Salmonella/microsome assay (Wehner 

    et al., 1979).  Extracts from medicinal plants and noxious weeds 
    were mutagenic towards  Salmonella in the Ames assay (Pool, 1982; 
    White et al., 1983; Koletsky et al., 1978). 

        From the limited data available, it seems that the carcinogenic 
    activity of individual alkaloids parallels their mutagenic 
    behaviour, but not their relative hepatotoxicities (Culvenor & 
    Jago, 1979). 

    6.4.7.1  Chromosome damage

        Pyrrolizidine alkaloids have been shown to be capable of 
    damaging chromosomes in plants, fungi, bacteria, tissue cell 
    cultures, and the fruit fly  (Drosophila melanogaster).  Literature 
    on this topic has been reviewed by Bull et al. (1968), McLean 
    (1970), and Mattocks (1986). 

        Several PAs are known for their ability to damage the 
    chromosomes of growing plant cells (Mattocks, 1986).  Similar 
    properties have been demonstrated in leukocyte cultures from the 
    marsupial  (Potorus tridactylus) (Bick & Jackson, 1968; Bick, 1970).  
    Bick & Culvenor (1971) found dehydroheliotridine, a metabolite of 
    heliotrine, to be 10 times more active than the alkaloid. 

        Infusions of  Symphytum officinale L., described in Polish 
    pharmacopoeia as  Radix symphyti, are recommended as expectorants, 
    especially for children.  Furmanowa et al. (1983) demonstrated the 
    mutagenic effects of an alkaloidal fraction and infusion in this 
    plant in the meristematic cells of the lateral roots of  Vicia faba 
    L. var  minor.  Lasiocarpine, a proven carcinogen, served as a 
    positive control. 

        Chromosome damage by PAs in the hamster lung cell line was 
    demonstrated by Takanashi et al. (1980).  Stoyel & Clark (1980) 
    used the transplacental micronucleus test in pregnant female mice 
    and showed the chromosome damaging properties of heliotrine (225 mg/kg
    body weight) and lasiocarpine (86 mg/kg) within 20 h of the injection. 

        The genotoxicity of heliotrine, monocrotaline, seneciphylline, 
    and senkirkine was studied by Bruggeman & Van der Hoeven (1985) 
    using the sister-chromatid exchange (SCE) assay in V79 Chinese 
    hamster cells co-cultured with primary chick embryo hepatocytes.  
    Exposure to these PAs resulted in the high induction of SCEs, a 
    more than 5-fold increase in the SCE rate with 2.5 mg 
    heliotrine/litre, 4-fold with monocrotaline at 5 mg/litre, 8-fold 
    with seneciphyline at 1.2 mg/litre, and more than 5-fold response 
    with senkirkine at 2.5 mg/litre.  For all compounds, a dose-
    response relationship was observed at concentrations that did not 
    seriously affect survival.  PAs are also known to induce DNA repair 
    in rodent hepatocytes (Green et al., 1981; Mori et al., 1985).  DNA 
    repair synthesis was elicited by 15 alkaloids, including 11 of 
    unknown carcinogenic potential (Mori et al., 1985). 

        There are also a few reports of chromosome damage by PAs in 
    man.  Martin et al. (1972) found chromosome damage in the blood 
    cells of children with veno-occlusive disease, probably caused by 
    fulvine.  It has also been shown by Ord et al. (1985) that 
    dehydroretronecine, is able to induce  SCE in human lymphocytes.  
    Kraus et al. (1985) studied the PAs senkirkine and tussilagine, 
    which occur in a medicinal plant  Tussilago farfara, for their 
    ability to induce chromosome damage in human lymphocytes  in vitro.  
    They were not found to enhance the number of chromosome aberrations 
    up to concentrations of 1000 µmol.  However, heliotrine, used for 
    comparison, induced chromosomal aberrations at concentrations of 
    100 µmol.  In addition, heliotrine was also found to be capable of 
    damaging unstimulated eg Go-phase lymphocytes. 

    6.4.8  Carcinogenesis

        Carcinogenesis has been reviewed by McLean (1970), IARC (1976, 
    1983), and Mattocks (1986).  A number of purified PAs, purified or 
    crude extracts of plants containing them in a mixture or the actual 
    plant, dried and milled, and several PA metabolites or synthetic 
    analogue compounds have been tested for carcinogenecity.  However, 
    these include only relatively few of the known cytotoxic PAs.  Data 
    relating to some of the representative studies on rats are 
    summarized in Table 13.  Studies on liver tumours found in rats 
    given PAs and plant materials are summarized in Table 14.  All 
    experimental animal studies, with the exception of one on chickens 
    (Campbell, 1956) and one on Syrian golden hamsters (Fushimi et al., 
    1978), have been carried out on rats. 

        The liver is the most common organ involved in experimental 
    studies.  Tumours produced are mostly of epithelial origin, but a 
    significant number are also vascular.  Lack of precision and 
    diversity of terms used to describe similar or identical tumours 
    makes it difficult to compare the types of carcinogenic effect in 
    different studies.  Some terms have been used interchangeably, 
    e.g., hepatomas, hepatocellular carcinomas, haemangiogenic and 
    cholangiogenic tumours; nodular hyperplasia, pre-neoplasma, 
    neoplastic nodules, and hepatocellular tumours.  In most studies, 
    there are no supporting photomicrographs to draw any inference as 
    to whether the tumours were malignant.  Difficulties in the 
    interpretation of data have been commented on by Schoental et al. 
    (1954) and McLean (1970). 

        Lasiocarpine has produced the largest yield of tumours.  In the 
    studies of Svoboda & Reddy (1972), 16/18 animals surviving for more 
    than 56 weeks after receiving ip multidoses of lasiocarpine 
    developed malignant tumours of the liver.  Of these, 10 animals had 
    more than one tumour.  Continuous feeding of rats on a regimen 
    containing lasiocarpine resulted in all animals (24/24) developing 
    tumours (NCI, 1978).  In one study, a single oral administration of 
    retrorsine (Schoental & Bensted, 1963) to weanling rats resulted in 
    7 of the 29 animals that survived for more than one year developing 
    11 tumours of a wide variety, at least of 5 which were malignant. 
    It is of note that this PA is known to have caused two cases of 
    human toxicity together with riddelline in two cases, though the 

    total intake was proportionately lower (Stillman et al., 1977; Fox 
    et al., 1978; Huxtable, 1980) (Table 15). 

        Tumours produced covered a very wide range in unrelated tissues 
    and organs, for example, the pancreas, urinary bladder, pituitary, 
    bone, retro-peritoneal tissues, and skin, among others.  
    Hepatocellular carcinoma and haemangiosarcoma were the most common. 

        Crude extracts of plants or whole plants have also been 
    demonstrated to produce a variety of tumours.  For example,  Senecio 
     longilobus has been shown to produce tumours in a significantly 
    large proportion of experimental animals (Harris & Chen, 1970).  
    Hirono et al. (1973, 1976, 1978, 1979b) demonstrated the 
    carcinogenic properties of a number of plants, used as food in 
    Japan, in the rat. 

        A summary of the relevant experimental data follows. 

    6.4.8.1  Purified alkaloids

        Kuhara et al. (1980) administered clivorine in the drinking-
    water at a concentration of 0.05 g/litre to 12 rats of both sexes, 
    continuously for 340 days followed by plain water.  There were 20 
    control rats.  All the treated rats survived 440 days.  Eight of 
    the 12 animals developed liver tumours including 2 
    haemangioendothelial sarcomas and 6 that were described as 
    "neoplastic nodules".  No liver tumours were seen in the control 
    animals (Table 13).

        Schoental (1975) tested the carcinogenic properties of 
    heliotrine, with and without prior administration of nicotinamide.  
    Nicotinamide protects against liver necrosis and so may enhance 
    tumour yeild, as shown by Rakietin et al. (1971), who studied 
    pancreatic tumours in rats given streptozootocin. 

        Heliotrine was administered intragastrically to 4 groups of 26 
    male weanling rats in 1 or 2 doses of 230 mg/kg and 300 mg/kg body 
    weight; 2 groups also received nicotinamide at 350 - 500 mg/kg body 
    weight, administered ip 10 - 15 min before, and 2.5 h after 
    administration of heliotrine, as per the dosing regimen shown in 
    Table 13.  There were 8 controls.  All animals administered the 
    higher dose of heliotrine (300 mg/kg body weight) died within 5 
    months of the PA administration.  The livers showed lesions 
    characteristic of PA toxicity.  No tumours were seen.  Only 1 out 
    of 4 animals receiving heliotrine alone, survived 27 months and it 
    showed an islet cell adenoma as well as adenoma of pituitary, but 
    so did 3 of the 8 controls.  In the group receiving 230 mg 
    heliotrine/kg and also treated with nicotinamide, 4 animals died 
    within 5.5 months (one with a fibrosarcoma and the other in a 
    moribund condition), chiefly from toxic liver disease, and two more 
    had to be killed.  Of the 6 animals surviving more than 22 months 
    after heliotrine treatment, islet cell adenoma was seen in 3 
    together with other tumours as shown in the table.  This tumour is 
    stated to be extremely rare in the animal strain used.  Hepatoma 
    was seen in only one animal.  The role of nicotinamide in this 
    study is not clear. 

        Table 13.  Summary of data on the carcinogenic action of PAs and PA-containing plants in rats (in chronological order)
                                                                                                                                                   
    Material     Strain,        Route of            Other          Duration       Period of      Tumours           Tumour       Reference
    tested       number,        administration      treatment      of             observation    produced          incidence
                 and sex of     and dosing                         treatment                                       in
                 animals        regimen                                                                            surviving
                                                                                                                   animals
                                                                                                                                                   
    Alkaloids    Wistar rat     drinking-water                     1 week         until death    only nodular                   Schoental
    of Senecio   (13 males,     (0.05 g/litre)                     (males)                       hyperplasia                    et al. (1954)
    jacobaea     12 females)                                       2 weeks                       of liver
                                                                   (females);
                                followed by                        gap of
                                                                   7 weeks

                 9 males,       0.03 g/litre,                      until
                 1 female       3 days/week                        death
                 (surviving)

    Retrorsine   10 males,      0.03 g/litre,                      until                         hepatomas         4/14
                 4 females      3 days/week                        death

    Isatidine    8 males,       0.05 g/litre
                 14 females     followed by
                                0.03 g/litre,                      20 months      until death    hepatomas         10/22
                                3 days/week

                 3 males,       as above            choline        until                         hepatomas         3/7
                 4 females                          (0.5% in       death
                                                    drinking-
                                                    water), 4
                                                    days/week
                                                                                                                                                   

    Table 13.  (contd.)
                                                                                                                                                   
    Material     Strain,        Route of            Other          Duration       Period of      Tumours           Tumour       Reference
    tested       number,        administration      treatment      of             observation    produced          incidence
                 and sex of     and dosing                         treatment                                       in
                 animals        regimen                                                                            surviving
                                                                                                                   animals
                                                                                                                                                   
    Isatidine    2 males,       single ip                                                                                       Schoental
    (contd.)     3 females      injection of                                                                                    et al. (1954)
                                2 mg in 0.2 ml
                                tricaprilyn
                                followed by
                                skin application                   15 months      15 months      hepatoma (?)      1/5
                                tion of 0.5%
                                solution, 3
                                days/week

                 controls                                                                        not mentioned
                 (7 males,
                 7 females)

    Retrorsine   Porton         single              400 r                         until death    hepatomas         5/25         Schoental &
                 Wistar         intragastric        radiation                                                                   Bensted (1963)
                 weanling       dose of 30 mg/kg    to 31/50                                     hepatocellular    1/25
                 rat            body weight         surviving
                 (50 males)                         for 100                                      carcinoma
                                                    days; 4/13                                   with
                                                    had head                                     metastases
                                                    shielded
                                                                                                 mammary           2/25
                                                                                                 tumours
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material     Strain,        Route of            Other          Duration       Period of      Tumours           Tumour       Reference
    tested       number,        administration      treatment      of             observation    produced          incidence
                 and sex of     and dosing                         treatment                                       in
                 animals        regimen                                                                            surviving
                                                                                                                   animals
                                                                                                                                                   
    Retrorsine                                                                                   lung carcinoma    1/25
    (contd.)
                                                                                                 renal             2/25
                                                                                                 carcinomas

                                                                                                 colonic           1/25
                                                                                                 carcinomas

                 Porton         as above            as above                                     splenic           1/25         Schoental &
                 Wistar rat                                                                      haemangio-                     Bensted (1963)
                 (50 males)                                                                      endothelioma

                                                                                                 osteosarcoma      1/25
                                                                                                 bone

                                                                                                 leukaemia         1/25

                                                                                                 "spindle cell"    1/25
                                                                                                 tumour (neck)
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material     Strain,        Route of            Other          Duration       Period of      Tumours           Tumour       Reference
    tested       number,        administration      treatment      of             observation    produced          incidence
                 and sex of     and dosing                         treatment                                       in
                 animals        regimen                                                                            surviving
                                                                                                                   animals
                                                                                                                                                   
    Retrorsine   95 males,      single oral dose                                  until death    hepatomas         5/29
    (contd.)     95 females     of 30 mg/kg body
                 (weanling)     weight                                                           mammary tumour    1/29
                 (controls)
                                                                                                 lung carcinoma    1/29

                                                                                                 splenic           1/29
                                                                                                 haemangio-
                                                                                                 endothelioma

                                                                                                 uterine           1/29
                                                                                                 carcinoma

                 Porton                                                                          retroperitoneal   1/29         Schoental &
                 Wistar rat                                                                      sarcoma                        Bensted (1963)
                 (controls)

                                                                                                 squamous cell     1/29
                                                                                                 carcinoma
                                                                                                 (jaw)
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material     Strain,        Route of            Other          Duration       Period of      Tumours           Tumour       Reference
    tested       number,        administration      treatment      of             observation    produced          incidence
                 and sex of     and dosing                         treatment                                       in
                 animals        regimen                                                                            surviving
                                                                                                                   animals
                                                                                                                                                   
    Retrorsine   6 males        no PAs              400 r                         until death    leukaemia         2/6
    (contd.)     (weanling)                         radiation

                                                                                                 osteosarcoma      1/6

                                                                                                 renal adenoma     1/6

                 10 males       single              partial                       until death    hepatomas         2/9
                 (weanling)     intragastric        hepatectomy
                                dose (30 mg/kg                                                   squamous cell     1/9
                                body weight)                                                     carcinoma
                                (9 days after                                                    (jaw)
                                partial
                                hepatectomy)
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,      Route of            Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,      administration      treatment      of             observation    produced          incidence
                   and sex of   and dosing                         treatment                                       in
                   animals      regimen                                                                            surviving
                                                                                                                   animals
                                                                                                                                                   
    Mixed PAs      Randomly     single                                            more than 1    pancreatic        1/15         Schoental et al.
    from seeds     bred from    intragastric dose                                 year?          islet cell                     (1970)
    of             Porton       at 500-1500 mg/kg                                                adenomaa
    Amsinckia      Wistar       body weight
    intermedia     weanling                                                                      "papillary        1/15
    (intermedine   rat                                                                           tumour"a of
    medine and     (15 males)                                                                    urinary
    lycopsamine)                                                                                 bladder
                                                                                                 pituitary         1/15
                                                                                                 adenomaa

                   as above     as above                                                         pancreatic        1/15         Schoental et al.
                                                                                                 islet cell        (1970)
                                                                                                 adenocarcinoma

                                                                                                 exocrine                       1/15
                                                                                                 pancreatic
                                                                                                 adenoma

    Leaves and     2 males      10% mixed with                     1 month        until death    pancreatic        1/2
    stems of       (weanling)   diet                                              (period not    islet cell
    Heliotropium                                                                  stated)        adenoma
    supinum L.
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,      Route of            Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,      administration      treatment      of             observation    produced          incidence
                   and sex of   and dosing                         treatment                                       in
                   animals      regimen                                                                            surviving
                                                                                                                   animals
                                                                                                                                                   
    Heliotropium   6 males      single                                            until death    pancreatic        1/6
    supinum L.                  intragastric dose                                 (longer        islet cell
    (contd.)                    at 200-300 mg/kg                                  than 1         adenoma
                                body weight of                                    year)
                                crude alkaloidal
                                fraction

                   controls                                                                      not stated

    Senecio        Harlan rat   0.75% of diet                                     until death    none                           Harris & Chen
    longilobus     50 males,                                                      within 131                                    (1970)
                   50 females                                                     days

                   50 males,    0.5% of diet                                      until death    none                           Harris & Chen
                   50 females                                                     within 200                                    (1970)
                                                                                  days
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of            Other       Duration        Period of       Tumours           Tumour       Reference
    tested         number,       administration      treatment   of              observation     produced          incidence
                   and sex of    and dosing                      treatment                                         in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Senecio        40 males,     0.5% of diet for                1 year          428-657         liver cell        4/23
    longilobus     40 females    1 month                                         days            carcinomas
    (contd.)                     alternating with
                                 normal diet for                                                 peritoneal        1/23
                                 2 weeks                                                         mesothelioma

                   50 males,     0.5% of diet                    54 weeks        217-470         liver cell        16/47
                   50 females    for 1 week                                      days            carcinomas
                                 alternating with
                                 normal diet for                                                 angiosarcoma      1/47
                                 1 week

                   controls                                                                      not stated
                   (10 males,
                   10 females)

    Lasiocarpine   Fischer rat   intraperitoneal                 till            60-76           hepatocellular    10/18        Svoboda & Reddy
                   (25 males)    injection at                    moribund        weeks           carcinomas                     (1972)
                                 7.8 mg/kg body                  or had
                                 weight, twice                   palpable
                                 weekly for 4                    tumours                         cholangio-        1/18
                                 weeks, then                                                     carcinoma
                                 once a week
                                 for an additional                                               lung adenomas     5/18
                                 52 weeks
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Lasiocarpine                                                                                 skin squamous     6/18
    (contd.)                                                                                     cell
                                                                                                 carcinomas

                   Fischer rat   as above                                                        ileal             2/18         Svoboda & Reddy
                   (25 males)                                                                    adenocarcinoma                 (1972)

                                                                                                 ileal             1/18
                                                                                                 adenomyoma

                                                                                                 testicular        1/18
                                                                                                 interstitial
                                                                                                 cell tumour

                   controls                                                                      lung adenomas     2/25
                   (25 males)
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material        Strain,       Route of          Other          Duration       Period of      Tumours           Tumour       Reference
    tested          number,       administration    treatment      of             observation    produced          incidence
                    and sex of    and dosing                       treatment                                       in
                    animals       regimen                                                                          surviving
                                                                                                                   animals
                                                                                                                                                   
    Retronecine,    Porton        single sc                                                      spinal cord       1/10         Schoental &
    hydrochlorine   Wistar        injection of                                                   ependymo-                      Cavanagh (1972)
                    newborn rat   300-1000 mg/kg                                                 blastoma
                    (6 males,     body weight
                    6 females)

                                                                                                 "pituitary        5/10
                                                                                                 tumour"

                                                                                                 "mammary          1/10
                                                                                                 tumour"

    Hydroxy-        5 males       single ip                                                      brain             1/5          Schoental &
    senkirkine      (weanling)    injection of                                                   astrocytoma                    Cavanagh (1972)
                                  100-300 mg/kg
                                  body weight
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Heliotropium   5 females     5% of diet fed                                                  Schwann cell      1/5
    ramosissimum                 to 1 pregnant                                                   tumour of
                                 rat during 1st                                                  spinal cord
                                 15 days of
                                 pregnancy and
                                 from 10th day
                                 of parturition
                                 until weaning;
                                 female off-
                                 spring (5) fed
                                 on experimental
                                 diet for 10 days
                                 at 6 months of
                                 age

                   controls                                                                      not stated
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
                                                                                                                   Groups
                                                                                                                   I      II
    Petasites      ACI rat       4% of diet for                    until          480 days       liver cell                     Hirono et al.
    japonicus      Group I:      6 months;                         death          or until       adenomas          6/27   4/19  (1973)
    (young         12 males,     subsequently,                                    moribund
    flower         15 females    8% of diet                                       liver                            3/27   8/19
    stalks)                      alternating                                                     haemangio-
                                 weekly with                                                     endotheliomas
                                 normal diet
                                                                                                 liver cell        2/27   1/19
                                                                                                 carcinomas
                   Group II:     4% of diet
                   11 males,
                   8 females

                   controls      normal diet                                                     none
                   7 females/
                   8 females
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material     Strain,        Route of              Other            Duration     Period of      Tumours         Tumour       Reference
    tested       number,        administration        treatment        of           observation    produced        incidence
                 and sex of     and dosing                             treatment                                   in
                 animals        regimen                                                                            surviving
                                                                                                                   animals
                                                                                                                                                   
    Mono-        Sprague        gastric intubation                                  72 weeks       liver cell      10/42        Newberne &
    crotaline    Dawley rat     weekly of 25 mg/kg                                                 carcinomas                   Rogers (1973)
                 (50 males)     body weight for
                                4 weeks, then
                                8 mg/kg body
                                weight for
                                38 weeks 

                 50 males       as above, with                                                     liver cell      14/33
                                a diet deficient                                                   carcinomas
                                in lipotropes

    Heliotrine   Porton         intragastric                                                                                    Schoental (1975)
                 Wistar
                 weanling
                 rat

                 4 males        300 mg/kg body                                      until death    none
                                weight; repeated                                    (5 months)
                                after 3 weeks to
                                2 surviving rats
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material     Strain,        Route of              Other            Duration     Period of      Tumours         Tumour       Reference
    tested       number,        administration        treatment        of           observation    produced        incidence
                 and sex of     and dosing                             treatment                                   in
                 animals        regimen                                                                            surviving
                                                                                                                   animals
                                                                                                                                                   
    Heliotrine   6 males        300 mg/kg body        nicotinamide                  until death    none                         Schoental (1975)
    (contd.)                    weight; repeated      (500 mg/kg                    (5 months)
                                after 3 weeks         body weight),
                                                      ip, before
                                                      and after
                                                      each heliotrine
                                                      administration

                 4 males        230 mg/kg body                                      27 months      pancreatic      1/1
                                weight; repeated                                                   islet cell
                                to 2/4 surviving                                                   adenoma
                                after 5 days

                                                                                                   pituitary       1/1
                                                                                                   adenoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material     Strain,       Route of             Other           Duration      Period of        Tumours         Tumour       Reference
    tested       number,       administration       treatment       of            observation      produced        incidence
                 and sex of    and dosing                           treatment                                      in
                 animals       regimen                                                                             surviving
                                                                                                                   animals
                                                                                                                                                   
    Heliotrine   12 males      230 mg/kg body       nicotinamide                  until death      fibrosarcoma    1/8
    (contd.)                   weight; repeated     (350 mg/kg                    or killed        of jaw
                               6.5 days later       body weight),                 when moribund
                                                    ip, before,                   up to 27.5       pancreatic      3/6
                                                    plus 2 doses                  months           islet cell
                                                    after each                                     adenomasb
                                                    heliotrine
                                                    administration                                 hepatomab       1/6

                                                                                                   urinary         1/6
                                                                                                   bladder
                                                                                                   papillomab

                 12 males                                                                          testicular      1/6
                                                                                                   interstitial
                                                                                                   cell tumourb

                 2 males       ip injection         nicotinamide                  19-27.5          pituitary       1/2          Schoental (1975)
                                                    at 350 mg/kg                  months           adenoma
                                                    body weight
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of              Other          Duration    Period of       Tumours          Tumour       Reference
    tested         number,       administration        treatment      of          observation     produced         incidence
                   and sex of    and dosing                           treatment                                    in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Heliotrine     controls      no treatment                                     19-27.5         pituitary        3/6
    (contd.)       (6 males)                                                      months          adenomas

    Tussilago      ACI rat       32% in diet for                      600 days    600 days        haemangio-       8/12         Hirono et al.
    farfara        (6 males,     4 days, then 16%                                                 endothelioma                  (1976)
    (coltsfoot)    6 females)    until end of 
    (preblooming   (1.5 months   study                                                            liver cell       1/12
    flowers)       old)                                                                           adenomac

                                                                                                  hepatocellular   1/12
                                                                                                  carcinomac

                                                                                                  bladder          1/12
                                                                                                  papillomac
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of              Other          Duration    Period of       Tumours          Tumour       Reference
    tested         number,       administration        treatment      of          observation     produced         incidence
                   and sex of    and dosing                           treatment                                    in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Tussilago      5 males,      8% in diet                           600 days    600 days        liver            1/9
    farfara        5 females                                                                      haemangio-
    (contd.)                                                                                      endothelioma

                   6 males,      4% in diet                           600 days    600 days        none                          Hirono et al.
                   5 females                                                                                                    (1976)

                   controls      none                                 600 days    600 days        none
                   (8 males,
                   8 females)

    Dehydro-       Sprague       sc injection          partial        12 months   10 months       rhabdomyo-       36/60        Allen et al.
    retronecine    Dawley rat    bi-weekly for 4       hepatectomy                                sarcomas (5                   (1975)
                   Group I:      months at             on 15                                      with
                   75 males      20 mg/kg body         animals                                    metastases)
                                 weight, followed      after 4
                                 by 10 mg/kg body      months
                                 weight for 8
                                 months
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material        Strain,      Route of              Other          Duration    Period of       Tumours          Tumour       Reference
    tested          number,      administration        treatment      of          observation     produced         incidence
                    and sex of   and dosing                           treatment                                    in
                    animals      regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Monocrotaline   Group II:    sc injection          partial        12 months   10 months       rhabdomyo-       2/60
                    75 males     bi-weekly at          hepatectomy                                sarcomas
                                 5 mg/kg body          on 15
                                 weight for 12         animals                                    hepatocellular   2/60
                                 months                after 4                                    carcinomas
                                                       months

                                                                                                  acute myeloid    2/60
                                                                                                  leukaemia

                                                                                                  pulmonary        2/60
                                                                                                  adenomas

                    controls     sc injection          partial        12 months   10 months       none mentioned                Allen et al.
                    (50 males)   bi-weekly,            hepatectomy                                                              (1975)
                                 0.1 mol               on 5 animals
                                 phosphate             after 4 
                                 buffer, pH7           months
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material        Strain,      Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested          number,      administration     treatment      of             observation    produced          incidence
                    and sex of   and dosing                        treatment                                       in
                    animals      regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Monocrotaline   Sprague      sc injection on                   12 months                     any tumours       17/60        Shumaker et al.
    (contd.)        Dawley rat   alternate weeks                                                 (several                       (1976)
                    (60 males)   (5 mg/kg body                                                   animals had
                                 weight)                                                         more than 1
                                                                                                 tumour)

                                                                                                 pulmonary         11/60
                                                                                                 carcinomas

                                                                                                 hepatocellular    5/60
                                                                                                 carcinomas

                                                                                                 acute myeloid     3/60
                                                                                                 leukaemia

                                                                                                 rhabdomyo-        4/60
                                                                                                 sarcomas

                                                                                                 adrenal           8/60
                                                                                                 adenomas

                                                                                                 kidney adenoma    1/60
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Dehydro-       60 males      sc injection on                   12 months      12 months      rhabdomyo-        39/60
    retronecine                  alternate weeks                                  until          sarcomas at
                                 at 20 mg/kg body                                 moribund       site of
                                 weight followed                                                 injection
                                 by 10 mg/kg
                                 body weight,
                                 alternate weeks
                                 for 8 months

                   controls                                                                      adrenal           2/45
                   (45 males)                                                                    adenomas
                   (same group
                   as above)

    Mono-          Sprague       single sc                                                       pancreatic        16/23        Hayashi et al.
    crotaline      Dawley rat    injection of                                                    insulinomas                    (1977)
                   (80 males)    40 mg/kg body
                                 weight

    Petasitenine   ACI rat       drinking-water                    45 days        72 days        none                           Hirono et al.
                   (3 males)     (0.05% solution)                                                                               (1977)
                   (1 month
                   old)
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Petasitenine   5 males,      0.01% solution                    until          up to 16       haemangio-        5/10d
    (contd.)       6 females                                       death          months         endothelial
                                                                   or                            sarcomas
                                                                   moribund

                                                                                                 liver adenomas    5/10d

                   controls      none                                                            fibrosarcoma      1/10         Hirono et al.
                   (10 males,                                                                    (subcutaneous)                 (1977)
                   9 females)

    Lasiocarpine   Fischer rat   mixed with                        55 weeks       59 weeks       liver             9/20         Rao & Reddy 
                   (20 males)    feed at a                                                       angiosarcomas                  (1978)
                                 concentration of
                                 50 mg/kg                                                        hepatocellular    7/20
                                                                                                 carcinomas

                                                                                                 malignant         1/20e
                                                                                                 adnexal tumour
                                                                                                 of skin

                                                                                                 malignant         1/20e
                                                                                                 lymphoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Lasiocarpine   controls                                                                      none
    (contd.)       (10 males)

    Petasites      ddN mice      4% of diet as                     480 days       480 days       lung adenomas     24/39        Fushimi et al.
    japonicus      24 males      dried flower                                                                                   (1978)
    (flower        21 females    stalks                                                          lung              6/39
    stalks)                                                                                      adenocarcinoma

                                                                                                 liver             4/39
                                                                                                 reticullum
                                                                                                 cell sarcoma

                                                                                                 liver             1/39
                                                                                                 haemangio-
                                                                                                 endothelial
                                                                                                 sarcoma

                                                                                                 thymoma           1/39

                                                                                                 leukemia          2/39
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Petasites      control       nil                               480 days       480 days       lung              1g
    japonicus      23 males                                                                      kidney
    (contd.)       27 females                                                                    haemangio-
                                                                                                 endothelial
                                                                                                 sarcoma           1g

                                                                                                 spleen
                                                                                                 haemangioma       1g

                   Swiss         4% diet as                        480 days       480 days       lung              5/26         Fushimi et al.
                   strain mice   dried flower                                                    adenoma                        (1978)
                   20 males      stalks
                   20 females                                                                    leukemia          1/26

                   controls      nil                               480 days       480 days       liver             1g
                   23 males                                                                      haemangio-
                   20 females                                                                    endothelioma

                                                                                                 breast            1g
                                                                                                 carcinoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Petasites                                                                                    lung adenoma      3g
    japonicus
    (contd.)
                                                                                                 leukemia          2g

                   C57BL/6       4% diet as                        480 days       480 days       no tumours
                   mice          dried flower
                   20 males      stalks
                   20 females

                   controls      none                              480 days       480 days       lung adenoma      1g
                   20 males
                   20 females

                   Syrian        4% diet as                        480 days       480 days       adrenal           1/25         Fushimi et al. 
                   golden        flower stalks                                                   cortical                       (1978)
                   hamsters                                                                      adenoma &
                   13 males                                                                      breast
                   17 females                                                                    carcinoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Petasites      controls      none                              480 days       480 days       no tumours
    japonicus      12 males
    (contd.)       9 females

    Symphytum      ACI rat       33% of diet as                    480 days       until death    liver adenomas    5/19         Hirono et al. 
    officinale     (1 - 1.5      leaves                                           or moribund                                   (1979b)
    (leaves or     months old)
    root)                                                                                        urinary           1/19
                   Group I.1:                                                                    bladder
                   11 males,                                                                     papilloma
                   8 females               

                                                                                                 urinary           2/19
                                                                                                 bladder
                                                                                                 carcinomas

                                                                                                 (rats with        5/19
                                                                                                 tumours)
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Symphytum      Group I.2:    33% of diet as                    600 days       until death    liver adenomas    11/19
    officinale     10 males,     leaves                            or             moribund
    (contd.)       10 females

                                                                                                 urinary           2/19
                                                                                                 bladder
                                                                                                 papillomas

                                                                                                 (rats with        11/19
                                                                                                 tumours)

                   Group II:     16% of diet as                    600 days       until death    liver adenomas    7/21         Hirono et al. 
                   11 males,     leaves                                           or moribund                                   (1979b)
                   10 females
                                                                                                 haemangio-        1/21
                                                                                                 endothelial
                                                                                                 sarcoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Symphytum                                                                                    urinary           2/21
    officinale                                                                                   bladder
    (contd.)                                                                                     papillomas

                                                                                                 urinary           1/21
                                                                                                 bladder
                                                                                                 carcinoma

                                                                                                 lymphatic         1/21
                                                                                                 leukaemia

                                                                                                 colonic           1/21
                                                                                                 adenoma

                                                                                                 pituitary         1/21
                                                                                                 adenoma

                                                                                                 (rats with        7/21
                                                                                                 tumours)
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Symphytum      Group III:    8% of diet as                     600 days       until death    liver adenoma     1/25         Hirono et al.
    officinale     14 males,     leaves                                           or moribund                                   (1979b)
    (contd.)       14 females

                   Group IV:     8% of diet as                     until                         liver adenomas    19/22
                   12 males,     root                              death
                   12 females
                                                                                                 urinary           2/19
                                                                                                 bladder
                                                                                                 papilloma

                                                                                                 (rats with        19/22
                                                                                                 tumours)

                   Group V:      4% of diet as                     until          until death    liver adenomas    16/42
                   24 males,     root reduced by                   death          or moribund
                   24 females    stages to basal
                                 diet after 180                                                  urinary           1/42
                                 days                                                            bladder
                                                                                                 carcinoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Symphytum                                                                                    adrenal           2/42
    officinale                                                                                   cortical
    (contd.)                                                                                     adenomas

                                                                                                 (rats with        16/42
                                                                                                 tumours)

                   Group VI:     2% of diet as                     280 days                      liver adenomas    10/23
                   12 males,     root for 190
                   12 females    days reduced by
                                 stages to basal
                                 diet

                   Group VII:    1% of diet as                     until                         liver adenomas    16/24        Hirono et al.
                   15 males,     root for 275                      death                                                        (1979b)
                   15 females    days and
                                 subsequently a                                                  liver             4/24
                                 basal diet                                                      haemangio-
                                 alternating                                                     sarcomas
                                 with 0.5% at
                                 3-week intervals                                                cholangio-        1/24
                                                                                                 carcinoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Symphytum                                                                                    adrenal           1/24
    officinale                                                                                   cortical
    (contd.)                                                                                     adenomas

                                                                                                 (rats with        17/24
                                                                                                 tumours)

                   Group VIII:   0.5% of diet as                   entire                        liver adenomas    8/30
                   15 males,     root                              study
                   15 females
                                                                                                 liver             9/30
                                                                                                 haemangio-
                                                                                                 sarcomas

                                                                                                 pituitary         1/30
                                                                                                 adenoma

                                                                                                 uterine           1/30
                                                                                                 adrenocarcinoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Symphytum                                                                                    gonadal           1/30         Hirono et al.
    officinale                                                                                   stromal tumour                 (1979b)
    (contd.)

                                                                                                 (rats with        10/30
                                                                                                 tumours)

                   controls      none                              till the                      urinary           1
                   (65 males,                                      end                           bladder
                   65 females)                                                                   papilloma

                                                                                                 subcutaneous      1
                                                                                                 fibrosarcoma
                                                                                                 caecal adenoma    1

                                                                                                 mammary           1
                                                                                                 fibroadenoma

                                                                                                 retroperitoneal   1
                                                                                                 teratoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Senkirkine     ACI rat       ip injection of    4 weeks,                      650 days       liver adenomas    9/20         Hirono et al.
                   (20 males)    22 mg/kg body      52 weeks                      or till                                       (1979a,b)
                                 weight, twice                                    moribund       myeloid           1/20
                                 weekly, then                                                    leukaemia
                                 once weekly
                                                                                                 testis
                                                                                                 interstitial      1/20
                                                                                                 tumour

    Symphytine     20 males      ip injection of    4 weeks,                      650 days       liver adenoma     1/20
                                 22 mg/kg body      52 weeks                      or till
                                 weight, twice                                    death or       liver             3/20
                                 weekly, then                                     moribund       haemangio-
                                 once weekly                                                     sarcomas

                   controls                                                                      myeloid           1/20
                   (20 males)                                                                    leukemia
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Symphytine                                                                                   fibroma-soft      1/20
    (contd.)                                                                                     tissues

                                                                                                 testis-           1/20
                                                                                                 interstitial
                                                                                                 tumour

    Clivorine      ACI rat       drinking-water                    340 days       480 days       liver             2/12         Kuhara et al. 
                   (6 males,     (0.005%)                                                        haemangio-                     (1980)
                   6 females)                                                                    sarcomas

                   ACI rat       drinking-water                                                  hepatic           6/12
                   (6 males,     (0.005%)                                                        neoplastic
                   6 females)                                                                    nodules

                                                                                                 testicular        3/12
                                                                                                 interstitial
                                                                                                 cell tumour
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Clivorine      10 males,                                                                     pituitary         1/20
    (contd.)       10 females                                                                    adenoma

                                                                                                 testicular        3/20f        Kuhara et al.
                                                                                                 interstitial
                                                                                                 cell tumour

                                                                                                 adreno-           1/20f
                                                                                                 cortical
                                                                                                 adenoma

                                                                                                 pancreatic        1/20
                                                                                                 aunar cell
                                                                                                 adenoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Crude          Sprague       intragastric                      104 weeks      114 weeks      liver tumours     13/40 (2     Habs et al.
    alkaloidal     Dawley rat    dose of 8 mg/kg                                                 (all types)       males, 11    (1982)
    extract        (20 males,    body weight, 5                                                                    females)
    from           20 females)   times per week
    Senecio                                                                                      other tumours     11/40
    numorensis                                                                                   (all types)       (5 males,
    fuchsii                                                                                                        6 females)

                   20 males,     intragastric                      104 weeks      114 weeks      liver tumours     34/40
                   20 females    dose of 40 mg/kg                                                (all types)       (5 males,
                                 body weight, 5                                                                    29 females)
                                 times per week
                                                                                                 other tumours     10/40
                                                                                                 (all types)       (7 males,
                                                                                                                   3 females)

                   controls                                                                      liver tumour      1/40
                   (20 males,                                                                                      (male)
                   20 females)
                                                                                                 other tumours     10/40
                                                                                                 (all types)       (4 males,
                                                                                                                   6 females)
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material       Strain,       Route of           Other          Duration       Period of      Tumours           Tumour       Reference
    tested         number,       administration     treatment      of             observation    produced          incidence
                   and sex of    and dosing                        treatment                                       in
                   animals       regimen                                                                           surviving
                                                                                                                   animals
                                                                                                                                                   
    Farfugium      AC1 rat       diet (20%)                        480 days       till death     liver             6/29         Hirono et al. 
    japonicum      (15 males,                                                     or end of      haemangio-                     (1983)
    (leaves        14 females)                                                    study          sarcomas
    and
    stalks)                                                                                      liver adenomas    7/29

                                                                                                 adrenalcortical   7/29
                                                                                                 adenomas

                                                                                                 adrenal           1/29
                                                                                                 phaeochromo-
                                                                                                 cytoma

                                                                                                 urinary           2/29
                                                                                                 bladder
                                                                                                 papillomas

                   ACI rat       diet (20%)                                                      testicular        2/29
                   (15 males,                                                                    interstitial
                   14 females)                                                                   tumours

                                                                                                 ileal             1/29
                                                                                                 adenocarcinoma
                                                                                                                                                    
    Table 13.  (contd.)
                                                                                                                                                   
    Material        Strain,       Route of          Other          Duration       Period of      Tumours           Tumour       Reference
    tested          number,       administration    treatment      of             observation    produced          incidence
                    and sex of    and dosing                       treatment                                       in
                    animals       regimen                                                                          surviving
                                                                                                                                                   
    Senecio         15 males,     diet (8%)                        none                                                         Hirono et al
    cannabifolius   15 females                                     survived                                                     (1983)
    (leaves and                                                    > 240 days
    stalks)
                    14 males,     diet (4%)                        none
                    14 females                                     survived
                                                                   > 240 days

                    12 males,     diet (1%)                        480 days       till dead      liver             1/23
                    12 females                                                                   haemangio-
                                                                                                 sarcoma

                                                                                                 liver adenomas    13/23

                                                                                                 adrenal           5/23
                                                                                                 cortical
                                                                                                 adenomas

                                                                                                 urinary           1/23
                                                                                                 bladder
                                                                                                 papilloma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material        Strain,       Route of          Other          Duration       Period of      Tumours           Tumour       Reference
    tested          number,       administration    treatment      of             observation    produced          incidence
                    and sex of    and dosing                       treatment                                       in
                    animals       regimen                                                                          surviving
                                                                                                                   animals
                                                                                                                                                   
    Senecio                                                                                      testicular        1/23
    cannabifolius                                                                                interstitial
    (contd.)                                                                                     tumour

                    12 males,     diet (0.2%)                      480 days       till death     liver             8/24
                    12 females                                                                   haemangio-
                                                                                                 sarcomas

                                                                                                 liver cell        3/24
                                                                                                 adenomas

                                                                                                 adreno-           3/24
                                                                                                 cortical
                                                                                                 adenomas

                                                                                                 adrenal           1/24         Hirono et al.
                                                                                                 phaeochromo-                   (1983)
                                                                                                 cytoma

                                                                                                 testicular        2/24
                                                                                                 interstitial
                                                                                                 tumours

                                                                                                 pituitary         1/24
                                                                                                 adenoma

                                                                                                 caecal            1/24
                                                                                                 fibrosarcoma
                                                                                                                                                   
    Table 13.  (contd.)
                                                                                                                                                   
    Material        Strain,       Route of          Other          Duration       Period of      Tumours           Tumour       Reference
    tested          number,       administration    treatment      of             observation    produced          incidence
                    and sex of    and dosing                       treatment                                       in
                    animals       regimen                                                                          surviving
                                                                                                                   animals
                                                                                                                                                   
    Senecio         controls      basal diet                       560 days                      cortical          3/49
    cannabifolius   (25 males,                                                                   adenomas of
    (contd.)        24 females                                                                   adrenal

                    controls      basal diet                                                     testicular        3/49         Hirono et al.
                    (25 males,                                                                   interstitial                   (1983)
                    24 females)                                                                  tumours

                                                                                                 pituitary         2/49
                                                                                                 adenomas
                                                                                                                                                   

    a  These tumours were present in the same animal.
    b  Each coexisted in one animal (seen in animals surviving 22 - 27.5 months).
    c  Together with haemangioendothelioma of the liver.
    d  Two animals had both tumours.
    e  Found in the same animal with angiosarcoma.
    f  In the same animal.
    g  No data available on number of surviving animals in the control groups.

    Note:   Figures in column 2 indicate the number of animals used at the start of the study. In column 8, animals developing a tumour out of
            the number surviving up to the end of study/or dying with tumour are indicated. Animals developing more than one tumour are
            indicated separately in a footnote. Different terms have been used in different studies to describe the same tumour in the liver,
            e.g., haemangiosarcoma, angiosarcoma, haemangioendothelioma, haemangioendothelial sarcoma, and haemangiogenic sarcoma.

    Table 14.  Liver tumours found in rats given PAs and plant materials (condensed results from various authors)a

                                                                                                                                                   

    Alkaloid or plant            Alkaloid type             Route of         Number of    Number and types of liver          Reference
    given                  Necine           Type of        administration   rats at             tumours found
                                            ester                           autopsy
                                                                                                                                                   

    Clivorine              otonecine        macrocyclic    oral                12        2 haemangiosarcomas                Kuhara et al. (1980)
                                            diester                                      6 neoplastic nodules               

    Lasiocarpine           heliotridine     "open"         ip                  18        11 hepatocellular carcinomas       Svoboda & Reddy
                                            diester        oral                20        9 angiosarcomas                    (1972);
                                                                                         7 hepatocellular carcinomas        Rao & Reddy (1978)

    Monocrotaline          retronecine      macrocyclic    oral                75        24 hepatocellular carcinomas       Newberne & Rogers
                                            diester                                                                         (1973)

    Petasitenine           otonecine        macrocyclic    oral                11        5 haemangiosarcomas                Hirono et al. (1977)
                                            diester                                      5 adenomas

    Retrorsine             retronecine      macrocyclic    various             14        4 "hepatomas"                      Schoental et al.
                                            diester                                                                         (1954);
                                                                                                                            Schoental & Head
                                                                                                                            (1957)

    Isatidine              retronecine      macrocyclic    various              7        4 "hepatomas" and                  Schoental et al.
                                            diester                                      "nodular hyperplasia"              (1954);
                                                                                                                            Schoental & Head
                                                                                                                            (1957)

    Senecionine            retronecine      macrocyclic    oral                80        19 hepatocellular tumours          Habs et al. (1982)
    (+ fuchsisenecionine)  (platynecine)    diester,                                     16 cholangiogenic tumours
                                            monoester                                    12 "haemangiogenic" tumours
                                                                                                                                                   

    Table 14 (cont'd)

                                                                                                                                                   

    Alkaloid or plant            Alkaloid type             Route of         Number of    Number and types of liver          Reference
    given                  Necine           Type of        administration   rats at             tumours found
                                            ester                           autopsy
                                                                                                                                                   

    Senkirkine             otonecine        macrocyclic    ip                  20        9 adenomas                         Hirono et al.
                                            diester                                                                         (1979a)

    Symphytine             retronecine      "open"         ip                  20        1 adenoma                          Hirono et al.
                                            diester                                      3 haemangiosarcomas                (1979a)

    Senecio longilobus                      macrocyclic    oral                47        16 hepatocellular carcinomas       Harris & Chen
    (seneciphylline,       (retronecine)    diester                                      1 angiosarcoma                     (1970)
    retrorsine, etc.)

    Petasites japonicus                     macrocyclic    oral                46        11 haemangiosarcomas               Hirono et al.
    (petasitenine)         (otonecine)      diester                                      10 adenomas                        (1973)
                                                                                         3 hepatocellular carcinomas

    Tussilago farfara                       macrocyclic    oral                12        8 haemangiosarcomas                Hirono et al.
    (senkirkine)           (otonecine)      diester                                                                         (1976)

    Symphytum officinale                    monoester +    oral               175        81 adenomas                        Hirono et al.
    (various)              (retronecine)    "open"                                       3 haemangiosarcomas                (1978)
                                            diester

    Farfugium japonicum    senkirkine                      oral                29        7 adenomas                         Hirono et al.
                                                                                         6 haemangioangiosarcomas           (1983)

    Senecio cannabifolius  petasitenine                    oral                21        16 adenomas                        Hirono et al.
                                                                                         9 haemangioangiosarcomas           (1983)
                                                                                                                                                   

    a  From:  Mattocks (1986).
            Svoboda & Reddy and their group have carried out 2 studies 
    using lasiocarpine (Svoboda & Reddy, 1972, 1974; Rao & Reddy, 
    1978).  Svoboda & Reddy (1972, 1974) gave repeated ip injections to 
    25 rats at a dose of 7.8 mg/kg body weight (0.1 LD50) for 56 weeks 
    as per regimen shown in Table 13.  Three rats died of acute liver 
    necrosis in the initial 4 weeks.  Eighteen rats survived 56 weeks, 
    by which time each animal had received an average cumulative dose 
    of 125 mg lasiocarpine.  Of these, 16 animals developed a variety 
    of tumours 60 - 76 weeks after the beginning of the study (Table 
    13).  Ten of the 16 animals had more than 1 tumour.  Hepatocellular 
    carcinoma was the most common.  The squamous cell carcinoma of the 
    skin was found to be transplantable.  When the same PA, mixed with 
    the diet at the rate of 50 mg/kg (Rao & Reddy, 1978) (Table 13), 
    was administered to 20 rats for 55 weeks, 17 animals developed 
    tumours.  Angiosarcoma of the liver emerged as the most common 
    tumour (9/20 animals), even though hepatocellular carcinomas were 
    also frequently seen (7/20 animals); squamous cell carcinoma of the 
    skin was not found, but there was a malignant adnexal tumour of the 
    skin.  The average cumulative dose of the alkaloid was estimated to 
    be 190 - 200 mg per rat. 

        Monocrotaline has been studied for its carcinogenic activity in 
    rats by Schoental & Head (1955), Newberne & Rogers (1973), Allen et 
    al. (1975), Shumaker et al. (1976), and Hayashi et al. (1977), 
    using different routes of administration. 

        Allen et al. (1975) studied the long-term effects on rats of 
    repeated sc injections of monocrotaline or its major detectable 
    metabolite, dehydroretronecine.  Male Sprague Dawley rats were 
    given biweekly injections of monocrotaline, at 5 mg/kg body weight 
    (75 animals) for 12 months, or dehydroretronecine at 20 mg/kg body 
    weight (75 animals) for 4 months followed by 10 mg/kg body weight 
    for 8 months.  Fifty control animals received phosphate buffer 
    (Table 13).  Partial hepatectomy was performed on 15 animals in 
    each of the treated groups and 5 in the control group.  They were 
    observed for 10 months following cessation of the injections.  Of 
    the 60 animals surviving in each of the treated groups, those 
    receiving monocrotaline showed rhabdomyosarcoma at the injection 
    site (2 animals), hepatocellular carcinoma (2 animals), acute 
    myelocytic leukaemia (2 animals), and pulmonary adenoma (2 
    animals).  In the group receiving dehydroretronecine, 36 animals 
    developed rhabdomyosarcomas, and 5 of these animals developed 
    metastases.  None of the control group developed tumours.  Tissues 
    obtained from partial hepatectomies showed that both compounds 
    caused inhibition of mitotic division in regenerating liver.  The 
    results of the study illustrate the dual alkylating and antimitotic 
    properties of these agents, commented on by Culvenor et al. (1969). 

        In a similar study by Shumaker et al. (1976), rats were 
    administered monocrotaline at 5 mg/kg body weight on alternate 
    weeks or its metabolite dehydroretronecine at 20 mg/kg body weight, 
    on alternate weeks for 4 months, followed by a dose of 10 mg/kg on 
    alternate weeks in the succeeding 8 months (Table 13).  Of the 60 
    rats receiving monocrotaline, 17 developed one or more tumours, but 
    not until after the treatment was discontinued.  The time interval 

    was not stated.  The most common tumour was carcinoma of the lung 
    (11 animals) followed by hepatocellular carcinoma (5 animals).  A 
    wide variety of other tumours was also seen.  A notable feature was 
    that the metabolite dehydroretronecine did not produce any tumours 
    by systemic action, but only at the site of injection, where 
    significant numbers of rhabdomyosarcomas (39/60 animals) were seen.  
    The marked difference in tumour sites is explained by the fact that 
    the parent alkaloid monocrotaline has to be metabolized before it 
    becomes a carcinogen.  For this reason, the tumours are distributed 
    in several organs of the body, whereas dehydroretronecine is itself 
    carcinogenic and so acts at the site of injection.  When 
    monocrotaline was delivered in a higher dose, but as a single 
    subcutaneous injection (40 mg/kg body weight) (Hayashi et al., 
    1977) to 40 rats there were no malignant tumours but only adenomas 
    of the islets in the pancreas in 16/23 surviving animals.  The 
    results of the above studies indicate that monocrotaline is 
    tumorigenic, but the type of tumour and the malignancy both depend 
    on the route of administration and the dosage used. 

        Hirono et al. (1977) studied petasitenine, the pure alkaloid 
    isolated from the flower stalks of the plant,  Petasites japonicus  
    Maxim, which has been found to be carcinogenic for rats (Hirono et 
    al., 1973).  Two groups of ACI rats of both sexes, Group I of 3 
    animals and Group II of 11 animals, were given the alkaloid in in 
    the drinking-water, at concentrations of 0.5 g/kg and 0.1 g/kg, 
    respectively (Table 13).  There were 19 controls.  All 3 animals in 
    group I died within 72 days showing marked hepatocellular damage; 
    no tumours were seen.  In Group II, 10 out of 11 rats survived for 
    more than 160 days.  Eight of the 10 animals developed tumours - 
    liver cell adenomas (5) and haemangiosarcomas (5) (Table 10).  Two 
    animals had both types of tumours.  The authors concluded that the 
    carcinogenicity of the plant was due to petasitenine. 

        Retrorsine and its  N-oxide (isatidine) have been used in 
    several studies.  Schoental et al. (1954) administered retrorsine 
    at a concentration of 3 mg/litre in the drinking-water to 14 rats 
    and isatidine at concentrations of 5 mg/litre followed by 3 mg/litre
    to 22 rats until death.  Twenty-five rats were administered the
    alkaloid mixture from  Senecio jacobaea Lin at a concentration of
    500 mg/litre followed by 300 mg/litre in the drinking-water.  Dosing
    regimens are shown in Table 13.  One group of 7 animals receiving
    isatidine received supplementary 0.5% choline in the drinking-water
    and another group of 5 animals received isatidine 2 mg as a single ip
    administration in 0.2 ml of tricaprilyn, followed by skin application
    of alkalids as 0.5% solution for 3 days/week. 

        The animals receiving mixed alkaloids of the plant showed 
    extensive liver damage followed by marked nodular hyperplasia; no 
    tumours developed.  The nodules were earlier interpreted as 
    hepatomas, but later only as an early stage in the progression from 
    hyperplasia to neoplasia. 

        The retrorsine group showed extensive liver damage associated 
    with cirrhosis and nodular hyperplasia.  In 4 rats, they were 
    interpreted as hepatomas. 

        In the isatidine group, 10 out of the 22 rats developed 
    hepatomas.  Tumours were present in 3 out of 7 animals receiving 
    isatidine plus choline indicating that the latter had no protective 
    role.  One out of the 5 animals receiving the alkaloid ip and then 
    through dermal application developed a tumour, which was also 
    interpreted as a hepatoma. 

        In another study (Schoental & Bensted, 1963), 95 weanling rats 
    were administered a single dose of retrorsine at 30 mg/kg body 
    weight, by stomach tube (Group II).  One comparable group of 50 
    weanling rats (Group I) received 400 r radiation in animals 
    surviving 100 days after retrorsine administration (31/50).  A 
    third group (Group III) of 6 animals received 400 r radiation 
    alone.  Another group of 10 weanling rats received the PA, 9 days 
    after partial hepatectomy (Group IV) (Table 13).  The additional 
    treatments were given to study whether they would act as
    co-carcinogens and induce neoplasia in hepatocytes, which are known to
    show injurious effects for long periods following PA treatment.  In 
    Group I, 19 of the 50 animals receiving a single dose of the PA 
    died before radiation could be given.  Of the 31 remaining animals 
    that received radiation after the PA, 25 survived 12 months.  Among 
    these, 19 tumours of a wide variety were seen (Table 13).  Of the 6 
    tumours in the liver, only 1 was malignant, having metastasized.  
    Most of the other tumours were malignant, including those of the 
    breast, one of which had also metastasized. 

        In Group II, 29 out of 95 animals that had received one dose of 
    PA and no radiation survived for more than a year, with a mean 
    survival time of 23 months.  Among these, 7 animals developed 
    tumours of a wide variety (Table 13).  Five tumours in the liver 
    were benign.  Most of the others were malignant.  Two tumours, a 
    cystic tumour of the breast and a carcinoma of the uterus, were 
    present in one animal.  Tumours seen in Groups III and IV, which 
    were found in animals surviving 12 months or more after the start 
    of the study, are shown in the Table 13.  The 2 tumours of the 
    liver in Group IV were also benign.  The number of control animals, 
    if any, were not indicated. 

        The authors concluded that the above studies did not provide 
    definite evidence of synergistic action in the carcinogenicity of 
    retrorsine by whole body radiation or partial hepatectomy. 

        Hirono et al. (1979a) studied the carcinogenic properties of 
    senkirkine extracted from the dried milled buds of  Tussilago 
     farfara (coltsfoot) and symphytine extracted from dried milled 
    roots of  Symphytum officinale (comfrey).  Both  Tussilago farfara 
    (Hirono et al., 1976) and  Symphytum officinale (Hirono et al., 
    1978) had earlier been demonstrated to have carcinogenic 
    properties. 

        Sixty inbred ACI strain male rats were divided into 3 groups of 
    20 animals each and received repeated ip injections of senkirkine 
    at 22 mg/kg body weight or symphytine at 13 mg/kg body weight as 
    per schedule given in Table 13.  All animals treated with 
    senkirkine survived 290 days.  Nine out of 20 rats developed liver 
    adenomas mostly after 350 - 450 days from start of the study.  
    Cirrhosis of liver was frequently observed.  Of the symphytine 
    group, all animals survived 330 days after the start of the study.  

        Three animals developed haemangioendothelial sarcoma and one,
    liver adenoma.  The sarcomas were noted at least 518 days after the
    start of the study. 

        Schoental & Cavanagh (1972) used two alkaloids, retronecine and 
    hydroxysenkirkine isolated from  Crotalaria laburnifolia, which was 
    injected ip in single doses ranging from 100 to 300 mg/kg body 
    weight in 5 weanling Porton Wistar rats (Table 13).  One animal 
    that had received the 300 mg/kg dose developed astrocytoma of the 
    brain after 14 1/2 months.  Retronecine hydrochloride was 
    administered in doses ranging from 300 to 1000 mg/kg body weight by 
    single subcutaneous injection to 10 new-born rats.  One male rat, 
    which was found to be paraplegic 6.6 months after receiving a dose 
    of 600 mg PA/kg, was also found to have ependymoblastoma of the 
    spinal cord.  Among the litter mates of this rat, 1 male that had 
    received a dose of 1000 mg/kg died.  The remaining 6 females and 2 
    males were killed within 22 months of being dosed.  Of the females, 
    5 had pituitary tumours and 1 had a mammary tumour (type not 
    stated).  Two males did not show any significant abnormalities. 

        One group of 5, 6-month-old female rats, born to dams that had 
    been fed on a diet containing 50 g dried and powdered  Heliotropium 
     ramosissimum/kg diet, a tribal remedy, used during pregnancy and 
    parturition, were then themselves fed on the same diet at 6 months 
    of age, as per the regimen indicated in Table 13.  One female rat 
    was found to be paraplegic at 7 months of age.  A tumour, possibly 
    of Schwann or satellite cell origin, was found. 

    6.4.8.2  Plant materials

        A number of plant materials have been tested for their 
    carcinogenicity by administering either a mixture of extracted 
    alkaloids (Cook et al., 1950; Schoental et al., 1954) or, more 
    often, the dried and milled plant mixed with the diet.  The only 
    study in which the plant alone was fed was that of Campbell (1956), 
    who produced tumours in chickens by feeding dried and milled 
     Senecio jacobaea plant.  It is notable that the plants tested are 
    almost all those that have been reported to be used as herbal 
    medicines and/or food, some of which have been reported to cause 
    human toxicity. 

        Schoental et al. (1970) used mixed PAs (intermedine and 
    lycopsamine) extracted from seeds of  Amsinckia intermedia, known to 
    cause livestock losses in the USA, and leaves and stems from 
     Heliotropium supinum L., known to be used by women in East Africa 
    as a herbal medicine, after childbirth.  The dosing regimen and 
    mode of administration are indicated in Table 13.  Of the 15 male 
    weanling rats that had received a single treatment with the 
     Amsinckia PAs and survived for more than 1 year, 3 rats showed one 
    adenoma, one adenocarcinoma of the islet cells, and one adenoma of 
    the exocrine pancreas, respectively (Table 13).  In addition, one 
    of the animals also developed a pituitary adenoma and papillary 
    tumour of the urinary bladder.  Eight animals received the 
    treatment with  Heliotropium supinum L. (Table 13).  One out of the 
    2 weanling rats fed on the plant with the diet and one out of the 6 
    animals that received a single intragastric dose of the crude 
    alkaloid fraction developed islet cell adenoma of the pancreas.  
    The number of control animals, if any, used in the study is not 
    stated. 

        Harris & Chen (1970) tested the carcinogenicity of  Senecio 
     longilobus, which has been associated with cases of human toxicity 
    (Stillman et al., 1977; Huxtable, 1980; Fox et al., 1978).  Harlan 
    rats were divided into 4 groups (equal numbers of both sexes) and 
    fed diets containing dried and powdered stems and leaves of the 
    plant in the proportion of 5 -7.5 g/kg.  The number of animals in 
    each group and the feeding regimen are shown in Table 13.  
    Continuous feeding did not produce any tumours, presumably because 
    of the comparatively low survival rate of animals.  Significant 
    results were obtained when the animals were fed contaminated diet 
    alternating with normal diet (Group IV).  Of the 100 animals fed on 
    this regimen for 54 weeks, 47 survived for more than 200 days.  
    Seventeen of these animals developed malignant tumours in the 
    livers - hepatocellular carcinomas in 16 and an angiosarcoma in 1 
    animal after a minimum feeding period of 217 days.  In Group III, 
    fed a contaminated diet for 1 year, 23 rats lived for more than 200 
    days.  Four rats (3 males and 1 female) developed hepatocellular 
    carcinomas and one, a peritoneal mesothelioma, after a minimum 
    feeding period of more than 428 days.  The tumour-bearing animals 
    were predominantly male.  The authors emphasized the relative 
    rarity of liver tumours in the strain of animals used.  Results 
    demonstrated that  S. longilobus is carcinogenic for rats. 

        For a study of Schoental & Cavanagh (1972), using dried and 
    powdered  Heliotropium ramosissimum, see section 6.4.8.1. 

        Hirono et al. tested the carcinogenic effects of 3 widely-used 
    herbs containing PAs.  Hirono et al. (1973) studied the possible 
    carcinogenic effects of the young flower stalks of  Petasites 

     japonicus Maxim, which has long been used in Japan as food or 
    herbal remedy as well as the PA, petasitenine, isolated from it.  
    The flower stalks of the plant were dried, milled, and fed to young 
    ACI rats of both sexes mixed with the basal diet in the proportion 
    of 40 - 80 g/kg, as indicated in the feeding regimen in Table 13, 
    until the animals were moribund or dead.  One group of 27 rats was 
    fed the plant mixed in the proportion of 40 g/kg diet for 6 months 
    followed by 80 g/kg diet on alternate weeks.  The second group of 
    19 rats was fed a contaminated diet (40 g/kg) continually.  Three 
    animals in Group I died of pneumonia.  Eleven out of the remaining 
    24 rats in this group developed liver tumours after 15 - 16 months 
    of feeding.  There were 2 hepatocellular carcinomas, 6 liver cell 
    adenomas, and 3 haemangiosarcomas, of which 2 had metastasized.  In 
    Group II, 2 animals died from non-tumorous causes.  Of the 
    remaining 17, 8 developed haemangiosarcomas, 4 liver cell adenomas 
    and 1 had a hepatocellular carcinoma.  The incidence of 
    haemangiosarcoma was statistically higher in Group II. 

        In a similar study on mice and hamsters (Fushimi et al. (1978), 
    groups comprising 20 - 24 male and 20 - 21 female 6-week-old ddN, 
    Swiss, and C57BL/6 mice, and 13 male and 17 female hamsters,  were 
    fed a diet combining 4% young, dried, and milled flower stalks of 
     Petasites japonicus Maxim for 480 days.  All surviving animals were 
    killed at the end of the study.  Lung adenomas and adenocarcinomas 
    were found in 30/39 surviving male and female ddN mice combined 
    (compared with 1/50 in the respective controls) in addition to 
    other tumours (Table 13).  No significant differences in tumour 
    incidence were observed between treated Swiss and C57BL/6 mice and 
    hamsters, and the corresponding controls.  No data were given on 
    the tumour incidence according to sex, in treated and control 
    animals or on survival in the controls. 

        In studies on rats (Hirono et al., 1976), the dried and 
    powdered flower buds of  Tussilago farfara (coltsfoot) were mixed 
    with the diet.  Forty-nine inbred ACI strain rats were divided into 
    4 groups and fed diets containing 40 - 320 g  T. farfara/kg for up 
    to 600 days.  The number of animals in each group and the dosage 
    and feeding regimen are given in Table 13.  Two groups receiving a 
    diet containing 80 - 320 g/kg on a continual basis developed 
    tumours in the liver of the same types as animals in the studies 
    using  Petasites japonicus, e.g., haemangioendotheliomas, liver 
    cell adenomas, and hepatocellular carcinomas (Table 13).  All 12 
    animals in Group I survived for more than 380 days after the start 
    of the study.  Of these, 8 (5 males, 3 females) developed 
    haemangioendothelial sarcoma of the liver.  In addition, 3 of the 8 
    rats developed simultaneously hepatocellular adenoma, hepatocellular
    carcinoma, or urinary bladder papilloma.  In Group II receiving
    coltsfoot at 80 g/kg in the diet, 9 out of 10 animals that survived
    for more than 420 days developed a haemangioendothelial sarcoma. 

        Hirono et al. (1978) fed  Symphytum officinale, similarly dried 
    and milled, to the ACI strain of inbred rats of both sexes at 
    different levels ranging from 80 to 330 g/kg as leaves or 5 - 40 g/kg
    as root, for 280 days or more.  Eight groups of animals of 
    19 - 48 animals each were used on different regimens of feeding.  
    The feeding regimen, the number of animals in each group, and the 
    duration of treatment are given in Table 13.  Sixty-five males and 
    64 females served as controls.  Tumours were induced in all groups 
    receiving leaves or roots.  The most common tumour was liver 
    adenoma.  Haemangiosarcomas were observed but infrequently (3 
    animals in the whole study).  No carcinomas of the liver were seen, 
    but there was a wide variety of other tumours.  It is noteworthy 
    that, in Group VII, 14 of 15 animals fed the lowest dose of 10 g/kg 
    for 275 days followed by 5 g/kg or basal diet alternating every 3 
    weeks, developed tumours, 2 of which were malignant (haemangiosarcomas).
    In the control group, single animals each had papilloma of urinary
    bladder, caecal adenoma, subcutaneous fibrosarcoma, mammary
    fibroadenoma, or retroperitoneal teratoma.  The livers of animals that
    did not have the tumours showed other features commonly encountered in
    animals administered PAs, e.g., megalocytosis, liver cirrhosis,
    hyperplastic nodules, etc., suggesting that they were induced by the
    PAs contained in the plant.  The authors concluded that the
    carcinogenic activity of this plant was weaker than that observed in
    animals fed  Petasites (coltsfoot).  Hirono et al. (1979b) have
    summarized the above studies on PAs found in edible plants in Japan.

        Habs (1982) and Habs et al. (1982) tested a crude alkaloid 
    extract from the plant  Senecio numorensis sp.  fuchsii containing 
    fuchsisenecionine (500 g/kg) and senecionine (10 g/kg).  The 
    extract was administered intragastrically in 2 doses of 8 mg and
    40 mg/kg body weight, respectively, to 2 groups of rats of both sexes,
    comprising 40 animals each, 5 times a week for 104 weeks (Table 
    13).  A large, dose-related number of liver tumours was produced, 
    originating in the liver cell and the sinusoidal system, and 
    predominantly affecting the female animals.  The tumour incidence 
    was higher in Group II (dose, 40 mg/kg) with 34 tumours compared 
    with 13 in Group I.  In Group I (dose, 8 mg/kg), 2 tumours were 
    found in 20 males compared with 11 tumours among 20 female rats. 
    Similarly, in Group II, only 6 tumours were found in 20 males 
    compared with 29 among 20 female rats.  The tumours included 19 of 
    "hepatocellular origin", 16 of "cholangigenic origin", and 12 of 
    "haemangiogenic origin" (Table 14).  Besides the liver tumours, 12 
    males and 9 females in the treated groups and 4 males and 6 females 
    in the control group developed a variety of extra-hepatic tumours.  
    Senecionine is known to be hepatotoxic and capable of being 
    converted in the rat liver into cytotoxic metabolites.  
    Fuchsisenecionine is a saturated PA, not previously known to be 
    cytotoxic.  It is therefore likely that senecionine was the 
    hepatotoxic component and that perhaps the two PAs acted

    synergistically with each other, though there is no actual evidence 
    for synergism.  However, this needs confirmation.  Moreover, there 
    appeared to have been other unknown components in the mixture 
    tested (Mattocks, 1986). 

        Hirono et al. (1983) studied the carcinogenicity of 2 more 
    plants  (Farfugium japonicum and  Senecio cannabifolius) of the tribe 
    Senecioneae in the family Compositae, the leaves and stalks of 
    which are used in Japan as human food.  Fresh leaves and stalks of 
    the plants were dried, milled, and mixed with the basal diet.  
    Inbred strain ACI rats of both sexes, with preponderance of 
    females, 1.5 months old, were divided into 6 groups.  They were fed 
    diets containing various proportions of the dried plant materials, 
    as indicated in Table 13.  The study was terminated at 480 days, 
    except for one group, which was studied for 560 days.  Besides the 
    groups shown in the tables, 2 more groups of 30 and 28 animals of 
    equal numbers of both sexes were fed 8% and 4% of  Senecio 
     cannabifolius, respectively.  None of these animals survived more 
    than 177 days and all died of hepatotoxicity.  A wide range of 
    tumours was observed, mostly in the liver, as shown in Table 13, 
    the most common being haemangiosarcomas, which were not encountered 
    in the control group. 

        The carcinogenicity of  Farfugium japonicum is considered to be 
    due to senkirkine and petasitenine.   Senecio cannabifolius contains 
    senecicannabine, a new macrocyclic PA, seneciphylline, and 
    jacozine.  It is probable that the carcinogenicity of  Senecio 
     cannabifolius is due to these PAs (Hirono, et al., 1983). 

        Hayes et al. (1985) studied the biological mechanisms by which 
    PAs initiate carcinogenesis in male Fischer 344 rats.  Lasiocarpine 
    (single or double injection of up to 80 µmol/kg body weight) and 
    senecionine (single or double injection of up to 160 µmol/kg) were 
    inactive as initiators of Y-GT-positive nodules in rats exposed to 
    2-acetylaminofluorene and partial hepatectomy.  Administration of 
    lasiocarpine or senecionine 12 h after partial hepatectomy resulted 
    in the development of very few nodules.  Lasiocarpine given in a 
    single or double dose (up to 80 µmol/kg) delayed hepatic 
    regeneration by at least 8 weeks after partial hepatectomy, and 
    pretreatment with this PA reduced the initiating capacity of 
    diethylnitrosamine and  N-nitrosomethylurea in rats subsequently 
    selected with 2-acetylaminofluorene and partial hepatectomy.  
    Resistant nodules selected with lasiocarpine also had the typical 
    resistant nodule phenotype (positive for Y-GT and epoxide 
    hydrolase) and also lacked PA-induced megalocytosis.  Lasiocarpine 
    treatment also resulted in small regenerative nodules that were 
    distinct from resistant nodules, because they were negative for 
    Y-GT and epoxide hydrolase. 

    6.4.8.3  Pyrrolizidine alkaloid metabolites and analogous synthetic 
             compounds

        The subject has been reviewed by Mattocks (1986).  The 
    pyrrolizidine alkaloids are converted into pyrrolic esters in the 
    hepatocytes.  These may then be hydrolysed into pyrrolic alcohols, 
    which are more water soluble and less active than the esters.  The 
    esters may be widely distributed throughout the body.  Some of 
    these compounds and their analogues have been tested for 
    carcinogenicity. 

        (a)  Pyrrolic esters

        (i)   Dehydromonocrotaline (monocrotaline pyrrole)

        Mattocks & Cabral (1979) tested dehydromonocrotaline on the 
    skin of mice.  In their first study on male BALB/c mice, they 
    applied the compound on the back at 1- to 2-week intervals.  
    Thirty-three applications of 1 µmol did not produce any skin 
    tumours in 16 mice, but 2 developed lung adenomas; no tumours 
    occurred in 14 control mice treated with the solvent (acetone).  In 
    the second study (Mattocks & Cabral, 1982), 11 female LACA mice 
    each received 47 applications of 2.5 µmol dehydromonocrotaline; one 
    animal developed a malignant skin tumour.  A second batch of 10 
    mice was given similar treatment and subsequently received 61 
    twice-weekly applications of croton oil at the same site.  Half of 
    these animals developed tumours.  In the control group of 10 mice, 
    only 1 tumour was seen.  The results of these studies indicate that 
    dehydromonocrotaline requires the action of a promoter to manifest 
    carcinogenic potential. 

        (ii)   Dehydroretrorsine

        This supposed pyrrolic metabolite of retrorsine was applied to 
    the skin of male BALB/c mice at 1- to 2-week intervals; 33 
    treatments of 0.5 or 1 µmol failed to produce tumours in 15 mice 
    that survived for up to 60 weeks (Mattocks & Cabral, 1979). 

        (iii)   1-Methyl-2,3-bistrimethylacetoxymethylpyrrole

        Mattocks & Cabral (1979, 1982) made 2 studies on this compound.  
    In the second study, which was more significant, 22 female LACA 
    mice received 47 dermal applications of 0.5 µmol each.  This caused 
    marked skin damage with ulceration and scarring.  Malignant skin 
    tumours developed in 19 out of 21 surviving animals.  Two 
    hydrolysis products of this ester, pivalic acid and 1-methyl-2,3-
    bishydroxymethylpyrrole, were similarly tested (Mattocks, 1986).  
    Tumours were produced, though fewer. 

        The results of the above studies suggest that the intact 
    pyrrolic ester is a carcinogen. 

        (b)  Pyrrolic alcohols

        Studies have been conducted using dehydroretronecine 
    (retronecine pyrrole) and dehydroheliotridine, which are secondary 
    metabolites of monocrotaline and heliotridine-based PAs, 
    respectively.  Originally they were regarded as (+)- and (-)- 
    forms, respectively, of dihydro-7-hydroxy-1-hydroxy-methyl-5H-
    pyrrolizine.  Kadzierski & Buhler (1985, 1986) showed that the 
    metabolite from monocrotaline is racemic and concluded that the 
    product from all heliotridine and retro-necine esters is the same 
    (±)- form. 

        Johnson et al. (1978) painted the skin on the back of 16 female 
    Swiss mice with dehydroretronecine, each dose equalling 20 mg/kg 
    body weight or about 5 µmol per mouse, once a week for 4 weeks and 
    then twice more after 6 months.  Six mice developed skin tumours.  
    Subcutaneous injections of the same dose yielded tumours in 13 out 
    of 21 mice.  Twenty-eight out of 55 mice given both topical 
    applications and sc injections developed skin tumours. 

        When the same study was repeated on 34 BALB/c mice, only one 
    skin tumour developed (Mattocks & Cabral, 1982).  When 17 animals 
    were given the same dose at more frequent intervals, e.g., 65 
    weekly doses, no tumours developed. 

        Similar results were obtained in a study by Shumaker et al. 
    (1976), already desecribed in section 6.4.8.1, in which 39/60 rats 
    given repeated subcutaneous injections of dehydro-retronecine 
    developed rhabdomyosarcomas at the site of injection.  The compound 
    appears to be a direct-acting carcinogen for rats and mice, though 
    the susceptibility of various strains of mice varies. 

        Peterson et al. (1983) gave 9 ip injections (60 - 76.5 mg/kg 
    body weight) of dehydroheliotridine to rats over a 32-week period.  
    A large variety of tumours was produced.  It was concluded that 
    this metabolite may be responsible for the carcinogenicity of its 
    parent PA. 

    6.4.8.4  Molecular structure and carcinogenic activity 

        Mattocks (1986) has reviewed the present position concerning 
    the relationship between molecular structure and carcinogenic 
    activity.  Data available at present are not adequate for any 
    strict correlation to be established between the molecular 
    structure of PAs and the types of tumours produced by them in the 
    rat.  However, the common determinants in the molecular structure

    of all carcinogenic PAs are that they are macrocyclic or "open" 
    diesters, in which the amino-alcohol moiety is retronecine, 
    heliotridine, or otonecine.  These are all esters of unsaturated 
    necines and are capable of being metabolized to pyrrolic esters in 
    the mammalian liver.  Studies on pyrrolic esters, the toxic 
    metabolic product of PAs, have yielded equivocal results.  
    Monocrotaline has been shown to bind covalently to DNA, which is 
    associated with the carcinogenic activity of the pyrrolizidine 
    alkaloids (Robertson, 1982).  Dehydromonocrotaline, the primary 
    metabolite of monocrotaline has been found to be an incomplete 
    dermal carcinogen (Hooson & Grasso, 1976; Mattocks & Cabral, 
    1982), whereas the synthetic compound 1-methyl-2,3-
    bistrimethylacetoxymethylpyrrole, which is chemically similar to 
    dehydromonocrotaline, is clearly carcinogenic (Mattocks & Cabral, 
    1982).  Dehydroretronecine (DHR), a second metabolite of 
    monocrotaline and possibly other retronecine-based PAs, is also 
    carcinogenic for the skin and is considered a proximate 
    carcinogenic metabolite, since, unlike monocrotaline, it acts at 
    the site of application directly and not at remote sites. 

    6.4.9  Antimitotic activity

        Literature on this phenomenon has been reviewed by Jago (1969), 
    McLean (1970), and Mattocks (1986).  The most characteristic 
    feature of the chronic hepatotoxicity of the PAs is the presence in 
    the liver of megalocytes (Bull & Dick, 1959; McLean, 1970), which 
    are generally enlarged hepatocytes containing large, hyperchromatic 
    nuclei (section 6.4.1.5).  These appear to be the result of a 
    combined action of PAs on the hepatocytes, a stimulus to regenerate 
    following parenchymal cell injury, and the powerful antimitotic 
    action of the pyrrole metabolites of the PAs (Mattocks, 1986; Jago, 
    1969).  This property has served as the basis for using a PA 
    (indicine- N-oxide) as a chemotherapeutic agent for cancer (Letendre 
    et al., 1981, 1984).  Peterson (1965) showed that the number of 
    mitoses following partial hepatectomy was reduced to 50% or less of 
    normal values by prior administration of hepatotoxic alkaloids, and 
    that the effect was dose dependent.  The hepatocytes seemed to 
    continue to grow without dividing.  The effect can be produced by a 
    single sublethal dose of the alkaloids (Schoental & Magee, 1957) or 
    can be a cumulative effect of small doses (Bull & Dick, 1959).  The 
    lesion appears within a few weeks and may persist for the lifetime of
    the animal (Mattocks, 1986).  It was characteristically described in
    the liver of the rat, but has also been reported in a number of other
    animals, e.g., mouse, sheep, horse, and pig, and in some other organs,
    e.g., kidney and lung (McLean, 1970).  It has not been observed in
    human livers (Tandon, H.D. et al., 1978; Mattocks, 1986), but it has
    been observed that cultured human fetal liver cells become enlarged
    when exposed to PAs (Armstrong et al., 1972) indicating a
    susceptibility to the antimitotic effect of PAs.  The lesion is not
    specific for PAs but has been reported to be produced by a number of

    toxins (McLean, 1970), including semisynthetic derivatives of PAs but
    not non-hepatotoxic PAs, such as platyphyline (Jago, 1970).  The 
    ultrastructural features of megalocytes are controversial.  
    However, consistent with other functional and metabolic features, 
    the cells show morphological characters suggesting increased 
    metabolic activity with increased exchange of material between the 
    nucleus and cytoplasm. 

        This unique reaction of the hepatocytes to PA has been used by 
    Jago (1970) to develop a method for assessing hepato-toxicity and 
    by Culvenor et al. (1976a) for screening 62 PAs for acute and 
    chronic hepatotoxicity and pneumotoxicity. 

        Mattocks (1986) suggested a scheme for the antimitotic action 
    of PAs  in vivo, consistent with observations of the phenomenon in 
    animals.  The PA is irreversibly metabolized by the hepatic 
    microsomes into a pyrrolic ester, which can be hydrolysed to a 
    pyrrolic alcohol.  The latter is the agent that inhibits mitosis.  
    The more reactive primary metabolite may also do this, by reacting 
    with tissue constituents to give products identical to pyrrolic 
    alcohols, inhibiting mitosis.  On the other hand, it can also 
    produce acute injury to the cells, which stimulates regeneration.  
    Thus administration of the PA or the pyrrolic ester can induce 
    megalocytosis to a much greater extent than a secondary metabolite 
    alone. 

        Antimitotic activity does not seem to be directly related to 
    inhibition of DNA synthesis, since the latter recovers within a 
    week while mitotic inhibition continues for up to a period of 4 
    weeks (Peterson, 1965; Armstrong & Zuckerman, 1970).  Mattocks & 
    Legg (1980) have shown that the level of DNA synthesis is reduced 
    in cells that do not divide, but is not totally inhibited.  Samuel 
    & Jago (1975) investigated the position in the cell cycle of the 
    antimitotic action of lasiocarpine and of its pyrrolic metabolite, 
    dehydroheliotridine.  Their studies indicated that the alkaloid 
    acts during the late S or early G2 phase of the cell cycle. 

    6.4.10  Immunosuppression

        Dehydroheliotridine (DHH), a pyrrolic metabolite, has a 
    significant immunosuppressant activity on the primary response in 
    young mice; when injected ip shortly before the antigenic stimulus 
    (Percy & Pierce, 1971).  The secondary response to antigenic 
    stimuli; as measured by the reduction in the number of 7S and 19S 
    specific antibody-synthesizing cells of the spleen, was suppressed 
    when DHH was administered at the time of secondary stimulus, but 
    not when it was given 24 or 36 h after the antigenic stimulus.  It 
    was suggested that dehydroheliotridine selectively destroys or 
    inactivates cells involved in the initial stages of antigen 
    recognition and processing. 

    6.4.11  Effects on mineral metabolism

        Aberrations of mineral metabolism have been observed in several 
    species of animals.  The most notable among them relate to 
    haemolysis and copper metabolism.  Anaemia has been reported to 
    occur in rats following PA poisoning (Schoental & Magee, 1959; 
    Schoental, 1963) and the kidney and liver show haemosiderosis 
    (Hayashi & Lalich, 1967).  Besides the haemolysis, PAs have been 
    found to exert a direct inhibitory effect on haematopoiesis in the 
    livers of new-born rats (Sundaresan, 1942).  Disturbances of iron 
    metabolism and haematopoiesis have also been demonstrated in rats 
    fed  Senecio jacobaea and supplementary copper (Swick et al., 1982d) 
    and they have been found to develop raised copper levels in the 
    liver and spleen, when fed on this plant (Swick et al., 1982b).  
    Miranda et al. (1979) reported elevated levels of iron in the liver 
    and spleen and of copper in the liver in rats fed on tansy ragwort.  
    Studies with radioactive iron also indicated a specific inhibitory 
    effect of PAs on haematopoiesis.  High copper levels in the liver 
    associated with haemoglobinuria have been reported in sheep grazing 
    on heliotrope (Bull et al., 1956), signs of disease closely 
    resembling those of chronic copper poisoning.  St. George-Grambauer 
    & Rac (1962) reported a similar outbreak of fatal jaundice due to 
    haemolytic crisis of chronic copper poisoning in sheep that had 
    grazed  Echium plantagineum over 2 or more seasons.  The 
    pathological changes in the liver were indistinguishable from those 
    of Heliotropium, and the livers had a high copper content.  Studies 
    of Miranda et al. (1981b) indicate that dietary copper can enhance 
    PA hepatotoxicity in rats.  It has been suggested that the 
    hepatotoxic effects of some PAs may interfere with the excretion of 
    copper (Bull & Dick, 1959; Farrington & Gallagher, 1960).  Similar 
    effects have been observed in pigs fed  Senecio (Harding et al., 
    1964).  White et al. (1984) did not observe any rise in hepatic 
    copper levels in sheep fed  Senecio jacobaea. 

    6.4.12  Methods for the assessment of chronic hepatotoxicity and
            pneumotoxicity

        Pyrrolizidine alkaloids produce acute as well as chronic liver 
    damage.  The acute effect is seen as extensive necrosis (Schoental 
    & Magee, 1957), while the chronic effect in rats is manifested 
    characteristically by the presence of greatly enlarged parenchymal 
    cells (Schoental & Magee, 1957; Bull & Dick, 1959), which persist 
    long after a single exposure (Schoental & Magee, 1959).  The latter 
    effect of megalocytosis may manifest without the liver cells going 
    through the process of necrosis (Schoental & Magee, 1959).  This 
    property has been used by Jago (1970) to develop a method for the 
    assessment of the relative chronic hepatotoxicity of different 
    alkaloids.  It has since been used by other investigators (Culvenor 
    et al., 1976a).  It consists of the intraperitoneal administration 

    of a single dose of between 0.025 and 3.2 µmol of the alkaloid per 
    kg body weight in 0.2 ml aqueous solution to 14-day-old suckling 
    hooded Wistar rats of both sexes.  The litters are randomized among 
    the mothers, one day before the administration of the toxin, and 
    then weaned at 28 days.  Animals are killed 4 weeks after the 
    injection.  The relative acute toxicity is indicated by the dose 
    levels that cause death within approximately a week and chronic 
    hepatotoxicity by those that produce hepatic megalocytosis within 4 
    weeks of the injection.  With this method, it is not only possible 
    to evaluate the hepatotoxicity of a given alkaloid but also to 
    compare the hepatotoxic effects of different compounds in relation 
    to each other on a molar basis.  Chronic effects on the lungs can 
    also be assessed by the same method.  Culvenor et al. (1976a) found 
    this method satisfactory for compounds of medium to high 
    hepatotoxicity but failed to detect toxicity in certain compounds 
    of known, low hepatotoxicity. 

    6.5  Effects on Wild-Life

    6.5.1  Deer

        There is very little information on the consumption of 
    PA-containing plants by non-domesticated animals, birds, and other 
    wild-life.  In one instance, the deaths of white-tailed deer in 
    coastal marshes in Louisiana in 1967, was ascribed to the 
    consumption of  Crotalaria and/or  Heliotropium species in a period 
    of feed scarcity (Seger et al., 1969).  The animals had thin and 
    watery blood, abnormal bone marrow, and serious atrophy of cardiac 
    and mesenteric adipose tissue.  In a 9-year-old doe, the liver was 
    dull and somewhat granular and evinced megalocytosis of the 
    hepatocytes.  In another 4-year-old animal, the liver appeared 
    normal but with microscopic evidence of early megalocytosis, with 
    considerable vacuolization in the centrilobular hepatocytes.  
    Plants of the genera  Crotalaria and  Heliotropium were abundant and 
    there were some Senecio species.  There were signs of ingestion of 
    the plants by the deer. 

         Senecio jacobaea (tansy ragwort) has been fed experimentally to 
    black-tailed deer  (Odocoileus hemionus columbianus) in Oregon to 
    determine their susceptibility to poisoning (Dean & Winward, 1974).  
    The ragwort was given  ad libitum together with different levels of 
    basal ration (85% alfalfa, 10% barley, 5% molasses) to captive 
    deer.  The ragwort was eaten, only when the basal ration was 
    inadequate.  One group, not given any basal ration for 6 days, 
    began eating ragwort and consumed 5.4 kg dry weight per animal in 
    42 days.  This represented 24% of the animal body weight.  The 
    animals did not show any toxic signs, and blood levels of SGOT and 
    bilirubin were normal. 

    6.5.2  Fish

        The effects of  S. jacobaea alkaloids on rainbow trout  (Salmo 
     gairdneri) fingerlings has been investigated (Hendricks et al., 
    1981).  Duplicate groups of 80 fingerlings were fed for up to 12 
    months on diets containing 20 or 100 mg alkaloid/kg.  The alkaloid 
    comprised 91% jacobine, 3% jacazine, 2.5% senecionine, and 2.5% 
    seneciphylline.  The 100 mg/kg diet resulted in severe growth 
    depression and mortality, which began at 3 - 4 months.  Both levels 
    of PAs produced severe hepatic lesions.  The livers from these fish 
    were shrivelled, mottled yellow or whitish in colour, nodular, 
    fibrous, and sometimes haemorrhagic.  Microscopically, there was 
    megalocytosis, severe fibrosis, and bile-duct proliferation. 
    Characteristic veno-occlusive changes were seen in the centrilobular
    and hepatic veins, which, in the case of fish receiving the 100 mg/kg
    diet, appeared after only 2 months on the diet. 

    6.5.3  Insects

        There are no reports of the toxicity of PAs for insects, but 
    there is substantial literature on the use of PAs by certain insect 
    families that have evolved with the ability to store the alkaloids 
    as defensive chemicals and to convert them into pheromones and 
    other signalling chemicals (see recent reviews by Brown (1984) and 
    Boppré (1986), and an earlier complementary paper by Edgar (1982)).  
    In some species, such as moths of the family Arctiidae, the larvae 
    feed on PA-containing plants.  In other families, such as Nymphalid 
    butterflies of the sub-families Danainae and Ithomiinae, the larvae 
    of most species live on other plants, but the adult males seek out 
    PA-containing species and contrive to ingest alkaloids from 
    wilting, dead, or damaged plant material or from nectar.  The 
    alkaloids so acquired have a functional role as defensive chemicals 
    against predators and, in some species, are also converted into 
    pheromones and other signalling chemicals involved in mating.  The 
    alkaloid derivatives may be pyrrolic compounds related to 
    dehydroretronecine or derivatives of the esterifying acids.  In one 
    Arctiid genus,  Creatonotus, the alkaloids have a morphogenetic or 
    hormonal effect, determining the size of the pheromone-disseminating
    organ.  Thus, for some insect species, PA-containing plants may be
    necessary for survival. 

    7.  EFFECTS ON MAN

        The toxic effects of pyrrolizidine alkaloids are principally on 
    the liver.  The toxic disease, produced by consuming PAs derived 
    from certain plants, is called veno-occlusive disease (VOD), the 
    pivotal and pathognomonic lesion being the occlusion of the central 
    and sublobular hepatic veins in the liver.  The larger hepatic vein 
    tributaries are characteristically unaffected in contrast with the 
    findings in Budd-Chiari syndrome (Bras, 1973). 

    7.1  Clinical Features of Veno-Occlusive Disease (VOD)

        There are several good clinical accounts of the disease, mostly 
    in the earlier reports from Jamaica, in children (Jelliffe et al., 
    1954a,b), and adults (Stuart & Bras, 1955), which have been 
    summarized by Stuart & Bras (1957).  Maksudov (1952) has described 
    the clinical features among children in outbreaks in the USSR, 
    where it was called toxic hepatitis with ascites, and Ismailov 
    (1948a,b), Mnushkin (1949, 1952), and Zheltova (1952) have 
    described them among adults.  Srivastava et al. (1978) also 
    described the clinical findings among children in a large outbreak.  
    Children seem to be particularly vulnerable as is evident from the 
    report of Stuart & Bras (1957) and that of Mohabbat et al. (1976) 
    concerning a large outbreak, though the disease is rare before the 
    age of one or two years.  Frequently, more than one member of the 
    family becomes affected (Stuart & Bras, 1957; Mohabbat et al., 
    1976; Tandon et al., 1976; Arora et al., 1981) within days or weeks 
    of each other. 

        The disease generally has an acute onset, characterized by 
    rapidly developing and progressing symptoms of upper abdominal 
    discomfort, dragging pain in right hypochondrium, ascites, and 
    sometimes oliguria and oedema of the feet.  Nausea and vomiting may 
    be present.  Jaundice and fever are rare.  There is generally 
    gross, tender, smooth hepatomegaly often accompanied by massive 
    pleural effusion, and sometimes slight splenomegaly and minimal 
    ankle oedema.  Liver function tests may show only mild disturbance.  
    The acute disease is associated with high mortality and a subacute 
    or chronic onset may lead to cirrhosis.  Death often occurs after 
    oesophageal haemorrhage. 

        Stuart & Bras (1957) summarized the clinical data of 84 
    patients ranging in age from 6 months to 53 years.  The highest 
    incidence occurred between the ages of 1 and 3 years (39%), and 26% 
    of patients belonged to the 3- to 6-year age group.  Thus, children 
    up to 6 years accounted for 65% of total cases.  Although the VOD 
    was relatively uncommon at the 2 extremes of age, early infancy and 
    adult life, mortality was highest in these groups, being 60% and 
    54%, respectively.  Hepatomegaly and some degree of ascites were 
    invariably present in acute cases; in 48 out of 64 patients, 
    ascites was acute enough to require paracentesis.  In 38 patients, 
    hepatomegaly was grossly severe, reaching more than half way down 
    to the umbilicus.  Jaundice was relatively uncommon.  Among the 
    liver function tests, the most significant and consistent changes 
    were found in the serum-cholinesterase (t = 2.67, 0.02 >  P > 0.01)

    and serum-albumin levels (t = 2.82, 0.01 >  P).  Mortality rates 
    associated with signs of parenchymal liver damage were 74% with 
    clinical jaundice or high levels of serum-bilirubin, 62% with 
    diminished serum-cholinesterase, and 58% with considerable anorexia 
    and apathy.  The mortality rates among cases with acute, subacute, 
    or chronic disease were reported to be 27%, 17%, and 57%, 
    respectively.  Death was mostly due to hepatocellular failure (71%) 
    in the acute phase and haemetemesis in the chronic phase (75%).  
    More recent publications (Lyford et al., 1976; McGee et al., 1976; 
    Tandon, B.N. et al., 1977; Datta et al., 1978a,b) also describe the 
    haemodynamic data of the hepatic blood flow and the results of 
    portovenographic studies that suggest outflow tract obstruction in 
    the liver at the post-sinusoidal level, and irregularity and 
    obstruction/distortion of the hepatic venous radicles, 
    respectively. 

        The clinical course of the disease has been shown schematically 
    by Stuart & Bras (1957) (Fig. 12).  However, the temporal 
    relationships in the different phases of the disease are not 
    precise, and their account, at best, represents a trend. 

    FIGURE 12

        The onset of the disease may be sudden (acute) or insidious 
    (subacute or chronic).  The acute disease may recover completely or 
    result in death.  A few patients may go on to the subacute phase, 
    with almost none or very few symptoms, but a persistent 
    hepatomegaly.  The patient may subsequently recover completely, or 
    may, after or without apparent clinical improvement, go on to the 
    chronic phase of disease, mostly ending up in cirrhosis.  Some 
    patients with the acute disease may go on to the subacute phase, 
    even after clinical recovery, or as postulated by the authors, 
    after a latent period of several years, progress to cirrhosis. 

        Braginskii & Bobokhadzaev (1965) related an experience 
    concerning the evolution of this disease that was similar to that 
    observed in the USSR.  About 50 - 60% of cases made a full recovery 
    and 35% made a partial recovery with continuing hepatomegaly.  
    About 2% of cases developed persistent ascites with "loss of 
    working capacity".  It has been suggested that these cases may 
    develop cirrhosis.

    7.2  Salient Pathological Features of Veno-Occlusive Disease

        The pathological features of the disease at different stages 
    have been described in detail (Terekhov, 1939, 1952; Dolinskaya, 
    1952; Bras et al., 1954; Bras & Watler, 1955; Bras & Hill, 1956; 
    Stuart & Bras, 1957; Stirling et al., 1962; Aikat et al., 1978; 
    Tandon, B.N. et al., 1978; Tandon, H.D. et al., 1978).  The 
    following description of Bras & Watler (1955) characterizes the 
    morphological changes at different stages of the disease described 
    by most investigators. 

        Morphological features of the liver at autopsy in 19 patients 
    from Jamaica who died at various stages of VOD have been described.  
    The ages ranged from 10 months to 45 years.  Fourteen patients were 
    below the age of 14 years.  Of the 14 patients, 9 had developed 
    cirrhosis, 3 died of acute disease, and 7 had various levels of 
    fibrosis superimposed over acute VOD.  In the acute stages, there 
    was acute centrilobular congestion.  The centrilobular and 
    sublobular veins showed different degrees of thickening of the wall 
    and occlusion of the lumen, mainly due to subintimal swelling 
    composed of loose reticular tissues and a few cells including 
    endothelial cells, occasional histocytes, lymphocytes, and 
    polymorphs, suggesting an acute exudative process.  In addition, 
    there was a small amount of fibrin.  Organization of this exudate 
    gradually led to collagenization and thickening of the wall.  The 
    centrilobular congestion resulted in compression and even 
    disappearance of liver cell cords.  The reticular framework of the 
    liver lobule was frequently preserved but was ruptured at places.  
    Clearly recognizable thrombi occurred sporadically but were not 
    commonly seen.  The portal veins and hepatic arteries were normal. 

        Stirling et al. (1962) in their description of the early lesion 
    of VOD of the liver also emphasized that thrombosis of the hepatic 
    veins was not an important histogenetic factor in the evolution of 
    the lesion.  In the later stages of the disease, hepatic venous 
    occlusion was chronically established.  Perivenous reticular 
    collapse and the resultant condensation and reduplication of the 
    reticular framework resulted in non-portal fibrosis and later 
    cirrhosis.  Macroscopically, the cirrhosis looked like Laennec's 
    cirrhosis.  The cirrhotic process, which was non-portal to begin 
    with, involved portal tracts which also became incorporated in the 
    connective tissue septa.  The presence of oesophageal varices was a 
    common finding.  Extra-hepatic collateral vessels and congestive 
    splenomegaly were frequently seen.  Within the liver, intrahepatic 
    collateral circulation was established with the coalescence of 
    sinusoids.  The larger hepatic veins and inferior vena cava did not 
    show any thrombi or other pathological change.  The subintimal 

    swelling observed in the hepatic veins was not seen in any other 
    vessels in the body.  The authors concluded that acute VOD 
    gradually leads to Laennec's cirrhosis, but, in the initial phases, 
    it is non-portal. 

        No clear dose or temporal relationships between the liver and 
    parenchymal and vascular changes in the liver lobules are evident 
    from the available human case reports. 

        It is notable that megalocytosis, which is the hallmark of 
    chronic PA toxicity in experimental animals and a morphological 
    manifestation of the antimitotic effect of PAs, has not been 
    observed in human subjects (Tandon, H.D. et al., 1978) (section 
    6.4.9). 

        Tandon H.D. (personal communication) has analysed all published 
    and unpublished observations including liver biopsies and autopsies 
    derived from 3 outbreaks of VOD of which two occurred in India 
    (Tandon, B.N. et al., 1976, 1977; Tandon, R.K. et al., 1976; 
    Tandon, H.D. et al., 1977) and one occurred in Afghanistan (Tandon, 
    B.N. et al., 1978; Tandon, H.D. et al., 1978).  He commented on the 
    frequency with which the characteristic veno-occlusion may not be 
    seen in the needle biopsy in the acute phase of the disease, though 
    it is invariably observed in the autopsy material.  Haemorrhages 
    may persist for up to 1 year after subsidence of acute symptoms. 
    There is no significant inflammation accompanying the veno-
    occlusive changes.  Cirrhosis was reported to have developed within 
    3 months in one patient, after an initial biopsy had shown no 
    fibrosis (Stuart & Bras, 1957). 

        Brooks et al (1970) made an ultrastructural study of the liver 
    in VOD in Jamaican children.  They found extensive damage in the 
    sinusoidal epithelium resulting in entra-vasation of red cells in 
    Disse's space and between hepatocytes.  The closed structure of the 
    vessels, the absence of fenestrations, the existence of a basement 
    membrane, and the presence of collagen in the wall contribute to 
    the resistance to cellular debris and erythrocytes which track up 
    Disse's space and tend to narrow the lumen of the sinusoid where it 
    enters the vein.  Fibrin may be present in this location and 
    occasionally in the sinusoids, but not within hepatic veins 
    themselves.  The venous block, therefore, does not appear to be the 
    result of thrombosis. 

        Pancreatic changes similar to those in Kwashiorkor were stated 
    to be commonly observed (Bras & Hill, 1956; Stuart & Bras, 1957). 

    7.3  Human Case Reports of Veno-Occlusive Disease

        Available data on human cases are summarized in Table 15, with 
    the countries they have been reported from in alphabetical order.  
    The first report of this disease in man and its relationship with 
    consumption of wheat flour contaminated by seeds of a plant of the 
    genus  Senecio was made by Albertjin in 1918 in a report made to the 
    government of South Africa, as quoted by Wilmot & Robertson (1920), 
    who gave the first account of this disease in scientific literature

    from that country.  It is possible that it may have existed even 
    earlier, as its occurrence in farm animals had been described since 
    the beginning of the century in veterinary literature (Bull et al., 
    1968).  Willmot & Robertson (1920) recorded the occurrence of 
    'bread poisoning' in South Africa for a period of about 10 years, 
    during which 80 cases had been observed, mostly resulting in death.  
    The detailed account of clinical data and pathological findings in 
    11 cases was consistent with what is known as VOD today.  The flour 
    from which the bread was prepared was found to be contaminated with 
    the flower heads and seeds of  Senecio spp. 

        Isolated case reports are available in the literature (Hashem, 
    1939; Wurm, 1939) describing changes in childrens' liver 
    characteristic of veno-occlusive disease; however, in these 
    studies, no mention was made of possible etiological agents. 

        A pattern of disease described in the Soviet literature as 
    dystrophy of the liver, and later as toxic hepatitis with ascites 
    has been known in the Central Asian republics of the USSR 
    especially Uzbekistan and Tadjikistan for a long time (Terekhov, 
    1952).  The local inhabitants called the disease, characterized by 
    rapidly filling ascites, the "camel-belly" syndrome and are 
    reported to have long distrusted a weed with fine seeds, known 
    locally as "Kharmyk", as a source of the disease (Dubrovinskii, 
    1952) (it is notable that, in the Afghan outbreak, which occurred 
    close to the area in Uzbekistan where the disease had been known, 
    the toxic seeds were known as "Charmak" among the local 
    population).  The "camel-belly" disease was associated with the use 
    of bread with a bitter taste (presumably caused by the mixing and 
    grinding of toxic seeds with the wheat grains).  Two waves of 
    outbreaks occurred, the first between 1931-35 (Terekhov, 1939) and 
    the second between 1945 - 46 (Dubrovinskii, 1947; Ismailov, 
    1948a,b).  In the first wave of outbreaks, approximately 1000 - 
    1500 subjects were estimated to have been affected with an overall 
    mortality rate of 13 - 15%.  The age of the affected subjects 
    ranged from 3 to 50 years.  In the second wave of outbreaks, 60 - 
    70% of the population in agricultural areas were estimated to have 
    been affected (Ismailov, 1948a,b).  Domestic animals also suffered 
    from the disease (Dubrovinskii, 1947).  During the second wave of 
    outbreaks, the disease was found to have been caused by 
    contamination of food crops with seeds of  Heliotropium lasiocarpum 
    (Dubrovinskii, 1947; Khanin, 1948).  The etiology was confirmed by 
    studies on a variety of experimental animals using the suspect seed 
    (Khanin, 1948; Kampanzev, 1952).  Pathological features of the 
    disease in children have been described by Dolinskaya (1952) and in 
    all age groups by Terekhov (1939, 1952).  First an acute form of 
    the disease was recognized, followed by hepatic cirrhosis, which 
    was found to result as a consequence of the initial disease 
    (Ismailov, 1948a,b; Savvina, 1952).  The disease has since been 
    largely eradicated by the agricultural and public health measures 
    taken. 

        Table 15.  Pyrrolizidine alkaloid poisoning and human reports of veno-occlusive disease (VOD)
               (in alphabetical order of the countries reported from)
                                                                                                                                              
    Country/         Age         Suspected      Name of          Alkaloid         Alkaloid         Nature of      Outcome        Reference
    number           group       vehicle of     plant                             concentration    lesion         (cirrhosis/
    cases                        intoxication                                     and intake                      died)
                                                                                                                                              

    Afghanistan

    8000             <14-80      contaminated   Heliotropium     mainly           1.32-1.49 g/kg   all stages;    many died      Tandon &
    (approximately)  years       wheat flour    popovii          heliotrine,      (in the          mostly acute                  Tandon (1975);
                                                gillianum        in addition      seeds) daily     to subacute                   Mohabbat
                                                                 to 1 or 2        intake, 2 mg;                                  et al. (1976)
                                                                 alkaloids        total
                                                                 similar to       consumption,
                                                                 lasiocarpine     up to 1.46 g
                                                                 (75 - 100%
                                                                 as N-oxides)

    Barbados

    3                2-4         herbal         Crotalaria       not analysed                      acute lesions  no details     Stuart & Bras
                     years       infusion       retusa                                                                           (1956)

    China, People's Republic of

    2                49-57       herbal         Gynura           not analysed                      acute lesions  died           Hou (1980)
                     years       infusion       segetum
                                                (Compositae)

    Ecuador

    1                35 years    herbal         Crotalaria       not analysed                      acute lesions  recovered      Lyford et al.
                                 infusion       juncea                                                                           (1976)
                                                                                                                                              

    Table 15 (cont'd)
                                                                                                                                              
    Country/         Age         Suspected      Name of          Alkaloid         Alkaloid         Nature of      Outcome        Reference
    number           group       vehicle of     plant                             concentration    lesion         (cirrhosis/
    cases                        intoxication                                     and intake                      died)
                                                                                                                                              

    Egypt

    3                            not known                                                                                       Hashem (1939)

    59               1-12        plant seed     not                                                acute lesions  cirrhosis      Safouh &
                     years       decoction      identified                                                        in one         Shehata (1965)
                                 in 17

    Federal Republic of Germany

    8                75-116      not known                                                         acute lesions  all died       Wurm (1939)
                     days        (alimentary
                                 toxaemia)

    Hong Kong

    4                23-28       herbal         Heliotropium     pyrrolizidine    herbs-alkaloid   acute VOD      1 died         Kumana et al.
                     years       infusion       lasiocarpum      alkaloids        base 0.42 g/kg;                                (1983, 1985);
                                                                                  N-oxides                                       Culvenor et al.
                                                                                  1.4 g/kg; daily                                (1986)
                                                                                  intake, 12 mg
                                                                                  base, 18 mg
                                                                                  N-oxide; total
                                                                                  intake, 570 -
                                                                                  1350 mg up to
                                                                                  development of
                                                                                  symptoms in 3
                                                                                  patients over
                                                                                  19-45 days,
                                                                                  1300 mg up to
                                                                                                                                              

    Table 15 (cont'd)
                                                                                                                                              
    Country/         Age         Suspected      Name of          Alkaloid         Alkaloid         Nature of      Outcome        Reference
    number           group       vehicle of     plant                             concentration    lesion         (cirrhosis/
    cases                        intoxication                                     and intake                      died)
                                                                                                                                              

    Hong Kong (cont'd)

                                                                                  death in 1
                                                                                  patient. 630 mg
                                                                                  in one case who
                                                                                  remained
                                                                                  asymptomatic.

    India

    2                25 and      herbal         not              unknown                           acute          recovered      Gupta et al.
                     35 years    decoction      identified                                                                       (1963)
                                 and pills

    25               12-38       possibly       not              not analysed                      acute          12 died;       Tandon, R.K.
                     years 6     contaminated   identified                                         lesions;       1 cirrhosis    et al. (1976);
                     patients    cereal                                                            cirrhosis                     Tandon, H.D.
                     studied)                                                                      in one                        et al. (1977)

    108              3-60+       contaminated   Crotalaria       crotananine      0.81 g/kg        various        up to 63%      Tandon, B.N.
                                 cereal         nana             and                               stages of      died           et al.
                                                                 crotaburmine                      disease                       (1976, 1977)a
                                                                                                                                 Siddiqui
                                                                                                                                 et al.
                                                                                                                                 (1978a,b)
                                                                                                                                              
    Table 15 (cont'd)
                                                                                                                                              
    Country/         Age         Suspected      Name of          Alkaloid         Alkaloid         Nature of      Outcome        Reference
    number           group       vehicle of     plant                             concentration    lesion         (cirrhosis/
    cases                        intoxication                                     and intake                      died)
                                                                                                                                              

    India (cont'd)

    35               1- > 25     contaminated   Crotalaria       as above         5.3 g/kg seed    acute          as above       Krishnamachari
                     years       cereal                                           (0 - 1.9% of                                   et al.
                                                                                  seeds in                                       (1977)a;
                                                                                  cereal); daily                                 Siddiqui et al.
                                                                                  intake, 40 mg                                  (1978a,b)

    6                20-70       herbal         Heliotropium     heliotrine       12 - 20 g/kg;    acute          3 died         Aikat et al.
                                 medicine in    eichwaldii       as N-oxide       total intake,    lesions;       (2 with        (1978);
                                 4 cases        by 3                              4 g and 10 g     cirrhosis      fulminant      Datta et al.
                                                patients                          in patients who  in one         disease)       (1978a,b)
                                                                                  died of          organizing
                                                                                  fulminant        thrombi in
                                                                                  disease          hepatic veins
                                                                                                   in 4 cases

    Iraq

    9                3-10        possibly       possibly         not                               acute          1 died         Al-Hasany &
                     years       wheat flour    Senecio          identified                                                      Mohamed (1970)

    South Africa

    11               11-19       wheat flour    Senecio          not                               centrilobular  "majority      Wilmot &
    (only 5          years       as bread       ilicifolius;     identified                        haemorrhages;  died"          Robertson
    described)                                  S. burchelli     senecionine?                      central vein                  (1920)
                                                                                                   "dilated"
                                                                                                                                              
    Table 15 (cont'd)
                                                                                                                                              
    Country/         Age         Suspected      Name of          Alkaloid         Alkaloid         Nature of      Outcome        Reference
    number           group       vehicle of     plant                             concentration    lesion         (cirrhosis/
    cases                        intoxication                                     and intake                      died)
                                                                                                                                              

    South Africa (cont'd)

    12                           wheat          not              not                               centrilobular                 Selzer & Parker
                                 flour          identified;      identified                        haemorrhages;                 (1951)
                                                "possibly                                          organizing
                                                Senecio"                                           thrombi in
                                                                                                   hepatic veins;
                                                                                                   3 cases of VOD

    12               5-30        herbal         not              none found                        "acute" in     3 died of      Stein &
                     months      medicines      identified       in herbal                         5 cases;       disease;       Isaacson (1962)
                                 admitted in                     medicines                         "subacute"     no follow-up   Stein (1957)
                                 4 cases;                                                          in 7 cases;    on 5
                                 food not                                                          no detail
                                 analysed

    United Kingdom

    1                26 years    herbal         Maté-Paraguay    identified       "trace           acute with     died           McGee et al.
                                 infusion       tea              only as          amounts"         veno-occlusion                (1976)
                                                                 pyrrolizidine                     going on to
                                                                 alkaloids                         chronic with
                                                                                                   fibrosis

    1                13 years    herbal         Symphytum        not analysed     not known        "thrombotic"                  Weston et al.
                                 infusion       officinale                                         variant of                    (1987)
                                                                                                   veno-occlusive
                                                                                                   disease
                                                                                                                                              
    Table 15 (cont'd)
                                                                                                                                              
    Country/         Age         Suspected      Name of          Alkaloid         Alkaloid         Nature of      Outcome        Reference
    number           group       vehicle of     plant                             concentration    lesion         (cirrhosis/
    cases                        intoxication                                     and intake                      died)
                                                                                                                                              

    USA

    1                2 months    herbal         Senecio          riddelline       15 g/kg as       acute lesions  died           Fox et al.
                                 infusion       longilobus       and N-oxides     alkaloids;                                     (1978)
                                                                 of retrorsine,   total intake,
                                                                 seneciphylline,  66 mg
                                                                 and senecionine

    1                6 months    herbal         Senecio          as above         3 g free         acute lesions  cirrhosis      Stillman et al.
                                 infusion       longilobus                        alkaloid plus                                  (1977);
                                                                                  10.5 g N-oxide                                 Huxtable (1980)
                                                                                  per kg; total
                                                                                  intake,
                                                                                  70-147 mg of
                                                                                  combined
                                                                                  alkaloid
                                                                                  and N-oxide

    1                49 years    herbal         Symphytum sp.    symphytum        14.1 µg/kg       acute lesions  recovered      Ridker et al.
                                 infusion                        pyrrolizidine    body weight                     after          (1985);
                                                                 alkaloids        per day; total                  short          Huxtable et al.
                                                                                  intake, 85 mg                   surgery        (1986)

    USSR

    1000 - 1500      3-50        contamination  Heliotropium     heliotrine,                       acute lesions  13-15%         Mirochnik
                     years       of wheat       lasiocarpum      lasiocarpine                                     died;          (1938);
                                 flour                                                                            cirrhosis      Terekhov
                                                                                                                  in many        (1939)
                                                                                                                                              

    Table 15 (cont'd)
                                                                                                                                              
    Country/         Age         Suspected      Name of          Alkaloid         Alkaloid         Nature of      Outcome        Reference
    number           group       vehicle of     plant                             concentration    lesion         (cirrhosis/
    cases                        intoxication                                     and intake                      died)
                                                                                                                                              

    USSR (cont'd)

    "large                       contamination  Heliotropium     heliotrine                                                      Dubrovinskii
    number"                      of wheat       lasiocarpium     lasiocarpine                                                    (1947);
                                 flour                                                                                           Ismailov
                                                                                                                                 (1948a,b)

    Venezuela

    1                5 years     "earth and     not                                                acute lesions  died           Grases & Beker
                                 plant eating   identified                                                                       (1972)
                                 habits"

    West Indies

    11               14 months   not known,                                                        acute to       data           Jelliffe et al.
                     to          possibly                                                          cirrhosis      unclear;       (1954b)
                     9 years     herbal                                                                           cirrhosis
                                 infusions                                                                        in some

    5                23 months   not known,     not                                                               1              Bras et al.
                     to 18       possibility    identified                                                        cirrhosis      (1954)
                     years       of "bush
                                 teas"

    4                16-45       history of     not                                                acute to       3 died         Stuart & Bras
                     years       herbal         identified                                         acute-on-                     (1955)
                                 infusion in                                                       chronic
                                 one case
                                                                                                                                              
    Table 15 (cont'd)

                                                                                                                                              
    Country/         Age         Suspected      Name of          Alkaloid         Alkaloid         Nature of      Outcome        Reference
    number           group       vehicle of     plant                             concentration    lesion         (cirrhosis/
    cases                        intoxication                                     and intake                      died)
                                                                                                                                              

    West Indies (cont'd)

    84               0.5-53      probably       not                                                               11% had        Stuart & Bras
                     years       "bush teas"    identified;                                                       cirrhosis;     (1957)
                                                (Senecio and                                                      27% died
                                                Crotalaria)

    23                           possibly       not stated       not analysed                      acute to                      Bras & Hill
                                 bush teas                                                         "cirrhosis"                   (1956)
                                                                                                   (no breakup)

    23               1-50                       no precise       no analysis                       cirrhosis                     Bras et al.
                     years                      identification;                                                                  (1961)
                                                "Crotalaria
                                                fulva and
                                                possibly
                                                other toxic
                                                factors"

    3                "children"  "toxic         not                                                acute          died           Stirling et al.
                                 substances     identified                                                                       (1962)
                                 in herbal
                                 remedies"
                                                                                                                                              

    a  Pertains to the same outbreak.
            Selzer & Parker (1951), from South Africa, described 12 cases, 
    10 of whom came from 3 families that had eaten bread made of flour 
    of "imperfectly winnowed wheat".  Five cases who were autopsied 
    showed characteristic occlusion of the central vein of the liver 
    lobules. 

        Attention on veno-occlusive disease as an entity followed a 
    spate of reports, mostly from Jamaica, on the occurrence of this 
    disease, mainly among children (Bras et al., 1954, 1961; Jelliffe 
    et al., 1954a,b; Bras & Watler, 1955; Bras & Hill, 1956; Stirling 
    et al., 1962) but also among adults (Stuart & Bras, 1955, 1957).  
    The youngest patient reported was a 3-month-old infant (Stein & 
    Isaacson, 1962), but the disease was stated even to have been 
    observed in the new-born (Stein, 1957).  Very often, several 
    members of the family were affected by disease, which presented 
    primarily as rapidly filling ascites, within a matter of days, 
    sometimes accompanied by fever, and leading to hepatic failure.  It 
    was considered an important cause of cirrhosis among children in 
    Jamaica (Jelliffe et al., 1954a,b).  On the basis of evidence of an 
    almost identical disease occurring among grazing animals (Hill, 
    1960), and a prevalent practice among the Jamaicans of using herbal 
    infusions for treating a variety of ailments as a home remedy, 
    herbs, notably of the  Senecio and  Crotalaria groups, were suspected 
    of being a contributory factor (Bras & Hill, 1956; Bras et al., 
    1957, 1961; Stuart & Bras, 1957). 

        Hill (1960) gave a comprehensive account of current knowledge 
    up to that time of the world-wide distribution of seneciosis in man 
    and animals in which mention is made of this disease only in Egypt, 
    the Federal Republic of Germany, South Africa, and the West Indies.  
    Pyrrolizidine alkaloids were identified as the active element in 
    the toxic factor in the plants. 

        However, there are earlier reports of this disease from 
    elsewhere.  Bras (1973) cited a number of reports from Europe from 
    1905 to 1949, including reports by Wurm (1939) and Teilum (1949), 
    of an entity in infants and children that was called Endophlebitis 
    hepatica obliterans.  He reported having studied some tissue 
    sections of liver in Austrian children aged 7 months - 1 year, who 
    had died with clinical and histological patterns of VOD.  Some of 
    these conformed to the Budd-Chiari syndrome whereas others were 
    consistent with VOD.  The case of a 36-year-old woman reported by 
    Teilum (1949) does not, however, fall in the usual category of 
    cases accepted as veno-occlusive, since thrombotic changes were 
    also seen in the larger hepatic veins and several, in the 
    peripheral parts of the body, as well as the portal vein. 

        A comprehensive account of all available published reports of 
    this disease to date from different parts of the world is given in 
    Table 15.  A clear distinction between the acute and chronic 
    effects of exposure is not always possible from the published data, 
    since, in most patients, the history of onset of disease with 
    intake of the toxic substance is not generally volunteered or 
    available, and so a precise temporal relationship is difficult to 

    establish.  However, this information is available in a reasonably 
    accurate form in more recent reports (Stillman et al., 1977; Datta 
    et al., 1978a,b; Fox et al., 1978; Kumana et al., 1985). 

        Gupta et al. (1963) from India described 2 cases who had drunk 
    some herbal infusions for the treatment of skin disorders.  The 
    liver biopsies confirmed changes typical of acute VOD.  However, 
    the herb was not identified and no analysis was made of the 
    infusion for alkaloids. 

        The cases of 59 children in Egypt who had symptoms of rapidly 
    developing abdominal distension and hepatomegaly were reported by 
    Safouh & Shehata (1965).  Their ages ranged between 1 and 12 years, 
    47 being below 4 years of age.  A dietary survey carried out on 17 
    patients indicated ingestion of drinks made by boiling some common 
    plant seeds, but these were not identified.  Wedge biopsies of 
    liver were performed in 6 patients and 16 postmortem examinations 
    were made.  In all the autopsy cases, there was thrombotic 
    obliteration of the main hepatic veins and their ostia.  The 
    central and sublobular veins were not uniformly thickened as they 
    were frequently dilated or disrupted and some contained fresh 
    thrombi.  There was centrilobular necrosis of the liver lobules.  
    One patient developed decompensated cirrhosis in 3 years.  The 
    authors observed that the clinical and pathological features 
    closely resembled those of VOD in Jamaica, but thrombosis was an 
    unusual feature of the latter disease. 

        Al-Hasany & Mohamed (1970) described a short outbreak in Iraq, 
    occurring during a 3-month period, affecting 9 children, all except 
    one being below 12 years of age, and belonging to 3 Bedouin 
    families living outside Baghdad.  One of the children died.  
    Autopsy of this case and biopsies on the others showed changes 
    characteristic of VOD.  Poisoning through the wheat flour 
    contaminated by seeds of some PA-containing plant was suspected, 
    but no analysis of the food was carried out. 

        Three of the largest outbreaks of the disease have been 
    reported from South Asia, two from the same site in central India 
    (Tandon, B.N. et al., 1976, 1977; Tandon, R.K. et al., 1976; 
    Krishnamachari et al., 1977; Tandon, H.D. et al., 1977) and one 
    from North-West Afghanistan (Tandon & Tandon, 1975; Mohabbat et 
    al., 1976; Tandon, B.N. et al., 1978; Tandon, H.D. et al., 1978).  
    The first Indian outbreak was reported by Tandon, B.N. et al. 
    (1976) and Tandon, R.K. et al. (1976) and Tandon, B.N. et al. 
    (1977) and Tandon, H.D. et al. (1977).  It occurred in a group of 5 
    tribal villages in central India in 1972 - 73.  The epidemiology of 
    the outbreak was later described by Arora et al. (1981).  Out of a 
    total population of 2060 in these villages, 71 households with 366 
    members were investigated.  Among these, 39 cases had developed and 
    19 had died before commencement of the investigations.  The 
    incidence rate was 1.1% and the case fatality rate was 50%.  All 
    cases occurred among 20 households.  In many households, several 
    members were affected.  In one household, 4 out of 5 cases died.  
    Six sick patients were investigated in detail with repeated liver 
    biopsies.  One patient died 17 months after onset and was 

    autopsied.  The clinical onset was characteristic of disease.  
    Haemodynamic and radiographic studies suggested a combined post and 
    perisinusoidal block.  Liver biopsy studies showed features 
    characteristic of acute centrilobular haemorrhagic necrosis with 
    progressive fibrosis, hepatic veno-occlusion, and non-portal 
    cirrhosis in one case who survived 17 months after the acute onset.  
    The etiological factor of this outbreak was not established, though 
    dietary contamination with pyrrolizidine alkaloids was considered. 

        A second outbreak occurred at the same site in 1975 and was 
    studied and reported independently by Krishnamachari et al. (1977) 
    and Tandon, B.N. et al. (1976, 1977).  A total population of 486 
    was affected, 67 cases were reported, of whom 28 (46%) had died.  
    There was a strong family history (Tandon, B.N. et al., 1976).  In 
    a later survey, 108 patients were studied and the mortality rate 
    was estimated to be 63% (Tandon, B.N. et al., 1977).  This time the 
    etiological factor was identified as the plant  Crotalaria nana 
    Burm, which had been growing in the fields of millet  (Panicum 
     miliare), their staple food crop.  The seeds of this plant became 
    mixed with the cereal grain during harvesting.  The toxic seeds 
    contained pyrrolizidine alkaloids that were identified as a 
    macrocyclic ester closely similar to monocrotaline.  The total 
    alkaloid content was estimated to be 5.3 g/kg of seed, expressed as 
    monocrotaline.  The levels of contamination of the millet with 
    seeds were reported to be 0 - 3.4 g/kg in the unaffected and
    0 - 19 g/kg in the affected households (Krishnamachari et al., 1977).
    A precise identification could not be made, but the same material,
    independently studied by Siddiqui et al. (1978a,b), were reported 
    to contain 2 new alkaloids, cronaburmine and crotananine.  The seed 
    contained an alkaloid level of 26 g/kg.  The levels of 
    contamination of the millet were up to 20 g/kg and the amount of 
    alkaloid ingested was estimated to be up to 40 mg per day. 

        The largest outbreak reported to date occurred in the Gulran 
    district of Herat Province in northwest Afghanistan, close to the 
    border of the USSR.  Tandon & Tandon (1975) identified the plant as 
    being causative factor of the disease, which was surveyed and 
    reported in detail by Mohabbat et al. (1976).  The outbreak, was 
    estimated to have affected a population of approximately 35 000 in 
    98 villages.  Examination of 7200 inhabitants of the affected 
    villages showed evidence of disease in 22.6%, which was more 
    serious in 15%.  Thus, it was estimated that approximately 8000 
    subjects suffered from the disease including 5000 who were 
    seriously affected.  All age groups were affected, but 46% of 
    subjects were below 14 years of age.  However, no sign of disease 
    was found in children below 2 years of age.  A detailed report 
    concerning the pathological material obtained from 14 liver 
    biopsies and 8 autopsies was made by Tandon, B.N. et al. (1978) and 
    Tandon, H.D. et al. (1978).  The time interval between the onset of 
    symptoms and the biopsy/autopsy was not indicated.  Pathological 
    findings were characteristic, ranging from acute disease with 
    characteristic veno-occlusion to non-portal cirrhosis, which was 
    observed in 5 of the above 22 cases.  The outbreak was ascribed to 
    massive contamination of wheat, the staple food crop, following 2 
    years of drought, with the seeds of  Heliotropium popovii H. Riedl 

    subsp.  gillianum H. Riedl, which had been growing profusely among
    the wheat crop.  The seeds contained pyrrolizidine alkaloids at 
    concentrations reported by 2 laboratories to be 7.2 and 13.2 -
    14.9 g/kg, identified as mainly as the  N-oxide of heliotrine (74%) 
    (Mattocks, personal communication), and one or two other compounds 
    similar in character to lasiocarpine.  Samples of wheat from 
    several villages contained an average of 40 seeds/kg, i.e., 0.03% 
    by weight.  It was estimated that an adult consumed at least 700 g 
    flour/day, containing approximately 2 mg alkaloid (based on a mean 
    of the seed analyses).  There is some uncertainty about the 
    estimate, since Mohabbat et al. (1976) also stated that the samples 
    of the wheat flour were assayed and contained 0.186 - 0.50% 
    alkaloid.  This analysis and the 0.72% result for alkaloid in the 
     H. popovii seed were from the same laboratory in Kabul (R.N. 
    Srivastava, personal communication), and together, imply 13 - 36% 
    seed in the wheat.  If correctly reported, the result for the flour 
    conflicts with the estimated proportion of the seed in the wheat 
    and can scarcely have been representative. 

        Tandon & Tandon (1975) stated in their report of the survey 
    during which the causative factor of the Afghan outbreak was 
    discovered, that such cases had always been observed by Government 
    physicians posted in this area in the past, sometimes in 
    significant numbers, but neither the population nor the physicians 
    remembered that the disease had occurred in the form of an 
    outbreak. 

        There was no mention of VOD in the hepatic lesions observed by 
    Sobin et al. (1969) among the 121 specimens of liver obtained at 
    medico-legal autopsies or by needle biopsies at Kabul, though 6.6% 
    of the 89 autopsy specimens showed "acute passive congestion with 
    necrosis".  The ages of these subjects was not stated and the 
    authors did not discuss the cause of the lesion.

        Following these outbreaks, a number of isolated cases have been 
    described following the use of herbal medicines.  McGee et al. 
    (1976) reported a case from the United Kingdom of a 26-year-old 
    woman who had consumed herbal tea containing pyrrolizidine 
    alkaloids.  The one sample examined contained only trace amounts of 
    alkaloids.  Neither the plant nor the alkaloids were further 
    characterized.  The patient had been taking very large quantities 
    of the tea for about 2 years and it is possible that some of the 
    earlier batches may have been more heavily contaminated.  Maté or 
    Paraguay tea ( Ilex species), which she was drinking, is stated to 
    be a popular drink in Brazil and is not believed to contain 
    pyrrolizidine alkaloids.  It has been stated (Huxtable, 1980) that 
    possibly she ingested the pyrrolizidine alkaloids from some other 
    unidentified source.  The clinical course of the disease progressed 
    rapidly.  Three biopsies carried out within one month and the 
    autopsy showed characteristic changes including centrizonal 
    fibrosis, but no cirrhosis.  It is notable that some involvement of 
    muscular pulmonary arteries was also seen. 

        Lyford et al. (1976) reported the case of a 35-year-old woman 
    from Ecuador, who had ingested herbal tea prepared from  Crotalaria 
     juncea.  She had consumed 1 - 2 litres of this infusion daily for 6 
    weeks, but no qualitative or quantitative analysis for 
    pyrrolizidine alkaloids was made.  She had had arthralgias for 3 
    years, for which she had received treatment with indomethacin and 
    phenylbutazone.  The liver biopsy showed characteristic changes of 
    acute VOD from which she recovered completely as proved by a repeat 
    biopsy carried out one year later. 

        The occurrence of acute disease was reported in 2 infants in 
    Arizona in the USA, aged 6 months and 2 months, respectively, 
    following ingestion of infusions of a herb, locally called the 
    Gordolobo Yerba by the Mexican-American population and identified 
    as  Senecio longilobus (Stillman et al., 1977; Fox et al., 1978; 
    Huxtable, 1980).  The plant from which this infusion had been 
    prepared was found to contain pyrrolizidine alkaloids identified as 
    riddelline and  N-oxides of retrorsine, seneciphylline, and 
    senecionine (Huxtable, 1980), in a concentration of 3 g free 
    alkaloid and 10.5 g  N-oxides/kg (Stillman et al., 1977).  It was 
    estimated that, during a period of 2 weeks, the 6-month-old infant 
    received a total dose of between 70 and 147 mg of combined alkaloid 
    and  N-oxide derivative.  The liver biopsy showed characteristic 
    features of acute disease, which had progressed to extensive 
    central, portal, and sinusoidal fibrosis (Stillman et al., 1977).  
    The child subsequently developed cirrhosis over the next 8-month 
    period.  The 2-month-old infant was administered an infusion of the 
    same herb for 4 days, after which he became progressively more ill 
    and stuporous (Fox et al., 1978).  On admission he was diagnosed as 
    a case of Reye's syndrome, but subsequently developed jaundice and 
    possibly ascites and died.  The sample of herb contained a 
    concentration of alkaloids of 15 g/kg.  It was estimated that the 
    infant had received a total of 66 mg of mixed alkaloids over the 
    4-day period.  At autopsy, extensive centrilobular haemorrhagic 
    necrosis of the liver was seen, which is characteristic of the 
    acute disease.  However, no occlusive lesions of the central vein 
    of the lobules were described, and no obstructive lesions were seen 
    in the larger hepatic veins or inferior vena cava.  No mention was 
    made of ascites. The basal ganglia showed kernicterus. 

        Datta et al. (1978 a,b) reported 6 cases that occurred between 
    1974 and 1977.  All of the patients had taken herbal medicines, 
    identified in 1 case as  Heliotropium eichwaldii, which contained 
     N-oxides of heliotrine.  Two patients took the herb as an extract 
    of the whole plant, which contained an alkaloid concentration of
    20 g/kg, for 20 and 50 days, respectively, and developed symptoms 
    after a time lag of 45 and 90 days, respectively.  They were both 
    estimated to have consumed 200 mg of heliotrine per day, the total 
    alkaloid intake being 4 g and 10 g,  respectively.  They had taken 
    the herb for treatment of epilepsy, and were admitted with acute 
    onset of symptoms of abdominal pain, ascites, jaundice, hepatic 
    encephalopathy, and gastrointestinal bleeding, which suggested 
    fulminant viral hepatitis.  They died within 2 - 12 weeks of the 
    onset of symptoms.  Only a brief description of the main autopsy 
    findings in the liver was given, which indicated that there were 

    changes characteristic of acute veno-occlusive disease of liver, 
    including marked centrilobular haemorrhagic necrosis of the liver 
    lobules, and occlusive lesions of the central and sublobular veins.  
    It is interesting to note that both patients had also been on long-
    term anticonvulsant phenobarbitone therapy.  The remaining 4 
    patients had a chronic insidious onset of disease suggesting 
    cirrhosis of the liver in 3, and alcoholic liver disease in one.  
    One of the former had been taking some indigenous powder, 
    presumably prepared from a herb, the indication and nature of which 
    are not known.  The patient died from hepatic encephalopathy.  No 
    detailed description of the autopsy findings was given, but it was 
    stated that the central and sublobular veins of the liver showed 
    chronic occlusive changes.  The inferior vena cava and large 
    hepatic veins were patent.  There was non-portal cirrhosis.  A 
    notable feature was that the arsenic levels in the liver tissue 
    were high (500 µg/kg; normal, 1 µg/kg).  There is no mention of 
    arsenic in the report on the analysis of the indigenous powder 
    taken by the patient.  Two of the remaining 3 patients with chronic 
    disease, had taken herbal medicine for vitiligo, and one for 
    diabetes mellitus.  The herb was identified as  Heliotropium 
     eichwaldii in the case of one of the vitiligo patients, who had 
    taken it for 10 days and in whom the onset of symptoms occurred 
    within 10 days.  The herb was taken in the form of seed with an 
    alkaloid content of 12 g/kg.  The daily intake was estimated to be 
    500 mg of alkaloid and the total intake, 5 g.  This patient was 
    admitted with a clinical diagnosis of cirrhosis of the liver.  The 
    liver biopsy showed changes characteristic of acute VOD.  Follow-up 
    data are not known.  The herb taken by the other 2 patients was not 
    identified.  The diabetic patient was being treated with oral 
    hypoglycaemic drugs and was also known to be an alcoholic.  The 
    indigenous powder being taken by him contained a high concentration 
    of arsenic (5 mg/kg).  The results of haemo-dynamic studies 
    suggested hepatic venous outflow tract obstruction of the 
    intrahepatic post-sinusoidal type in the smallest hepatic veins.  
    Liver biopsy showed characteristic centrilobular haemorrhages.  
    Central veins could not be recognized.  There were mild changes in 
    the liver cells, but no alcoholic hyaline was seen.  The liver 
    biopsy of the third patient showed characteristic features of acute 
    disease with veno-occlusion. 

        Two cases have been reported from China (Hou et al., 1980).  
    Both were adults who were taken ill after taking medicinal 
    infusions prepared from  Gynura segetum of the family Compositae 
    (tribe, Senecioneae).  The presenting symptoms and the cause of 
    disease, as well as the pathological findings, were characteristic, 
    except that one patient had jaundice and also portal vein 
    thrombosis, which is not a usual feature of the disease.  No 
    chemical analysis of the plant was made and the alkaloid was not 
    precisely identified.  Furthermore, the total intake of alkaloid 
    was not calculated.  It has been stated that this was the first 
    report of such a case, but that it was possible that the disease 
    might occur among adults more frequently without being reported. 

        Ghanem & Hershko (1981) reported 3 cases of Arabs, one 3-year-
    old child and 2 adults, who were diagnosed as having VOD of the 
    liver, on the basis of the clinical findings and morphological 
    features of liver biopsies.  One more patient had occlusive lesions 
    of both the small and large hepatic vein radicles.  They were among 
    29 patients with clinical features of hepatic vein thrombosis (HVT) 
    found on a retrospective analysis of data from 9 major hospitals in 
    Israel.  Of these patients, 15 were Jews and 14 were Arabs.  
    Notable features were that all Jewish patients were adults, whereas 
    the majority of Arab patients were children below 10 years of age 
    and primary HVT was 2.4 times more common among the latter.  No 
    analysis of the diet was made for PAs and their etiological role 
    was suspected only on a presumptive basis.  A survey of stored 
    wheat grain in 9 villages showed that 2 samples were contaminated 
    with seeds of  Lolium, belonging to the Graminae family, which were 
    found to contain 2 PAs (loline and norloline).  However, these PAs 
    are not known to be hepatotoxic.  The authors argued that, even 
    though in a classical case of VOD there should not be thrombosis of 
    the larger hepatic vein radicles, the difference in the anatomical 
    appearance of VOD and that of primary HVT of the near-east type is 
    not due to a different etiological agent but rather to a difference 
    in the dose and rate of absorption of the ingested toxic compounds. 

        A further report of the disease from Hong Kong, by Kumana et 
    al. (1985), described it in 4 young Chinese women with psoriasis 
    who took infusions of a herbal remedy, the toxic component of which 
    has since been identified as  Heliotropium lasiocarpum (Culvenor et 
    al., 1986).  They developed symptoms 19 - 45 days after starting 
    the herbal treatment, and were examined 61 - 68 days after its 
    initiation.  The condition of patient No. 2, who continued taking 
    the herb for 16 days after the onset of symptoms, deteriorated and 
    she died of hepatic failure and was autopsied.  The liver biopsies 
    and autopsy confirmed the presence of acute disease in all 
    patients.  Patient No. 4 stopped taking the herb after 21 days, on 
    account of a new rash.  When assessed 77 days later, she had mild 
    hepatomegaly only.  A detailed analysis of the alkaloid content was 
    carried out for each case.  The pyrrolizidine alkaloids were 
    quantified as if senecionine based.  The herb contained 0.42 g 
    alkaloid/kg and 1.4 g  N-oxide/kg.  The daily intakes of alkaloid 
    base and  N-oxide were estimated to be 12 ± 1 mg and 18 ± 4 mg, 
    respectively.  The respective cumulative doses of alkaloid (base 
    and  N-oxide) consumed by patients Nos 1, 2, and 3, up to onset of 
    symptoms, were calculated to be 1350 mg over 45 days, 900 mg over 
    30 days, and 570 over 19 days, respectively.  Patient No. 4 who had 
    irrefutable histological evidence of disease but did not develop 
    symptomatic disease, must have consumed 630 mg over 21 days. 
    Patient No. 2, who died, was estimated to have taken a total amount 
    of 1380 mg alkaloid over 46 days.  The estimated cumulative intake 
    per kg body weight before the development of symptoms for patients 
    Nos. 1, 2, 3, and 4 was 26, 15, 12, and 15 mg/kg, respectively.  It 
    should be noted that, in patient No. 2, who died, the cumulative 
    dose until the onset of symptoms was the same as in patient No. 4, 
    who was asymptomatic.  Moreover, the total intake by patient No. 2, 
    was 23 mg/kg, which was lower than the intake by patient No. 1, who 
    survived.  The authors compared the intake data of their patients

    with those of the Arizona children reported by Stillman et al. 
    (1977) and Fox et al. (1978).  The 6-month-old baby, who survived 
    but developed cirrhosis, and the 2-month-old baby, who died, are 
    estimated by comparison to have taken cumulative doses of 12 - 25 
    and 11 mg/kg, respectively.  The above data suggest marked 
    variation in susceptibility among individual subjects.  It is also 
    known from experimental animal studies that the young and new-born 
    animals are particularly vulnerable (Jago, 1970). 

        A case reported from the USA is ascribed to the consumption of 
    comfrey ( Symphytum sp.) powder, sold as a digestive aid (Ridker et 
    al., 1985).  A 49-year-old woman presented with classical symptoms 
    and signs of VOD.  The haemodynamic data showed a hepatic vein 
    wedge pressure of 3.07 kPa (23 mmHg) with a sinusoidal pressure of 
    2.27 kPa (17 mmHg).  Hepatic venograms showed near obliteration of 
    the smaller radicles of the hepatic veins during balloon distension 
    of one of the intrahepatic venous tributaries, and there was extra-
    vasation of the dye into the hepatic parenchyma.  A porta-caval 
    shunt was carried out and the operative findings confirmed the 
    presence of a post-sinusoidal block.  The liver biopsy showed 
    marked centrilobular necrosis and congestion with dilatation of the 
    central veins and sinusoids, consistent with hepatic venous outflow 
    tract obstruction.  According to the clinical history, the patient 
    had been a heavy consumer of food supplements.  Apart from several 
    vitamins and minerals, she had been drinking 3 cups of camomile tea 
    per week and for 6 months before admission had consumed 1 g/day of 
    a commercially available herbal tea.  For 4 months before 
    admission, she had taken 2 capsules of "comfrey-pepsin" pills with 
    each meal.  The herbal tea and the pills were analysed for PAs.  
    Pyrrolizidine alkaloids and their  N-oxides were found, but the 
    compounds were not precisely identified.  On the basis of the 
    analysis of the PA content, the patient was estimated to have 
    consumed a total of at least 85 mg of PA (Huxtable et al., 1986) 
    (14.1 µg/kg body weight per day, Ridker et al., 1985).  The authors 
    emphasized that the total PA consumption was relatively low.  It 
    was possible that the patient had other sources of exposure and 
    probably she had been consuming PA-containing supplements for 
    longer than the periods stated by her in the clinical history. 

        The latest is the report of a 13 year old boy from the U.K. who 
    is stated to have developed symptoms of toxicity following 
    administration of herbal tea prepared from comfrey leaf (Symphytum 
    officinale) for treatment of inflammatory bowel disease for two or 
    three years (Weston et al., 1987).  The exact quantity of leaves 
    consumed and frequency of administration were not known.  The liver 
    biopsy is stated to have shown a "thrombotic variant" of veno-
    occlusive disease, though the inferior vena cava and the major 
    hepatic veins were patent on Doppler ultrasound and percutaneous 
    phlebography.  He had earlier been treated with predinisolone 
    and sulphasalazine.  The case is unusual in so far as the 
    thrombosis of the central veins of the liver lobules, which is not 
    a usual feature of veno-occlusive disease of the liver. 

    7.4  VOD and Cirrhosis of the Liver

        Jelliffe et al. (1954b) were the first to draw attention to VOD 
    being an important cause of cirrhosis among Jamaican children.  
    Prior to this, Hashem (1939), while reviewing the records of all 
    cases of cirrhosis admitted to a children's hospital in Egypt since 
    1933, described 3 cases of a special type of cirrhosis that was 
    rare in adults.  The clinical features and pathological findings 
    were similar to those in cases of VOD.  They speculated that the 
    cause was some metabolic toxins of gastrointestinal origin.  Royes 
    (1948) suggested that the cirrhosis in the Jamaican children was 
    very like the disease described from India and Egypt. 

        The clinical and pathological features of 100 cases of VOD 
    among Jamaican children were described by Bras et al. (1954). 
    Sixty-five of the cases were below 12 years of age.  None of the 
    100 patients had cirrhosis initially, but 5 showed occlusive 
    lesions in hepatic veins and features of non-portal fibrosis.  Four 
    of these 5 cases later developed cirrhosis.  The authors concluded 
    that VOD contributed to a substantial number of all cases of 
    cirrhosis in this age group.  Stuart & Bras (1957) studied 84 
    patients with VOD including 64 acute, 6 subacute, and 14 chronic 
    cases.  Twenty-three patients were followed up, some for up to 5 
    years.  Autopsy performed on 21/26 cases showed cirrhosis in 11 
    cases.  Notable features were that 1 of the 6 cases of acute 
    disease described in detail, developed cirrhosis.  Of the 2 cases 
    with chronic disease, 1 developed cirrhosis within 3 months of a 
    liver biopsy for acute disease, at which time the liver had shown 
    hepatic venous occlusive lesions but no fibrosis.  Autopsy findings 
    were described by Bras & Watler (1955) in 19 patients aged 10 
    months - 45 years in different stages of the disease.  Nine 
    patients had cirrhosis that was non-portal to begin with but 
    finally became indistinguishable from Laennec's portal cirrhosis.  
    Rhodes (1957) studied the pattern of liver disease among Jamaican 
    children.  A total of 193 liver biopsies was studied derived from 
    39 children who had one biopsy and 59 who had more than one at 
    intervals of 1 week - 3 years.  Of the 14 autopsies on cases of 
    VOD, 12 had cirrhosis.  A notable feature was that the disease 
    could occur asymptomatically with hepatomegaly.  The autopsy 
    material from the University College Hospital, Jamaica was analysed 
    by Bras et al. (1961).  Of the 1560 autopsies, 28.5% cases 
    concerned infants of less than one year old, mostly from poor, 
    predominantly black families.  Cirrhosis was seen in 77 autopsies.  
    Approximately 30% of these 77 cases were diagnosed as post-VOD.  
    The authors postulated that they might have resulted from the 
    ingestion of  Crotalaria fulva or some other toxic substances. 

        In a follow-up study of 61 patients who developed the disease 
    in an outbreak in the USSR in 1958, 28 developed "Hepatoleinal 
    syndrome" (Braginski & Bobokhadzaev, 1965).  Two of these cases, in 
    whom the disease in the initial stages was not particularly severe, 
    developed cirrhosis within 4 years.

        Tandon, H.D. (personal communication) has analysed the 
    pathological data derived from the Afghan and Indian outbreaks on 
    the basis of 61 liver biopsies and 17 autopsies, including repeat 
    biopsy studies on 15 patients who were followed-up for 1 month - 3 
    years after onset in the Indian outbreaks.  Three of the 11 
    patients, followed up for 16 months or longer for persistent 
    clinical evidence of disease, ended up with cirrhosis and 2 more 
    had marked fibrosis with equivocal changes of cirrhosis in the 
    biopsy.  Notable features of the study were that the disease 
    progressed to cirrhosis in patients who were put on a normal diet, 
    free from alkaloids, after appearance of symptoms of acute disease 
    and did not receive any subsequent exposure.  Impact of alcohol 
    intake was excluded.  There was a poor correlation between the 
    clinical and pathological severity of disease.  Centrilobular 
    haemorrhages, which are a sign of acute disease, were seen to 
    persist for over one year in patients, some of whom were apparently 
    well.  At needle biopsy, characteristic hepatic venous occlusions 
    were not seen in many patients in the acute phase of the disease, 
    though they were seen in all livers at autopsy and most of them 
    showed persistent centrilobular haemorrhages.  Biopsy findings in 
    cirrhotic livers were often not histopathologically characteristic 
    for any specific form of cirrhosis.  Features that might suggest 
    the veno-occlusive etiology of cirrhosis at biopsy included 
    paraseptal dilatation of sinusoids and persistent haemorrhages or 
    haemosiderin in the septa. 

        In studies by Aikat et al. (1978) and Datta et al. (1978a,b), 6 
    cases of VOD were reported following ingestion of herbal medicines 
    containing PAs.  Four of the patients had symptoms of chronic 
    disease and one of them developed non-portal cirrhosis. 

        One of the 2 infants from Arizona, USA, who suffered from VOD 
    following the administration of PA-containing herbal medicine, 
    developed cirrhosis during the 8-month period following the 
    appearance of acute symptoms (Stillman et al., 1977; Fox et al., 
    1978; Huxtable, 1980).  Huxtable (1980) mentioned the death from 
    cirrhosis of liver of a 62-year-old woman, who had consumed the 
    same herb as the 2 infants for 6 months prior to her death.  
    However, there was no confirmation of the diagnosis of cirrhosis. 

    7.5  Differences Between VOD and Indian Childhood Cirrhosis (ICC)

        A type of cirrhosis of liver, peculiar to the people of Indian 
    origin, Indian Childhood Cirrhosis (ICC), has been ascribed to PA 
    toxic etiology (Bras et al., 1954; Rhodes, 1957), owing to the 
    observation of occlusive changes in the central and sublobular 
    veins of the liver, by some investigators (Radhakrishna Rao, 1935; 
    Prabhu, 1940; Jelliffe et al., 1957; Ramalingaswami & Nayak, 1969), 
    though this is not a characteristic feature of the disease.  The 
    presence of copper-positive granules in the hepatocytes (Salaspuro 
    & Sipponen, 1976) has added to such a conjecture (Tanner & 
    Portmann, 1981), because of the reported aberration of copper 
    metabolism in experimental animals exposed to PAs (section 6.4.11).  
    However, ICC and VOD are clinically and pathologically distinct.  
    ICC, confined to infants and children, often affects siblings.  

    Jaundice is a common sign and hepatosplenomegaly is a common 
    feature.  The disease is almost invariably fatal due to rapidly 
    developing hepatocellular failure.  Liver parenchymal changes are 
    characterized by marked ballooning degeneration of hepatocytes, 
    prominent deposits of alcoholic hyaline, severe cholestasis, and 
    aggressive, pericellular fibrosis (Nayak et al., 1969), all 
    features not characteristic of VOD.  Moreover, occlusive changes in 
    the hepatic veins are very rare and were not observed by any member 
    of the liver diseases subcommittee of the Indian Council of Medical 
    Research (1955), who made a critical study of the disease. 

    7.6  Chronic Lung Disease

        Heath et al. (1975) reported the case of a 19-year-old African 
    man who had died of congestive cardiac failure, and who was 
    suspected of having ingested a herbal remedy containing the seeds 
    of  Crotalaria laburnoides.  Histopathological examination of the 
    lungs showed characteristic vascular changes of severe primary 
    pulmonary hypertension.  Powdered seeds of the plant were fed in 
    the diet to Wistar albino rats for 60 days (Table 11).  
    Characteristic features of pulmonary hypertension including 
    hypertensive vascular changes in the lung and right ventricular 
    hypertrophy of the heart were produced in the animals showing that 
    the seeds contained an agent capable of inducing pulmonary 
    hypertension in rats.  Apart from this indirect evidence, there was 
    no proof of such a causal relationship with the pulmonary 
    hypertensive disease in the patient. 

        A brief mention is made by McGee et al. (1976) of finding 
    changes "somewhat similar but rather more mature" (than those seen 
    in the hepatic veins) involving some branches of the pulmonary 
    artery in the lower lobe of the left lung, in a case of veno-
    occlusive disease of the liver caused by ingestion of PA-containing 
    herbal teas.  The alkaloids were not further characterized.  The 
    changes were also stated to be similar to those produced in 
    experimental animals by PAs.  Apart from these 2 cases, there is no 
    mention of involvement of the pulmonary arterial system in any of 
    the case reports available.

        The possibility that diet-mediated agents might induce 
    pulmonary hypertension in man has been discussed at length by 
    Fishman (1974).  A parallel was drawn with the epidemic of 
    pulmonary hypertension that occurred in Austria, the Federal 
    Republic of Germany, and Switzerland between 1966 and 1968 (Kay et 
    al., 1971b), which was suspected of being caused by Aminorex, a 
    compound that resembles epinephrine and amphetamine in chemical 
    structure.  Although the etiological role of Aminorex could not be 
    conclusively proved on epidemiological or experimental grounds, 
    there continues to be a suspicion that agents taken by mouth can 
    evoke pulmonary hypertension in man.  Levine et al. (1973) reported 
    the cases of 3 children aged 5 1/2 - 13 years, and 11 months, 
    respectively, with portal hypertension, who developed progressive 
    pulmonary hypertension resulting in cor pulmonale and death.  In 
    all 3 cases, there was evidence of extra-hepatic portal vein 
    obstruction confirmed at autopsy, and the symptoms and signs of 

    portal obstruction had appeared in early childhood.  They developed 
    symptoms of cardio-respiratory failure.  Studies of pulmonary and 
    cardiovascular function including haemodynamic studies of pulmonary 
    circulation in 2 of the children suggested pulmonary vascular 
    obstructive disease.  At autopsy, no primary parenchymal lung 
    disease was found.  There were vascular changes of advanced 
    pulmonary hypertension (plexiform lesions), but no evidence of 
    thromboembolism was found.  No factor responsible for pulmonary 
    vasoconstriction was identified.  In one case, the liver was stated 
    to show coarse nodularity at autopsy, but the microscopic 
    examination showed only patchy areas of portal fibrosis and 
    regeneration.  In the other 2 cases, there were only non-specific 
    changes, with fibrosis in one.  However, centrilobular congestion 
    was present in 2 cases.  It is possible that some metabolites of 
    toxic agents, such as PAs, which are metabolized in the liver, 
    might have blocked a metabolic pathway that ordinarily exerts a 
    pulmonary antihypertensive effect, or that, by damaging the liver, 
    vasoactive substances, such as histamine, serotonin, and 
    catecholamines, might escape metabolic pathways to reach the lungs 
    and injure the pulmonary vessels.  However, no such agents were 
    identified. 

        Kay et al. (1971b) made a plea that, on the basis of the 
    experimental data available, including the ability of several 
    agents to produce pulmonary hypertension in experimental animals, 
    and, in spite of the fact that pulmonary vascular disease has never 
    been demonstrated in human cases of veno-occlusive disease, careful 
    enquiries should be made on the possibility of patients presenting 
    with unexplained pulmonary hypertension having ingested a plant 
    product.  A similar plea was made by Heath et al. (1975). 

    7.7  Trichodesma Poisoning

        The disease "Ozhalanger encephalitis", which occurred in the 
    Samarkand region of Uzbekistan, USSR in the period 1942 - 51, is 
    believed to have been caused by contamination of food grain with 
    the seeds of  Trichodesma incanum (Shtenberg & Orlova, 1955; 
    Ismailov et al., 1970), which contain 1.5 - 3.1% alkaloids, mainly 
    trichodesmine and incanine (Yunusov & Plekhanova, 1959).  Clinical 
    features of the disease have been described by Ismailov et al. 
    (1970).  This outbreak differed from the others described above in 
    that the primary symptomatology was extra-hepatic.  Over 200 
    patients were affected, not including children below the age of 10, 
    or the breast-fed infants of the affected mothers.  Following 
    exposure, there was a latent period of about 10 days, then vertigo 
    and recurring headaches developed leading to nausea and vomiting.  
    This was followed by generalized malaise, which progressed to 
    delirium and loss of consciousness.  Physical signs included 
    pathological reflexes in 59% of the patients and paresis of the 
    extremities and the facial nerve.  Death was stated to have been 
    caused mostly by respiratory depression.  Of the 200 patients 
    affected, 44 died.  Autopsy findings were relatively non-specific 
    degenerative and necrotic lesions scattered in several organs 
    including the central nervous system.  No report of such a disease 
    is available from outside the USSR.

    7.8  Relationship Between Dose Level and Toxic Effects

        In some recent human case reports, the PAs consumed have ben 
    identified and estimates made of the daily and total intakes (Table 
    16).  The relationship between these intake levels and the known 
    toxicity of the alkaloids in rats has been discussed by Culvenor 
    (1983) and Mattocks (1986). 

        Discussion of the relationship between the dose level and toxic 
    effects in human cases is complicated, because the poisoning is 
    generally due to a mixture of alkaloids found in naturally-
    occurring plant products, consumed as herbal remedies or food, and
    different plant species.  There are wide differences in the acute 
    toxicities of the alkaloids, which are the best available measure 
    of comparative effects due to long-term intake as well.  
    Furthermore, estimates of intake can, at best, be approximate.  
    When a large population is affected through the contamination of a 
    food crop, as happened in the Afghan and Indian outbreaks (section 
    7.3), no precise estimates are possible regarding the extent of 
    contamination in different households, the amount of the 
    contaminated grain consumed, the length of exposure resulting in 
    the appearance of symptoms or signs of toxicity, or death, in the 
    cases studied.  There may also be various other contributing 
    factors that are not apparent, e.g., food or cooking habits, on 
    account of which no conclusive generalizations regarding the 
    causative role of the toxic agent can be made.  Reports on chemical 
    analysis for the toxic agent and, hence, the amounts ingested, may 
    not always be reliable (refer to the controversy in the Afghan 
    outbreak in section 7.3).  In cases, such as the one reported by 
    Ridker et al. (1985), when the total dose of PAs estimated to have 
    been received by the patient before the disease developed, was only 
    a fraction of that of other alkaloids causing episodes of human 
    toxicity (Table 16), it is not certain whether that was the only 
    toxic alkaloid agent that the patient was exposed to, or whether 
    there were other contributing factors, particularly as the patient 
    was stated to be a heavy consumer of herbs, vitamins, and natural 
    food supplements. 

        In the known instances of human toxicity the principal 
    alkaloids involved are heliotrine from  Heliotropium, echimidine and 
    related alkaloids from  Symphytum, riddelline and retrosine from 
     Senecio longilobus, and crotananine and cronaburmine from 
     Crotalaria nana (Table 16).  Approximate acute toxicity values 
    (LD50 in rats) for these alkaloid mixtures are 300, 500, and
    50 mg/kg, respectively.  For the mixture from  C. nana, for which
    there are no experimental data, the acute toxicity was assumed to 
    be similar to that of monocrotaline, 100 mg/kg.  These relativities 
    need to be taken into account in discussing dose-effect 
    relationships for the PAs as a group.  This has been done by 
    discussing first the dose estimates for heliotrine, since it was 
    the main alkaloid in 1 epidemic and in 6 case reports (Table 16).  
    Then in discussing the other alkaloids, reference is made to a 
    heliotrine-equivalent dose as well as the actual dose.  The 

    heliotrine equivalent is: 

                             LD50 of heliotrine
        actual dose x ----------------------------------
                      LD50 of alkaloid mixture concerned

    The estimated daily intake in poisoning by heliotrine ranges from 
    0.033 mg/kg body weight in the Afghan epidemic (Mohabbat et al., 
    1976), which after a period of about 180 days and a total intake of 
    about 6 mg/kg caused fatalities, to 3.3 mg/kg, which led, in 2 
    cases in India (Datta et al., 1978a,b), to death after 20 and 50 
    days and total intakes of 67 and 167 mg/kg, respectively.  In 
    between are 4 cases in Hong Kong with estimated daily intakes 
    ranging from 0.49 to 0.71 mg/kg (Kumana et al., 1983, 1985; 
    Culvenor et al., 1986).  Three cases were non-fatal at total doses 
    of 11 - 27 mg/kg body weight and one was fatal at a total dose of 
    23 mg/kg.  These heliotrine cases imply that daily intakes are 
    cumulative down to 0.033 mg/kg and may be fairly rapidly fatal 
    above 0.5 mg/kg.  Above a total dose of 6 - 15 mg/kg, VOD may 
    become evident and sometimes fatal. 

        In the 2 cases due to riddelline and retrorsine in  Senecio 
     longilobus (Stillman et al., 1977; Fox et al., 1978; Huxtable, 
    1980), the estimated daily intakes were 0.8 - 0.17 and 3 mg/kg body 
    weight (equivalent to 3 and 1 mg heliotrine/kg, respectively, and 
    the total doses were 12 - 25 and 12 mg/kg (equivalent to 72 - 150 
    and 72 mg heliotrine/kg, respectively).  These levels are 
    comparable with the highest reported intakes of heliotrine and, in 
    infants, led to the rapid development of VOD and, in one case, 
    death. 

        In the epidemic due to mixed crotananine and cronaburmine in 
     Crotalaria nana (Tandon, B.N. et al., 1976; Tandon, R.K. et al., 
    1976; Krishnamachari et al., 1977), the estimated daily intake of 
    0.66 mg/kg and the total intake of 40 mg/kg (equivalent to 2 and 
    120 mg heliotrine/kg, respectively) also corresponded to the 
    highest intake of heliotrine. 

        In the case of  Symphytum poisoning (Ridker et al., 1985), the 
    estimated daily intake of echimidine and related alkaloids was 
    0.015 mg/kg and the total dose 1.7 mg/kg (equivalent to 0.009 and 
    1.0 mg/kg heliotrine, respectively).  The dose levels are lower in 
    equivalent terms than the lowest estimates in cases due to 
    heliotrine by a factor of about 4 for daily intake and 6 for total 
    intake.  The estimates were based on questioning of the patient and 
    assay of the material concerned.  It seems prudent to conclude that 
    a daily intake of pyrrolizidine alkaloid as low as the equivalent 
    of 0.01 mg/kg heliotrine may lead to disease in humans. 


        Table 16.  Estimated intakes of PAs in human beings
    -----------------------------------------------------------------------------------------
    Principal        Age of    Daily     Period     Total dose    Toxic    Reference
    alkaloids(s)a    subject   intake    (days)    mg      mg/kg  effectc
                               (years)   (mg/kg)b
    -----------------------------------------------------------------------------------------
    1. Heliotrine    variousd  0.033     180e      360     6e     VOD,     Mohabbat et al. 
                                                                  death    (1976)

    2. Heliotrine    (a) 20    3.3       20        4000    67     death    Datta et al. 
                     (b) 23    3.3       50        10000   167    death    (1978a,b)

    3. Riddelline,   0.5       0.8-1.7b  14        70-147  12-25  VOD      Stillman et al. 
       retrorsine                                                          (1977); Huxtable 
                                                                           (1980)

    4. Riddelline,   0.17      3.0b      4         66      12     death    Fox et al. (1978)
       retrorsine

    5. Crotananine,  variousd  0.66      c. 60e    2400    40     VOD,     Tandon, B.N. et al.
       crotaburmine                                               death    (1976); Tandon, 
                                                                           R.K. et al. (1976);
                                                                           Krishnamachari et 
                                                                           al. (1977)

    6. Heliotrine    (a) 28    0.59      45        1350    27     VOD      Kumana et al. 
                     (b) 26    0.49      46        1380    23     death    (1983, 1985); 
                     (c) 23    0.60      19        570     11     VOD      Culvenor et al. 
                     (d) 27    0.71      21        630     15     VOD      (1986)

    7. Echimidine    49        0.015     120       94      1.7    VOD      Ridker et al. 
                                                                           (1985)
    -----------------------------------------------------------------------------------------
    a The principal alkaloid is recorded as the free base, even if there was evidence of its 
      presence in the plant mainly as  N-oxide.
    b Calculated for a 60-kg adult, unless definite information available.  The 0.5-year 
      infant was said to be 6 kg, and
    c When mentioned, death was a consequence of severe liver damage.
    d Epidemic.
    e Estimate based on unpublished information available to the Task Group.
            There is substantial overlap between intake rates and total 
    intakes for fatal and non-fatal poisoning.  This presumably 
    reflects the influences of a number of factors, such as individual 
    sensitivity, age, nutritional status, and general health, but it is 
    also due to the progressive nature of pyrrolizidine toxicity and 
    the effects of time.  In the epidemics, in which some people died, 
    only an estimated average intake is available and some who were 
    alive at the time of investigation may have died later.  Comparing 
    the total intakes for human toxicity with the total doses up to 
    death observed in the long-term administration of PAs to rats, 
    1.2 - 10.9 times the LD50 dose, equivalent to 360 - 3270 mg 
    heliotrine/kg (Table 10, section 6.4.1.5), it is evident that human 
    beings are more susceptible to the acute and chronic effects of the 
    alkaloids than rats, sometimes markedly so. 

        These considerations of the toxic effects in human beings of 
    various intake levels could provide a basis for some assessment of 
    the likely hazard from other types of exposure to PAs.  For 
    example, the consumption of comfrey root tea, estimated by Roitman 
    (1981) to contain 8.5 mg alkaloid per cup, at the rate of 3 cups 
    per day, or the ingestion of comfrey leaf at the rate of one leaf 
    per day, could lead to alkaloid ingestion rates of 0.40 and 
    0.016 mg/kg.  These rates are respectively, much greater than, and
    equal to, the lowest daily rate causing veno-occlusive disease.  Lower
    levels of exposure arising from such sources as the consumption of 
    milk from cows eating PA-containing plants or of honey derived from 
    such plants, seems unlikely, in practice, to cause acute or 
    subacute liver disease.  However, care should be exercised. In an 
    experimental situation in which cows were fed  Senecio jacobaea,  
    the milk was reported to contain up to 0.84 mg alkaloid/litre.  A 
    30-kg child drinking 0.5 litre/day of this milk could ingest
    0.014 mg/kg alkaloid, equivalent to 0.028 mg heliotrine/kg (assuming 
    an LD50 of 150 mg/kg for  S. jacobaea alkaloid).  This is above the
    lowest daily rate leading to veno-occlusive disease and the lowest 
    estimated total toxic dose would be achieved in 36 days.  This 
    level of contamination of milk is undoubtedly extreme and there is 
    no knowledge of any contamination of commercial milk supplies. 
    Honey derived from  Echium plantagineum has been reported to contain 
    up to 1 mg alkaloid/kg (Culvenor et al., 1981).  A 30-kg child 
    consuming 30 g/day of honey (a high consumption rate) would ingest 
    0.001 mg alkaloid/kg body weight.  The lowest estimated total toxic 
    dose (1.7 mg comfrey alkaloid/kg, very similar to  Echium alkaloid) 
    would be achieved in 1700 days.  Although it seems likely that 
    consumption of contaminated milk and honey would lead to acute or 
    subacute liver disease, the possibility remains that they may 
    contribute to chronic liver disease or liver tumours. 

        The possibility of carcinogenic effects due to long-term 
    exposure to PA-containing plants has been discussed by Culvenor 
    (1983).  Some of the PAs involved in instances of human poisoning 
    have been found to be carcinogenic in experimental animals (Table 
    13).  Data from some of the significant experimental studies were 
    summarized by Culvenor (1983) with approximate estimates of PA 
    dosages administered to rats in terms of mg/kg body weight per day 
    (Table 17).  The dose rates that were carcinogenic for rats (Table 
    17) ranged from 2 to 6 mg/kg per day for an initial period and 
    0.2 - 3 mg/kg per day for a remaining period of about 12 months, 
    except in one study in which a dose of 10 mg/kg per day was used.  
    It can be seen that, in all except two instances of human 
    poisonings summarized in Table 16, the estimated daily rates of 
    intake ranging from 0.015 to 3.3 mg/kg body weight per day are 
    within close range of those known to induce tumours in rats.  In 
    other reports, the consumption rates are above and below this 
    range. 

        Epidemiological studies to assess the carcinogenic role of PAs 
    for man are not available.  In countries with a high incidence of 
    primary liver cancer, it is possible that PAs may have an additive 
    effect with those attributed to aflatoxin (Newberne & Rogers, 1973) 
    and hepatitis B virus.  The total evidence now available warrants 

    long-term studies of the survivors of poisoning outbreaks, 
    especially where a substantial number of people were affected, as 
    in the Afghanistan outbreak. 

    7.9  Pyrrolizidine Alkaloids as a Chemotherapeutic Agent for Cancer

        The PA, indicine  N-oxide derived from  Heliotropium indicum, a 
    widely used indigenous drug in Ayurvedic medicine, has been found 
    to have an antitumour activity and has been used in clinical trials 
    as a chemotherapeutic agent for leukaemia (Letendre et al., 1981, 
    1984; Cook et al., 1983) and solid tumours (Kovach et al., 1979a,b; 
    Nichols et al., 1981; Ohnuma et al., 1982; Taylor et al., 1983).  
    Dosing schedules typically were 5 consecutive intravenous doses of 
    0.15 - 3 g/m2 body surface area (approximately 2.5 - 5 mg/kg body 
    weight) repeated at 4- or 6-week intervals (Kovach et al., 1979a; 
    Letendre et al., 1981; Ohnuma et al., 1982).  Hepatic toxicity, as 
    judged by increased SGOP levels, was infrequent and mild.  However, 
    subsequent trials with this agent have indicated more serious 
    hepatotoxicity.  In a more recent report by the same workers 
    (Letendre et al., 1984), 5 out of 22 cases of refractory acute 
    leukaemia, treated with indicine  N-oxide, had severe 
    hepatotoxicity, presumably induced by the drug.  One of these 
    patients had been treated for 18 months with methyl-testosterone, 4 
    months prior to receiving indicine  N-oxide.  Symptoms of severe 
    hepatocellular failure appeared in 3 patients after the initial 
    course of treatment.  This occurred after 4 daily doses of 3 g/m2 
    surface area in one patient and after 5 daily doses of 3.75 g/m2 
    surface area in 2 patients.  Two other patients who had received 
    3 g/m2 surface area daily for 5 days developed hepatocellular failure
    after the second course of treatment, one at 3.3 g/m2 and the other 
    at 3.75 g/m2 surface area, daily, for 5 days.  In each patient, the 
    onset of hepatic disease was rapid and the course was downhill.  
    Livers of 4 of these patients examined at post-mortem showed severe 
    centrilobular vascular congestion with necrosis of parenchymal 
    cells, and, in one patient, a few sublobular veins were found to be 
    occluded.

        Miser et al. (1982) reported severe hepatotoxicity in 4 of 45 
    children treated with indicine  N-oxide for refractory leukaemia or 
    advanced solid tumours.  Similarly, Cook et al. (1983) reported the 
    case of a 5-year-old child with acute myeloid leukaemia who 
    developed severe hepatic failure within 3 days of starting the 
    treatment.  Autopsy showed massive hepatic necrosis.  

        However, it should be noted that no hepatic failure was 
    reported in more than 100 adults with solid tumours, treated with 
    the same agent (Kovach et al., 1979a,b; Nichols et al., 1981; 
    Taylor et al., 1983).  No hepatotoxic effects were reported by 
    Ohnuma et al. (1982) among 37 patients who received this drug for 
    solid tumours.  The major toxic effect was myelosuppression (Kovach 
    et al., 1979b). 

        Table 17.  Rates of administration of PAs leading to tumours in ratsa
    ------------------------------------------------------------------------------------------
    Alkaloid       Dosing schedule                Approximate       Number of   Reference
                                                  equivalent rateb  rats
                                                  (mg/kg per day)   developing
                                                                    tumours
    ------------------------------------------------------------------------------------------
    Lasiocarpine   (a) 7 mg/kg diet, 24 months    0.70              23/24       Nat. Cancer 
                                                                                Institute  
                                                                                (1978)

                   (b) 15 mg/kg diet, 24 months   1.50              24/24       Nat. Cancer 
                                                                                Institute 
                                                                                (1978)

                   (c) 7.8 mg/kg, ip, 2/week for  2.2 for 4 weeks,  16/18       Svoboda & 
                       4 weeks and 1/week for     then 1.1 for                  Reddy (1972)
                       52 weeks                   52 weeks

                   (d) 50 mg/kg diet, 55 weeks    5.0               18/20       Rao & Reddy 
                                                                                (1978)

                   (e) 0.39 mg/kg, ip, 3/week,    0.2               2/7         Culvenor & 
                       to death                                                 Jago (1979)

    Monocrotaline  (a) 25 mg/kg, ip, 1/week for   3.5 for 4 weeks,  10/50       Newberne &  
                       4 weeks, and 8 mg/kg,      then 1.1 for                  Rogers (1973)
                       for 38 weeks               38 weeks

                   (b) 5 mg/kg sc, once per       0.36              43/60       Shumaker et 
                       2 weeks for 52 weeks                                     al. (1976)

    Retrorsine     (a) 30 mg/kg, ip, single dose  -                 7/29        Schoental & 
                                                                                Bensted (1963)
                   (b) 30 mg/litre in water,      1.3               4/14        Schoental et 
                       3 days/week, to death                                    al. (1954)

                   (c) 30 - 50 mg  N-oxide/litre   1.3 - 2.0         10/22       Schoental et 
                       in water, 3 days/week                                    al. (1954)
                       for 20 months
    ------------------------------------------------------------------------------------------

    Table 17.  (contd)
    ------------------------------------------------------------------------------------------
    Alkaloid       Dosing schedule                Approximate       Number of   Reference
                                                  equivalent rateb  rats
                                                  (mg/kg per day)   developing
                                                                    tumours
    ------------------------------------------------------------------------------------------

    Petasitenine   0.1 g/litre in water,          10                8/10        Hirono et 
                   up to 16 months                                              al. (1977)

    Senkirkine     22 mg/kg, ip, 2/week for       6 for 4 weeks,    11/24       Hirono et 
                   4 weeks and 1/week for         then 3 for                    al. (1979a)
                   52 weeks                       25 weeks

    Symphytine     13 mg/kg, ip, 2/week for       3.7 for 4 weeks,  5/24        Hirono et 
                   4 weeks and 1/week for         then 1.9 for                  al. (1979a)
                   52 weeks                       52 weeks
    ------------------------------------------------------------------------------------------
    a From:  Culvenor (1983).
    b Where necessary, estimates assume a daily rat food intake of 100 g/kg body weight, and 
      water intake 100 ml/kg body weight.  Injected doses are given  pro rata, for daily 
      administration.
    
    7.10  Prevention of Poisoning in Man

        At present, prevention of poisoning can be achieved only by 
    reducing or eliminating ingestion of the alkaloids.  The two 
    effective procedures are control of PA-containing plants in 
    agricultural areas and educational programmes directed to the 
    populations at risk. 

        The control of plant populations for this purpose has been 
    carried out only in Uzbekistan, USSR, following the epidemics of 
    human disease due to contamination of grain by seeds of 
     Heliotropium lasiocarpum and  Trichodesma incanum.  The following 
    measures were introduced and have been effective in preventing 
    further outbreaks: 

    1.  A state standard was set for the quality of seed grain, which 
        must be certified by a State Seed Inspectorate.  Current 
        standards prohibit the sowing of wheat, rye, barley, or oats 
        contaminated by seed of  Heliotropium lasiocarpum or  Trichodesma 
         incanum. 

    2.  A state standard was set for the quality of grain stored for 
        food.  The limits for  Heliotropium lasiocarpum and 
         Trichodesma incanum seeds are 0.2% and zero, respectively. 

    3.  Agricultural (agritechnical) measures to ensure minimum
        contamination of crops and harvested grain, including
        specification of the most suitable methods and timing of
        cultivation, use of clean seed for sowing, weeding of crops 
        prior to maturing of the grain (towards the end of May), and 
        mechanical cleaning of grain. 

    4.  Methods for monitoring levels of contamination of flour, bread, 
        and similar products. 

    5.  Publication of educational booklets describing the biological, 
        environmental, and morphological characteristics of the toxic 
        weeds, their pathways of distribution and the causes of the 
        toxicoses experienced. 

    6.  Promotion of weed control by governmental authorities and
        provision of legislation to enforce the control measures.

        In other countries, the control of some PA-containing weeds in 
    crops is practised by cultivation and herbicide treatment, in order 
    to maximize yield and the general quality of the grain.  In 
    pastures, animal management and herbicide treatments are used to 
    increase pastures and reduce poisoning of animals.  Specific 
    treatment methods differ according to the plant species and the 
    circumstances.  General references were not available to the Task 
    Group. 

        In Australia, where  Heliotropium europaeum and  Echium
     plantagineum are widespread weeds in wheat-growing areas but where 
    normal agricultural practices prevent all but occasional minor 

    contamination, relevant tolerance standards for wheat delivered at 
    storage silos are not specific.   Heliotropium europaeum seed is 
    rarely seen and would form part of the "unmillable material" the 
    seed component of which can be up to 1% of the volume of the wheat. 
    Seed of  Echium plantagineum is occasionally seen in delivered grain 
    at levels of up to 10 seeds per half litre, the tolerance level for 
    this seed fraction being 50 seeds per half litre. 

    8.  BIOLOGICAL CONTROL

        Biological control methods have been investigated for several 
    PA-containing plant species, notably  Senecio jacobaea, Heliotropium 
     europaeum, Echium plantagineum, and  Trichodesma incanum.  The 
    effectiveness of such methods is variable and good results may be 
    confined to certain regions where favourable conditions exist.  For 
    example, in control programmes against  S. jacobaea in Australia, 
    Canada, New Zealand, and the USA, using 3 insect species, results 
    varied from virtually nil to nearly 100% control (Julien 1982).  
    The effects of the introduction of the cinnabar moth ( Tyria 
     jacobaea L.) on  S. jacobaea in these countries have been summarized 
    as in Table 18. 

    Table 18.  Results of the attempted control of  Senecio 
                jacobaea (ragwort) with the cinnabar moth 
    ------------------------------------------------------------
    Country or region               Result 
    ------------------------------------------------------------
    Australia          Establishment precluded by predation,
                       parasitism and disease

    Western Canada     Moth populations stabilized below that
                       required for control

    Eastern Canada     Establishment and subsequent notable
                       reductions in ragwort levels

    New Zealand        Marginal establishment, moth population
                       limited by predation and parasitism,
                       little impact on ragwort

    USA                Widespread establishment, ragwort levels
                       sometimes reduced at localities near the
                       limits of its distribution
    ------------------------------------------------------------

        Several agents are being tested in Australia for the control of 
     H. europaeum and one species, a flee beetle  Longitarsus albineus, 
    has been released (Julien, 1982; Delfosse, 1985).  There are good 
    prospects in this country for the biological control of  Echium 
     plantagineum and 2 other  Echium spp., with 8 insect species 
    approved for release, when legal restrictions are lifted (Delfosse 
    & Cullen, 1985a,b).  Preliminary studies have been made on the 
    biological control of  Amsinckia and other  Senecio species (e.g., 
    Pantone et al., 1985). 

        Given adequate funding, PA-containing plants are a suitable 
    target for biological control.

    9.  EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

    9.1  Human Exposure Conditions

    9.1.1  Reported sources of human exposure

        The two main sources of exposure of human beings to toxic PAs 
    that have led to major outbreaks of poisoning with high mortalities 
    as well as to individual cases in several countries are: 

        (a)  the contamination of cereal grains, such as wheat and 
             millet, with the seed or other parts of plants containing 
             alkaloids; and 

        (b)  the consumption for medicinal or dietary purposes of herbs 
             containing the alkaloids, either as the plant itself or as 
             infusions. 

    Consumption of contaminated grain is more likely to occur in 
    regions where food is in short supply, and particularly when 
    drought favours infestation of the grain crop by PA-containing 
    weeds.  A qualitative field test for detecting the presence of 
    toxic pyrrolizidine alkaloids in plant materials, using simple 
    laboratory methods, is now available (section 2.2.2.5). 

    9.1.2  Plant species involved

        Plant species containing toxic PAs occur throughout the world 
    and are known in 47 genera in 6 plant families.  As many as 6000 
    species are potentially PA-containing.  The most important genera 
    responsible for human and animal disease are  Senecio and other 
    genera of the tribe Senecioneae (family Compositae),  Crotalaria
    (family Leguminosae) and  Heliotropium, Trichodesma, and other 
    genera of the family Boraginaceae (sections 3.1 and 3.2). 

        Approximately 150 different toxic PAs have been isolated from 
    about 360 plant species that have been investigated and contain 
    this type of alkaloid.  Of these, about 12 have been involved in 
    instances of human toxicity (section 3.1).  The molecular 
    structures of almost all of these alkaloids are known and the main 
    outlines of structure-toxicity relationships have been established 
    (section 2.1). 

        The alkaloids may occur in all plant parts and are often 
    present as the  N-oxide derivatives, which are also toxic when 
    ingested orally.  Alkaloid contents vary from low (0.1 g/kg dry 
    weight) to very high (40 g/kg up to the maximum recorded of 
    180 g/kg in  Senecio riddelli).  Levels vary with stage of growth,
    locality, and other circumstances.  In some, but not all, species, 
    the alkaloid is partly decomposed during the drying or storage of 
    the plant.  The decomposition is largely enzymic and once the plant 
    material is dry, the alkaloid is fairly stable. 

    9.1.3  Modes and pathways of exposure

    9.1.3.1  Contamination of grain crops

        Large outbreaks of poisoning have occurred through 
    contamination of wheat crops in Afghanistan, India, and the USSR.  
    In particular, 3 species of Boraginaceae  (Heliotropium lasiocarpum, 
     H. popovii, and  H. europaeum) are well adapted to vigorous growth 
    under the climatic conditions in which wheat is usually grown.  
    Contamination can be effectively controlled in wheat produced using 
    modern harvesting techniques and grain seed that is inspected and 
    controlled for weed seed contamination, but control of 
    contamination is more difficult where these conditions cannot be 
    met.  Contamination of staple food grain is of particular concern, 
    since entire populations are exposed, and control may not be 
    possible, if the people are not aware of the hazard that 
    PA-containing weeds present. 

    9.1.3.2  Herbal medicines

        Herbal preparations containing PAs are used as tonics, 
    treatments, preventatives, and food supplements.  Such usages are 
    so widespread that they are nearly universal.  Many are 
    traditional, while others reflect a rejection of, or lack of access 
    to, standard health care services (section 3.3.2). 

        Veno-occlusive disease was first recognized as a clinical 
    entity in Jamaica as a result of the medicinal use of PA-containing 
    herbs prepared from  Crotalaria.  Crotalaria-containing herbs have 
    also been responsible for human poisonings in Barbados, Equador, 
    and other locations in the West Indies.   Heliotropium herbs have 
    been reported to cause poisoning in Hong Kong and India.  
     Symphytum- and  Senecio-containing herbs have given rise to case 
    reports in the USA.  Other reports of PA poisoning are known in 
    which the herbs used were not botanically identified.  PA-poisoning 
    has been associated with both home-prepared and commercially 
    available herbs, the latter including prescriptions by herbalists 
    (Weston et al., 1987). 

        Various other genera of PA-containing plants in the families 
    Boraginaceae, Compositae, and Leguminosae are also widely used as 
    herbs.  No case reports are available for these genera. 

        Reported cases of PA poisoning due to the use of the herbs are 
    geographically widespread, but few in number.  However, the scale 
    on which PA-containing plants are used as herbs, the typically 
    delayed effects of long-term exposure, and the difficulties of 
    diagnosis led the Task Group to conclude that there is every 
    indication of under-reporting of intoxications from the use of such 
    herbs.   Symphytum root preparations, in particular, represent a 
    hazard, and certain user groups are routinely exposed to levels of 
     Symphytum alkaloids that are higher than those at which 
    intoxications have been reported. 

        The risks associated with the use of PA-containing herbs are 
    accentuated by the difficulties of controlling this use. 

    9.1.3.3  PA-containing plants used as food and beverages

        Some PA-containing plants are used for food or the making of 
    beverages in many countries, including developed countries.  The 
    following species are known to be used (though many other plants 
    are also probably used in this way):   Cacalia yatabei, Symphytum 
    species,  Ligularia dentata, Petasites japonicus, Senecio burchellii,
     S. inadequidens, S. pierotti, Syneilesis palmata, Crotalaria
     anagyrodies, C. brevidens, C. juncea, C. laburnifolia, C. pumila,
     C. recta, and  C. retusa.  No information is available on 
    the extent to which the different types are consumed (section 3.3.3).

    9.1.3.4  Other foods contaminated by PAs

        Several species of Boraginaceae are nectar and pollen sources 
    for bees.   Echium plantagineum, in particular, is a widespread weed 
    in some countries and a substantial source of honey containing a 
    low level of alkaloid.   Senecio species are also visited by bees 
    and yield alkaloid-containing honey, though  Senecio-derived honey 
    is not known to be produced in quantity for sale.  Thus, some 
    regional and local populations are exposed to a low-level intake 
    through the presence of PAs in honey, and surveillance may be 
    desirable in countries producing honey (section 3.3.4). 

        Under experimental conditions, PAs are transmitted from the 
    feed of dairy cows and goats into the milk.  Some PA-containing 
    species, such as  Senecio jacobaea, S. lautus, and  Echium plantagineum,
    are weeds in dairy pastures in some countries and are eaten by cattle
    under certain conditions.  There are no published reports of alkaloid
    in milk supplies for human consumption (section 3.3.5). 

        The Task Group was not aware of any reported cases of 
    pyrrolizidine toxicity that had been ascribed to either honey or 
    dairy products. 

        No information was available to the Task Group on the possible 
    presence of alkaloids or their metabolites in meat from animals 
    that had consumed PA-containing plants shortly before slaughter.  
    The results of metabolic studies in rats have indicated that the 
    alkaloid is rapidly cleared from the body and, therefore, the 
    levels of PAs in meat are expected to be very low.  However, there 
    is no information on the possibility of alkaloid accumulating in 
    storage sites. 

    9.1.4  Levels of intake

        Reliable estimates of levels of intake of PAs, especially in 
    outbreaks of disease caused by the contamination of cereal crops 
    with the seeds of toxic plants, are extremely difficult to make.  
    Sampling of the contaminated grain may not be strictly 

    representative, since the extent of the contamination may vary in 
    different sites and households, as is evident from the estimates of 
    PA intake in the Indian and Afghan outbreaks reported in section 
    7.3.  Furthermore, no accurate record is possible of the amount of 
    contaminated food consumed over an uncertain length of time.  No 
    records of the levels of toxic PA intake are available in the 
    earlier reports of human toxicity.  Where available, estimates of 
    intake in outbreaks caused by the contamination of staple food 
    crops have been made on the basis of random sampling of the 
    contaminated grain in food stores, and rough estimates of daily 
    consumption by average adults.  Food-on-the-plate analyses have not 
    been made.  The estimated lengths of exposure, and hence the amount 
    of total intake, are also approximate. 

        The contamination of cereal grains with the seed of 
    PA-containing plants has caused major epidemics of human poisoning, 
    though, in the two instances where estimates of alkaloid intake are 
    available, the intakes were lower than in some exposures due to the 
    use of herbal medicines.  The estimated intakes are summarized in 
    Table 16.  In an outbreak in India, millet contaminated with 
     Crotalaria nana seed had an average alkaloid content of 0.5 g/kg, 
    and the estimated daily intake by the population was 0.66 mg/kg 
    body weight.  In a larger outbreak in Afghanistan, due to the seeds 
    of  Heliotropium popovii in wheat, the level of contamination was 
    probably variable; representative samples of wheat contained 
    alkaloid at 0.04 g/kg.  The estimated daily intake was 0.033 mg/kg 
    body weight.  These intakes, sustained for periods of approximately 
    2 and 6 months, respectively, resulted in typical acute and 
    subacute veno-occlusive disease. 

        The highest intake rates have been associated with the use or 
    misuse of medicinal herbs and have resulted in acute liver damage 
    and death.  In two occasions, the consumption of  Heliotropium 
     eichwaldii as a treatment for epilepsy led to an estimated intake 
    of 3.3 mg/kg body weight daily for 20 or 50 days, and the use of 
    extracts of  Senecio longilobus as medicine for young children led 
    to estimated intakes of 3 and 0.8 - 1.7 mg/kg body weight.  The 
    highest intake led to extensive liver necrosis.  However, it is 
    possible that, in the above case of poisoning by  Heliotropium 
     eichwaldii, the toxicity was enhanced due to simultaneous 
    administration of phenobarbitone, which has a potentiating effect 
    on the microsomal enzymes in the liver cells that convert the PAs 
    to toxic metabolites. 

        The use of  Heliotropium lasiocarpum as a component of a herbal 
    treatment for psoriasis involved somewhat lower daily intake rates 
    of 0.49 - 0.71 mg/kg body weight in 4 patients, who, after periods 
    of 19 - 46 days, developed veno-occlusive disease.  The patient 
    with the longest intake period and a total intake of 1.4 g alkaloid 
    or 23 mg/kg body weight died. 

        The lowest ingestion rate leading to a case of veno-occlusive 
    disease was also due to medicinal herbal treatment or, more 
    specifically, to the use of a digestive aid containing a 
    preparation of comfrey root.  Commercial herb and food supplement 

    preparations containing comfrey leaf or root are on sale in many 
    countries.  Limited assays of one comfrey-pepsin preparation 
    prepared from comfrey root indicated a PA content of 2.9 g/kg.  
    Another preparation made from comfrey leaf contained up to 0.27 g 
    alkaloid/kg.  The consumption of these preparations led to an 
    estimated daily intake of 0.015 mg/kg body weight.  Veno-occlusive 
    disease was diagnosed after a 4- to 6-month period. 

        The consumption of  Symphytum officinale (comfrey) and  S. x 
     uplandicum (Russian comfrey) in the form of food, infusions, or 
    other preparations is widespread, though the full extent cannot be 
    estimated.  A high level of consumption as salad appears to be 
    about 5 - 6 leaves per day and consumption as comfrey tea probably 
    reaches a similar level.  Limited assays indicate that the average 
    alkaloid content of the leaf is about 1 mg/leaf, the concentration 
    being higher in the younger, smaller leaves.  The alkaloid intake 
    from comfrey leaves could therefore vary from a low value, up to 
    about 6 mg/day, or 0.1 mg/kg body weight for an adult, an intake 
    within the range producing veno-occlusive disease.  However, the 
    Task Group noted that some people claim to have consumed comfrey at 
    such a rate without suffering any disease. 

        Overall, the estimates of intake of PAs by human beings (Table 
    16) indicate that ingestion rates above 0.015 mg/kg body weight for 
    the mixture of echimidine and related alkaloids in comfrey may lead 
    to acute or subacute liver disease.  If expressed in terms of 
    equivalent doses of heliotrine (section 7.8), the estimated total 
    doses in the known outbreaks or cases of veno-occlusive disease 
    range from 1 to 167 mg/kg body weight.  There is little real 
    difference in the ranges of estimated total doses in non-fatal 
    cases (1 - 120 mg/kg body weight) and those leading to death 
    (6 - 167 mg/kg).  These figures, when compared with the total 
    lethal dose of several PAs in rats, i.e., 1.2 - 10.9 times the LD50 
    dose (equivalent to 360 - 3270 mg heliotrine/kg) (Table 10), would 
    seem to indicate that man is markedly more sensitive than the rat 
    to the toxic effects of PAs with regard to the development of acute 
    and chronic effects on the liver.  It should be noted that these 
    estimates are based on limited raw data and a number of 
    assumptions, and so are of uncertain reliability. 

        The dose estimates indicate strongly that the effects of PAs in 
    human beings are cumulative at very low intake rates.  Lower rates 
    of intake of PAs may lead to chronic forms of intoxication, though, 
    at present, there is no evidence on which the degree of risk in 
    these circumstances can be evaluated.  The information available on 
    dose-response relationships is very limited, but the data support 
    the conclusion that even low rates of intake of PAs over a period 
    of time may present a health risk and that exposure should be 
    minimized wherever possible. 

        There has not been any systematic monitoring of PAs in cereal 
    grains, food products, and herbal medicines.  Analytical surveys of 
    these materials are feasible, but it would be difficult to design 
    surveys that would give direct estimates of the dietary intake of 
    PAs. 

    9.2  Acute Effects of Exposure

    9.2.1  Acute liver disease

        All cases of human intoxication in reported accounts have been 
    in the acute phase of the disease, the dominant symptom being 
    rapidly filling ascites.  The disease can affect large 
    subpopulations and, in one study, up to 22.6% of the population was 
    affected. 

        Children appear to be the most vulnerable group and mortality 
    can be high at the extremes of age.  The liver is the principal 
    target organ.  In the acute stage of the disease, the liver shows a 
    characteristic centrilobular haemorrhagic necrosis, which in man is 
    accompanied by occlusion of the hepatic veins.  However, 
    characteristic veno-occlusive lesions, seen in the central veins of 
    hepatic lobules, may not always be evident in the needle biopsy 
    examination of the liver, but are always apparent on examination of 
    the autopsy material. 

    9.3  Chronic Effects of Exposure

    9.3.1  Cirrhosis of the liver

        There is evidence that the administration of a single dose of 
    PA to experimental animals or a single acute episode of illness in 
    man, following brief consumption of PA-containing herbs or 
    PA-contaminated food, may lead to progressive chronic liver disease 
    resulting in cirrhosis.  Cirrhosis may also be a consequence of 
    long-term low-dose administration of PAs to experimental animals 
    and possibly also of long-term low intake of PAs by human beings, 
    though there is no proof of the latter.  Cirrhosis resulting from 
    the toxic effects of PAs in the advanced stage, may not be 
    distinguishable from that resulting from other causes (sections 
    6.4.1.5 and 7.4).  The Task Group did not find any evidence 
    suggesting that PAs are a causative factor of the specific disease, 
    Indian Childhood Cirrhosis (section 7.5). 

    9.3.2  Mutagenicity and teratogenicity

        Several PAs, PA-derivatives, and related compounds have been 
    shown to produce chromosome aberrations in plants and several cell 
    culture systems, mutagenic effects ( Salmonella ("Ame's"), sister 
    chromatid exchanges, and other tests), and teratogenic and 
    fetotoxic effects in experimental animals (sections 6.4.5, 6.4.6, 
    6.4.7).  Chromosomal aberrations have been reported in the blood 
    cells of children suffering from veno-occlusive disease, believed 
    to have been caused by fulvine.  The Task Group was not aware of 
    data on the teratogenic/fetotoxic effects of PAs on human beings 
    and was unable to evaluate the potential for these effects in PA 
    exposure. 

    9.3.3  Cancer of the liver

        A relatively large number of people have been exposed, in the 
    past, to PAs and have suffered acute and chronic toxic effects.  
    However, no information is available on the long-term follow-up of 
    these populations, to ascertain whether this type of exposure could 
    have resulted in an increased incidence of liver cancer or other 
    types of cancer.  Because of this lack of knowledge, it is not 
    possible, at present, to make an evaluation of the cancer risk due 
    to PAs.  However, various PAs have been shown to be carcinogenic 
    for experimental animals, which implies that a potential cancer 
    risk for human beings should be seriously considered. 

        Of several PAs evaluated for carcinogenicity by IARC (1976, 
    1983), there is "sufficient or limited evidence" for the 
    carcinogenicity in experimental animals (IARC, 1976) of 
    monocrotaline, retrorsine, isatidine, lasiocarpine, petasitenine, 
    senkirkine, and of extracts of the PA-containing plants  Petasites 
     japonicum, Tussilago farara, Symphytum officinale, Senecio 
     longilobus, Senecio numorensis, Farfugium japonicum, and  Senecio
     cannabifolius.  These studies were carried out mainly on rats, with 
    few studies on mice or hamsters (section 6.4.8).  The 
    carcinogenicity data obtained with other PAs are difficult to 
    evaluate, because of the limited number of treated animals and the 
    lack of adequate numbers as controls.  The main target organ is the 
    liver, where liver cell tumours and haemangioendothelial sarcomas 
    were observed.  In some instances, tumours in extra-hepatic tissues 
    (lung, pancreas, intestine) were also observed, namely with 
    monocrotaline, retrorsine, and lasiocarpine.  Some PAs, for 
    example, retrorsine, have been shown to be carcinogenic after a 
    single dose.  The pyrrolic metabolites have also been shown to be 
    carcinogenic for rats. 

        It may be recalled that several of the PAs involved in human 
    poisoning include the above compounds.  It is notable that the dose 
    rates that have been effective in inducing tumours in rats, mostly 
    equivalent to 0.2 - 3 mg/kg body weight per day (Table 17), are 
    roughly similar in magnitude to estimated intake rates (0.49 -
    3.3 mg/kg body weight per day) (Table 16) in several episodes of human 
    toxicity.  Comparison of the total intakes resulting in human 
    toxicity with the total doses to death observed in the chronic 
    toxicity studies on rats indicates that human beings are more 
    susceptible (section 7.8) and suggests that human beings may 
    survive for sufficient time to develop cancer after only a brief 
    exposure at this level or a longer exposure at a markedly lower 
    level. A more quantitative assessment is not possible on the basis 
    of the available information, and the Task Group stressed the need 
    for appropriate epidemiological studies. 

    9.3.4  Effects on other organs

        Substantiated reports of PA-induced extra-hepatic injury in man 
    are limited to  Trichodesma intoxication, in which symptoms and 
    signs were predominantly neurological.  The range of organs 

    affected by other PAs in experimental and farm animals suggests 
    that exposure of human beings to other PAs may also carry the 
    potential for extra-hepatic injury. 

        There are extensive reports of experimental studies in which 
    PAs have been demonstrated to produce the characteristic vascular 
    changes of primary pulmonary hypertension and consequent right 
    ventricular hypertrophy of the heart in rats and non-human primates 
    (section 6.4.2).  Susceptibility is age dependent, weanling rats 
    being more vulnerable than older animals.  There is only 
    circumstantial evidence of PA-induced pulmonary vascular disease in 
    one patient (section 7.6), but judging by the experimental evidence 
    available, it is possible that human beings may be susceptible to 
    PA-induced cardiopulmonary changes. 

        In the opinion of the Task Group, the neurological involvement 
    which is a dominant feature in PA-intoxicated horses and is also 
    seen in cows and sheep, cannot be explained solely as a consequence 
    of liver damage.  Central nervous system lesions have been 
    demonstrated in sheep, pigs, and rats.  Distribution studies of the 
    radiolabelled metabolite, 3H-dehydroretronecine, show increasing
    accumulation of radioactivity in the brain with time. 

         Trichodesma alkaloids, structurally related to monocrotaline, 
    are neurotoxic agents.   Trichodesma toxicosis in man has been 
    reported only from the USSR, together with several studies on 
    experimental animals.  Detailed reports on the pathological 
    findings were not available to the Task Group, but the information 
    available indicated that the central nervous system was the primary 
    target organ (sections 6.4.3 and 7.7). 

        Stomach and intestinal lesions have been shown in PA-exposed 
    sheep, mice, cows, and rats.  Distribution studies with 
    radiolabelled pyrroles showed a high retention of radioactivity in 
    the stomach, consistent with the acid-sensitive nature of the 
    pyrroles.  In rats, pyrrolic metabolites are secreted in high 
    concentrations in the bile. 

        Kidney changes following to PA administration have been shown 
    in mice, pigs, horses, sheep, and monkeys.  Pyrrolizidine 
    metabolites have been found covalently bound to kidney DNA in rats.  
    Urinary excretion is a major route of excretion of metabolic 
    products of PAs in rats. 

        There is no evidence of involvement of organs other than the 
    liver and central nervous system ascribed primarily to PA toxicity 
    in any of the published human case reports.  It is possible that 
    under some circumstances, other major organ systems may also be at 
    risk.  As bioactivation of PAs has been demonstrated only in the 
    liver, the risk of damage should be expected to be lower in the 
    organs. 

    9.4  Effects on the Environment

    9.4.1  Agriculture

        In some countries, PA-containing weeds densely cover areas of 
    up to thousands of square kilometres.  Their adverse effects 
    include the covering of pastures, additional costs in agricultural 
    production, and the poisoning of farm animals.  The toxicity of PAs 
    for farm animals, including sheep, cattle, horses, pigs, goats, and 
    poultry, which has been the inspiration for much of the investigation
    of PA toxicity.  In Australia, for example,  Heliotropium europaeum
    and  Echium plantagineum cause the death of thousands of animals
    annually (section 6.2). 

    9.4.2  Wild-life

        By contrast, little is known about the consumption of PA-containing
    plants by wild-life, or of their individual sensitivities.  The death
    of deer in Louisiana has been ascribed to eating  Heliotropium or
     Crotalaria species, and an experimental study has shown that the
    rainbow trout  (Salmo gairdneri) is sensitive to  Senecio jacobaea
    alkaloids (sections 6.5.1 and 6.5.2). 

        There is no information on the effects of the alkaloids on 
    field rodents or other seed-eating mammals and birds that might be 
    expected to consume seeds of PA-containing plants and to suffer 
    toxic effects. 

    9.4.3  Insects

        Many species of insects, such as some moths of the family 
     Arctidae and butterflies of the sub-families  Danainae and 
     Ithominae, have become dependent on PA-containing plants, using the 
    alkaloids as defensive chemicals and derivatives of them as 
    pheromones and other signalling chemicals.  Thus, complete 
    elimination of PA-containing plants in a region might lead to a 
    marked reduction in the local population of insects of this type 
    (section 6.5.3). 

    9.4.4  Soil and water

        There have not been any studies on the fate of PAs when the 
    plants in which they occur wilt and age.  If alkaloid is leached 
    into soil or water, it is probably readily degraded by microorganisms
    since, as a base and ester, it is subject to oxidative and hydrolytic
    reactions. 

    REFERENCES

    AFZELIUS, B.A. & SCHOENTAL, R.  (1967)  The ultrastructure of 
    enlarged hepatocytes induced in rats with a single oral dose of 
    retrorsine, pyrrolizidine  (Senecio) alkaloids.  J. ultra-struct. 
     Res., 20: 328-345. 

    AIKAT, B.K., BHUSNURMATH, S.R., DATTA, D.V., & CHHUTTANI, P.N. 
    (1978)  Veno-occlusive disease in north-west India.  Indian J. 
     Pathol. Microbiol., 21: 203-211. 

    AL-HASANY, M. & MOHAMED, A.S. (1970)  Veno-occlusive disease of the 
    liver in Iraq.  Arch. dis. Child., 45: 722-724. 

    ALLEN, J.R. & CARSTENS, L.A. (1968)  Veno-occlusive disease in 
    rhesus monkeys.  Am. J. vet. Res., 29: 1681-1694. 

    ALLEN, J.R. & CARSTENS, L.A. (1970)  Pulmonary vascular occlusions 
    initiated by endothelial lysis in monocrotaline-intoxicated rats.  
     Exp. mol. Pathol.., 13: 159-171. 

    ALLEN, J.R. & CARSTENS, L.A. (1971)  Monocrotaline induced Budd-
    Chiari syndrome in monkeys.  Am. J. dig. Dis., 16: 111-121. 

    ALLEN, J.R. & CHESNEY, C.F. (1972)  Effect of age on development of 
    cor pulmonale in non-human primates following pyrrolizidine 
    alkaloid intoxication.  Exp. mol. Pathol., 17: 220-232. 

    ALLEN, J.R., CHILDS, G.R., & CRAVENS, W.W. (1960)  Crotalaria 
     spectabilis toxicity in chickens.  Proc. Soc. Exp. Biol. Med., 104: 
    434-436. 

    ALLEN, J.R., LALICH, J.J., & SCHMUTTLE, S.M. (1963)  Crotalaria 
     spectabilis induced cirrhosis in turkeys.  Lab. Invest., 12: 512-517. 

    ALLEN, J.R., CARSTENS, L.A., & OLSON, B.E. (1967)  Veno-occlusive 
    disease in Macaca speciosa monkeys.  Am. J. Pathol., 50: 653-667. 

    ALLEN, J.R., CARSTENS, L.A., & KATAGIRI, G.J. (1969)  Hepatic veins 
    of monkeys with veno-occlusive disease-sequential ultrastructural 
    changes.  Arch. Pathol., 87: 279-289. 

    ALLEN, J.R., CHESNEY, C.F., & FRAZEE, W.J. (1972)  Modifications of 
    pyrrolizidine alkaloids intoxication resulting from altered 
    microsomal enzymes.  Toxicol. appl. Pharmacol., 23: 470-479. 

    ALLEN, J.R., HSU, I.-C., & CARSTENS, L.A. (1975) Dehydroretronecine 
    induced rhabdomyosarcomas in rats.  Cancer Res., 35: 997-1002. 

    AMES, M.M. & POWIS, G. (1978)  Determination of indicine  N-oxide 
    and indicine in plasma and urine by electron-capture gas-liquid 
    chromatography.  J. Chromatogr., 166: 519-526. 

    ARMSTRONG, S.J. & ZUCKERMAN, A.J. (1970)  Production of pyrroles 
    from pyrrolizidine alkaloids by human embryo tissue.  Nature 
     (Lond.), 228: 569-570. 

    ARMSTRONG, S.J., ZUCKERMAN, A.J., & BIRD, R.G. (1972)  Induction of 
    morphological changes in human embryo liver cells by the 
    pyrrolizidine alkaloid lasiocarpine.  Br. J. exp. Pathol., 53: 
    145-149. 

    ARORA, R.R., PYARELAL, GHOSH, T.K., MATHUR, K.K., & TANDON, B.N. 
    (1981)  Epidemiology of veno-occlusive disease in tribal population 
    of Madhya Pradesh and Bihar.  J. commun. Dis. (India), 13: 147-151. 

    ARSECULERATNE, S.N., GUNATILAKA, A.A.L., & PANABOKKE, R.G. (1981)  
    Studies on medicinal plants of Sri Lanka: occurrence of 
    pyrrolizidine alkaloids and hepatotoxic properties in some 
    traditional medicinal herbs.  J. Ethnopharmacol., 4: 159-177. 

    ASADA, Y., FURUYA, T., & MURAKAMI, N. (1981)  Pyrrolizidine 
    alkaloids from  Ligularia japonica. Planta Med., 42: 202-203. 

    ASADA, Y., SHIRAISHI, M., TAKEUCHI, T., OSAWA, Y., & FURUYA, T. 
    (1985)  Pyrrolizidine alkaloids from  Crassocephalum crepidioides. 
     Planta Med., 51: 539-540. 

    BARNES, J.M., MAGEE, P.N., & SCHOENTAL, R. (1964)  Lesions in the 
    lungs and livers of rats poisoned with pyrrolizidine alkaloids 
    fulvine and its  N-oxide.  J. Pathol. Bacteriol., 88: 521-531. 

    BERRY, D.M. & BRAS, G. (1957)  Venous occlusion of the liver in 
     Crotalaria and  Senecio poisoning.  North Am. Vet., 38: 323-327. 

    BHATTACHARYA, K.J. (1965)  Foetal and neonatal responses to 
    hepatotoxic agents.  J. Pathol. Bacteriol., 90: 151-161. 

    BICCHI, C., D'AMATO, A., & CAPPELLETTI, E. (1985)  Determination 
    of pyrrolizidine alkaloids in  Senecio inaequidens D.C. by 
    capillary gas chromatography.  J. Chromatogr., 349: 23-29. 

    BICK, Y.A.E. (1970)  Comparison of the effects of LSD, heliotrine, 
    and X-irradiation on chromosome breakage and the effects of LSD on 
    the rats of cell division.  Nature (Lond.), 226: 1165-1167. 

    BICK, Y.A.E. & CULVENOR, C.C.J. (1971)  Effects of dehydro- 
    heliotridine, a metabolite of pyrrolizidine alkaloids on chromosome 
    structure and cell division in cultures of animal cells.  Cytobios, 
    3: 245-255. 

    BICK, Y.A.E. & JACKSON, W.D. (1968)  Effects of the pyrrolizidine 
    alkaloid heliotrine on cell division and chromosome breakage in 
    cultures of leucocytes from the marsupial  Potorus tridactylus. 
     Aust. J. biol. Sci., 21: 469-481. 

    BICK, Y.A.E., CULVENOR, C.C.J., & JAGO, M.V. (1975)  Comparative 
    effects of pyrrolizidine alkaloids and related compounds on 
    leukocyte cultures from  Potorus tridactylus. Cytobios, 14: 151-160. 

    BIRECKA, H., FROHLICH, M.W., HULL, L., &  CHASKES, M.J. (1980)  
    Pyrrolizidine alkaloids of  Heliotropium from Mexico and adjacent 
    USA.  Phytochemistry, 19: 421-426. 

    BIRECKA, H., CATALFAMO, J.L., & EISEN, R.N. (1981)  A sensitive 
    method for detection and quantitative determination of 
    pyrrolizidine alkaloids.  Phytochemistry, 20: 343-344. 

    BLACK, D.N. & JAGO, M.V.  (1970)  Interaction of 
    dehydroheliotridine, a metabolite of heliotridine based 
    pyrrolizidine alkaloids, with natural and heat denatured RNA. 
     Biochem. J., 118: 347-353. 

    BOHLMANN, F., KLOSE, W., & NIKISCH, K. (1979)  [Synthesis of 
    dehydroheliotridine and 3-oxy-dehydroheliotridine.]  Tetrahedr. 
     Lett., 1979: 3699-3702 (in German). 

    BOHLMANN, F., ZDERO, C., JAKUPOVIC, J., GRENZ, M., CASTRO, V., 
    KING, R.M., ROBINSON, H., & VINCENT, L.P.D. (1986)  Further 
    pyrrolizidine alkaloids and furoeremophilanes from  Senecio species. 
     Phytochemistry, 15: 1151-1159. 

    BOPPRE, M. (1986)  Insects pharmacophagously utilizing defensive 
    plant chemicals (pyrrolizidine alkaloids).  Naturwiss enschaften, 73: 
    17-26. 

    BRAGINSKII, B.M. & BOBOKHADZAEV, I. (1965)  [Hepatosplenomegaly 
    against the background of heliotropic toxicosis.]  Sov. Med., 28: 
    57-60 (in Russian). 

    BRAS, G. (1973)  Aspects of hepatic vascular diseases. In: Gall, 
    E.A. & Mostofi, F.K., ed.  The liver: International Academy of 
     Pathology monograph, Baltimore, Maryland, Williams and Wilkins, 
    pp. 406-530. 

    BRAS, G. & HILL, K.R. (1956)  Veno-occlusive disease of the liver - 
    essential pathology.  Lancet, 2: 161-163. 

    BRAS, G. & WATLER, D.C. (1955)  Further observations on the 
    morphology of veno-occlusive disease of the liver in Jamaica.  West 
     Indian med. J., 4: 201-211. 

    BRAS, G., JELLIFFE, D.B., & STUART, K.L. (1954)  Veno-occlusive 
    disease of the liver with nonportal type of cirrhosis occurring in 
    Jamaica.  Arch. Pathol., 57: 285-300. 

    BRAS, G., BERRY, D.M., & GYORGI, P. (1957)  Plants as etiological 
    factor in veno-occlusive disease of liver.  Lancet, 1: 960-962. 

    BRAS, G., BROOKS, S.E.H., & WATLER, D.C. (1961)  Cirrhosis of liver 
    in Jamaica.  J. Pathol. Bacteriol., 82: 503-512. 

    BRAUCHLI, J., LUTHY, J., ZWEIFEL, U., & SCHLATTER, C. (1982)  
    Pyrrolizidine alkaloids from  Symphytum officinale L. and their 
    percutaneous absorption in rats.  Experientia (Basel), 38: 
    1085-1087. 

    BRIGGS, L.H., CAMBIE, R.C., CANDY, B.J., O'DONOVAN, G.M., RUSSELL, 
    R.A., & SEELYE, R.N. (1965)  Alkaloids of New Zealand  Senecio  
    species. Part II: senkirkine.  J. Chem. Soc., C1965: 2492-2498. 

    BRINK, N.G. (1982)  Somatic and teratogenic effects induced by 
    heliotrine in  Drosophilia. Mutat. Res., 104: 105-111. 

    BROOKS, W.E.H., MILLER, C.G., MCKENZIE, K., AUDRETSCH, J.J., & 
    BRAS, G. (1970)  Acute veno-occlusive disease of the liver. Fine 
    structure in Jamaican children.  Arch. Pathol., 89: 507-529. 

    BROWN, K.S. (1984)  Chemical ecology of dehydropyrrolizidine 
    alkaloids in adult Ithomiinae (Lepidoptera: Nymphalidae).   Rev. 
     Bras. Biol., 44: 435-440. 

    BRUGGEMAN, I.M. & VAN DER HOEVEN, J.C.M. (1985)  Induction of SCEs 
    by some pyrrolizidine alkaloids in V79 Chinese hamster cells co-
    cultured with chick embryo hepatocytes.  Mutat. Res., 142: 209-212. 

    BRUNER, L.H., CARPENTER, L.J., HAMLOW, P., & ROTH, R.A. (1986)  
    Effect of a mixed-function oxidase inducer and inhibitor on 
    monocrotaline pyrrole pneumotoxicity.  Toxicol. appl. Pharmacol., 85: 
    416-427. 

    BUCKMASTER, G.W., CHEEKE, P.R., ARSCOTT, G.H., DICKINSON, E.D., 
    PIERSON, M.L., & SHULL, L.R. (1977)  Response of Japanese quail to 
    dietary and injected pyrrolizidine  (Senecio) alkaloid. J. anim. 
     Sci., 45(6): 1322-1325. 

    BULL, L.B. (1955)  The histological evidence of liver damage from 
    pyrrolizidine alkaloids.  Aust. vet. J., 31: 33-40. 

    BULL, L.B. & DICK, A.T. (1959)  The chronic pathological effects on 
    the liver of the rat of the pyrrolizidine alkaloids heliotrine, 
    lasiocarpine, and their  N-oxides.  J. Pathol. Bacteriol., 78: 483-502. 

    BULL, L.B. & DICK, A.T. (1960)  The function of total dose in the 
    production of chronic lethal disease in rats by periodic injections 
    of the pyrrolizidine alkaloid heliotrine.  Aust. J. exp. Biol. med. 
     Sci., 38: 515. 

    BULL, L.B., DICK, A.T., KEAST, J.C., & EDGAR, G. (1956)  An 
    experimental investigation of the hepatotoxic and other effects on 
    sheep of consumption of  Heliotropium europaeum L. Heliotropium 
    poisoning of sheep.  Aust. J. agric. Res., 7: 281-332. 

    BULL, L.B., DICK, A.T., & MCKENZIE, J.S. (1958)  The acute effects 
    of heliotrine and lasiocarpine and their  N-oxides on the rat.  J. 
     Pathol. Bacteriol., 75: 17-25. 

    BULL, L.B., CULVENOR, C.C.J., & DICK, A.T. (1968)   The 
     pyrrolizidine alkaloids, Amsterdam, North Holland Publishing Co. 

    BURGUERA, J.A., EDDS, G.T., & OSUNA, O. (1983)  Influence of 
    selenium on aflatoxin B1 or  Crotalaria toxicity in turkey poults. 
     Am. J. vet. Res., 44: 1714-1717. 

    BURNS, J. (1972)  The heart and pulmonary arteries in rat fed on 
     Senecio jacobaea. J. Pathol., 106: 187-194. 

    BUTLER, W.H., MATTOCKS, A.R., & BARNES, J.M. (1970)  Lesions in the 
    liver and lungs of rats given pyrrole derivatives of pyrrolizidine 
    alkaloids.  J. Pathol., 100: 169-175. 

    CAMPBELL, J.G. (1956)  An investigation of the hepatotoxic effects 
    in the fowl of ragwort ( Senecio jacobaea Linn) with special 
    reference to the induction of liver tumours with seneciphylline. 
     Proc. R. Soc. Edinburgh, B66: 111-130. 

    CAMPBELL, J.G. (1957a)  Studies on the influence of sex hormones on 
    the avian liver. II. Acute liver damage in the male fowl and the 
    protective effect of oestrogen, as determined by a liver function 
    test.  J. Endocrinol., 15: 346-350. 

    CAMPBELL, J.G. (1957b)  Studies on the influence of sex hormones on 
    avian liver. III. Oestrogen-induced regeneration of the chronically 
    damaged liver.  J. Endocrinol., 15: 351-354. 

    CANDRIAN, U., LUTHY, J., GRAF, U., & SCHLATTER, CH. (1984a)  
    Mutagenic activity of the pyrrolizidine alkaloids seneciphylline 
    and senkirkine in  Drosophila and their transfer into rat milk. 
     Food chem. Toxicol., 22: 223-225. 

    CANDRIAN, U., LUTHY, J., SCHMID, P., SCHLATTER, CH., & GALLASZ, E. 
    (1984b)  Stability of pyrrolizidine alkaloids in hay and silage.  J. 
     agric. food Chem., 32: 935-937. 

    CANDRIAN, U., LUTHY, J., & SCHLATTER, CH.  (1985)   In vivo binding 
    of retronecine-labelled (3H), seneciphylline, and 3H-senecionine to 
    DNA of rat liver, lung and kidney.  Chem.-biol. Interact., 54: 57-69. 

    CARILLO, L. & AVIADO, D.M. (1969)  Monocrotaline induced pulmonary 
    hypertension and  p-chlorophenylalanine.  Lab. Invest., 20: 213-218. 

    CARSTENS, L.A. & ALLEN, J.R. (1970)  Arterial degeneration and 
    glomerular hyalinization in the kidney of monocrotaline intoxicated 
    rats.  Am. J. Pathol., 60: 75-92. 

    CHALMERS, A.H., CULVENOR, C.C.J., & SMITH, L.W. (1965)  
    Characterization of pyrrolizidine alkaloids by gas, thin layer, and 
    paper chromatography.  J. Chromatogr., 20: 270-277. 

    CHEEKE, P.R. & GORMAN, G.R. (1974)  Influence of dietary protein 
    and sulphur amino-acid levels on the toxicity of  Senecio jacobaea 
    (tansy ragwort) to rats.  Nutr. Rep. Int., 9: 197-207. 

    CHEEKE, P.R. & PIERSON-GOEGER, M.L. (1983)  Toxicity of  Senecio 
     jacobaea and pyrrolizidine alkaloids in various laboratory animals 
    and avian species.  Toxicol. Lett., 18: 343-349. 

    CHEN, K.K. (1945)  Pharmacology. Hepatotoxic alkaloids.  Ann. Rev. 
     Physiol., 7: 695-697. 

    CHEN, K.K., HARRIS, P.N., & ROSE, C.L. (1940)  The action and 
    toxicity of platyphylline and seneciphylline.  J. Pharmacol. exp. 
     Ther., 68: 130-140. 

    CHESNEY, C.F. & ALLEN, J.R. (1973a)  Resistance of the guinea pig 
    to pyrrolizidine alkaloid intoxication.  Toxicol. appl. Pharmacol., 
    26: 385-392. 

    CHESNEY, C.F. & ALLEN, J.R. (1973b)  Endocardial fibrosis 
    associated with monocrotaline induced pulmonary hypertension in 
    non-human primates  (Macaca arctoides). Am. J. vet. Res., 34: 
    1577-1581. 

    CHESNEY, C.F., HSU, I.C., & ALLEN, J.R. (1974)  Modifications of 
    the  in vitro metabolism of the hepatotoxic pyrrolizidine alkaloid, 
    monocrotaline.  Res. Commun. chem. Pathol. Pharmacol., 8: 567-570. 

    CHOPRA, R.N., ed. (1933)   Indigenous drugs of India, Calcutta, The 
    Art Press. 

    CLARK, A.M. (1959)  Mutagenic activity of the alkaloid heliotrine 
    in  Drosophila. Nature (Lond.), 183: 731-732. 

    CLARK, A.M. (1976)  Naturally occurring mutagens.  Mutat. Res., 32: 
    361-174. 

    COOK, B.A., SINNHUBER, J.R., THOMAS, P.J., OLSON, T.A., SILVERMAN, 
    T.A., JONES, R., WHITEHEAD, V.M., & ROYMANN, F.B. (1983)  Hepatic 
    failure secondary to indicine  N-oxide toxicity. A pediatric 
    oncology group study.  Cancer, 52: 61-63. 

    COOK, J.W., DUFFY, E., & SCHOENTAL, R. (1950)  Primary liver 
    tumours in rats following feeding with alkaloids of  Senecio 
     jacobaea. Br. J. Cancer, 4: 405-410. 

    CRAIG, A.M., SHEGGEBY, G., & WICKS, C.E. (1984)  Large scale 
    extraction of pyrrolizidine alkaloids from tansy ragwort  (Senecio 
     jacobaea). Vet. hum. Toxicol., 26: 108-111. 

    CULVENOR, C.C.J. (1968)  Tumour inhibitory activity of 
    pyrrolizidine alkaloids.  J. pharm. Sci., 57: 1112-1117. 

    CULVENOR, C.C.J. (1978)  Pyrrolizidine alkaloids: occurrence and 
    systematic importance in angiosperms.  Bot. Notiser, 131: 473-486. 

    CULVENOR, C.C.J. (1980)  Alkaloids and human disease. In: Smith, 
    R.L. & Bababunmi, E.A., ed.  Toxicology in the tropics, London, 
    Taylor & Francis Ltd., pp. 124-141. 

    CULVENOR, C.C.J. (1983)  Estimated intakes of pyrrolizidine 
    alkaloids by humans. A comparison with dose rates causing tumours 
    in rats.  J. Toxicol. environ. Health, 11: 625-635. 

    CULVENOR, C.C.J. (1985)  Pyrrolizidine alkaloids: some aspects of the 
    Australian involvement.  Trends pharmacol. Sci., 6: 18-22. 

    CULVENOR, C.C.J. & JAGO, M.V. (1979)  Carcinogenic plant products 
    and DNA. In: Grover, P.L., ed.  Chemical carcinogens and DNA, Boca 
    Raton, Florida, CRC Press, Vol. 1, 161 pp. 

    CULVENOR, C.C.J., DOWNING, D.T., EDGAR, J.A., & JAGO, M.V. (1969)  
    Pyrrolizidine alkaloids as alkylating and antimitotic agents.  N.Y. 
     Acad. Sci., 163: 837-847. 

    CULVENOR, C.C.J., EDGAR, J.A., SMITH, L.W., &  TWEEDDALE, H.J. 
    (1970a)  Dihydropyrrolizines. III. Preparation and reactions of 
    derivatives related to pyrrolizidine alkaloids.  Aust. J. Chem., 23: 
    1853-1867. 

    CULVENOR, C.C.J., EDGAR, J.A., SMITH, L.W., & TWEEDDALE, H.J. 
    (1970b)  Dehydrolizines. IV. Manganese dioxide oxidation of 
    1,2-dihydropyrrolizidines.  Aust. J. Chem., 23: 1869-1879. 

    CULVENOR, C.C.J., EDGAR, J.A., JAGO, M.V., OUTTERIDGE, A., PETERSON 
    J.E., & SMITH, L.W. (1976a)  Hepato- and pneumotoxicity of 
    pyrrolizidine alkaloids and derivatives in relation to molecular 
    structure.  Chem.-biol. Interact., 12: 299-324. 

    CULVENOR, C.C.J., EDGAR, J.A., SMITH, L.W., & HIRONO, I. (1976b)  
    The occurrence of senkirkine in  Tussilago farfara. Aust. J. Chem.,
    29: 229-233. 

    CULVENOR, C.C.J., CLARKE, M., EDGAR, J.A., FRAHN, J.L., JAGO, M.V., 
    PETERSON, J.E., & SMITH, L.W. (1980a)  Structure and toxicity of 
    the alkaloids of Russian comfrey ( Symphytum x uplandicum Nyman), a 
    medicinal herb and item of human diet.  Experientia (Basel), 36: 
    377-389. 

    CULVENOR, C.C.J., EDGAR, J.A., FRAHN, J.L., &  SMITH, L.W. (1980b)  
    The alkaloids of  Symphytum x uplandicum (Russian comfrey).  Aust. 
     J. Chem., 33: 1105-1113. 

    CULVENOR, C.C.J., EDGAR, J.A., & SMITH, L.W. (1981)  Pyrrolizidine 
    alkaloids in honey from  Echium plantagineum L.  J. agric. food 
     Chem., 29: 958-960. 

    CULVENOR, C.C.J., JAGO, M.V., PETERSON, J.E., SMITH, L.W., PAYNE, 
    A.L, CAMPBELL, D.G., EDGAR, J.A., & FRAHN, J.L. (1984)  Toxicity of 
     Echium plantagineum (Paterson's curse). I. Marginal toxic effects 
    of Merino wethers from long-term feeding.  Aust. J. agric. Res., 35: 
    293-304. 

    CULVENOR, C.C.J., EDGAR, J.A., SMITH, L.W., KUMANA, C.R., & LIN, 
    H.J. (1986)   Heliotropium lasiocarpum Fisch and Mey identified as 
    cause of veno-occlusive disease due to a herbal tea.  Lancet, 1: 978. 

    DANN, A.T.  (1960)  Detection of  N-oxides of the pyrrolizidine 
    alkaloids.   Nature, 4730: 1051. 

    DANNINGER, T., HAGEMANN, U., SCHMIDT, V., & SCHOENHOEFER, P.S. 
    (1983)  Toxicity of pyrrolizidine alkaloid-containing medicinal 
    plants.  Pharm. Ztg, 128: 289-303. 

    DATTA, D.V., KHUROO, M.S., MATTOCKS, A.R., AIKAT, B.K., & 
    CHHUTTANI, P.N. (1978a)  Veno-occlusive disease of liver due to 
    Heliotropium plant used as medicinal herb (report of six cases with 
    review of literature).  J. Assoc. Phys. India, 26(5): 383-393. 

    DATTA, D.V., KHUROO, M.S., MATTOCKS, A.R., AIKAT, B.K., & 
    CHHUTTANNI, P.N. (1978b)  Herbal medicines and veno-occlusive 
    disease in India.  Postgrad. med. J., 54: 511-515. 

    DAVIDSON, C.S. (1963)  Plants and fungi as etiologic agents of 
    cirrhosis.  New Engl. J. Med., 268: 1072-1073. 

    DAVIDSON, J. (1935)  The action of retrorsine on rat's liver.  J. 
     Pathol. Bacteriol., 40: 285-295. 

    DEAGEN, J.T. & DEINZER, M.L. (1977)  Improvement in the extraction 
    of pyrrolizidine alkaloids.  Lloydia, 40: 395-397. 

    DEAN, R.E. & WINWARD, A.H. (1974)  An investigation into the 
    possibility of tansy ragwort poisoning of black-tailed deer.  J. 
     wildl. Dis., 10: 166-169 

    DEINZER, M.L., THOMSON, P.A., BURGETT, D.M., & ISAACSON, D.L. 
    (1977)  Pyrrolizidine alkaloids: their occurrence in honey from 
    tansy ragwort  (S. jacobaea). Science, 195: 497-499. 

    DEINZER, M.L., THOMSON, P.A., GRIFFIN, D., & DICKINSON, E. (1978)  
    Sensitive analytical method for pyrrolizidine alkaloids. The mass 
    spectra of retronecine derivatives.  Biomed. mass Spectrom., 5: 
    175-179. 

    DELFOSSE,  E.S. (1985)  Re-evaluation of the biological control 
    program for  Heliotropium europaeum in Australia. In: Delfosse, 
    E.S., ed.  Proceedings of the VI International Symposium on 
     Biological Control of Weeds, 19-25 August 1984, Vancouver, Canada,  
    Ottawa Agriculture Canada, pp. 735-42. 

    DELFOSSE, E.S. & CULLEN, J.M. (1985a)  CSIRO division of Entomology 
    Submission to the enquiries into biological control of  Echium 
     plantagineum L., Paterson's curse/Salvation Jane.  Plant Prot. Q., 
    1: 24-40. 

    DELFOSSE, E.S. & CULLEN, J.M. (1985b)   Echium plantagineum: 
    catalyst for conflict and change in Australia. In: Delfosse, E.S., 
    ed.  Proceedings of the VI International Symposium on Biological 
     Control of Weeds, 19-25 August, 1984, Vancouver, Canada, Ottawa 
    Agriculture Canada pp. 249-292. 

    DELORME, P., JAY, M., & FERRY, S. (1977)  Phytochemical inventory 
    in indigenous Boraginaceae: study of the alkaloids and polyphenolic 
    compounds (anthocyanic and flavonoid compounds).  Plant. Méd. 
     Phytothér., 11: 5-11.  (in french) 

    DE WAAL, H.L. & VAN TWISK, P.  (1964)  The chemical composition of 
    four  Senecio species from Kruger National Park.   Koedoe (South 
     Africa), 7: 40-42. 

    DICK, A.T., DANN, A.T., BULL, L.B., & CULVENOR, C.C.J. (1963)  
    Vitamin B12 and the detoxication of hepatotoxic pyrrolizidine 
    alkaloids in rumen liquor.  Nature (Lond.), 197: 207-208. 

    DICKINSON, J.O. (1980)  Release of pyrrolizidine alkaloids into 
    milk.  Proc. West. Pharmacol. Soc., 23: 377-379. 

    DICKINSON, J.O., COOKE, M.P., KING, R.R., & MOHAMED, P.A. (1976)  
    Milk transfer of pyrrolizidine alkaloids in cattle.  J. Am. Vet. 
     Med. Assoc., 169: 1192-1196. 

    DIMENNA, G.P., KRICK, T.P., & SEGALL, H.J. (1980)  Rapid high 
    performance liquid chromatography isolation of monoesters, 
    diesters, and macrocyclic diesters and pyrrolizidine alkaloids from 
     Senecio jacobaea  and Amsinckia intermedia.  J. Chromatogr., 192: 
    474-478. 

    DOLINSKAYA, K.N. (1952)  [Pathomorphology of heliotropic hepatic 
    dystrophy in children.]  In: Milenkov, S.M. & Kizhaikin, Y., ed. 
     [Collection of scientific papers on Toxic Hepatitis with Ascites,]  
    Tashkent, Publishing House of the University of Central Asia,
    pp. 165-172 (in Russian). 

    DREIFUSS, P.A.  (1984)  A study of the mass spectra of 
    pyrrolizidine alkaloids by negative ion chemical ionization and 
    ms/ms:  the identification of a monoester pyrrolizidine alkaloid in  
     Eupatorium rugosum.  Diss. Abstr. Int. B., 45:'1183-1184. 

    DRIVER, H.E. & MATTOCKS, A.R. (1984)  The toxic effects in rats of 
    some synthanecine carbamate and phosphate esters analogous to 
    hepatotoxic pyrrolizidine alkaloids.  Chem.-biol. Interact., 51: 
    201-218. 

    DUBROVINSKII, S.B. (1946)  [About the alimentary toxicosis caused 
    by heliotrope.]  J. Sov. Prot. Health, 6: 17-21 (in Russian). 

    DUBROVINSKII, S.B. (1952)  [The etiology of toxic hepatitis with 
    ascites.] In: Millenkov, S.M. & Kizhaikin, Y., ed.  [Collection of 
     scientific papers on Toxic Hepatitis with Ascites, Tashkent, USSR,]  
    Tashkent, Publishing House of the University of Central Asia,
    pp. 9-25 (in Russian). 

    EASTMAN, D.F. & SEGALL, H.J. (1982)  A new pyrrolizidine alkaloid 
    metabolite, 19-hydroxysenecionine, isolated from  in vitro mouse 
    hepatic microsomes using high performance liquid chromatography. 
     Drug Metab. Disp., 10: 696-699. 

    EASTMAN, D.F., DIMENNA, G.P., & SEGALL, H.J. (1982)  Covalent 
    binding of two pyrrolizidine alkaloids, senecionine, and 
    seneciphylline to hepatic macromolecules and their distribution, 
    excretion and transfer into milk of lactating mice.  Drug Metab. 
     Disp., 10: 236-240. 

    EDGAR, J.A. (1982)  Pyrrolizidine alkaloids sequestered by Solomon 
    Island Danaine butterflies. The feeding preferences of the Danainae 
    and Ithomiinae.  J. Zool. (Lond.), 196: 385-399. 

    EDGAR, J.A. (1985)  Gas chromatography of pyrrolizidine alkaloids. 
    In: Seawright, A.A., Hegarty, M.P., James, L.F., & Keeler, R.F., 
    ed.  Plant toxicology. Proceedings of the Australia - USA Poisonous 
     Plants Symposium, Brisbane, Australia, 14-18 May 1984, Brisbane, 
    Queensland Poisonous Plants Committee, pp. 227-234. 

    EISENSTEIN, D. & HUXTABLE, R.J. (1979)  Approaches to the treatment 
    of pyrrolizidine toxicosis. In: Cheeke, P.R., ed.  Proceedings of 
     the Symposium on Pyrrolizidine (Senecio) Alkaloids: Toxicity, 
     Metabolism, and Poisonous Plant Control Measures, Corvallis, 
     Oregon, USA, 23-24 February 1979, Oregon, Nutrition Research 
    Institute, pp. 109-113. 

    EL DAREER, S.M., TILLERY, K.F., LLOYD, H.H., & HILL, D.L. (1982)  
    Disposition of indicine  N-oxide in mice and monkeys.  Cancer Treat. 
     Rep., 66: 183-186. 

    EMMEL, M.W. (1948)   Crotalaria poisoning in cattle.  J. Am. Vet. 
     Med. Assoc., 113: 164. 

    EMMEL, M.W., SANDERS, D.A., & HENLEY, W.W. (1935)   Crotalaria 
     spectabilis seed poisoning in swine.  J. Am. Vet. Med. Assoc., 86: 
    43-49. 

    EVANS, J.V., PENG, A., & NIELSEN, C.J. (1979)  The gas 
    chromatographic mass spectrometric analysis of the new anti-tumour 
    drug indicine-N-oxide utilizing a novel reaction accompanying 
    trimethysilylation.  Biomed. mass Spectrom., 6: 38-43. 

    EVANS, J.V., DALEY, S.K., MCCLUSKY, G.A., & NIELSEN, C.J. (1980)  
    Direct quantitative analysis of indicine N-oxide in cancer patient 
    samples by gas chromatography using the internal standard 
    heliotrine  N-oxide including a mass spectral comparison of their 
    trimethylsilyl derivatives.  Biomed. mass Spectrom., 7: 65-73. 

    FARRINGTON, K.J. & GALLAGHER, C.H. (1960)  Complexes of copper with 
    some pyrrolizidine alkaloids and with some of their esterifying 
    acids.  Aust. J. biol. Sci., 13: 600-603. 

    FISH, M.S., SWEELEY, C.C., JOHNSON, N.M., LAWRENCE, E.P., & 
    HORNING, E.C. (1956)  Chemical and enzymic rearrangements of 
     N,N-dimethylaminoacid oxides.  Biochem. Biophys. Acta, 21: 196-197.

    FISHMAN, A.P. (1974)  Dietary pulmonary hypertension.  Circ. Res., 
    35: 657-660. 

    FORSYTH, A.A. (1968)   British poisonous plants, London, Her 
    Majesty's Stationery Office. 

    FOX, D.W., HART, M.C., BERGESON, P.S., JARRETT, P.B., STILLMAN, 
    A.E., & HUXTABLE, R.J. (1978)  Pyrrolizidine  (Senecio)  
    intoxication mimicking Reye's syndrome.  J. Paediatr., 93: 980-982. 

    FRAHN, J.L., CULVENOR, C.C.J., & MILLS, J.A. (1980)  Preparative 
    separation of the pyrrolizidine alkaloids, intermedine and 
    lycopsamine, as their borate complexes.  J. Chromatogr., 195: 
    379-383. 

    FURMANOWA, M., GUZEWSKA, J., & BELDOWSKA, B. (1983)  Mutagenic 
    effects of aqueous extracts of  Symphytum officinale L. and of its 
    alkaloidal fractions.  J. appl. Toxicol., 3: 127-130. 

    FURUYA, T. & HIKICHI, M. (1971)  Alkaloids and triterpenoids of 
     Symphytum officinale. Phytochemistry, 10: 2217-2220. 

    FUSHIMI, K., KATO, K., KATO, T., MATSUBARA, N., & HIRONO, I. (1978) 
    Carcinogenicity of flower stalks of  Petasites japonicus Maxim in 
    mice and Syrian golden hamsters.  Toxicol. Lett., 1: 291-294. 

    GADELLA, T.W.J., KLIPHUIS, E., & HUIZING, H.J. (1983)  Cyto- and 
    chemotaxonomical studies on the sections  officinalia and  coerulea  
    of the genus  Symphytum. Bot. Helv., 93: 169-192. 

    GANEY, P.E., FINK, G.D., & ROTH, R.A. (1985)  The effect of dietary 
    restriction and altered sodium intake on the cardiopulmonary 
    toxicity of monocrotaline pyrrole.  Toxicol. appl. Pharmacol., 78: 
    55-62. 

    GARDINER, M.R., ROYCE, R., & BOKOR, A. (1965)  Studies on 
     Crotalaria crispata, a newly recognized cause of Kimberley horse 
    disease.  J. Pathol. Bacteriol., 89: 43-55. 

    GHANEM, J. & HERSHKO, C. (1981)  Veno-occlusive disease and primary 
    hepatic vein thrombosis in Israeli Arabs.  Isr. J. med. Sci., 17: 
    339-347. 

    GHODSI, F. & WILL, J.A. (1981)  Changes in pulmonary structure and 
    function induced by monocrotaline intoxication.  Am. J. Physiol., 
    240: H149-H155. 

    GILRUTH, J.A. (1903)   Hepatic cirrhosis affecting horses and cattle 
     (so-called "Winton disease"), Wellington, New Zealand Department 
    of Agriculture, pp. 228-279 (11th annual report). 

    GOEGER, D.E., CHEEKE, P.R., SCHMITZ, J.A., & BUHLER, D.R. (1982)  
    Toxicity of tansy ragwort  (Senecio jacobaea) to goats.  Am. J. vet. 
     Res., 43: 252-254. 

    GOEGER, D.E., CHEEKE, P.R., RAMSDELL, H.S., NICHOLSON, S.S., & 
    BUHLER, D.R. (1983)  Comparison of the toxicities of  Senecio 
     jacobaea, Senecio vulgaris and  Senecio glabellus in rats. 
     Toxicol. Lett., 15: 19-23. 

    GONZALEZ, A.G., DE LA FUENTE, G., REINA, M., & LOYOLA, L.A. (1986a)  
    Pyrrolizidine alkaloids from  Senecio phillipicus and  Senecio 
     illinitus. Planta Med., 52: 160. 

    GONZALEZ, E., GARCIA, R., LEMUS, I., & ERAZO, S. (1986b)  
    Pharmacological studies on senecionine, a pyrrolizidine alkaloid 
    from  Senecio fistulosus Poepp. Ex less. (hualtata).  An. R. Acad. 
     Farm., 52(1): 123-132. 

    GRASES, P.J. & BEKER, S.G. (1972)  Veno-occlusive disease of the 
    liver. A case from Venezuela.  Am. J. Med., 53: 511-516. 

    GRAY, A.I., NIC, A.N., TSAOIR, E., & WATERMAN, P.G. (1983)  
    Hepatotoxic alkaloids and allantoin in  Symphytum tuberosum. J. 
     Pharm. Pharmacol., 35(Suppl): 13. 

    GREEN, C.R. & CHRISTIE, G.S. (1961)  Malformations in foetal rats 
    induced by the pyrrolizidine alkaloid, heliotrine.  Br. J. exp. 
     Pathol., 42: 369-378. 

    GREEN, M.H.L. & MURIEL, W.J. (1975)  Use of repair-deficient 
    strains of  Escherichia coli and liver microsomes to detect and 
    characterise DNA damage caused by the pyrrolizidine alkaloids 
    heliotrine and monocrotaline.  Mutat. Res., 28: 331-336. 

    GREEN, C.E., SEGALL, H.J., & BYARD, J.L. (1981)  Metabolism, 
    cytotoxicity, and genotoxicity of the pyrrolizidine alkaloid 
    senecionine in primary cultures of rat hepatocytes.  Toxicol. appl. 
     Pharmacol., 60: 176-185. 

    GUENGERICH, F.P. (1977)  Separation and purification of multiple 
    forms of microsomal cytochrome P-450.  J. biol. Chem., 252: 
    3970-3979. 

    GUENGERICH, F.P. & MITCHELL, M.B. (1980)  Metabolic activation of 
    model pyrroles by cytochrome P-450.  Drug Metab. Disp., 8: 34-38. 

    GUIDUGLI, F.H., PESTCHANKER, M.J., DESALMERON, M.S.A., & GIORDANO, 
    O.S. (1986)  1-hydroxyplatyphyllide, a norsequiterpene lactone 
    from  Senecio gilliesiano. Phytochemistry, 25: 1923-1926. 

    GUNER, N. (1986)  Alkaloids of  Heliotropium suaveolens. J. nat. 
     Prod., 49: 369. 

    GUPTA, P.S., GUPTA, G.D., & SHARMA, M.L. (1963)  Veno-occlusive 
    disease of the liver.  Br. med. J., 1: 1184-1186. 

    HABS, H.  (1982)   Senecio numorensis fuchsii. Carcinogenic and 
    mutagenic activity of an alkaloidal extract from a phytotherapeutic 
    drug.  Dtsch. Apoth. Ztg, 122: 799-804. 

    HABS, H., HABS, M., MARQUARDT, H., RODER, E., SCHMAHL, D., & 
    WIEDENFELD, H. (1982)  Carcinogenic and mutagenic activity of an 
    alkaloidal extract of  Senecio numorensis spp. fuchsii. 
     Arzneimittelforsch, 32: 144-148. 

    HAGGLUND, K.M., L'EMPEREUR, K.M., ROBY, M.R., & STERMITZ, F.R. 
    (1985)  Latifoline and latifoline  N-oxide from  Hackelia 
     floribunda, the western false forget-me-not.  J. nat. Prod., 48: 
    638-639. 

    HAMMOUDA, F.M., RIZK, A.M., ISMAIL, S.I., ATTEYA, S.Z., GHALEB, 
    H.A., MADKOUR, M.K., POWAND, A.E., & WOOD, G. (1984)  Poisonous 
    plants contaminating edible ones and toxic substances in plant 
    foods. Part 3: pyrrolizidine alkaloids from  Heliotropium digynum  
    Forssk (= H. luteum, Poir).  Pharmazie, 39: 703-705. 

    HARDING, J.D.J., LEWIS, G., DONE, J.T., & ALLCROFT, R. (1964)  
    Experimental poisoning by  Senecio jacobaea in pigs.  Pathol. Vet., 
    1: 204-220. 

    HARRIS, C., REDDY, J., CHIGA, M., & SVOBODA, D. (1969)  
    Polyribosomal disaggregation by lasiocarpine.  Biophys. Acta 180: 
    587-589. 

    HARRIS, P.N. & CHEN, K.K. (1970)  Development of hepatic tumours in 
    rats following ingestion of  Senecio longilobus.  Cancer Res., 30: 
    2881-2886. 

    HARRIS, P.N., ROSE, C.L., & CHEN, K.K. (1957)  Hepatotoxic and 
    pharmacological properties of heliotrine.  Arch. Pathol., 64: 
    152-157. 

    HARTMANN, T. & ZIMMER, M. (1986)  Organ-specific distribution and 
    accumulation of pyrrolizidine alkaloids during the life history of 
    2 annual  Senecio species.  J. plant Physiol., 122: 67-80. 

    HASHEM, M. (1939)  Etiology and pathology of types of liver 
    cirrhosis in Egyptian children.  J. Egypt. Med. Assoc., 22: 319-354. 

    HAYASHI, Y. (1966)  Excretion and alteration of monocrotaline in 
    rats after a subcutaneous injection (abstract).  Fed. Proc., 25: 688. 

    HAYASHI, Y. & LALICH, J.J. (1967)  Renal and pulmonary alterations 
    induced by a single injection of monocrotaline.  Proc. Soc. Exp. 
     Biol. Med., 124: 392-396. 

    HAYASHI, Y. & LALICH, J.J. (1968)  Protective effect of 
    mercaptoethylamine and cysteine against monocrotaline intoxication 
    in rats.  Toxicol. appl. Pharmacol., 12: 36-43. 

    HAYASHI, Y., HUSSA, J.F., & LALICH, J.J. (1967)  Cor pulmonale in 
    rats.  Lab. Invest., 16: 875-881. 

    HAYASHI, Y., KATO, M., OTSUKA, H.  (1979)  Inhibitory effects of 
    diet reduction on Monocrotaline intoxication in rats.   Toxicol. 
     Lett., 3: 151-155. 

    HAYASHI, Y., SHIMADA, M., & KATAYAMA, H. (1977)  Experimental 
    insulinoma in rats after a single administration of monocrotaline. 
     Toxicol. Lett., 1: 41-44. 

    HAYASHI, Y., KOKUBO, T., TAKAHASHI, M., FURUKAWA, F., OTSUKA, H., & 
    HASHIMOTO, K. (1984)  Correlative morphological and biochemical 
    studies on monocrotaline-induced pulmonary alterations in rats. 
     Toxicol. Lett., 21: 65-77. 

    HAYES, M.A., ROBERTS, E., & FARBER, E. (1985)  Initiation and 
    selection of resistant hepatocyte nodules in rats given the 
    pyrrolizidine alkaloids lasiocarpine and senecionine.  Cancer Res., 
    45: 3726-3734. 

    HEATH, D., SHABA, J., WILLIAMS, A., SMITH, P., & KOMBE, A. (1975)  
    A pulmonary hypertension-producing plant from Tanzania.  Thorax, 30: 
    399-404. 

    HENDRICKS, J.D., SINNHUBER, R.O., HENDERSON, M.D., & BUHLER, D.R. 
    (1981)  Liver and kidney pathology in rainbow trout  (Salmo 
     gairdneri)  exposed to dietary pyrrolizidine  (Senecio) alkaloids. 
     Exp. mol. Pathol., 35: 170.183. 

    HILL, K.R. (1960)  Worldwide distribution of seneciosis in man and 
    animals.  Proc. R. Soc. Med., 53: 281-282. 

    HILLIKER, K.S. & ROTH, R.A. (1984)  Alteration of monocrotaline 
    pyrrole-induced cardiopulmonary effects in rats by hydrallazine, 
    dexamethasone or sulphinpyrazone.  Br. J. Pharmacol., 82: 375-380. 

    HILLIKER, K.S., BELL, T.G., & ROTH, R.A. (1983)  Monocrotaline 
    pyrrole-induced pulmonary hypertension in fawn-hooded rats with 
    platelet storage pool deficiency: 5 hydroxytryptamine uptake by 
    isolated, perfused lungs.  Thromb. Haemostasis (Stuttgart), 50: 
    844-847. 

    HILLIKER, K.S., BELL, T.G., LORIMER, D., & ROTH, R.A. (1984)  
    Effects of thrombocytopaenia in monocrotaline pyrrole-induced 
    pulmonary hypertension.  Am. J. Physiol., 246: H747-H753. 

    HIRONO, I., SHIMIZU, M., FUSHIMI, K., MORI, H., & KATO, K. (1973)  
    Carcinogenic activity of  Petasites japonicus Maxim, a kind of 
    coltsfoot.  Gann Monogr. Cancer Res., 64: 527-528. 

    HIRONO, I., MORI, H., & CULVENOR, C.C.J. (1976)  Carcinogenic 
    activity of coltsfoot,  Tussilago farfara L.  Gann Monogr. Cancer 
     Res., 67: 125-129. 

    HIRONO, I., MORI, H., YAMADA, K., HIRATA, Y., HAGA, M., TATEMATSU, 
    H., & KANIE, S. (1977)  Carcinogenic activity of petasitenine, a 
    new pyrrolizidine alkaloid isolated from  Petasites japonicus  
    Maxim.  J. Natl Cancer Inst., 58: 1155-1157. 

    HIRONO, I., MORI, H., & HAGA, M. (1978)  Carcinogenic activity of 
     Symphytum officinale. J. Natl Cancer Inst., 61: 865-869. 

    HIRONO, I., HAGA, M., FUJII, M., MATSUURA, S., MATSUBARA, N., 
    NAKAYAMA, M., FURUYA, T., HIKICHI, M., TAKANASHI, H., UCHIDA, E., 
    HOSAKA, S., & UENO, I. (1979a)  Induction of hepatic tumours in 
    rats by senkirkine and symphytine.  J. Natl Cancer Inst., 63: 469-472. 

    HIRONO, I., MORI, H., HAGA, M., FUJII, M., YAMADA, K., TAKANASHI, 
    H., UCHIDA, E., HOSAKA, S., UENO, I., MATSUSHIMA, I., UMEZAVA, K., 
    & SHIRAI, A. (1979b)  Edible plants containing carcinogenic 
    pyrrolizidine alkaloids in Japan. In: Miller, B.C. ET AL., ed. 
     Naturally occurring carcinogens, mutagens, and modulators of 
     carcinogenesis, Baltimore, Maryland, University Park Press, pp. 
    79-87. 

    HIRONO, I., UENO, I., AISO, S., YAMAJI, T., & HAGA, M. (1983)  
    Carcinogenic activity of  Farfagium japonicum and  Senecio 
     cannabifolius. Cancer Lett., 20: 191-198. 

    HOET, P., ASTIGUETA, M., & FRISQUE, A.M. (1981)  Phytochemical 
    study of  Crotalaria nitens. Rev. Latinoam. Quim., 12: 34-35. 

    HOOPER, P.T. (1972)  Spongy degeneration in the brain in relation 
    to hepatic disease and ammonia toxicity in domestic animals.  Vet. 
     Res., 90: 37-38. 

    HOOPER, P.T. (1974)  The pathology of  Senecio jacobaea poisoning of 
    mice.  J. Pathol., 113: 227-230. 

    HOOPER, P.T. (1975a)  Spongy degeneration in the central nervous 
    system of domestic animals. Part I: morphology.  Acta neuropathol. 
     (Berlin), 31: 325-334. 

    HOOPER, P.T. (1975b)  Spongy degeneration in the central nervous 
    system of domestic animals. Part III: occurrence and pathogenesis - 
    hepatocerebral disease caused by hyper-ammonaemia.  Acta 
     neuropathol. (Berlin), 31: 343-351. 

    HOOPER, P.T. (1975c)  Experimental acute gastro-intestinal disease 
    caused by the pyrrolizidine alkaloid, lasiocarpine.  J. comp. 
     Pathol., 85: 341-349. 

    HOOPER, P.T. (1978)  Pyrrolizidine alkaloid poisoning-pathology 
    with particular reference to differences in animal and plant 
    species. In: Keeler, R.F., Van Kampen, K.R., & James, L.F., ed. 
     Effects of poisonous plants on livestock, New York, Academic 
    Press, pp. 161-176. 

    HOOPER, P.T. & SCANLAN, W.A. (1977)   Crotalaria retusa poisoning in 
    pigs and poultry.  Aust. vet. J., 53: 109-114. 

    HOOPER, P.T., BEST, S.M., & MURRAY, D.R. (1974)  Hyper-ammonaemia 
    and spongy degeneration of the brain in sheep affected with hepatic 
    necrosis.  Res. vet. Sci., 16: 216-222. 

    HOOSON, J. & GRASSO, P. (1976)  Cytotoxic and carcinogenic response 
    to monocrotaline pyrrole.  J. Pathol., 118: 121-127. 

    HOU JINQUI, XIA YUDIN, YU ZHANSHI, AN YAN, & TANG YIXAI  (1980)  
    [Veno-occlusive disease of the liver with report of 2 cases.]  Chung 
     Hua Nei ko Tsa Chih, 19: 187-191 (in Chinese). 

    HSU, I.C., ALLEN, J.R., & CHESNEY, C.F. (1973)  Identification and 
    toxicological effects of dehydroretronecine, a metabolite of 
    monocrotaline.  Proc. Soc. Exp. Biol. Med., 144: 834-838. 

    HSU, I.C., SHUMAKER, R.C., & ALLEN, J.R. (1974)  Tissue 
    distribution of tritium labelled dehydroretronecine.  Chem.-biol. 
     Interact., 8: 163. 

    HUIZING, H.J., DE BOER, R., & MALINGRE, T.M. (1981)  Preparative 
    ion-pair high performance liquid chromatography and gas 
    chromatography of pyrrolizidine alkaloids from comfrey.  J. 
     Chromatogr., 214: 257-262. 

    HURLEY, J.V. & JAGO, M.V. (1975)  Pulmonary oedema in rats given 
    dehydromonocrotaline: a topographic and electron-microscopic 
    study.  J. Pathol., 117: 23-32. 

    HUXTABLE, R.J. (1980)  Herbal teas and toxins: novel aspects of 
    pyrrolizidine poisoning in the United States.  Perspect. Biol. Med., 
    24: 1-14. 

    HUXTABLE, R., PAPLANUS, S., & LAUGHARN, J. (1977)  The prevention 
    of monocrotaline induced right ventricular hypertrophy.  Chest, 71: 
    308-310. 

    HUXTABLE, R.J., CIARAMITARO, D., & EISENSTEIN, D. (1978)  The 
    effect of a pyrrolizidine alkaloid, monocrotaline and a pyrrole, 
    dehydroretronecine, on the biochemical functions of the pulmonary 
    endothelium.  Mol. Pharmacol., 14: 1189-1203. 

    HUXTABLE, R.J., LUTHY, J., & ZWEIFEL, V. (1986)  Toxicity of 
    comfrey-pepsin preparations: letters to the editor.  New Engl. J. 
     Med., 315: 1095. 

    IARC  (1976)  Pyrrolizidine alkaloids. In:  Some naturally occurring 
     substances, Lyons, International Agency for Research on Cancer, 
    pp. 265-342 (IARC Monograph on the Evaluation of Carcinogenic Risk 
    of Chemicals to Man, Vol. 10). 

    IARC  (1983)   Some food additives, feed additives and naturally 
     occurring substances, Lyons, International Agency for Research on 
    Cancer, pp. 207-245 (IARC Monograph on the Evaluation of 
    Carcinogenic Risk of Chemicals to Humans, Vol. 31). 

    INDIAN COUNCIL OF MEDICAL RESEARCH  (1955)  Infantile cirrhosis of 
    the liver in India.  Indian J. med. Res., 43: 723-747. 

    ISMAILOV, N.I. (1948a)  [On clinical features, etiology and 
    pathogenesis of heliotropic dystrophy (toxic hepatitis with 
    ascites).]  Clin. Med., 8: 28 (in Russian). 

    ISMAILOV, N.I. (1948b)   [Heliotropic toxicosis (toxic hepatitis 
     with ascites),]  Tashkent, Academy of Sciences of Uzbekistan,
    pp. 1-120 (in Russian). 

    ISMAILOV, N.I., MADZHIDOV, N.M., MAGRUPOV, A.I., MAKHKAMOV, G.M., & 
    MUKMINOVA, S.G. (1970)  [Clinical signs, diagnosis and treatment of  
     Trichodesma toxicosis (alimentary toxic encephalopathy),]  
    Tashkent, Meditsina, p. 85 (in Russian). 

    JAGO, M.V. (1969)  The development of the hepatic megalocytosis 
    of chronic pyrrolizidine alkaloid poisoning.  Am. J. Pathol., 56: 
    405-422. 

    JAGO, M.V. (1970)  A method for the assessment of the chronic 
    hepatotoxicity of pyrrolizidine alkaloids.  Aust. J. exp. Biol. med. 
     Sci., 48: 93-103. 

    JAGO, M.V. (1971)  Factors affecting the chronic hepatotoxicity 
    of pyrrolizidine alkaloids.  J. Pathol., 105: 1-11. 

    JAGO, M.V., LANIGAN, G.W., BINGLEY, J.B., PIEREY, D.W.T., WHITTEN, 
    J.H., & TITCHEN, D.A. (1969)  Excretion of the pyrrolizidine 
    alkaloid heliotrine in the urine and bile of sheep.  J. Pathol., 98: 
    115-128. 

    JAGO, M.V., EDGAR, J.A., SMITH, L.W., & CULVENOR, C.C.J. (1970)  
    Metabolic conversion of heliotrine based pyrrolizidine alkaloids to 
    dehydroheliotridine.  Mol. Pharmacol., 6: 402-406. 

    JELLIFFE, D.B., BRAS, G., & STUART, K.L. (1954a)  Veno-occlusive 
    disease of the liver.  Paediatrics, 14: 334-339. 

    JELLIFFE, D.B., BRAS, G., & STUART, K.L. (1954b)  The clinical 
    picture of veno-occlusive disease of the liver in Jamaican 
    children.  Ann. trop. Med. Parasitol., 48: 386-396. 

    JELLIFFE, D.B., BRAS, G., & MUKHERJEE, K.L. (1957)  Veno-occlusive 
    disease of the liver and Indian childhood cirrhosis.  Arch. dis. 
     Child., 32: 369-385. 

    JOHNSON, A.E. (1976)  Changes in calves and rats consuming milk 
    from cows fed chronic lethal doses of  Senecio jacobaea (tansy 
    ragwort).  Am. J. vet. Res., 37: 107-110. 

    JOHNSON, A.E. (1979)  Toxicity of tansy ragwort to cattle.  In: 
    Cheeke, P.R., ed.  Proceedings of the Symposium on Pyrrolizidine 
     (Senecio) Alkaloids: Toxicity, Metabolism, and Poisonous Plant 
     Control Measures, Corvallis, Oregon, USA, 23-24 February 1979,  
    Oregon, Nutrition Research Institute, pp. 129-134. 

    JOHNSON, A.E. (1982)  Failure of mineral-vitamin supplements to 
    prevent tansy ragwort  (Senecio jacobaea) toxicosis in cattle.  Am. 
     J. vet. Res., 43: 718-723. 

    JOHNSON, A.E. & MOLYNEUX, R.J. (1984)  Toxicity of thread leaf 
    groundsel  (Senecio douglasii var.  longilobus) to cattle.  Am. J. 
     vet. Res., 45: 26-31. 

    JOHNSON, A.E., MOLYNEUX, R.J., & STUART, L.D. (1985)  Toxicity of 
    Riddel's groundsel  (Senecio riddelli) to cattle.  Am. J. vet. Res., 
    46: 577-582. 

    JOHNSON, W.D. (1981)  Mechanism of  in vitro acute toxicity of 
    dehydromonocrotaline a metabolite of pyrrolizidine alkaloid 
    monocrotaline.  Toxicologist, 1: 107-108. 

    JOHNSON, W.D., ROBERTSON, K.A., POUNDS, J.G., & ALLEN, J.R. (1978)  
    Dehydroretronecine-induced skin tumours in mice.  J. Natl Cancer 
     Inst., 61: 85-89. 

    JULIEN, M.H., ed.  (1982)   Biological control of weeds. A world 
     catalog of agents and their target weeds, Curepe, Trinidad and 
    Toabago, Commonwealth Institute of Biological Control. 

    JUNEJA, T.R., GUPTA, R.L., & SAMANTA, S. (1984)  Activation of 
    monocrotaline, fulvine and their derivatives to toxic pyrroles by 
    some thiols.  Toxicol. Lett., 21: 185-189. 

    KAMPANZEV, N.N. (1952)  [Experimental heliotropic hepatitis.] In: 
    Milenkov, S.M. & Kizhaikin, Y., ed.  [Collection of scientific 
     papers on Toxic Hepatitis with Ascites,] Tashkent, Publishing 
    House of the University of Central Asia, pp. 165-172 (in Russian). 

    KAY, J.M. & HEATH, D. (1966)  Observation on the pulmonary arteries 
    and heart weight of rats fed on  Crotalaria spectabilis seeds.  J. 
     Pathol., 92: 385-394. 

    KAY, J.M. & HEATH, D. (1969)   Crotalaria spectabilis: the pulmonary 
     hypertension plant, Springfield, Illinois, Charles C. Thomas, p. 
    38. 

    KAY, J.M., HARRIS, P., & HEATH, D. (1967a)  Pulmonary hypertension 
    produced in rats by ingestion of  Crotalaria spectabilis seeds. 
     Thorax, 22: 176-179. 

    KAY, J.M., GILLUND, T.D., & HEATH, D. (1967b)  Mast cells in the 
    lungs of rats fed on  Crotalaria spectabilis seeds.  Am. J. Pathol., 
    51: 1031-1044. 

    KAY, J.M., HEATH, D., SMITH, P., BRAS, G., & SUMMERELL, J. (1971a)  
    Fulvine and pulmonary circulation.  Thorax, 26: 249-261. 

    KAY, J.M., SMITH, P., & HEATH, D. (1971b)  Aminorex and the 
    pulmonary circulation.  Thorax, 26: 262-270. 

    KAY, J.M., KEANE, P.M., & SUYAMA, K.L. (1985)  Pulmonary 
    hypertension induced in rats by monocrotaline and chronic hypoxia 
    is reduced by  p-chlorophenylalanine.  Respiration, 47: 48-56. 

    KEDZIERSKI, B. & BUHLER, D.R. (1985)  Configuration of necine 
    pyrroles - toxic metabolites of pyrrolizidine alkaloids.  Toxicol. 
     Lett., 25: 115-119. 

    KEDZIERSKI, B. & BUHLER, D.R. (1986)  The formation of 6,7-di- 
    hydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, a metabolite of 
    pyrrolizidine alkaloids.  Chem.-biol. Interact., 57: 217-222. 

    KHANIN, M.N. (1948)  [The etiology of toxic hepatitis with 
    ascites.]  Arch. Pathol. USSR, 1: 42-47 (in Russian). 

    KIM, H.L. & JONES, L.P. (1982)  Protective effects of butylated 
    anisole, ethoxyquin and disulfiran on acute pyrrolizidine alkaloid 
    poisoning in mice.  Res. Comm. chem. Pathol. Pharmacol., 36: 341-344. 

    KIRFEL, A., WILL, G., WIEDENFELD, H. & RODER, E.  (1980)  
    (1aR,6bR,10R,11R)-9,15-dioxo-10-hydroxy-10,11,13-trimethyl-1a,2, 
    3,6b-tetrabydro-5H-pyrrolizino-[1a,6b,6a,c],8-dioxa-15-cis-tride 
    cene,C18H25NO5.   Cryst. Styruct. Comm., 9: 353-361. 

    KNIGHT, A.P., KIMBERLING, C.V., STERMITZ, F.R., & ROBY, M.R. (1984)  
     Cynoglossum officinale (hound's tongue) - a cause of pyrrolizidine 
    alkaloid poisoning in horses.  J. Am. Vet. Med. Assoc., 185: 647-650. 

    KOEKEMOER, M.J. & WARREN, F.L. (1951)  The occurrence and 
    preparation of the  N -oxides. An improved method of extraction of 
    the  Senecio alkaloids.  J. Chem. Soc., C1951: pp. 66-68. 

    KOLETSKY, A., OYASU, R., & REDDY, J.K. (1978)  Mutagenicity of the 
    pyrrolizidine  (Senecio) alkaloid, lasiocarpine in the 
    salmonella/microsome test.  Lab. Invest., 38: 352 (Abstract). 

    KOVACH, J.S., MOERTEL, C.G., & HAHN, R.G. (1979a)  A phase 1 study 
    of indicine- N-oxide.  Proc. Am. Assoc. Cancer Res., 20: 357. 

    KOVACH, J.S., AMES, M.M., POWIS, G., MOERTEL, C.G., HAHN, R.G., & 
    CREAGAN E.T.  (1979b)  Toxicity and pharmacokinetics of a 
    pyrrolizidine alkaloid, indicine- N-oxide, in humans.  Cancer Res., 
    39: 4540-4544. 

    KRAUS, C., ABEL, G., & SCHIMMER, O. (1985)  [Studies on the 
    chromosome damaging effect of some pyrrolizidine alkaloids in human 
    lymphocytes  in vitro.]  Planta Med., 51: 89-91 (in German). 

    KRISHNAMACHARI, K.A.V.R., BHAT, R.V., KRISHNAMURTHY, D., 
    KRISHNASWAMY, K., & NAGARAJAN  (1977)  Aetiopathogenesis of endemic 
    ascites in Sarguja district of Madhya Pradesh.  Indian J. med. Res., 
    65: 672-678.

    KUHARA, K., TAKANASHI, H., HIRONO, I., FURUYA, T., & ASADA, Y. 
    (1980)  Carcinogenic activity of clivorine, a pyrrolizidine 
    alkaloid isolated from  Ligularia dentata. Cancer Lett., 10: 117-122. 

    KUMANA, C.R., NG, M., LIN, H.J., KO, W., WU, P.C., & TODD, D. 
    (1983)  Hepatic veno-occlusive disease due to toxic alkaloid herbal 
    tea - letter to the editor.  Lancet, 10 December: 1360-1361. 

    KUMANA, C.R., NG, M., LIN, H.J., KO, W., WU, P.C., & TODD, D. 
    (1985)  Herbal tea induced hepatic veno-occlusive disease: 
    quantification of toxic alkaloid exposure in adults.  Gut, 26: 
    101-104. 

    KUROZUMI, T., TANAKA, K., KIDO, M., & SHOYAMA, Y. (1983)  
    Monocrotaline-induced renal lesions.  Exp. mol. Pathol., 39: 377-386. 

    LAFRANCONI, W.M. & HUXTABLE, R.J. (1983)  Changes in angiotensin-
    converting enzyme activity in lungs damaged by the pyrrolizidine 
    alkaloid, monocrotaline.  Thorax, 38: 307-309. 

    LAFRANCONI, W.M. & HUXTABLE, R.J. (1984)  Hepatic metabolism and 
    pulmonary toxicity of monocrotaline using isolated perfused liver 
    and lung.  Biochem. Pharmacol., 33: 2479-2484. 

    LAFRANCONI, W.M., DUHAMEL, R.C., BRENDEL, K., & HUXTABLE,R.J.  
    (1984)  Differentiation of the cardiac and pulmonary toxicity of 
    monocrotaline, a pyrrolizidine alkaloid.  Biochem. Pharmacol., 33: 
    191-197. 

    LAFRANCONI, W.M., OHNUMA, S., & HUXTABLE, R.S.  (1985)  Biliary 
    excretion of novel pneumotoxic metabolites of the pyrrolizidine 
    alkaloid monocrotaline.  Toxicon, 23: 903-992. 

    LALICH, J.J. & EHRHART, L.A. (1962)  Monocrotaline induced 
    pulmonary arteritis in rats.  J. atheroscler. Res., 2: 482-492. 

    LALICH, J.J. & MERKOW, L. (1961)  Pulmonary arteritis produced in 
    rats by feeding  Crotalaria spectabilis. Lab. Invest., 10: 744-750. 

    LANCET  (1984)  Pyrrolizidine alkaloids (editorial).  Lancet, 1: 
    201-202. 

    LANGLEBEN, D. & REID, L.M. (1985) Effect of methylprednisolone on 
    monocrotaline-induced pulmonary vascular disease and right 
    ventricular hypertrophy.  Lab. Invest., 52: 298-303. 

    LANIGAN, G.W. (1971)  Metabolism of pyrrolizidine alkaloids in the 
    ovine rumen. III. The competitive relationship between heliotrine 
    metabolism and methanogenesis in rumen fluid  in vitro. Aust. J. 
     agric. Res., 22: 123-130. 

    LANIGAN, G.W. (1972)  Metabolism of pyrrolizidine alkaloids in 
    ovine rumen. IV. Effects of chloral hydrate and halogenated 
    methanes on rumen methanogenesis and alkaloid metabolism in 
    fistulated sheep.  Aust. J. agric. Res., 23: 1085-1091. 

    LANIGAN, G.W. & SMITH, L.W. (1970a)  Metabolism of pyrrolizidine 
    alkaloids in the ovine rumen. I. Formation of 7 alpha-hydroxy-
    1alpha-methyl-8alpha-pyrrolizidine from heliotrine and 
    lasiocarpine.  Aust. J. agric. Res., 21: 493-500. 

    LANIGAN, G.W. & SMITH, L.W. (1970b)  Metabolism of pyrrolizidine 
    alkaloids in the ovine rumen. II. Formation of 7alpha-hydroxy-
    1alpha-methyl-8alpha-pyrrolizidine from heliotrine and 
    lasiocarpine.  Aust. J. agric. Res., 22: 123-130. 

    LANIGAN, G.W. & WHITTEM, J.H. (1970)  Cobalt pellets and 
     Heliotropium europaeum poisoning in penned sheep.  Aust. vet. J., 
    46: 17-21. 

    LANIGAN, G.W., PAYNE, A.L., & PETERSON, J.E. (1978)  Anti- 
    methanogenic drugs and  Heliotropium europaeum poisoning in penned 
    sheep.  Aust. J. Agric. Res., 29: 1281-1292. 

    LAWS, L.  (1968)  Toxicity of  Crotalaria mucronata to sheep.  Aust. 
     vet. J., 44: 453-455. 

    LETENDRE, L., SMITHSON, W.A., GILCHRIST, G.S., BURGERT, E.O., Jr, 
    HOGLAND, C.H., AMES, M.M., POWIS, G., & KOVACH, J.S. (1981)  
    Activity of indicine- N-oxide in refractory acute leukemia.  Cancer, 
    47: 437-441. 

    LETENDRE, L., LUDWIG, J., PERRAULT, J., SMITHSON, W.A., & KOVACH, 
    J.S. (1984)  Hepatocellular toxicity during the treatment of 
    refractory acute leukemia with indicine- N-oxide.  Cancer, 54: 
    1256-1259. 

    LEVINE, O.R., HARRIS, R.C., BLANCE, W.A., & MELLINS, R.B. (1973)  
    Progressive pulmonary hypertension in children with portal 
    hypertension.  J. Pediatr., 83: 964-972. 

    LIN, J.J., LIU, C., & SVOBODA, D.J. (1974)  Long-term effects of 
    aflatoxin B1 and vocal hepatitis on marmoset liver: a preliminary 
    report.  Lab. Invest., 30: 267-278. 

    LUTHY, J., ZWEIFEL, U., SCHLATTER, C., & BENN, M.H. (1980)  
    [Pyrrolizidine alkaloids in coltsfoot ( Tussilago farfara L.) of 
    various sources.]  Mitt. Geb. Lebensm. Hyg., 71: 73-80 (in German 
    with English summary). 

    LUTHY, J., ZWEIFEL, U., KARLHUBER, B., & SCHLATTER, C. (1981)  
    Pyrrolizidine alkaloids of  Senecio alpinus L. and their detection 
    in feedstuffs.  J. agric. food Chem., 29: 302-305. 

    LUTHY, J., HEIM, TH., & SCHLATTER, CH. (1983)  Transfer of [3H] 
    pyrrolizidine alkaloids from  Senecio vulgaris L. and metabolites 
    into rat milk and tissues.  Toxicol. Lett., 17: 283-288. 

    LUTHY, J., BRAUCHLI, J., ZWEIFEL, U., SCHMID, P., & SCHLATTER, CH. 
    (1984)  [Pyrrolizidine alkaloid in arzneipflanzen der Boraginaceen: 
     Borago officinalis  L. and  Pulmonaria officinalis L.]  Pharm. Acta 
     Helv., 59: 242-246 (in German). 

    LYFORD, C.L., VERGAVA, G.G., & MOELLER, D.D. (1976)  Hepatic veno-
    occlusive disease originating in Ecuador.  Gastro-enterology, 70: 
    105-108. 

    MCCOMISH, M., BODEK, I., & BRAUFMAN, A.R. (1980)  Quantitation of 
    the antineoplastic agent indicine- N-oxide in human plasma by 
    differential pulse polarography.  J. pharmacol. Sci., 69: 727-729. 

    MCCOY, J.W., ROBY, M.R., & STERMITZ, F.R. (1983)  Analysis of plant 
    alkaloid mixtures by ammonia chemical ionization mass spectroscopy. 
     J. nat. Prod., 46: 894-900. 

    MCGEE, J.O'D., PATRICK, R.S., WOOD, C.B., & BLUMGART, L.H. (1976)  
    A case of veno-occlusive disease of the liver in Britain associated 
    with herbal tea consumption.  J. clin. Pathol., 29: 788-794. 

    MCGRATH, J.P.M., DUNCAN, J.R., & MUNNELL, J.F. (1975)   Crotalaria 
     spectabilis toxicity in swine: characterization of the renal 
    glomerular lesion.  J. comp. Pathol., 85(2): 185-194. 

    MCLEAN, E.K. (1969)  The early sinusoidal lesion in experimental 
    veno-occlusive disease.  Br. J. exp. Pathol., 50: 223-229. 

    MCLEAN, E.K. (1970)  The toxic actions of pyrrolizidine  (Senecio)  
    alkaloids.  Pharmacol. Rev., 22: 429-483. 

    MCLEAN, E.K. (1974)   Senecio and other plants as liver poisons. 
     Isr. J. med. Sci., 10: 436-440. 

    MCLEAN, E.K. & HILL, K.R. (1969)  Portal hypertension in acute 
    experimental veno-occulsive disease of the liver in rats.  Br. J. 
     exp. Pathol., 50: 37-41. 

    MCLEAN, E.K., BRAS, G., & GYORGY, P. (1964)  Veno-occlusive lesions 
    in livers of rats fed  Crotalaria fulva. Br. J. exp. Pathol., 45: 
    242-247. 

    MAKSUDOV, A.M. (1952)  [Heliotropic toxic hepatitis with ascites.] 
    In: Milenkov, S.M. & Kizhaikin, Y., ed.  [Collection of scientific 
     papers on Toxic Hepatitis with Ascites,] Tashkent, Publishing House 
    of the University of Central Asia, pp. 103-116 (in Russian). 

    MARTIN, P.A., THORBURNE, M.K., HUTCHINSON, S., BRAS, G., & MILLER, 
    C.G. (1972)  Preliminary findings of chromosome studies in rats and 
    humans with veno-occlusive disease.  Br. J. exp. Pathol., 53, 374-380. 

    MATTOCKS, A.R. (1961)  Extraction of heat-labile alkaloids from 
    plants.  Nature (Lond.), 191: 1281-1282. 

    MATTOCKS, A.R. (1967a)  Spectrophotometric determination of 
    unsaturated pyrrolizidine alkaloids.  Anal. Chem., 39: 443-447. 

    MATTOCKS, A.R. (1967b)  Detection of pyrrolizidine alkaloids on 
    thin-layer chromatograms.  J. Chromatogr., 27: 505-508. 

    MATTOCKS, A.R. (1968a)  Toxicity of pyrrolizidine alkaloids.  Nature 
     (Lond.), 217: 723-728. 

    MATTOCKS, A.R. (1968b)  Spectrophotometric determination of 
    pyrrolizidine alkaloids: some improvements.  Anal. Chem., 40: 1749. 

    MATTOCKS, A.R. (1969)  Dehydropyrrolizine derivatives from 
    unsaturated pyrrolizidine alkaloids.  J. Chem. Soc., C1969: 
    1155-1162. 

    MATTOCKS, A.R. (1971a)  Synthetic compounds with toxic properties 
    similar to those of pyrrolizidine alkaloids and their pyrrolic 
    metabolites.  Nature (Lond.), 232: 476. 

    MATTOCKS, A.R. (1971b)  The occurrence and analysis of 
    pyrrolizidine alkaloid  N-oxides.  Xenobiotica, 1: 451-453. 

    MATTOCKS, A.R. (1971c)  Hepatotoxic effects due to pyrrolizidine 
    alkaloid  N-oxides.  Xenobiotica, 1: 563-565. 

    MATTOCKS, A.R. (1971d)  A field test for  N-oxides of unsaturated 
    pyrrolizidine alkaloids.  Trop. Sci., 13: 65-70. 

    MATTOCKS, A.R. (1972a)  Toxicity and metabolism of  Senecio 
    alkaloids. In: Harborne, J.B., ed.  Phytochemical ecology, London, 
    New York, Academic Press, pp. 179-200. 

    MATTOCKS, A.R. (1972b)  Acute hepatotoxicity and pyrrolic 
    metabolites in rats dosed with pyrrolizidine alkaloids.  Chem.-biol. 
     Interact., 5: 227-242. 

    MATTOCKS, A.R. (1973)  Mechanisms of pyrrolizidine alkaloid 
    toxicity. Pharmacology and the future of man. In:  Proceedings of 
     the 5th International Congress on Pharmacology, San Francisco,  
     1972, Basel, Karger, Vol. 2, pp. 114-123. 

    MATTOCKS, A.R. (1977)  Tissue distribution of radioactivity in rats 
    given tritiated analogues of hepatotoxic pyrrolizidine alkaloids. 
     Xenobiotica, 7: 665-670. 

    MATTOCKS, A.R. (1980)  Toxic pyrrolizidine alkaloids in comfrey. 
     Lancet, 22 November: 1136-1137. 

    MATTOCKS, A.R. (1981a)  Liver cell enlargement in rats given 
    hydroxymethyl pyrroles analogous to pyrrolizidine alkaloid 
    metabolites, followed later by the hepatotoxin dimethylnitrosamine. 
     Toxicol. Lett., 8: 201-205. 

    MATTOCKS, A.R. (1981b)  Relation of structural features to pyrrolic 
    metabolites in livers of rats given pyrrolizidine alkaloids and 
    derivatives.  Chem.-biol. Interact., 35: 301-310. 

    MATTOCKS, A.R. (1981c)  A simple preparation of dehydroretronecine 
    using potassium nitrosodisulphonate.  Chem. Ind., 7: 251. 

    MATTOCKS, A.R. (1982)  Hydrolysis and hepatotoxicity of retronecine 
    diesters.  Toxicol. Lett., 14: 111-116. 

    MATTOCKS, A.R. (1986)   Chemistry and toxicology of pyrrolizidine 
     alkaloids, London, New York, Academic Press. 

    MATTOCKS, A.R. & BIRD, I. (1983)  Pyrrolic and  N-oxide metabolites 
    formed from pyrrolizidine alkaloids by hepatic microsomes  in vitro:  
    relevance to  in vivo hepatotoxicity.  Chem.-biol. Interact., 43: 
    209-222. 

    MATTOCKS, A.R. & CABRAL, J.R.P. (1979)  Effects of some pyrrolic 
    and dehydropyrrolizine esters on mouse skin: a preliminary study.
     Tumori, 65: 289-293. 

    MATTOCKS, A.R. & CABRAL, J.R.P. (1982)  Carcinogenicity of some 
    pyrrolizidine alkaloid metabolites and analogues.  Cancer Lett., 17: 
    61-66. 

    MATTOCKS, A.R. & DRIVER, H.E. (1983)  A comparison of the 
    pneumotoxicity of some pyrrolic esters and similar compounds 
    analogous to pyrrolizidine alkaloid metabolites given intravenously 
    to rats.  Toxicology, 27: 159-177. 

    MATTOCKS, A.R. & DRIVER, H.E.  (1987)  Toxic actions of senaetnine, 
    a new pyrrolizidine alkaloid, in rats.  Toxicol. Lett., 38: 315-319. 

    MATTOCKS, A.R. & JUKES, R. (1987)  New improved field test for 
    toxic pyrrolizidine alkaloids.  J. nat. Prod., 50: 161-166. 

    MATTOCKS, A.R. & LEGG, R.F. (1980)  Antimitotic activity of 
    dehydroretronecine, a pyrrolizidine alkaloid metabolite, and some 
    analogous compounds in a rat liver parenchymal cell line.  Chem.-
     biol. Interact., 30: 325-336. 

    MATTOCKS, A.R. & WHITE, I.N.H. (1970)  Estimation of metabolites 
    of pyrrolizidine alkaloids in animal tissues.  Anal. Biochem., 38: 
    529-535. 

    MATTOCKS, A.R. & WHITE, I.N.H. (1971a)  The conversion of 
    pyrrolizidine alkaloids to dihydropyrrolizine derivatives by rat-
    liver microsomes  in vitro. Chem.-biol. Interact., 3: 383-396. 

    MATTOCKS, A.R. & WHITE, I.N.H. (1971b)  Pyrrolic metabolites from 
    non-toxic pyrrolizidine alkaloids.  Nat. new Biol., 231: 114-115. 

    MATTOCKS, A.R. & WHITE, I.N.H. (1973)  Toxic effects and pyrrolic 
    metabolites in the liver of young rats given the pyrrolizidine 
    alkaloid retrorsine.  Chem.-biol. Interact., 6: 297-306. 

    MATTOCKS, A.R. & WHITE, I.N.H. (1976)  The distribution of
    [3H]-synthanecine A bis- n-ethylcarbamate and its metabolites
    in the rat.  Chem.-biol. Interact., 15: 173-184. 

    MEYRICK, B.O. & REID, L.M. (1979)  Development of pulmonary 
    arterial changes in rats fed  Crotalaria spectabilis. Am. J. 
     Pathol., 94: 37-50. 

    MEYRICK, B.O. & REID, L.M. (1982)  Crotalaria-induced pulmonary 
    hypertension: uptake of 3H-thymidine by the cells of the pulmonary 
    circulation and alveolar walls.  Am. J. Pathol., 106: 84-94. 

    MILKOWSKY, A.S. (1985)  The synthesis and anti-tumour activity of 
    monocyclic analogs of indicine- N-oxide.  Diss. Abstr. Int., 45(3): 
    844. 

    MILLER, W.C., RICE, D.L., KREUSEL, R.G., & BEDROSSIAN, C.W.M. 
    (1978)  Monocrotaline model of non-cardiogenic pulmonary edema in 
    dogs.  J. appl. Physiol., 45: 962-965.

    MIRANDA, C.L., BUHLER, D.R., & CHEEKE, P.R. (1979)  The effect of 
    tansy ragwort consumption on mineral metabolism in the rat. In: 
    Cheeke, P.R., ed.  Proceedings of the Symposium on Pyrrolizidine  
     (Senecio) Alkaloids: Toxicity, Metabolism, and Poisonous Plant 
     Control Measures, Corvallis, Oregon, USA, 23-24 February 1979,  
    Oregon, Nutrition Research Institute, pp. 61-64. 

    MIRANDA, C.L., CARPENTER, H.M., CHEEKE, P.R., & BUHLER, D.R. 
    (1981a)  Effect of ethoxyquin on the toxicity of pyrrolizidine 
    alkaloid monocrotaline and on hepatic long metabolism in mice. 
     Chem.-biol. Interact., 37: 95-107. 

    MIRANDA, C.L., HENDERSON, M.C., & BUHLER, D.R. (1981b)  Dietary 
    copper enhances the hepatotoxicity of  Senecio jacobaea in rats. 
     Toxicol. appl. Pharmacol., 60(3): 418-423. 

    MIRANDA, C.L., REED, R.L., CHEEKE, P.R., & BUHLER, D.R. (1981c)  
    Protective effects of butylated hydroxyanisole against the acute 
    toxicity of monocrotaline in mice.  Toxicol. appl. Pharmacol., 59: 
    424-430. 

    MIRANDA, C.L., BUHLER, D.R., RAMSDELL, H.S., CHEEKE, P.R., & 
    SCHMITZ, J.A. (1982a)  Modifications of chronic hepatotoxicity of 
    pyrrolizidine  (Senecio)  alkaloids by butylated hydroxyanisole 
    and cysteine.  Toxicol. Lett., 10: 177-182. 

    MIRANDA, C.L., HENDERSON, M.C., BUHLER, D.R., & SCHMITZ, J.A. 
    (1982b)  Comparative effects of antioxidants on the toxicity of 
    mixed pyrrolizidine alkaloids from  Senecio jacobaea in mice.  J. 
     Toxicol. environ. Health, 9(5-6): 933-939. 

    MIRANDA, C.L., HENDERSON, M.C., REED, R.L., SCHMITZ, J.A., & 
    BUHLER, D.R. (1982c)  Protective action of zinc against 
    pyrrolizidine alkaloid-induced hepatotoxicity in rats.  J. Toxicol. 
     environ. Health, 9: 359-366. 

    MIROCHNIK, M.F.  (1938)  [Clinical course and etiopathogenesis of 
    toxic hepatites with ascites.]   Medical Institute, scientific 
     papers of the second therapeutic clinic, Vol I, Tashkent, Medical 
    Literature Publishers. 

    MISER, J.S., MISER, A.W., SMITHSON, W.A., COCCIA, P.F., AMES, M.M., 
    DAVIS, D.M., HUGHES, C.S., & GILCHRIST, G.S.  (1982)  A phase I 
    trial of indicine- N-oxide in childhood malignancy.  Proc. Am. Soc. 
     Clin. Oncol., 1: 137. 

    MNUSHKIN, A.S. (1949)  [Some materials to clinical features and 
    pathogenesis of toxic hepatitis with ascites.]   Sov. Med., 1: 8-9 
    (in Russian).

    MNUSHKIN, A.S. (1952)  [The clinical features, pathogenesis and 
    treatment of toxic hepatitis with ascites.] In: Milenkov, S.M. & 
    Kizhaikin, Y., ed.  [Collection of scientific papers on Toxic 
     Hepatitis with Ascites,]  Tashkent, Publishing House of the 
    University of Central Asia, pp. 91-98 (in Russian). 

    MOHABBAT, O., SRIVASTAVA, R.N., YOUNOS, M.S., SEDIQ, G.G., MENZAD, 
    A.A., & ARAM, G.N. (1976)  An outbreak of hepatic veno-occlusive 
    disease in north-western Afghanistan.  Lancet, 7 August: 269-271. 

    MOLTENI, A., WARD, W.F., TS'AO, C.-S., PORT, C.D., & SOLLIDAY, N.H. 
    (1984)  Monocrotaline-induced pulmonary endothelial dysfunction in 
    rats.  Proc. Soc. Exp. Biol. Med., 176: 88-94. 

    MOLYNEUX, R.J. & ROITMAN, J.N. (1980)  Specific detection of 
    pyrrolizidine alkaloids on thin-layer chromatograms.  J. 
     Chromatogr., 195: 412-415. 

    MOLYNEUX, R.J., JOHNSON, A.E., ROITMAN, J.N., & BENSON, M.E. (1979)  
    Chemistry of toxic range plants. Determination of pyrrolizidine 
    alkaloid content and composition in  Senecio species by nuclear 
    magnetic resonance spectroscopy.  J. agric. food Chem., 27: 494-499. 

    MORI, H., SUGIE, S., YOSHIMI, N., ASADA, Y., FURUYA, T., & 
    WILLIAMS, G.M. (1985)  Genotoxicity of a variety of pyrrolizidine 
    alkaloids in the hepatocyte primary culture-DNA repair test using 
    rat, mouse, and hamster hepatocytes.  Cancer Res., 45: 3125-3129. 

    MUNIER, R. (1953)  Separation of alkaloids from their  N-oxides by 
    paper chromatography.  Bull. Soc. Chim. Biol., 35: 1225. 

    NARDI, N.B., GIMMLER, M.C., & LEITE, B.G. (1980)  Antimitotic 
    action of integerrimine in rats.  Rev. Bras. Genet., 3: 387-392. 

    NATIONAL CANCER INSTITUTE  (1978)   Bioassay of lasiocarpine for 
     possible carcinogenecity, Bethesda, Maryland, National Cancer 
    Institute, US Department of Health, Education and Welfare (Report 
    of Carcinogenesis Testing Programme, Division of Cancer Cause and 
    Prevention). 

    NAYAK, N.C., SAGREIYA, K., & RAMALINGASWAMI, V. (1969)  Indian 
    childhood cirrhosis - the nature and significance of cytoplasmic 
    hyaline of hepatocytes.  Arch. Pathol., 88: 631-637. 

    NEWBERNE, P.M. (1968)  The influence of a low isotrope diet on 
    response of maternal and fetal rats to lasiocarpine.  Cancer Res.,
    28: 2327-2337. 

    NEWBERNE, P.M. & ROGERS, A.E. (1973)  Nutrition, monocrotaline and 
    aflatoxin B1 in liver carcinogenesis.  Plant Food Man, 1: 23-31. 

    NEWBERNE, P.M., WILSON, R., & ROGERS, A.E. (1971)  Effects of a 
    low-lipotrope diet on the response of young male rats to the 
    pyrrolizidine alkaloid monocrotaline.  Toxicol. appl. Pharmacol., 18: 
    387-397. 

    NEWBERNE, P.M., CHAN, W.C., & ROGERS, A.E. (1974)  Influence of 
    light, riboflavin and carotene on the response of rats to the acute 
    toxicity of aflatoxin and monocrotaline.  Toxicol. appl. Pharmacol., 
    28: 200-208. 

    NICHOLS, W.C., MOERTL, C.G., RUBIN, J., SCHULT, A.J., & BRITTEL, 
    J.C. (1981)  Phase II trial of indicine- N-oxide (NSC132319) in 
    patients with advanced colorectal carcinoma.  Cancer Treat. Rep., 65: 
    337-339. 

    NIWA, H., ISHIWATA, H., & YAMADA, K. (1985)  Isolation of 
    petasitenine, a carcinogenic pyrrolizidine alkaloid from  Farfugium 
     japonicum. J. nat. Prod., 48: 10083-1004. 

    NOLAN, J.P., SCHEIG, R.L., & KLATSKIN, G. (1966)  Delayed hepatitis 
    and cirrhosis in weanling rats following a single small dose of the 
     Senecio alkaloid, lasiocarpine.  Am. J. Pathol., 49: 129-151. 

    OHNUMA, T., SRIDHAR, K.S., RATNER, L.H., & HOLLAND, J.F. (1982)  
    Phase I study of indicine- N-oxide in patients with advanced cancer. 
     Cancer Treat. Rep., 66(7): 1509-1515. 

    OHTSUBO, K., ITO, Y., SAITO, M., FURUYA, T., & HIKICHI, M. (1977)  
    Hypertrophy of pulmonary arteries and arterioles with cor pulmonale 
    in rats induced by seneciphylline, a pyrrolizidine alkaloid. 
     Experientia (Basel), 33: 498-499. 

    OLSON, J.W., HACKER, A.D., ALTIERE, R.J., & GILLESPIE, M.N. (1984)  
    Polyamines and the development of monocrotaline-induced pulmonary 
    hypertension.  Am. J. Physiol., 247: H682-H685. 

    OLSON, J.W., ATKINSON, J.E., HACKER, A.D., ALTIERE, R.J., & 
    GILLESPIE, M.N. (1985)  Suppression of polyamine biosynthesis 
    prevents monocrotaline induced pulmonary oedema and arterial medial 
    thickening.  Toxicol. appl. Pharmacol., 81: 91-99. 

    ORD, M.J., HERBERT, A., & MATTOCKS, A.R. (1985)  The ability of 
    bifunctional and monofunctional pyrrole compounds to induce sister 
    chromatid exchange (SCE) in human lymphocytes and mutations in 
     Salmonella typhimurium. Mutat. Res., 149: 485-493. 

    PANTONE, D.J., BROWN, S.M., & WOMERSLEY, C. (1985)  Biological 
    control of fiddleneck.  California Agric., 39: 4-9. 

    PECKHAM, J.C., SANGSTER, L.P., & JONES, O.H. (1974)   Crotalaria  
     spectabilis poisoning in swine.  J. Am. Vet. Med. Assoc., 165: 
    633-638. 

    PEDERSEN, E. (1975)  Pyrrolizidine alkaloids in Danish species of 
    the family Boraginaceae.  Arch. Pharm. Chem. Sci. Ed., 3: 55-64. 

    PERCY, J.J. & PIERCE, A.E. (1971)  Immunosuppressive activity of 
    the pyrrolizidine alkaloid metabolite dehydroheliotridine. 
     Immunology, 21: 273-280. 

    PERSAUD, J.V. & HOYTE, D.A. (1974)  Pregnancy and progeny in rats 
    treated with the pyrrolizidine alkaloid fulvine.  Exp. Pathol., 9: 
    59-63. 

    PESTCHANKER, M.J. & GIORDANO, O.S. (1986)  Pyrrolizidine alkaloids 
    from five  Senecio species.  J. nat. Prod., 49: 722-723. 

    PESTCHANKER, M.J., ASCHERI, M.S., & GIORDANO, O.S. (1985a)  
    Uspallatine, a pyrrolizidine alkaloid from  Senecio uspallatensis.  
     Phytochemistry, 24: 1622-1624. 

    PESTCHANKER, M.J., ASCHERI, M.S., & GIORDANO, O.S. (1985b)  
    Pyrrolizidine alkaloids from  Senecio subulatus and  S. glandulosus. 
     Planta Med., 51: 165-166. 

    PETERSON, J.E. (1965)  Effects of pyrrolizidine alkaloid 
    lasiocarpine  N -oxide on nuclear and cell division in the liver of 
    rats.  J. Pathol. Bacteriol., 89: 153-171. 

    PETERSON, J.E. & CULVENOR, C.C.J. (1983)  Plant and fungal toxins. 
    In: Keeler, R.F. & Tu, A.T., ed.  Handbook of natural toxins, New 
    York, Marcel Dekker, Vol. 1, pp. 637-681. 

    PETERSON, J.E. & JAGO, M.V. (1980)  Comparison of the toxic effects 
    of dehydroheliotridine and heliotrine in pregnant rats and their 
    embryos.  J. Pathol., 131: 339-355. 

    PETERSON, J.E. & JAGO, M.V. (1984)  Toxicity of  Echium plantagineum  
    (Paterson's curse): pyrrolizidine alkaloid poisoning in rats.  Aust. 
     J. agric. Res., 35: 305-316. 

    PETERSON, J.E., SAMUEL, A., & JAGO, M.V. (1972)  Pathological 
    effects of dehydroheliotridine, a metabolite of heliotridine-based 
    pyrrolizidine alkaloids in the young rat.  J. Pathol., 107: 107-189. 

    PETERSON, J.E., JAGO, M.V., REDDY, J.K., & JARRETT, R.G. (1983)  
    Neoplasia and chronic disease associated with the prolonged 
    administration of dehydroheliotridine to rats.  J. Natl Cancer 
     Inst., 70: 381-386. 

    PETRY, T.W., BOWDEN, G.P., HUXTABLE, R.J., & SIPES, I.G.  (1984)  
    Characterization of hepatic DNA damage induced in rats by the 
    pyrrolizidine alkaloid monocrotaline.  Cancer Res., 44: 1505-1509. 

    PETRY, T.W., BOWDEN, G.P., BUHLER, D.R., & SIPES, I.G.  (1986)  
    Genotoxicity of the pyrrolizidine alkaloid jacobine in rats. 
     Toxicol. Lett., 32: 275-281. 

    PIERCY, P.L. & RUSOFF, L.L. (1946)   Crotalaria spectabilis 
    poisoning in Louisiana livestock.  J. Am. Vet. Med. Assoc., 108: 
    69-73. 

    PIERSON, M.L., CHEEKE, P.R., & DICKINSON, E.O. (1977)  Resistance 
    of the rabbit to dietary pyrrolizidine  (Senecio) alkaloid. Res. 
     Commun. chem. Pathol. Pharmacol., 16: 561-564. 

    PIETERS, L.A.C. & VLIETINCK, A.J.  (1985)  Quantitative 'H Fourier 
    transform nuclear magnetic resonance spectroscopic analysis of 
    mixtures of pyrrolizidine alkaloids from  Senecio vulgaris. 
     Fresenius' Z. Anal. Chem., 321: 355-358. 

    PIETERS, L.A.C. & VLIETINCK, A.J. (1986)  Comparison of high-
    performance liquid chromatography with 1H nuclear magnetic 
    resonance spectrometry for the quantitative analysis of 
    pyrrolizidine alkaloids from  Senecio vulgaris. J. liq. Chromatogr., 
    9: 745-755. 

    PLESTINA, R. & STONER, H.B. (1972)  Pulmonary oedema in rats given 
    monocrotaline pyrrole.  J. Pathol., 106: 235-249. 

    POOL, B.L. (1982)  Genotoxic activity of an alkaloidal extract of 
     Senecio nemorensis spp.  fuchsii in  Salmonella typhimurium and 
     Escherichia coli systems (letter to the editor).  Toxicology, 24: 
    351-355. 

    POWIS, G., AMES, M.M., & KOVACH, J.S. (1979)  Metabolic conversion 
    of indicine- N-oxide to indicine in rabbits and humans.  Cancer 
     Res., 39: 3564-3570. 

    PRABHU, M.B. (1940)  Infantile cirrhosis of liver.  Indian J. 
     Pediatr., 7: 121-134. 

    RADHAKRISHNA RAO, M.V. (1935)  Histopathology of the liver in 
    infantile biliary cirrhosis.  Indian J. med. Res., 23: 69-90. 

    RAJAGOPALAN, T.R. & NEGI, R.K.S. (1985)  Alkaloids from  Doronicum 
     pardalianches Linn.  Indian J. Chem., 24B, 882. 

    RAKIETEN, N., GORDON, B.S., BEATTY, A., COONEY, D.A., DAVIS, R.D., 
    & SCHIEN, P.S. (1971)  Pancreatic islet cell tumours produced by 
    the combined action of streptozotocin and nicotinamide.  Proc. Soc. 
     Exp. Biol. Med., 137: 280-283. 

    RAMALINGASWAMI, V. & NAYAK, N.C. (1969)  Liver disease in India. 
    In: Popper, H. & Schaffner, F., ed.  Progress in liver diseases,  
    New York, London, Grune and Stratton, pp. 222-235. 

    RAMSDELL, A.S. & BUHLER, D.R. (1981)  High performance liquid 
    chromatographic analysis of pyrrolizidine  (Senecio) alkaloids 
    using a reversed phase styrene-divinylbenzene resin column.  J. 
     Chromatogr., 210: 154-158. 

    RAO, M.S. & REDDY, J.K. (1978)  Malignant neoplasms in rats fed 
    lasiocarpine.  Br. J. Cancer, 37: 289-293. 

    RAPPAPORT, A.M., KNOBLAUCH, M., SUMMERSKILL, J., & BRAS, G. (1967)  
    Experimental veno-occlusive disease.  Gastroenterology, 54: 164. 

    RATNOFF, O.D. & MIRICK, G.S. (1949)  Influence of sex upon the 
    lethal effects of an hepatotoxic alkaloid, monocrotaline.  Bull. 
     Johns Hopkins Hosp., 84: 507-25. 

    RECHICIGL, M., Jr, ed. (1983)   CRC Handbook on naturally occurring 
     food toxicants, Boca Raton, Florida, CRC Press, 266 pp. 

    RESCH, J.F. & MEINWALD, J.  (1982)  A revised structure for 
    acetylheliosupine.  Phytochemistry, 21: 2340-2431. 

    RHODES, K. (1957)  Two types of liver disease in Jamaican children. 
     West Indian med. J., 6: 1-29. 

    RIDKER, P.M., OHKUMA, S., MCDERMOTT, W.V., TREY, C., & HUXTABLE, 
    R.J. (1985)  Hepatic veno-occlusive disease associated with 
    consumption of pyrrolizidine alkaloid containing dietary 
    supplements.  Gastroenterology, 88: 1050-1054. 

    RIZK, A.M., HAMMOUDA, F.M., ISMAIL, S.I., GHALEB, H.A., MADKOUR, 
    M.K., POHLAND, A.E., & WOOD, G. (1983)  Poisonous plants 
    contaminating edible ones and toxic substances in plant foods. I. 
    Alkaloids from  Senecio desfontainei. Fitoterapia, 54: 115-121. 

    ROBERTSON, K.A. (1982)  Alkylation of N2 in deoxyguanosine by 
    dehydroretronecine, a carcinogenic metabolite of the pyrrolizidine 
    alkaloid monocrotaline.  Cancer Res., 42: 8-14. 

    ROBINS, D.J. (1982)  The pyrrolizidine alkaloids. In: Herz, W., 
    Grisebach, H., & Kirby, G.W., ed.  Progress in the chemistry of 
     organic natural products, Vienna, New York, Springer-Verlag, pp. 
    115-203. 

    RODER, E., WEIDENFELD, H., & STENGL, P. (1981)  Pyrrolizidine 
    alkaloids senecionine and retrorsine from  Senecio inaequidens. 
     Planta Med., 41: 412-413. 

    RODER, E., WIEDENFELD, H., & KNOZINGER-FISCHER  (1984a)  
    Pyrrolizidine alkaloid from  Senecio abrotanifolius  spp. 
     abroatanifolius, spp.  abrotanifolius, and var.  tiroliensis. 
     Planta Med., 41: 412-413. 

    RODER, E., WIEDENFELD, H., & BRITZ-KIRSTGEN, R. (1984b)  
    Pyrrolizidine alkaloids from  Senecio cacaliaster. Phytochemistry, 
    23: 1761-1763. 

    ROGERS, A.E. & NEWBERNE, P.M. (1971)  Lasiocarpine factors 
    influencing its toxicity and effects in liver cell division. 
     Toxicol. appl. Pharmacol., 18: 356-366. 

    ROITMAN, J.N. (1981)  Comfrey and liver damage.  Lancet, 25 April: 
    944. 

    ROITMAN, J.N. (1983)  Ingestion of pyrrolizidine alkaloids: a 
    health hazard of global proportions. In: Finlay, J.W. & Schwass, 
    D.E., ed.  Xenobiotics in foods and feeds, Washington DC, American 
    Chemical Society, pp. 345-378 (ACS Series). 

    ROSE, A.L., GARDNER, C.A., MCDONNELL, J.D., & BULL, L.B. (1957a)  
    Field and experimental investigation of "walk-about" disease of 
    horses (Kimberley horse disease) in northern Australia:  Crotalaria  
    poisoning in horses. Part II.  Aust. vet. J., 33: 49-62. 

    ROSE, A.L., GARDNER, C.A., MCDONNELL, J.D., & BULL, L.B. (1957b)  
    Field and experimental investigation of "walk about" disease of 
    horses (Kimberley horse disease) in northern Australia. Part I. 
     Aust. vet. J., 33: 25-33. 

    ROSE, C.L., HARRIS, P.N., & CHEN, K.K. (1959)  Some pharmacological 
    action of supinine and lasiocarpine.  J. Pharmacol. exp. Ther., 126: 
    179-184. 

    ROSE, E.F. (1972)   Senecio species: toxic plants used as food and 
    medicine in the Transkei.  S. Afr. Med. J., 46: 1039-1043. 

    ROSENFIELD, I. & BEATH, O.A. (1945)  Tissue changes induced by 
     Senecio riddellii. Am. J. clin. Pathol., 15: 407-412. 

    ROTH, R.A., DOTZLAF, L.A., BARANYI, B., & HOOK, J.B. (1981)  Effect 
    of monocrotaline ingestion on liver, kidney and lung of rat. 
     Toxicol. appl. Pharmacol., 60: 193-203. 

    ROYES, K. (1948)  Infantile hepatic cirrhosis in Jamaica.  Caribb. 
     med. J., 10: 16-48. 

    SAFOUH, M. & SHEHATA, A.H. (1965)  Hepatic vein occlusion disease 
    of Egyptian children.  J. Pediatr., 67: 415-432. 

    ST. GEORGE-GRAMBAUER, T.D. & RAC, R. (1962)  Hepatogenous chronic 
    copper poisoning in sheep in south Australia due to consumption of 
     Echium plantagineum L. (Salvation Jane).  Aust. vet. J., 38: 288-293. 

    SALASPURO, M. & SIPPONEN, P. (1976)  Demonstration of an 
    intracellular copper-binding protein in orcein staining in long 
    standing cholestatic liver diseases.  Gut, 17: 787-790. 

    SAMUEL, A. & JAGO, M.V. (1975)  Localization in the cell cycle of 
    the antimitotic action of the pyrrolizidine alkaloid, lasiocarpine, 
    and its metabolite, dehydroheliotridine.  Chem.-biol. Interact., 10: 
    185-197. 

    SANDERS, D.A., SHEALY, A.L., & EMMEL, M.W. (1936)  The pathology of 
     Crotalaria spectabilis Roth poisoning in cattle.  J. Am. Vet. Med. 
     Assoc., 89: 150-156. 

    SAVIN, I.G.,  (1983)   [Research of the influence of heliotrin on 
     the microsomal mono oxygenase systems of the rat liver.]  N.E. 
    Pyrogov 2nd Moscow State Medical Institute.  (Thesis in biological 
    chemistry) (in Russian). 

    SAVVINA, K.I. (1952)  Pathological anatomy of atrophic hepatic 
    cirrhosis.  Arch. Pathol., 14: 65-70. 

    SCHOENTAL, R. (1959)  Liver lesions in young rats suckled by 
    mothers treated with the pyrrolizidine  (Senecio) alkaloids, 
    lasiocarpine, and retrorsine.  J. Pathol. Bacteriol., 77: 485-504. 

    SCHOENTAL, R. (1961)  Herbal medicines and liver disease.  J. exp. 
     med. Sci., 4: 126-135. 

    SCHOENTAL, R. (1963)  Liver disease and 'natural' hepatotoxins. 
     Bull. World Health Organ., 29: 823-833. 

    SCHOENTAL, R. (1968)  Toxicology and carcinogenic action of 
    pyrrolizidine alkaloids.  Cancer Res., 28: 2237-2246. 

    SCHOENTAL, R. (1970)  Hepatotoxic activity of retrorsine, 
    senkirkine and hydroxysenkirkine in newborn rats, and the role of 
    epoxides in carcinogenesis by pyrrolizidine alkaloids and 
    aflatoxins.  Nature (Lond.), 227: 401-402. 

    SCHOENTAL, R. (1975)  Pancreatic islet-cell and other tumours in 
    rats given heliotrine, a monoester pyrrolizidine alkaloid, and 
    nicotinamide.  Cancer Res., 35: 2020-2024. 

    SCHOENTAL, R. & BENSTED, J.P.M. (1963)  Effects of whole body 
    irradiation and of partial hepatectomy on the liver lesions induced 
    in rats by a single dose of retrorsine, a pyrrolizidine  (Senecio)  
    alkaloid.  Br. J. Cancer, 17: 242-251. 

    SCHOENTAL, R. & CAVANAGH, J.B. (1972)  Brain and spinal cord 
    tumours in rats treated with pyrrolizidine alkaloids.  J. Natl 
     Cancer Inst., 49: 665-671. 

    SCHOENTAL, R. & COADY, A. (1968)  The hepatotoxicity of some 
    Ethiopian and east African plants including some used as 
    traditional medicines.  East Afr. med. J., 45: 577-579. 

    SCHOENTAL, R. & HEAD, M.A. (1955)  Pathological changes in rats as 
    result of treatment with monocrotaline.  Br. J. Cancer, 9: 229-237. 

    SCHOENTAL, R. & HEAD, M.A. (1957)  Progression of liver lesions 
    produced in rats by temporary treatment with pyrrolizidine 
     (Senecio) alkaloids, and the effects of betaine and high casein 
    diet.  Br. J. Cancer, 11: 535-544. 

    SCHOENTAL, R. & MAGEE, P.N. (1957)  Chronic liver changes in rats 
    after a single dose of lasiocarpine, a pyrrolizidine  (Senecio)  
    alkaloid.  J. Pathol. Bacteriol., 74: 305-319. 

    SCHOENTAL, R. & MAGEE, P.N. (1959)  Further observation on the 
    subacute and chronic liver changes rats after a single dose of 
    various pyrrolizidine  (Senecio) alkaloids.  J. Pathol. Bacteriol., 
    78: 471-482. 

    SCHOENTAL, R. & MATTOCKS, A.R. (1960)  Hepatotoxic activity of 
    semisynthetic analogues of pyrrolizidine alkaloids.  Nature (Lond.), 
    185: 842-843. 

    SCHOENTAL, R., HEAD, M.A., & PEACOCK, P.R. (1954)   Senecio 
    alkaloids: primary liver tumours in rats as a result of treatment 
    with (1) mixture of alkaloids from  S. jacobaea Lin, (2) 
    retrorsine, (3) isatidine.  Br. J. Cancer, 8: 458-465. 

    SCHOENTAL, R., FOWLER, M.E., & COADY, A. (1970)  Islet cell tumours 
    of the pancreas found in rats given pyrrolizidine alkaloids from 
     Amsinckia intermedia Fisch and Mey and from  Heliotropium supinum. 
     Cancer Res., 30: 2127-2131. 

    SEGALL, H.J. (1979a)  Reversed phase isolation of pyrrolizidine 
    alkaloids.  Liq. Chromatogr., 2: 429-436. 

    SEGALL, H.J. (1979b)  Preparative isolation of pyrrolizidine 
    alkaloids derived from  Senecio vulgaris. Liq. Chromatogr., 2: 
    1319-1323. 

    SEGALL, H.J., DALLAS, J.L., & HADDON, W.F. (1984)  Two 
    dihydropyrrolizine alkaloid metabolites isolated from mouse hepatic 
    microsomes  in vitro. Drug Metab. Disp., 12: 68-71. 

    SEGALL, H.J., WILSON, D.W., DALLAS, J.L., & HADDON, W.F. (1985)  
    Trans-4-hydroxy-2-hexenol: a reactive metabolite from the 
    macrocyclic pyrrolizidine alkaloid senecionine.  Science, 229: 472-475. 

    SEGER, C.L., NEWSON, J.D., ROTH, E.E., & HUTCHINSON, W.R. (1969)  
    Chronic toxic hepatitis in deer from a Louisiana coastal marsh. 
     Bull. Wildlife Disease Assoc. Proc. Ann. Conf., pp. 295-6. 

    SELZER, G. & PARKER, R.G.F. (1951)   Senecio poisoning exhibiting 
    as Chiari's syndrome: a report of 12 cases.  Am. J. Pathol., 27: 
    885-907. 

    SENER, B., TEMIZER, H., TEMIZER, A., & KARAKAYA, A.E.  (1986)  High 
    performance liquid chromatographic determination of alkaloids in 
     Senecio vernalis. J. Pharm. Belg., 41: 115-117. 

    SHARMA, R.K., KHAJURIA, G.S., & ATAL, C.K. (1965)  Thin layer 
    chromatography of pyrrolizidine alkaloids.  J. Chromatogr., 19: 
    433-434. 

    SHERLOCK, S. (1968)   Diseases of liver and biliary system, 4th 
    ed., Oxford, Edinburgh, Blackwell Scientific Publishers, 250 pp. 

    SHTENBERG, A.I. & ORLOVA, N.V.  (1955)  [The question of the 
    etiology of the so-called "Ozhalangar Encephalitis".]  Vopr. Pitan., 
    14: 27-31 (in Russian). 

    SHULL, L.R., BUCKMASTER, G.W., & CHEEKE, P.R. (1976)  Factors 
    influencing pyrrolizidine  (Senecio) alkaloid metabolism: species, 
    liver sulphydryls and rumen fermentation.  J. anim. Sci., 43: 1247-1253. 

    SHUMAKER, R.C., ROBERTSON, K.A., HSU, I.C., & ALLEN, J.R. (1976)  
    Neoplastic transformation in tissues of rats exposed to 
    monocrotaline or dehydroretronecine.  J. Natl Cancer Inst., 56: 787-789. 

    SIDDIQI, M.A., SURI, K.A., SURI, M.P., & ATAL, C.K. (1978a)  Novel 
    pyrrolizidine alkaloid from  Crotalaria nana. Phytochemistry, 17: 
    2143-2144. 

    SIDDIQI, M.A., SURI, K.A., SURI, O.P., & ATAL, C.K. (1978b)  Genus 
     Crotalaria. Part 34. Cronaburmine, a new pyrrolizidine alkaloid 
    from  Crotalaria nana Burm.  Indian J. Chem., 16B: 1132-1133. 

    SMITH, L.W. &  CULVENOR, C.C.J. (1981)  Plant sources of 
    hepatotoxic pyrrolizidine alkaloids.  J. nat. Prod., 44: 129-152. 

    SMITH, T.E., WEISBACH, H., &  UDENFRIEND S.  (1962)  Studies on the 
    mechanism of monoaminooxidase: metabolism of  N,N-dimethyltryptamine
    and  N,N-dimethyltryptamine- N-oxide.  Biochemistry, 1: 137.

    SOBIN, L.H., FETRAT, M.E., & ANWER, M.A. (1969)  Hepatic lesions in 
    Afghanistan.  Trop. geogr. Med., 21: 27-29. 

    SRIVASTAVA, R.N., MOHABBAT, O., GHANI, A.R., & ARAM, G.N. (1978)  
    Veno-occlusive disease of the liver.  Indian. Paediatr., 15: 143-146. 

    SRUNGBOONMEE, S. & MASKASAME, C.  (1981)  A preliminary study on 
    the toxicity of  Crotalaria juncea to cattle.  Sattawaphaet San, 32: 
    91-107. 

    STEIN, H. (1957)  Veno-occlusive disease of liver in African 
    children.  Br. med. J., 29 June: 1496-1499. 

    STEIN, H. & ISAACSON, C. (1962)  Veno-occlusive disease of the 
    liver.  Br. med. J., 10 February: 372-374. 

    STENMARK, K.R., MORGANROTH, M.L., REMIGIO, L.K., VOELKEL, N.F., 
    MURPHY, R.C., HENSON, P.M., MATHIAS, M.M., & REEVES, J. (1985)  
    Alveolar inflammation and arachidonate metabolism in monocrotaline-
    induced pulmonary hypertension.  Am. J. Physiol., 248: H859-H866. 

    STILLMAN, A.E., HUXTABLE, R.J., CONSROE, P., KOHNEN, P., & SMITH, 
    S. (1977)  Hepatic veno-occlusive disease due to pyrrolizidine 
    poisoning in Arizona.  Gastroenterology, 73: 349-352. 

    STIRLING, G.A., BRAS, G., & URQUHART, A.E. (1962)  The early 
    lesions in veno-occlusive disease of the liver.  Arch. dis. Child., 
    37: 535-538. 

    STOYEL, C. & CLARK, A.M. (1980)  The transplacental micronucleus 
    test.  Mutat. Res., 74: 393-398. 

    STUART, K.L. & BRAS, G. (1955)  Clinical observations on veno-
    occlusive disease of the liver in Jamaican adults.  Br. med. J., 2: 
    348-352. 

    STUART, K.L. & BRAS, G. (1956)  Veno-occlusive disease of the liver 
    in Barbados.  West Indian med. J., 5: 33-36. 

    STUART, K.L. & BRAS, G. (1957)  Veno-occlusive disease of the 
    liver.  Q. J. Med., 26: 291-315. 

    STYLES, J., ASHBY, J., & MATTOCKS, A.R. (1980)  Evaluation  in vitro  
    of several pyrrolizidine alkaloid carcinogens: observation on the 
    essential pyrrolic nucleus.  Carcinogenesis, 1: 161-164. 

    SUFFNESS, M. & CORDELL, G.A. (1985)  Antitumour alkaloids. In: 
    Brossi, A., ed.  The alkaloids, New York, Academic Press, 
    pp. 1-347. 

    SUGITA, T., HYERS, T.M., DAUBER, I.M., WAGNER, W.W., MCMURTRY, 
    I.F., & REEVES, J.T. (1983a)  Lung leak precedes right ventricular 
    hypertrophy in monocrotaline treated rats.  J. appl. Physiol., 54: 
    371-374. 

    SUGITA, T., STENMARK, K.R., WAGNER, W.W., HENSON, P.M., HENSON, 
    J.E., HYERS, T.M., & REEVES, J.T. (1983b)  Abnormal alveolar cells 
    in monocrotaline induced pulmonary hypertension.  Exp. lung Res., 
    5: 201-215. 

    SUNDARESON, A.E. (1942)  An experimental study of placental 
    permeability to cirrhogenic poisons.  J. Pathol. Bacteriol., 54: 
    289-298. 

    SVOBODA, D. & REDDY, J.K. (1972)  Malignant tumours in rats given 
    lasiocarpine.  Cancer Res., 32: 908-912. 

    SVOBODA, D. & REDDY, J.K. (1974)  Lasiocarpine induced, 
    transplantable squamous cell carcinoma of rat skin.  J. Natl Cancer 
     Inst., 53: 1415-1418. 

    SVOBODA, D. & SOGA, J. (1966)  Early effects of pyrrolizidine 
    alkaloids on the fine structure of rat liver cells.  Am. J. Pathol., 
    48: 347-373. 

    SWICK, R.A., CHEEKE, P.R., & BUHLER, D.R. (1979)  Factors affecting 
    the toxicity of dietary tansy ragwort to rats. In: Cheeke, P.R., 
    ed.  Proceedings of the Symposium on Pyrrolizidine (Senecio) 
     Alkaloids: Toxicity, Metabolism, and Poisonous Plant Control 
     Measures, Corvallis, Oregon, USA, 23-24 February 1979, Oregon, 
    Nutrition Research Institute. 

    SWICK, R.A., CHEEKE, P.R., GOEGER, D.E., & BUHLER, D.R. (1982a)  
    Effect of dietary  Senecio jacobaea  and injected  Senecio alkaloids 
    and monocrotaline on guinea pigs.  J. anim. Sci., 55: 1411-1416. 

    SWICK, R.A., CHEEKE, P.R., & BUHLER, D.R. (1982b)  Subcellular 
    distribution of hepatic copper, zinc and iron and serum 
    ceruloplasmin in rats intoxicated by oral pyrrolizidine  (Senecio)  
    alkaloids.  J. anim. Sci., 55(6): 1425-1430. 

    SWICK, R.A., CHEEKE, P.R., PATTON, N.M., & BUHLER, D.R. (1982c)  
    Absorption and excretion of pyrrolizidine  (Senecio) alkaloids and 
    their effects on mineral metabolism in rabbits.  J. anim. Sci., 55: 
    1417-1424. 

    SWICK, R.A., CHEEKE, P.R., MIRANDA, C.L., & BUHLER, D.R. (1982d)  
    The effect of consumption of the pyrrolizidine alkaloid containing 
    plant  Senecio jacobaea on iron and copper metabolism in the rat. 
     J. Toxicol. environ. Health, 10: 757-768. 

    SWICK, R.A., CHEEKE, P.R., RAMSDELL, H.S., & BUHLER, D.R. (1983)  
    Effect of sheep rumen fermentation and methane inhibition on the 
    toxicity of  Senecio jacobaea. J. anim. Sci., 56: 645-651. 

    TAKANASHI, H., UMEDA, M., & HIRONO, I. (1980)  Chromosomal 
    aberrations and mutation in cultured mammalian cells induced by 
    pyrrolizidine alkaloids.  Mutat. Res., 78: 67-77. 

    TAKEOKA, O., ANGEVINE, M., & LALICH, J. (1962)  Stimulation of mast 
    cells in rat fed various chemicals.  Am. J. Pathol., 40: 545-554. 

    TANDON, B.N., TANDON, H.D., TANDON, R.K., NARENDRANATHAN, M., & 
    JOSHI, Y.K. (1976)  Epidemic of veno-occlusive disease in central 
    India.  Lancet, 7 August: 271-272. 

    TANDON, B.N., TANDON, H.D., KOSHY, A., NARENDRANATHAN, M., JOSHI, 
    Y.K., TANDON, R.K., BHARGAVA, S., RAJANI, M., BHATIA, M.L., 
    MANCHANDA, S.C., & KASTURI, T.E. (1977)  Epidemiological, 
    clinical, biochemical and haemodynamic study of veno-occlusive 
    disease of the liver due to  Crotalaria alkaloids in India. J. All 
     Ind. Inst. Med. Sci., 3: 165-175. 

    TANDON, B.N., TANDON, H.D., & MATTOCKS, A.R. (1978)  Study of an 
    epidemic of veno-occlusive disease in Afghanistan.  Indian J. med. 
     Res., 68: 84-90. 

    TANDON, H.D. & TANDON, B.N. (1975)  Epidemic of liver disease - 
     Gulran District, Herat Province, Afghanistan, Alexandria, World 
    Health Organization, Regional Office for the Eastern Mediterranean 
    (Assignment report No. EM/AFG/OCD/001/RB). 

    TANDON, H.D., TANDON, B.N., TANDON, R., & NAYAK, N.C. (1977)  A 
    pathological study of the liver in an epidemic outbreak of veno-
    occlusive disease.  Indian J. med. Res., 65: 679-684. 

    TANDON, H.D., TANDON, B.N., & MATTOCKS, A.R. (1978)  An epidemic of 
    veno-occlusive disease of the liver in Afghanistan.  Am. J. 
     Gastroenterol., 72: 607-613. 

    TANDON, R.K., TANDON, B.N., TANDON, H.D., BHATIA, M.L., BHARGAVA, 
    S., LAL, P., & ARORA, R.R. (1976)  Study of an epidemic of veno-
    occlusive disease in India.  Gut, 17: 849-855. 

    TANNER, M.S. & PORTMANN, B. (1981) Indian childhood cirrhosis. 
     Arch. dis. Child., 56: 4-6. 

    TAYLOR, S., BELT, R.J., HAAS, C.D., & HOOGSTRATEN, B. (1983)  Phase 
    I trial of indicine- N-oxide on two dose schedules.  Cancer, 51: 
    1988-1991. 

    TEILUM, G. (1949)  Endophlebitis hepatica obliterans.  Acta pathol. 
     microbiol., 26: 147-166. 

    TEMIZER, A., ONAR, A.N., SENER, B., TEMIZER, H., & KARAKAYA, A.E.  
    (1985)  Determination of alkaloids by differential pulse 
    polarography. I.  Senecio alkaloids.  J. Pharm. Belg., 40(2): 75-78. 

    TEREKHOV, G.N. (1939)  [Pathomorphology of toxic hepatitis with 
    ascites.] In: Mirochnik, M.F., ed.  [Functional, diagnostic and 
     pathological changes of toxic hepatitis with ascites,]  Tashkent, 
    State Publishing House of Science Technology and Socio-economic 
    Literature of Uzbekistan, pp. 145-200 (in Russian). 

    TEREKHOV, G.N. (1952)  [The pathomorphology of alimentary toxic 
    dystrophy with ascites.] In: Milenkov, S.M. & Kizhaikin, Y., ed. 
     [Collection of scientific papers on Toxic Hepatitis with Ascites,]  
    Tashkent, Publishing House of the University of Central Asia, pp. 
    148-164 (in Russian). 

    THORPE, E. & FORD, E.J.H. (1968)  Development of hepatic lesions in 
    calves fed with ragwort  (Senecio jacobaea). J. comp. Pathol., 78: 
    195-205. 

    TITTEL, G., HINZ, H., & WAGNER, H. (1979)  Quantitative 
    determination of pyrrolizidine alkaloids in  Symphyti radix by 
    HPLC.  Planta Med., 37: 1-8. 

    TUCHWEBER, B., KOVAKS, K., JAGO, M.V., & BEAULIEU, T. (1974)  
    Effect of steroidal and non-steroidal microsomal enzyme inducers on 
    the hepatotoxicity of pyrrolizidine alkaloids in rats.  Res. Commun 
     chem. Pathol. Pharmacol., 7: 459-480. 

    TUCKER, A., BRYANT, S.E., FROST, H.H., & MIGALLY, N. (1983)  
    Chemical sympathectomy and serotonin inhibition reduce 
    monocrotaline-induced right ventricular hypertrophy in rats.  Can. 
     J. Physiol. Pharmacol., 61: 356-362. 

    TURNER, J.H. & LALICH, J.J. (1965)  Experimental cor pulmonale in 
    the rat.  Arch. Pathol., 79: 409-418. 

    VALDIVIA, E., SONNAD, J., HAYASHI, Y., & LALICH, J.J. (1967a)  
    Experimental interstitial pulmonary oedema.  Angiology, 18: 378-383. 

    VALDIVIA, E., LALICH, J., HAYASHI, Y., & SONNAD, J. (1967b)  
    Alterations in pulmonary alveoli after a single injection of 
    monocrotaline.  Arch. Pathol., 84: 64-76. 

    VAN DER WATT, J.J., PURCHASE, I.F.H., & TUSTIN, R.C. (1972)  The 
    chronic toxicity of retrorsine, a pyrrolizidine alkaloid, in vervet 
    monkeys.  J. Pathol., 107: 279-287. 

    VILLARROEL, L.H., TORRES, R.G., NAVARRO, J.M., & FAJARDO, V.M. 
    (1985)  Senecionine and seneciphylline from  Senecio patagonicus. 
     Fitoterapia, 56: 250-251. 

    VISCONTINI, M. & GILHOF-SCHAUFELBERGER, H. (1971)  Synthesis of (±) 
    dehydroheliotridine.  Helv. chim. Acta, 54: 449-456. 

    WAGENVOORT, C.A., DINGEMANS, K.P., & LOTGERING, G.G. (1974a)  
    Electron microscopy of pulmonary vasculature after application of 
    fulvine.  Thorax, 29: 511-521. 

    WAGENVOORT, C.A., WAGENVOORT, N., & DIJK, H.J. (1974b)  Effect of 
    fulvine on pulmonary arteries and veins of the rat.  Thorax, 29: 
    522-529. 

    WAGNER, H., NEIDHARDT, U., & TITTEL, G. (1981)  TLC and HPLC 
    analysis of pyrrolizidine  N-oxide alkaloids of  Symphyti radix. 
     Planta Med., 41: 232-239. 

    WAKIM, K.G., HARRIS, P.N., & CHEN, K.K. (1946)  The effects of 
    senecionine on the monkey.  J. Pharmacol. exp. Ther., 87: 38-45. 

    WALKER, K.H. & KIRKLAND, P.D. (1981)   Senecio lautus toxicity in 
    cattle.  Aust. vet. J., 57: 1-7. 

    WATT, J.M. & BREYER-BRANDWIJK, M.G. (1962)   Medicinal and poisonous 
     plants of southern and eastern Africa, Edinburgh, London, E. and 
    S. Livingstone, 584 p. 

    WEHNER, F.C., THIEL, P.G., & VAN RENSBURG, S.J. (1979)  
    Mutagenicity of alkaloids in the  Salmonella/microsome system. 
     Mutat. Res., 66: 187-190. 

    WESTON, C.F.M., COOPER, B.T., DAVIES, J.D., & LEVINE, D.F.  (1987)  
    Veno-occlusive disease of the liver secondary to ingestion of 
    comfrey.  Br. med. J., 295: 183. 

    WHITE, I.N.H. (1976)  The role of liver glutathion in the acute 
    toxicity of retrorsine to rats.  Chem.-biol. Interact., 13: 333-342. 

    WHITE, I.N.H. (1977)  Excretion of pyrrolic metabolites in bile of 
    rats given retrorsine or the bis- n-ethylcarbamate of synthanecine 
    A.  Chem.-biol. Interact., 16: 169-180. 

    WHITE, I.N.H. & MATTOCKS, A.R. (1971)  Some factors affecting the 
    conversion of pyrrolizidine alkaloids to  N-oxides and to pyrrolic 
    derivatives  in vitro. Xenobiotica, 1: 503-505. 

    WHITE, I.N.H. & MATTOCKS, A.R. (1972)  Reactions of dihydro-
    pyrrolizidine with deoxyribonucleic acid  in vitro. Biochem.
     J., 128: 291-297.

    WHITE, I.N.H., MATTOCKS, A.R., & BUTLER, W.H. (1973)  The 
    conversion of the pyrrolizidine alkaloid retrorsine to pyrrolic 
    derivatives  in vivo and  in vitro and its acute toxicity to 
    various animal species.  Chem.-biol. Interact., 6: 207-218. 

    WHITE, R.D., KRUMPERMAN, P.H., CHEEKE, P.R., & BUHLER, D.R. (1983)  
    An evaluation of acetone extracts from six plants in the Ames 
    mutagenicity test.  Toxicol. Lett., 15: 25-31. 

    WHITE, R.D., SWICK, R.A., CHEEKE, P.R.  (1984)  Effect of dietary 
    copper and molybdenum on tansy ragwort  (Senecio Jacobaea) toxicity 
    in sheep.   Am. J. vet. Res., 45: 159-161. 

    WHO  (1980)   Inventory of medicinal plants used in the different 
     countries, Geneva, World Health Organization (Document No. 
    DPM/80.3). 

    WICKRAMANAYAKE, P.P., ARBOGAST, B.L., BUHLER, D.R., DEINZER, M.L., 
    & BURLINGAME, A.L.  (1985)  Alkylation of nucleosides and 
    nucleotides by dehydroretronecine: characterization of covalent 
    adducts by liquid secondary ion mass spectrometry.  J. Am. Chem. 
     Soc., 107: 2485-2488. 

    WIEDENFELD, H. (1982)  Two pyrrolizidine alkaloids from  Gynura 
     scandens. Phytochemistry, 21: 2767-2768. 

    WIEDENFELD, H., PASTEWKA, U., STENGL, P., & ROEDER, E. (1981)  On 
    the gas chromatographical determination of the pyrrolizidine 
    alkaloids of some  Senecio species.  Planta Med., 41: 124-128. 

    WIEDENFELD, H., RODER, E., & ANDERS, E. (1985)  Pyrrolizidine 
    alkaloids from seeds of Crotalaria scassellatii.  Phytochemistry, 
    24: 376-378. 

    WILLIAMS, A.O., EDINGTON, G.M., & OBAKPONOVWE, P.C. (1967)  
    Hepatocellular carcinoma in infancy and childhood in Ibadan, 
    western Nigeria.  Br. J. Cancer, 21(3): 474-482. 

    WILMOT, F.C. & ROBERTSON, G.W. (1920)   Senecio disease or 
    cirrhosis of the liver due to  Senecio poisoning.  Lancet, 
    23 October: 848-849. 

    WITTIG, M. & STEPHEN, C.  (1964)  Modification of microsomal lipid 
    peroxidation and drug metabolism by cytoplasmic copper.   Res. 
     Commun. Chem. Pathol. Pharmacol., 44: 477-493. 

    WONG, R.Y. & ROITMAN, J.N.  (1984)  Structure and absolute 
    configuration of (+)-doronine-benzene  (1:1).   Acta Crystallogr., 
     Sect. C:  Cryst. Struc. Commun., C40: 163-166. 

    WURM, H. (1939)  [A cluster of endophlebilis hepatica obliterans in 
    the age group of suckling infants.]  Klin. Wochenschr., 18: 1527-1531 
    (in German). 

    YAMANAKA, H., NAGAO, M., SUGIMURA, T., FURUYA, T., SHIRAI, A., & 
    MATSUSHIMA, T. (1979)  Mutagenicity of pyrrolizidine alkaloids in 
    the  Salmonella/mammalian microsome test.  Mutat. Res., 68: 211-216. 

    YULDASHEVA, L.N. & SULTANOVA, R.G. (1983)  [Oxidative reactions in 
    rat liver tissue under conditions of chronic heliotrine hepatitis.] 
     Vopr. Med. Khim., 29: 81-85 (in Russian). 

    YUNUSOV, S.YU. & PLEKHANOVA, N.V.  (1959)  [The alkaloids of 
     Trichodesma incanum.  The structure of incanine and trichesmine.] 
     Zh. Obshch. Khim., 29: 677-684 (in Russian). 

    ZHELTOVA, L.I. (1952)  [The clinical course of toxic hepatitis.] 
    In: Milenkov, S.M. & Kizhaikin, Y., ed.  [Collection of scientific 
     papers on Toxic Hepatitis with Ascites,]  Tashkent, Publishing 
    House of the University of Central Asia, pp. 76-90 (in Russian). 

        APPENDIX I

    Pyrrolizidine Alkaloids and Their Plant Sources

    ISOLATIONS OF TOXIC PYRROLIZIDINE ALKALOIDS - (8701081148)

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    19-Acetoxysenkirkine            Senecio laricifolius H.B.K.                     Bohlmann et al. (1986)                       A

    6-Acetylanacrotine              Crotalaria agatiflora Schweinf.                 Culvenor & Smith (1972)                      B281

    6-Acetyl-trans-anacrotine       Crotalaria agatiflora Schweinf.                 Culvenor & Smith (1972)                      B281

    Acetylcrotaverrine              Crotalaria verrucosa L.                         O.P. Suri et al. (1976)                      B339
                                    C. walkeri Arnott                               K.A. Suri et al. (1976)                      B340

    7-Acetylechinatine              Cynoglossum amabile Stapf. and Drummond         Culvenor & Smith, unpubl.
                                    Lindelofia spectabilis Lehm.                    Rao et al. (1974)                            B62
                                    Symphytum asperum Lepech.                       Pedersen (1975b)                             A
                                    S. officinale Linn.                             Pedersen (1975b)                             A

    Acetylgynuramine                Gynura scandens O. Hoffm.                       Wiedenfeld (1982)                            C

    Acetylheliosupine               Cynoglossum officinale L.                       Pedersen (1970); Resch & Meinwald (1982)     B43, A
                                    Myosotis sylvatica Hoffm.                       Resch & Meinwald (1982)                      A
                                    Symphytum asperum Lepech.                       Pedersen (1975)                              A
                                    S. officinale Linn.                             Pedersen (1975)                              A

    Acetylindicine                  Heliotropium indicum L.                         Mattocks (1967a)                             B71

    7-Acetylintermedine             Borago officinale L.                            Luthy et al. (1984)                          A
                                    Symphytum aspera                                Roitman (1981)                               A
                                    S. officinale Linn.                             Roder et al. (1982)                          C
                                    S. x uplandicum Nyman                           Culvenor et al. (1980a), (1980b)             A, A
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Acetyllasiocarpine              Heliotropium europaeum L.                       Culvenor et al. (1975)                       B69

    7-Acetyllycopsamine             Amsinckia menziesii (Lehm.) Nels                Roitman, (1983a)                             C
                                    & Macbr.
                                    Anchusa officinalis L.                          Pedersen (1975); Broch-Due &                 A, B32
                                    Aasen (1980)
                                    Borago officinale L.                            Luthy et al. (1984)                          A
                                    Symphytum aspera                                Roitman (1981)                               A
                                    S. officinale Linn.                             Huizing & Malingre (1981)                    C
                                    S. x uplandicum Nyman                           Culvenor et al. (1980a, 1980b)               A, A

    3a'-Acetyllycopsamine           Amsinckia menziesii (Lehm.) Nels.               Roitman (1983a                               C
                                    & Macbr.

    7-Acetylmadurensine             Crotalaria agatiflora Schweinf.                 Culvenor & Smith (1972)                      B281

    7-Acetyl-cis-madurensine        Crotalaria agatiflora Schweinf.                 Culvenor & Smith (1972)                      B281

    7-Acetylscorpioidine            Myosotis scorpioides L.                         Resch et al. (1982)                          C

    Acetylseneciphylline            Senecio pterophorus DC.                         Edgar et al. (1976)                          B246

    18-Acetylsenkirkine             Senecio illinitus Phill.                        Gonzalez et al. (1986a)                      A
                                    S. kirkii Hook. f. ex Kirk                      Briggs et al. (1965)                         A
                                    S. tenuifolius Burm.                            Bhakuni & Gupta (1982)                       C

    Acetylsyneilesine               Syneilesis palmata Maxim.                       Hikichi & Furuya (1976)                      B279

    Amabiline                       Borago officinale L.                            Luthy et al. (1984)                          A
                                    Cynoglossum amabile Stapf. et Drummond          Culvenor & Smith (1967)                      B33
                                    C. glochidiatum Wall. ex Lindl.                 K.A. Suri et al. (1975a)                     B36
                                    Eupatorium cannabinum L.                        Luthy et al. (1984)                          A
                                    Lindelofia angustifolia (Schrenk) Brand.        K.A. Suri et al. (1975a)                     B36
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Anacrotine                      Crotalaria agatifolia Schweinf.                 Culvenor & Smith (1972)                      B281
                                    C. incana L.                                    Mattocks (1968)                              B302
                                    C. laburnifolia L.                              Snehelata et al. (1966); Sawhney             B309,
                                    et al. (1967)                                   291
                                    C. laburnifolia L. subsp. eldomae               Crout (1972)                                 B312
                                    C. micans Link.                                 Atal et al. (1966a)                          B280
                                    C. verrucosa L.                                 Subramanian & Nagarajan (1967)               B338

    Anadoline                       Symphytum orientale                             Ulubelen & Doganca (1971); Culvenor          B103,
                                                                                    et al. (1975)                                104
                                    S. tuberosum  L.                                Ulubelen & Ocal (1977)                       B105

    Angelylechimidine (or           Symphytum asperum Lepech.                       Gadella et al. (1983)                        A
    isomer)                         S. x uplandicum Nyman                           Gadella et al. (1983)                        A

    7-Angelylheliotridine           Heliotropium supinum L.                         Crowley & Culvenor (1959)                    B83
    trachelanthate

    7-Angelylheliotridine           Heliotropium supinum L.                         Crowley & Culvenor (1959)                    B83
    viridiflorate

    7-Angelylheliotrine             Heliotropium digynum Forssk.                    Hammouda et al. (1984)                       A
                                    H. eichwaldii Steud ex DC.                      O.P. Suri et al. (1975)                      B63

    7-Angelyl-9-sarracinyl-         Senecio triangularis Hook.                      Rueger & Benn (1983)                         C
    retronecine

    6-Angelyl-trans-anacrotine      Crotalaria agatiflora Schweinf.                 Culvenor & Smith (1972)                      B281

    Asperumine                      Echium vulgare L.                               Karimov et al. (1975)                        B50
                                    Symphytum asperum Lepech.                       Man'ko et al. (1969); Man'ko &               B95, 96
                                                                                    Kotowskii (1970); Man'ko et al. 1970         B97
                                    S. caucasicum Bieb.                             Man'ko et al. (1972); Mel'kumova             B98, 99
                                                                                    et al. 1974
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Axillaridine                    Crotalaria axillaris Ait.                       Crout (1968b, 1969)                          B287, 288
                                    C. scassellatii Chiov                           Wiedenfeld et al. (1985)                     A

    Axillarine                      Crotalaria axillaris Ait.                       Crout (1968b, 1969)                          B287, 288
                                    C. scassellatii Chiov                           Wiedenfeld et al. (1985)                     A

    Bisline                         Senecio othonniformis Fourcade                  Coucourakis & Gordon-Gray (1970)             B224
                                    S. petasitis DC.                                Gonzalez et al. (1973)                       B225

    Brachyglottine                  Brachyglottis repanda Forst. et Forst.          White, pers. commun.

    Carategine                      Lindelofia tschimganica                         Akramov et al. (1965)                        B87
                                    Rindera oblongifolia M. Pop.                    Akramov et al. (1965)                        B87
                                    Solenanthus karategenius Lipsky                 Akramov et al. (1964)                        B93

    Chlorodeoxysceleratine          Senecio latifolius DC. (S. sceleratus)          Gordon-Gray (1967)                           B261

    Clivorine                       Ligularia brachyphylla Hand. Mazz.              Klasek et al. (1971)                         B134
                                    L. clivorum                                     Klasek et al. (1967, 1969, 1970);            B135,
                                                                                    Birnbaum et al. (1971)                       136,
                                                                                                                                 137,
                                                                                                                                 138
                                    L. dentata (A. Gray) Hara                       Klasek et al. (1971)                         B134
                                    L. elegans (Cass.)                              Klasek et al. (1971)                         B134

    Crispatine                      Crotalaria candicans W. & A.                    Suri et al. (1982)                           C
                                    C. crispata F. Muell. ex Benth.                 Culvenor & Smith (1963)                      B294
                                    C. lunata Beddome ex Polhill                    Rothschild et al. (1979)                     C
                                    C. madurensis R. Wight                          Habib et al. (1971)                          B315

    Crobarbatine                    Crotalaria barbata R. Graham ex R. Wight        Puri et al. (1973)                           B289
                                    Walk.-Arn.
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Cromadurine                     Crotalaria madurensis Wight                     Rao et al. (1974, 1975b)                     B62, 316

    Cronaburmine                    Crotalaria nana Burm.                           Siddiqi et al. (1978b)                       A

    Crosemperine                    Crotalaria aegyptiaca Benth.                    Zalkow et al. (1979)                         B419
                                    C. semperflorens Vent.                          Atal et al. (1967)                           B331

    Crotaflorine                    Crotalaria agatiflora Schweinf.                 Culvenor & Smith (1972)                      B281

    Crotafoline                     Crotalaria laburnifolia L. subsp. eldomae       Crout (1972)                                 B312

    Crotalarine                     Crotalaria burhia Buch-Ham.                     Ali & Adil (1973); Rao et al. (1975a)        B292,
                                                                                                                                 293

    Crotaleschenine                 Crotalaria leschenaultii                        Suri & Atal (1967); Smith et al. (1988)      B313

    Crotananine                     Crotalaria nana Burm.                           Siddiqi et al. (1978a)                       A

    Crotastriatine                  Crotalaria pallida Ait. (syn. C.                Gandhi et al. (1968); Batra et al. (1975)    B323,
                                    mucronata Desv., C. striata DC.)                324

    Crotaverrine                    Crotalaria verrucosa L.                         O.P. Suri et al. (1976)                      B339
                                    C. walkeri Arnott                               K.A. Suri et al. (1976)                      B340

    Cruentine A                     Senecio cruentus DC.                            Chu & Chu (1964)                             B172

    Cruentine B                     Senecio cruentus DC.                            Chu & Chu (1964)                             B172

    Curassavinine                   Heliotropium curassavicum Linn.                 Mohanraj et al. (1982a)                      C

    Cynaustine                      Borago officinale L. (or amabiline ?)           Larson et al. (1984)                         C
                                    Cynoglossum australe R. Br.                     Culvenor & Smith, 1967                       B33
                                    C. lanceolatum Forsk.                           Suri et al. 1975a                            B36
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Deoxyaxillarine                 Crotalaria scassellatii Chiov                   Wiedenfeld et al. 1985                       A

    Diacetyllycopsamine             Amsinckia menziesii (Lehm.) Nels. & Macbr.      Roitman, 1983a                               C

    Dibenzoylretronecine            Caccinea glauca Savi.                           Siddiqi et al. 1978a                         B346

    Dicrotaline                     Crotalaria dura J.M. Wood et Evans              Marais, 1944; Adams & Van Duuren, 1953a      B295, 296

                                    C. globifera E. Mey.                            Marais, 1944; Adams & Van                    B295,
                                                                                    Duuren, 1953a                                296

    N-(Dihydropyrrolizino-          Heliotropium europaeum L.                       Culvenor & Smith, 1969                       B68
    methyl) heliotrine chloride

    15,20-Dihydroxyerucifoline      Senecio dolichodoryius Cuatr.                   Bohlmann et al. 1986                         A

    Dihydroxytriangularine          Alkanna tinctoria Tausch                        Roder et al. 1984b                           C

    Doronenine                      Senecio abrotanifolius ssp.                     Roder et al. 1984a                           A
                                    abrotanifolius
                                    S. abrotanifolius ssp. abrotanifolius           Roder et al. 1984a                           A
                                    var tiroliensis
                                    S. doronicum L.                                 Roder et al. 1979a, 1980; Kirfel             B176, C
                                                                                    et al. 1980                                  A

    Doronine                        Doronicum macrophyllum                          Alieva et al. 1976                           B122
                                    Senecio abrotanifolius ssp.                     Roder et al. 1984a                           A
                                    abrotanifolius
                                    S. abrotanifolius ssp. abrotanifolius           Roder et al. 1984a                           A
                                    var tiroliensis
                                    S. clevelandii E.L. Greene                      Wong & Roitman, 1984                         A
                                    S. othonnae Bieb.                               Khalilov et al. 1977                         B223
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    H. suaveolens Bieb.                             Guner, 1986                                  A
                                    H. supinum L.                                   Crowley & Culvenor, 1959                     B83
                                    Lappula glochidiata                             Suri et al. 1978                             B84
                                    Lindelofia angustifolia (Schrenk) Brand.        Rao et al. 1974; Suri et al. 1975a           B62, 36
                                    L. spectabilis Lehm.                            Rao et al. 1974; Suri et al. 1975a           B62, 36
                                    L. stylosa (Kar. et Kir.) Brand.                Kiyamitdinova et al. 1967                    B75
                                    L. tschimganica                                 Akramov et al. 1965                          B87
                                    Paracynoglossum imeritinum (Kusn.) M. Pop.      Man'ko & Marchenko, 1971b                    B88
                                    Prestonia sp.                                   Edgar, 1985                                  A
                                    Rindera austroechinata M. Pop.                  Akramov et al. 1965                          B87
                                    R. baldshuanica Kusnezov                        Akramov et al. 1965                          B87
                                    R. cyclodonta                                   Akramov et al. 1967a                         B59
                                    R. echinata Regel                               Men'shikov & Denisova, 1953                  B92
                                    R. oblongifolia M. Pop.                         Akramov et al. 1965                          B87
                                    Solenanthus circinnatus Ledeb.                  Akramov et al. 1964                          B93
                                    S. coronatus                                    Kiyamitdinova et al. 1967                    B75
                                    S. karateginus Lipsky                           Akramov et al. 1964                          B93
                                    Symphytum asperum Lepech.                       Man'ko et al. 1970b                          B97
                                    S. caucasicum Bieb.                             Man'ko et al. 1972                           B98
                                    S. officinale Linn.                             Man'ko et al. 1970a; Huizing &               B101, C
                                    Malingre, 1979

    Echiumine                       Amsinckia hispida (Ruiz et Pav.)                Culvenor & Smith, 1966a                      B30
                                    I.M. Johnston
                                    A. intermedia Fisch et C. Mey.                  Culvenor & Smith, 1966a                      B30
                                    A. lycopsoides Lehm.                            Culvenor & Smith, 1966a                      B30
                                    Echium plantagineum L.                          Culvenor, 1956                               B48

    Emiline                         Emilia flammea Cass.                            Kohlmunzer & Tomczyk, 1969; Tomczyk &        B123, 124
                                                                                    Kohlmunzer, 1971; Kohlmunzer                 B125
                                                                                    et al. 1971
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    13,19-Epoxyseneciphylline       Senecio megaphyllus Green.                      Bohlmann et al. 1986                         A

                                    S. usgorensis Cuatr.                            Bohlmann et al. 1986                         A

    13,19-Epoxyspartiodine          Senecio megaphyllus Green.                      Bohlmann et al. 1986                         A

    Erucifoline                     Senecio aegypticus L.                           Klasek et al. 1968a                          B145
                                    S. erraticus Berthol. subsp.                    Schroter & Santavy, 1960; Sedmera            B179,
                                    barbaraeifolius Krock.                          et al. 1972                                  180
                                    S. erucifolius L.                               Kompis & Santavy, 1962; Sedmera              B183,
                                                                                    et al. 1972                                  180

    Europine                        Heliotropium arbainense                         Zalkow et al. 1979                           B419
                                    H. digynum Forssk.                              Hammouda et al. 1984                         A
                                    H. europaeum L.                                 Culvenor, 1954                               B66
                                    H. lasiocarpum Fisch and Mey.                   Culvenor et al. 1986                         A
                                    H. maris-mortui                                 Zalkow et al. 1978                           B74
                                    H. rotundifolium                                Zalkow et al. 1978                           B74
                                    H. suaveolens Bieb.                             Guner, 1986                                  A
                                    Trichodesma africana                            Zalkow et al. 1979                           B419

    Floricaline                     Cacalia floridana                               Cava et al. 1968                             B119

    Floridanine                     Cacalia floridana                               Cava et al. 1968                             B119
                                    Doronicum macrophyllum                          Alieva et al. 1976                           B122
                                    Senecio aureus L.                               Roder et al. 1983                            C
                                    S. erraticus Berthol.                           Gaiduk et al. 1974                           B177
                                    S. othonnae Bieb.                               Khalilov & Telezhenetskaya, 1973b            B222

    Florosenine                     Cacalia floridana                               Cava et al. 1968                             B119
                                    Senecio aureus L.                               Roder et al. 1983                            C
                                    S. fluviatilis Wallr.                           Klasek et al. 1973b                          B185
                                    S. quebradensis Greem.                          Bohlmann et al. 1986                         A
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Fulvine                         Crotalaria berteroana DC. (C. fulva Roxb.)      Schoental, 1963                              B297
                                    C. crispata F. Muell. ex Benth.                 Culvenor & Smith, 1963                       B294
                                    C. madurensis R. Wight                          Atal et al. 1966a; Habib et al. 1971         B280, 315
                                    C. paniculata Willd.                            Subramanium et al. 1968                      B325

    Globiferine                     Crotalaria globifera E. Mey.                    Brown et al. 1984                            C

    Grahamine                       Crotalaria grahamiana R. Wight et               Atal et al. 1969                             B299
                                    Walk.-Arn.

    Grantaline                      Crotalaria globifera E. Mey                     Brawn et al., 1984                           C
                                    C. virgulata subsp. grantiana (Harv.)           Smith & Culvenor, 1984                       C
                                    Polhill (C. grantiana Harvey)

    Grantianine                     Crotalaria globifera E. Mey.                    Brown et al. 1984                            C
                                    C. virgulata subsp. grantiana (Harv.)           Adams et al. 1942b; Adams &                  B301
                                    Polhill (C. grantiana Harvey)                   Gianturco, 1956b; Smith &                    B259, C
                                    Culvenor, 1984

    Gynuramine                      Gynura scandens O. Hoffm.                       Wiedenfeld, 1982                             A

    Heleurine                       Heliotropium europaeum L.                       Culvenor, 1954                               B66
                                    H. indicum L.                                   Hoque et al. 1976                            B72
                                    H. lasiocarpum Fisch and Mey.                   Culvenor et al. 1986                         A

    Heliosupine                     Cynoglossum creticum                            Zalkow et al. 1979                           B419
                                    C. officinale                                   Man'ko & Borisyuk, 1957; Man'ko, 1959;       B38, 39
                                                                                    Sykulska, 1961                               B40
                                    C. pictum                                       Man'ko & Marchenko, 1971b, 1972b             B44, 45
                                    C. viridiflorum Pallas ex Lehm.                 Man'ko, 1972                                 B34
                                    Echium vulgare L.                               Man'ko, 1964                                 B49
                                    Heliotropium supinum L.                         Denisova et al. 1953; Crowley &              B82, 83
                                                                                    Culvenor, 1959
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    Myosotis sylvatica Hoffm.                       Culvenor & Smith, unpubl.
                                    Paracynoglossum imeritinum (Kusn.)              Man'ko & Marchenko, 1971                     B88
                                    Symphytum asperum Lepech.                       Man'ko et al. 1970b                          B97
                                    S. officinale Linn.                             Man'ko et al. 1970a                          B101

    Heliotrine                      Heliotropium acutiflorum                        Akramov et al. 1968                          B51
                                    H. arbainense                                   Zalkow et al. 1979                           B419
                                    H. arguzioides Kar. et Kir.                     Zolotavina, 1963                             B53
                                    H. curassavicum Linn.                           Rajagopalan & Batra, 1977b                   B56
                                    H. dasycarpum Ledeb.                            Akramov et al. 1961a                         B54
                                    H. digynum Forssk.                              Hammouda et al. 1984                         A
                                    H. eichwaldi Steud. ex DC.                      Gandhi et al. 1966a                          B60
                                    H. europaeum L.                                 Trautner & Neufeld, 1949; Culvenor           B64, 65
                                                                                    et al. 1954
                                    H. indicum L.                                   Hoque et al. 1976                            B72
                                    H. lasiocarpum Fisch et C. Mey.                 Men'shikov, 1932                             B73
                                    H. olgae                                        Kiyamitdinova et al. 1967; Sheveleva         B75, 76
                                                                                    et al. 1969
                                    H. popovii H. Riedl. subsp. gillianum           Mohabbet et al. 1976                         B77
                                    H. ramosissimum                                 Habib, 1975; Schoental &                     B79, 78
                                    Cavanagh, 1972
                                    H. suaveolens Bieb.                             Guner, 1986                                  A
                                    H. supinum L.                                   Pandey et al. 1983                           C
                                    H. transoxanum                                  Akramov et al. 1968                          B51

    Heliovinine                     Heliotropium curassavicum Linn.                 Mohanraj et al. 1982c                        C

    Heterophylline                  Parsonsia heterophylla A. Cunn.                 Edgar et al. 1980                            B418
                                    P. spiralis                                     Edgar et al. 1980                            B418

    18-Hydroxysenkirkine            Crotalaria laburnifolia L. subsp.               Crout, 1972                                  B312

    19-Hydroxysenkirkine            Senecio laricifolius H.B.K.                     Bohlmann et al. 1986                         A
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Incanine                        Heliotropium olgae                              Sheveleva et al. 1969                        B76

                                    Trichodesma incanum Alph. DC.                   Yunusov & Plekhanova, 1953, 1957,            B110, 111
                                                                                    1959; Tashkhodzhaev et al. 1979a             B112, C

    Indicine                        Heliotropium amplexicaule Vahl.                 Ketterer et al. 1987, (in press)
                                    H. indicum L.                                   Mattocks et al. 1961                         B70
                                    Prestonia sp.                                   Edgar, 1985                                  A

    Integerrimine                   Cacalia hastata L. subsp. orientalis            Hayashi et al. 1972                          B120
                                    Kitamura
                                    Crotalaria brevidens Benth. var.                Suri et al. 1975b                            B304
                                    intermedia (Kotschy) Polhill
                                    C. brevifolia                                   Sawhney & Atal, 1966                         B290
                                    C. incana L.                                    Adams & Van Duuren, 1953b                    B187
                                    C. pallida Ait.                                 Sawhney et al. 1967                          B291
                                    C. tetragona Roxb.                              Puri et al. 1974                             B314
                                    C. zanzibarica Benth. (C. usaramoensis)         Culvenor & Smith, 1966b                      B337
                                    Petasites hybridus L.                           Luthy et al. 1983                            C
                                    Senecio alpinus L.                              Luthy et al. 1981                            A
                                    S. antieuphorbium (L.) Sch. Bip.                Rodriguez & Gonzalez, 1969                   B152
                                    S. brasiliensis DC.                             Motidome & Ferreira, 1966a                   B163
                                    S. brasiliensis Less. var tripartitus           Nardi et al. 1980                            A
                                    S. durieui Gay                                  Panizo & Rodriguez, 1974                     B157
                                    S. erraticus Berthol. subsp.                    Santavy et al. 1962                          B182
                                    barbaraeifolius Krock.
                                    S. faberi Hemsl.                                Wei et al. 1982                              C
                                    S. formosus                                     Munoz Quevedos, 1976                         B186
                                    S. glandulosus Don ex Hook. et Arn.             Pestchanker et al. 1985b                     A
                                    S. inaequidens DC.                              Bicchi et al. 1985                           A
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    S. incanus L. subsp. carniolicus                Klasek et al. 1968b                          B147
                                    (Willd.) Br.-Bl.
                                    S. integerrimus                                 Manske, 1939a; Roitman et al. 1979           B156, 420
                                    S. kleinia Sch. Bip.                            Gonzalez & Calero, 1958b                     B205
                                    S. leucostachys Baker                           Pestchanker & Giordano, 1986                 A
                                    S. magnificus F. Muell.                         Gellert & Mate, 1964                         B216
                                    S. morrisonensis Hayata                         Lu, Sheng-Teh, 1972                          B217
                                    S. nebrodensis L. var sicula                    Plescia et al. 1976                          B218
                                    S. ragonesei Cabr.                              Pestcharnker & Giordano, 1986                A
                                    S. spathulatus A. Rich.                         White, 1969                                  B158
                                    S. squalidus L.                                 Kropman & Warren, 1950; Gonzalez &           B265,
                                    Calero, 1958a                                   205
                                    S. tenuifolius Burm.                            Bhakuni & Gupta, 1982                        C
                                    S. triangularis Hook.                           Roitman, 1983b                               C
                                    S. vernalis Walst. et Kit.                      Sener et al. 1986; Hartmann &                A, A
                                    Zimmer, 1986
                                    S. viscosus L.                                  Barger & Blackie, 1936; Santavy et al.       B264,
                                                                                    1962                                         182
                                    S. vulgaris L.                                  Pieters & Vlietinck, 1986                    A

    Intermedine                     Amsinckia hispida (Ruiz et Pav.)                Culvenor & Smith, 1966a                      B30
                                    I.M. Johnston
                                    A. intermedia Fisch et C. Mey.                  Culvenor & Smith, 1966a                      B30
                                    A. lycopsoides Lehm.                            Culvenor & Smith, 1966a                      B30
                                    A. menziesii (Lehm.) Nels. & Macbr.             Roitman, 1983                                C
                                    Borago officinale L.                            Luthy et al. 1984                            A
                                    Conoclinium coelestinium (L.) DC                Herz et al. 1981                             C
                                    Eupatorium compositifolium Walt.                Herz et al. 1981                             C
                                    Symphytum aspera                                Roitman, 1981                                A
                                    S. officinale Linn.                             Roder et al. 1982                            C
                                    S. x uplandicum Nyman                           Culvenor et al. 1980a, 1980b                 A, A
                                    Trichodesma africana                            Zalkow et al. 1979                           B419
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Isocromadurine                  Crotalaria candicans W. and A.                  Suri et al. 1982                             C
                                    C. madurensis R. Wight                          ]Rao et al. 1975c                            B317

    Isoline                         Senecio othonniformis Fourcade                  Coucourakis & Gordon-Gray, 1970;             B224
                                    Coucourakis et al. 1972                         B225

    Jacobine                        Crassocephalum crepidioides                     Asada et al. 1985                            A
                                    Senecio alpinus L.                              Luthy et al. 1981                            A
                                    S. brasiliensis DC.                             Adams & Gianturco, 1956e                     B148
                                    S. cineraria DC.                                Barger & Blackie, 1937                       B167
                                    S. jacobaea L.                                  Manske, 1931; Blackie, 1937;                 B194, 153
                                    Bradbury & Culvenor, 1954                       B198
                                    S. paludosus L.                                 Blackie, 1937; Dorosh & Alekseev, 1960       B153, 227
                                    Alekseev, 1961b                                 B228

    Jacoline                        Crassocephalum crepidioides                     Asada et al. 1985                            A
                                    Senecio alpinus L.                              Luthy et al. 1981                            A
                                    S. jacobaea L.                                  Bradbury & Culvenor, 1954                    B198

    Jaconine                        Senecio alpinus L.                              Luthy et al. 1981                            A
                                    S. jacobaea L.                                  Bradbury & Culvenor, 1954                    B198

    Jacozine                        Senecio alpinus L.                              Luthy et al. 1981                            A
                                    S. cannabifolius Less                           Asada et al. 1982                            C
                                    S.jacobaea L.                                   Bradbury & Culvenor, 1954; Culvenor,         B198,
                                                                                    1964                                         202

    Junceine                        Crotalaria juncea L.                            Adams & Gianturco, 1956a, 1956b, 1956c       B305,
                                                                                                                                 B306
                                                                                                                                 B307
                                    C. wightiana Grah. ex Wight & Arn.              Atal et al. 1966b                            B329
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Lasiocarpine                    Heliotropium arbainense                         Zalkow et al. 1979                           B419
                                    H. arborescens L.                               Carcamo-Marquez, 1961                        B52
                                    H. curassavicum Linn.                           Rajagopalan & Batra, 1977b                   B56
                                    H. digynum Forssk.                              Hammouda et al. 1984                         A
                                    H. eichwaldii Steud. ex DC.                     Rao et al. 1974                              B62
                                    H. europaeum L.                                 Culvenor et al. 1954                         B65
                                    H. indicum Linn.                                Hoque et al. 1976                            B72
                                    H. lasiocarpum Fisch. et C. Mey.                Men'shikov, 1932                             B73
                                    H. maris mortui                                 Zalkow et al. 1979                           B419
                                    H. suaveolens Bieb.                             Guner, 1986                                  A
                                    H. supinum L.                                   Pandey et al. 1983                           C
                                    Lappula intermedia                              Man'ko & Vasil'kov, 1968                     B85
                                    Symphytum caucasicum                            Man'ko et al. 1969                           B95
                                    S. officinale Linn.                             Man'ko et al. 1969, 1970a                    B95, 101

    Latifoline                      Cynoglossum latifolium R. Br.                   Crowley & Culvenor, 1962                     B37
                                    Hackelia floribunda                             Hagglund et al. 1985                         A

    Ligudentine                     Ligularia brachyphylla Hand.-Mazz.              Klasek et al. 1971                           B134
                                    L. dentata (A. Gray) Hara.                      Klasek et al. 1971                           B134

    Ligularidine                    Ligularia dentata (A. Gray) Hara                Hikichi et al. 1979, Asada &                 B436, C
                                    Furuya, 1984a

    Ligularine                      Ligularia brachyphylla Hand.-Mazz.              Klasek et al. 1971                           B134
                                    L. dentata (A. Gray) Hara.                      Klasek et al. 1971                           B134
                                    L. elegans Cass.                                Klasek et al. 1971                           B134

    Ligularizine                    Ligularia dentata (A. Gray) Hara.               Asada & Furuya, 1984a                        C
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Lycopsamine                     Amsinckia hispida (Ruiz et Pav.)                Culvenor & Smith, 1966a                      B30
                                    I.M. Johnston
                                    A. intermedia Fisch. et C. Mey.                 Culvenor & Smith, 1966a                      B30
                                    A. lycopsoides Lehm.                            Culvenor & Smith, 1966a                      B30
                                    A. menziesii (Lehm.) Nels. & Macbr.             Roitman, 1983a                               C
                                    Anchusa officinalis L.                          Broch-Due & Aasen, 1980                      B32
                                    Borago officinale L.                            Larson et al. 1984; Luthy et al. 1984        C, A
                                    Eupatorium compositifolium Walt.                Herz et al. 1981                             C
                                    Heliotropium steudneri Vatke                    Schneider et al. 1975                        B8O
                                    Messerschmidia sibirica                         Hikichi et al. 1980                          C
                                    Parsonsia eucalyptophylla F. Muell.             Edgar & Culvenor, 1975                       B28
                                    P. straminea (R. Br.) F. Muell.                 Edgar & Culvenor, 1975                       B28
                                    Prestonia sp.                                   Edgar, 1985                                  A
                                    Symphytum asperum Lepech.                       Roitman, 1981                                A
                                    Symphytum officinale Linn.                      Huizing & Malingre, 1981                     C
                                    Symphytum x uplandicum Nyman                    Culvenor et al. 1980a, 1980b                 A, A

    Madurensine                     Crotalaria agatiflora Schweinf.                 Atal et al. 1966a                            B280
                                    C. laburnifolia L. subsp. eldomae               Crout, 1972                                  B312
                                    C. madurensis R. Wight                          Atal et al. 1966a; Mahran et al. 1979        B280, 426

    Merenskine                      Senecio latifolius DC.                          Bredenkamp et al. 1985                       C

    Monocrotaline                   Crotalaria aegyptiaca Benth.                    Mahran et al. 1979; Zalkow et al. 1979       B426, 419
                                    C. assamica Benth.                              Crotalaria Research Group, 1974              B286
                                    C. burhia                                       Rao et al. 1975                              B293
                                    C. cephalotes Steud. ex A. Rich                 Pilbeam et al. 1983                          C
                                    C. crispata F. Muell. ex Benth.                 Culvenor & Smith, 1963                       B294
                                    C. cunninghamii R. Br.                          Pilbeam et al. 1983                          C
                                    C. grahamiana R. Wight ex Walk.-Arn.            Gandhi et al. 1966b; Atal et al. 1969        B298, 299
                                    C. leschenaultii DC.                            Suri & Atal, 1967                            B313
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    C. leiloba Bartl.                               Puri et al. 1974                             B314
                                    C. mitchellii Benth.                            Culvenor et al. 1967b                        B318
                                    C. mysorensis Roth.                             Sawhney & Atal, 1968                         B319
                                    C. nitens Kunth.                                Hoet et al. 1981                             A
                                    C. novae-hollandiae DC. subsp.                  Culvenor et al. 1967b                        B318
                                    lasiophylla (Benth.) A. Lee
                                    C. paulina schrank.                             Pilbeam et al. 1983                          C
                                    C. quinquefolia L.                              Pilbeam et al. 1983                          C
                                    C. recta Steud. ex A. Rich                      Crout, 1968a                                 B326
                                    C. retusa L.                                    Adams & Rogers, 1939; Culvenor &             B327,
                                                                                    Smith, 1957a                                 328
                                    C. sagittalis L.                                Willette & Cammarata, 1972                   B330
                                    C. spectabilis Roth.                            Neal et al. 1935; Adams & Rogers,            B325,
                                                                                    1939                                         327
                                    C. stipularia Desv.                             Puri et al. 1974                             B314
                                    Lindelofia spectabilis Lehm.                    Rao et al. 1974                              B62

    Monocrotalinine                 Crotalaria grahamiana Wight et Arn.             Rajagopalan & Batra, 1977a                   B300

    Myoscorpine                     Myosotis scorpioides L.                         Resch et al. 1982                            C
                                    Symphytum officinale Linn.                      Resch et al. 1982C                           C

    Neoligularidine                 Ligularia dentata (A. Gray) Hara.               Asada & Furuya, 1984a                        C

    Neopetasitenine                 Ligularia japonica                              Asada et al. 1981                            A
                                    Petasites japonicus Maxim.                      Yamada et al. 1976a                          B19

    Neosenkirkine                   Senecio auricola Bourg.                         Panizo & Rodriguez, 1974                     B157
                                    S. grandifolius Less.                           Bohlmann et al. 1986                         A
                                    S. pierotii                                     Asada & Furuya, 1982                         C

    Neotriangularine                Senecio triangularis Hook.                      Roitman, 1983b                               C
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Nilgirine                       Crotalaria pallida Ait.                         Atal et al. 1968                             B322

    Onetine                         Senecio othonnae Bieb.                          Danilova et al. 1962                         B221

    Otosenine                       Cacalia floridana                               Cava et al. 1968                             B119
                                    Doronicum macrophyllum                          Alieva et al. 1976                           B122
                                    D. pardalianches Linn.                          Rajagopalan & Negi, 1985                     A
                                    Emilia flammea Cass.                            Kohlmunzer & Tomczyk, 1969                   B123
                                    Senecio aegypticus L.                           Klasek et al. 1968a                          B145
                                    S. aureus L.                                    Resch et al. 1983, Roder et al. 1983         C, C
                                    S. cineraria DC.                                Habib, 1974                                  B170
                                    S. desfontainei Druce                           Haddad et al. 1963; Klasek                   B173,
                                                                                    et al. 1968a                                 145
                                    S. erraticus Berthol.                           Gaiduk et al. 1974                           B177
                                    S. erraticus subsp. barbaraeifolius Krock.      Santavy, 1958; Schroter & Santavy, 1960      B178, 179
                                    S. fluviatilis Wallr.                           Klasek et al. 1973b                          B185
                                    S. jacobaea L.                                  Akramov et al. 1968                          B51
                                    S. othonnae Bieb.                               Zhdanovich & Men'shikov, 1941;               B220
                                                                                    Danilova et al. 1962                         B221
                                    S. renardii Winkl.                              Danilova & Konovalova, 1950                  B249
                                    S. tomentosus                                   Adams et al. 1956; Schroter &                B271,
                                                                                    Santavy, 1960                                179

    Parsonsine                      Parsonsia heterophylla A. Cunn.                 Eggers & Gainsford, 1979; Edgar et al.       B417,
                                                                                    1980                                         418
                                    P. spiralis                                     Edgar et al. 1980                            B418

    Petasinine                      Petasites japonicus Maxim.                      Yamada et al. 1978b                          B141

    Petasitenine                    Farfugium japonicum Kitam                       Niwa et al. 1985                             A
                                    Petasites japonicus Maxim.                      Yamada et al. 1976a, 1976b;                  B19, 139
                                                                                    Furuya et al. 1976                           B20
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Retroisosenine                  Senecio nemorensis L. var. bulgaricus           Nghia et al. 1976                            B219
                                    (Vel.) Stoj. et Stef.
                                    S. nemorensis L. var subdecurrens               Klasek et al. 1980a                          B422

    Retrorsine                      Crotalaria spartioides DC.                      Bruemmerhoff & de Waal, 1961                 B334
                                    C. zanzibarica Benth. (C. usaramoensis)         Culvenor & Smith, 1966b                      B337
                                    Senecio ambrosioides                            Adams & Gianturco, 1956e                     B148
                                    S. ampullaceus Hook.                            Adams & Govindachari, 1949b; Adams &         B149, 151
                                    Looker, 1951; Warren et al. 1950                B150
                                    S. bipinnatisectus Belcher                      White, 1969                                  B158
                                    S. brasiliensis DC.                             Motidome & Ferreira, 1966a                   B163
                                    S. bupleuroides DC.                             Sapiro, 1949                                 B164
                                    S. cineraria DC.                                Klasek et al. 1975                           B171
                                    S. cruentus DC.                                 Asada et al. 1982                            C
                                    S. cymbaroides                                  Roitman et al. 1979                          B420
                                    S. desfontainei Druce                           Rizk et al. 1983                             A
                                    S. discolor DC.                                 Schoental, 1960; Hennig, 1961                B174, 175
                                    S. douglasii DC.                                Adams & Govindachari, 1949b; Adams           B149,
                                                                                    & Looker, 1951                               151
                                    S. eremophilus Richards                         Adams & Govindachari, 1949b; Adams           B149,
                                                                                    & Looker, 1951                               151
                                    S. erucifolius L.                               Ferry & Brazier, 1976                        B184
                                    S. filaginoides (H. et A.) DC.                  Pestchanker & Giordano, 1986                 A
                                    S. formosus                                     Munoz Quevedo, 1976                          B186
                                    S. gilliesiano                                  Guidugli et al. 1986                         A
                                    S. glaberrimus DC.                              Blackie, 1937                                B153
                                    S. glandulosus Don ex Hook. et Arn.             Pestchanker et al. 1985b                     A
                                    S. graminifolius N.J. Jacq.                     de Waal, 1941                                B188
                                    S. griesbachii                                  Motidome & Ferreira, 1966b                   B190
                                    S. ilicifolius Thunb.                           de Waal, 1940a, 1940b, 1941; Culvenor        B191, 192
                                                                                    & Smith, 1954                                B188, 127
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    S. inaequidens DC.                              Roder et al. 1981                            A
                                    S. isatideus DC.                                Blackie, 1937; de Waal, 1939                 B153, 193
                                    S. jacobaea L.                                  Ferry & Brazier, 1976                        B184
                                    S. latifolius DC.                               Watt, 1909; Barger et al. 1935               B211, 212
                                    S. longilobus Benth.                            Adams & Govindachari, 1949b; Adams &         B149, 151
                                                                                    Looker, 1951; Warren et al. 1950             B150
                                    S. paucicalyculatus Klatt                       Pretorius, 1949                              B230
                                    S. phillipicus Roegel et Koern                  Gonzalez et al. 1986a                        A
                                    S. pterophorus DC.                              de Waal, 1940b, 1941; Culvenor &             B192, 188
                                                                                    Smith, 1954                                  127
                                    S. quadridentatus Labill.                       Culvenor & Smith, 1955                       B247
                                    S. ragonesei Cabr.                              Pestchanker & Giordano, 1986                 A
                                    S. retrorsus DC.                                Manske, 1931; de Waal, 1939                  B194, 193
                                    S. riddellii Torr. et A. Gray                   Roitman et al. 1979                          B420
                                    S. riddellii Torr. et A. Gray var.              Adams & Govindachari, 1949b                  B149
                                    parksii Cory.
                                    S. ruderalis Harvey                             Leisegang, 1950                              B254
                                    S. seratophiloides Griseb.                      Pestchanker & Giordano, 1986                 A
                                    S. spartioides                                  Roitman et al. 1979                          B420
                                    S. subulatus Don ex Hook. et Arn                Pestchanker et al. 1985b                     A
                                    var erectus
                                    S. swaziensis Compton                           Gordon-Gray et al. 1972; Gordon-Gray         B268,
                                                                                    & Wells, 1974                                270
                                    S. triangularis Hook.                           Roitman, 1983b                               C
                                    S. uspallatensis                                Pestchanker et al. 1985a                     A
                                    S. venosus Harvey                               Blackie, 1937                                B153
                                    S. vernalis Waldst. et Kit.                     Roder et al. 1979b                           C
                                    S. viminalis Bremek.                            de Waal & van Twisk, 1964                    A
                                    S. vulgaris L.                                  Tschu Shun et al. 1960                       B277
                                    S. werneriaefolius                              Roitman et al. 1979                          B420
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Retusamine                      Crotalaria mitchellii Benth.                    Culvenor et al. 1967b                        B318
                                    C. mitchellii Benth. subsp. laevis              Culvenor et al. 1967b                        B318
                                    A. Lee
                                    C. novae-hollandiae DC. subsp.                  Culvenor et al. 1967b                        B318
                                    lasiophylla Benth. A. Lee
                                    C. novae-hollandiae DC. subsp.                  Culvenor et al. 1967b                        B318
                                    novae-hollandiae
                                    C. retusa L.                                    Culvenor & Smith, 1957a; Wunderlich,         B328, C
                                                                                    1962

    Riddelliine                     Crotalaria juncea L.                            Adams & Gianturco, 1956a                     B305
                                    Senecio aegypticus L.                           Klasek et al. 1968a                          B145
                                    S. ambrosioides                                 Roitman et al. 1979                          B420
                                    S. cruentus DC.                                 Asada et al. 1982                            C
                                    S. cymbaroides                                  Roitman et al. 1979                          B420
                                    S. desfontainei Druce                           Haddad et al. 1963; Klasek et al.            B173,
                                                                                    1968a                                        145
                                    S. douglassi DC.                                Adams & Govindachari, 1949b; Adams &         B149,
                                                                                    Looker, 1951                                 151
                                    S. eremophilus Richards                         Adams & Govindachari, 1949b; Adams &         B149,
                                                                                    Looker, 1951                                 151
                                    S. longilobus Benth.                            Adams & Govindachari, 1949b; Adams &         B149,
                                                                                    Looker, 1951;                                151
                                                                                    Warren et al. 1950                           B150
                                    S. riddellii Torr. et A. Gray                   Manske, 1939a; Adams et al. 1942c            B156, 252
                                    S. riddellii Torr. et A. Gray var.              Adams & Govindachari, 1949b                  B149
                                    parksii (Cory)
                                    S. spartioides                                  Roitman et al. 1979                          B420
                                    S. vernalis Walst. et Kit.                      Sener et al. 1986                            A
                                    S. vulgaris                                     Roitman et al. 1979                          B420
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Rinderine                       Eupatorium altissimum L.                        Herz et al. 1981                             C
                                    E. serotinum Michx.                             Locock et al. 1966                           B131
                                    Prestonia sp.                                   Edgar, 1985                                  A
                                    Rindera baldshuanica Kusnezov                   Akramov et al. 1961c                         B91
                                    Solenanthus turkestanicus Regel                 Akramov et al. 1962                          B94
                                    et Smirnov
                                    (Kusnezov)

    Sceleratine                     Senecio latifolius DC. (S. sceleratus)          de Waal & Pretorius, 1941; de Waal           B257,
                                                                                    et al. 1963                                  260

    Scorpioidine                    Myosotis scorpioides L.                         Resch et al. 1982                            C

    Sencalenine                     Senecio cacaliaster (Lam.)                      Roder et al. 1984b                           A

    Senecicannabine                 Senecio cannabifolius Less.                     Asada et al. 1982                            C

    Senecionine                     Brachyglottis repanda Forst. et Forst.          Mortimer & White, 1967                       B117
                                    Caltha biflora                                  Stermitz & Adamovics, 1977                   B341
                                    C. leptosepala                                  Stermitz & Adamovics, 1977                   B341
                                    Castilleja rhexifolia Rydb.                     Stermitz & Suess, 1978                       B342
                                    Castilleja "rhexifolia aff. miniata"            Roby & Stermitz, 1984                        C
                                    Crotalaria juncea L.                            Adams & Gianturco, 1956a                     B305
                                    C. micans Link.                                 Sethi & Atal, 1964                           B284
                                    C. zanzibarica Benth. (C. usaramoensis)         Culvenor & Smith, 1966b                      B337
                                    Emilia sonchifolia DC.                          Culvenor, unpubl.
                                    Erechtites hieracifolia (L.) Raf. ex DC.        Manske, 1939b; Culvenor & Smith, 1954        B126, 127
                                    Gynura segetum (Lour.) Merr.                    Hua et al. 1983; Liang & Roder, 1984         C, C
                                    Ligularia japonica                              Asada et al. 1981                            A
                                    Petasites hybridus L.                           Luthy et al. 1983                            C
                                    P. laevigatus (Willd.) Reichenb.                Massagetov & Kuzovkov, 1953                  B142
                                    Senecio aegypticus L.                           Klasek et al. 1968a; Gharbo &                B145,
                                                                                    Habib, 1969                                  146
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    S. alpinus (L.) Scop.                           Luthy et al. 1981                            A
                                    S. ambrosioides                                 Adams & Gianturco, 1956e                     B148
                                    S. ampullaceus Hook.                            Adams & Govindachari, 1949b; Adams &         B149,
                                                                                    Looker, 1951;                                151
                                                                                    Warren et al. 1950                           B150
                                    S. argentino Baker (vira-vira Hieron)           Pestchanker & Giordano, 1986                 A
                                    S. aureus L.                                    Manske, 1936, 1939a                          B155, 156
                                    S. brasiliensis DC.                             Fonseca, 1951; Novelli & De Varella,         B162,
                                                                                    1945; Adams & Gianturco, 1956e               161
                                                                                                                                 B148
                                    S. carthamoides Greene                          Adams & Govindachari, 1949b; Adams           B149,
                                                                                    & Looker, 1951                               151
                                    S. cineraria DC.                                Barger & Blackie, 1937; Adams &              B167,
                                                                                    Govindachari, 1949a;                         168
                                                                                    Alekseev et al. 1962a                        B169
                                    S. congestus (R.Br.) DC.                        Roder et al. 1982                            C
                                    S. cruentus DC.                                 Asada et al. 1982                            C
                                    S. cymbaroides                                  Roitman et al. 1979                          B420
                                    S. desfontainei Druce                           Klasek et al. 1968a                          B145
                                    S. discolor DC.                                 Schoental, 1960; Hennig, 1961                B174, 175
                                    S. douglasii DC.                                Adams & Govindachari, 1949b; Adams           B149,
                                                                                    & Looker, 1951                               151
                                    S. eremophilus Richards                         Adams & Govindachari, 1949b; Adams           B149,
                                                                                    & Looker, 1951                               151
                                    S. erraticus Berthol.                           Gaiduk et al. 1974                           B177

                                    S. erraticus Berthol. subsp.                    Santavy, 1958; Schroter & Santavy,           B178,
                                    barbaraeifolius Krock.                          1960; Kompis et al. 1960                     179
                                                                                                                                 B181
                                    S. erucifolius L.                               Kompis & Santavy, 1962                       B183
                                    S. filaginoides (H. et A.) DC.                  Pestchanker & Giordano, 1986                 A
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    S. fistulosus Poepp. ex Less.                   Gonzalez et al. 1986b                        A
                                    S. fremontii Torr. et A. Gray                   Adams & Gianturco, 1956e                     B148
                                    S. gilliesiano                                  Guidugli et al. 1986                         A
                                    S. glabellus Turcz. DC.                         Adams & Van Duuren, 1953b                    B187
                                    S. ilicifolius Thunb.                           de Waal, 1940a, 1940b, 1941; Culvenor        B191,
                                                                                    & Smith, 1954                                192
                                                                                                                                 B188,
                                                                                                                                 127
                                    S. illinitus Phill.                             Gonzalez et al. 1986a                        A
                                    S. inaequidens DC.                              Roder et al. 1981                            A
                                    S. integerrimus Nutt.                           Manske, 1939a                                B156
                                    S. jacobaea L.                                  Bradbury & Mosbauer, 1956                    B199
                                    S. laricifolius H.B.K.                          Bohlmann et al. 1986                         A
                                    S. lautus Forst. f. ex Willd.                   Culvenor unpubl.
                                    S. leucostachys Baker                           Pestchanker & Giordano, 1986                 A
                                    S. longiflorus Sch. Bip.                        de Waal & van Twisk, 1964                    A
                                    S. magnificus F. Muell.                         Culvenor, 1962                               B215
                                    S. multilobatus                                 MaCoy et al. 1983                            A
                                    S. multivenius Benth. in Oerst                  Bohlmann et al. 1986                         A
                                    S. nebrodensis L. var. sicula                   Plescia et al. 1976                          B218
                                    S. nemorensis L. subsp. fuchsii Gmel.           Wiedenfeld & Roder, 1979                     B423
                                    S. pampeanus Cabrera                            Novelli, 1958                                B229
                                    S. pancicii Degen var arnautorum                Jizba et al. 1982                            C
                                    (Velen.) Stoj. Stef. et Kit.
                                    S. pancicii Degen var pancicii                  Jizba et al. 1982                            C
                                    S. patagonicus Hook. and Arn.                   Villarroel et al. 1985                       A
                                    S. petasitis DC.                                Gharbo & Habib, 1969                         B146
                                    S. pimpinellifolius H.B.K.                      Bohlmann et al. 1986                         A
                                    S. pseudo-arnica Less.                          Manske, 1939a                                B156
                                    S. pterophorus DC.                              de Waal, 1940b, 1941; Culvenor &             B192,
                                                                                    Smith, 1954                                  188
                                                                                                                                 B127
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    S. quadridentatus Labill.                       Culvenor & Smith, 1955                       B247

                                    S. sandrasicus                                  Temizer et al. 1985                          A
                                    S. scandens                                     Batra & Rajagopalan, 1977                    B256
                                    S. seratophiloides Griseb.                      Pestchanker & Giordano, 1986                 A
                                    S. spartioides                                  Manske, 1939a; Adams & Gianturco,            B156,
                                                                                    1957b                                        262
                                    S. spathulatus A. Rich                          White, 1969                                  B158
                                    S. squalidus L.                                 Barger & Blackie, 1936; Kropman              B264,
                                                                                    & Warren, 1950                               265
                                    S. subalpinus C. Koch                           Trivedi & Santavy, 1963                      B267
                                    S. subulatus Don ex Hook. et Arn                Pestchanker et al. 1985b                     A
                                    var erectus
                                    S. tenuifolius Burm.                            Bhakuni & Gupta, 1982                        C
                                    S. tomentosus                                   Adams et al. 1956                            B271
                                    S. triangularis Hook.                           Kupchan & Suffness, 1967                     B272
                                    S. uintahensis                                  Roitman et al. 1979                          B420
                                    S. vernalis Waldst. et Kit.                     Roder et al. 1979b                           C
                                    S. viminalis Bremek.                            de Waal & van Twisk, 1964                    A
                                    S. viscosus L.                                  Barger & Blackie, 1936; Santavy et al.       B264,
                                                                                    1962                                         182
                                    S. vulgaris L.                                  Grandval & Lajoux, 1895; Barger &            B275,
                                                                                    Blackie, 1936; Konovalova &                  264
                                                                                    Orekhov, 1937a; Tschu Shun                   B276
                                                                                    et al. 1960                                  B277
                                    S. wernariaefolius                              Roitman et al. 1979                          B420
                                    Syneilesis palmata Maxim.                       Hikichi & Furuya, 1976                       B279
                                    Tussilago farfara                               Rosberger et al. 1981                        C

    7-Senecioyl-9-(2-hydroxy-       Senecio caudatus DC.                            Bohlmann et al. 1986                         A
    3-acetylbutyrl)retronecine
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    7-Senecioyl-9-(2-hydroxy-       Senecio caudatus DC.                            Bohlmann et al. 1986                         A
    methyl-2,3-dihydroxy-
    butyrylretronecine

    7-Senecioyl-9-(2-methyl-        Senecio caudatus DC.                            Bohlmann et al. 1986                         A
    2,3-dihydroxybutyryl-
    retronecine

    7-Senecioylretronecine          Senecio cacaliaster (Lam.)                      Roder et al. 1984b                           C
                                    S. caudatus DC.                                 Bohlmann et al. 1986                         A
                                    S. triangularis Hook.                           Rueger & Benn, 1983a                         C
                                    S. variabilis Sch. Bip.                         Bohlmann et al. 1986                         A

    9-Senecioylretronecine          Senecio caudatus DC.                            Bohlmann et al. 1986                         A
                                    S. variabilis Sch. Bip.                         Bohlmann et al. 1986                         A

    7-Senecioyl-9-sarracinyl-       Senecio cacaliaster                             Roder et al. 1984b                           C
    retronecine                     S. caudatus DC.                                 Bohlmann et al. 1986                         A
                                    S. triangularis Hook.                           Rueger & Benn, 1983                          C
                                    S. ungeniensis Thell.                           Bohlmann et al. 1986                         A
                                    S. variabilis Sch. Bip.                         Bohlmann et al. 1986                         A

    Seneciphylline                  Adenostyles alliarae                            Yakhontova et al. 1976                       B115
                                    A. glabra                                       Wiedenfeld et al. 1984                       C
                                    A. rhombifolius (Willd.) M. Pimen.subsp.        Pimenov et al. 1975                          B116
                                    platyphylloides
                                    Crotalaria juncea L.                            Adams & Gianturco, 1956a                     B305
                                    Erechtites hieracifolia (L.) Raf. ex DC.        Manske, 1939a; Culvenor & Smith, 1954        B126, 127
                                    Senecio alpinus (L.) Scop.                      Klasek et al. 1968b; Luthy et al. 1981       B147, A
                                    S. ambrosioides                                 Adams & Gianturco, 1956e                     B148
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    S. ampullaceus Hook.                            Adams & Govindachari, 1949b; Adams &         B149,
                                                                                    Looker, 1951; Warren et al. 1950             151
                                                                                                                                 B150
                                    S. aquaticus Hill                               Blackie, 1937; Evans & Evans, 1949           B153, 154
                                    S. borysthenicus                                Red'ko, 1956; Alekseev, 1961a                B159, 160
                                    S. brasiliensis DC.                             Fonseca, 1951; Novelli & de Varella,         B162,
                                                                                                                                 161
                                                                                    1945; Adams & Gianturco, 1956e               B148
                                    S. cannabifolius Less.                          Alekseev, 1964; Asada et al. 1982            B165, C
                                    S. carthamoides Greene                          Adams & Govindachari, 1949b; Adams &         B149,
                                                                                    Looker, 1951                                 151
                                    S. chrysanthemoides                             Wali & Handa, 1964                           B166
                                    S. cineraria DC.                                Barger & Blackie, 1937; Adams &              B167,
                                                                                    Govindachari, 1949a                          168
                                    S. cruentus DC.                                 Asada et al. 1982                            C
                                    S. cymbaroides                                  Roitman et al. 1979                          B420
                                    S. desfontainei Druce                           Gharbo & Habib, 1969                         B146
                                    S. douglasii DC.                                Adams & Govindachari, 1949b; Adams &         B149,
                                                                                    Looker, 1951                                 151
                                    S. eremophilus Richards                         Adams & Govindachari, 1949b; Adams &         B149,
                                                                                    Looker, 1951                                 151
                                    S. erraticus Berthol. subsp.                    Kompis et al. 1960; Santavy                  B181,
                                    barbaraeifolius Krock.                          et al. 1962                                  182
                                    S. erucifolius L.                               Kompis & Santavy, 1962                       B183
                                    S. fluviatilis Wallr.                           Klasek et al. 1973b                          B185
                                    S. fremontii Torr. et A. Gray                   Adams & Gianturco, 1956e                     B148
                                    S. grandifolia                                  Glonti, 1958                                 B189
                                    S. ilicifolius Thunb.                           de Waal, 1940a, 1940b, 1941;                 B191, 192
                                                                                    Culvenor & Smith, 1954                       B188,
                                                                                                                                 127
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    S. incanus L. subsp. carniolus                  Klasek et al. 1968b                          B147
                                    (Willd.) Br.-Bl.
                                    S. jacobaea L.                                  Blackie, 1937; Bradbury &                    B153,
                                                                                    Culvenor, 1954                               198
                                    S. krylovii                                     Sapunova & Ban'kovskii, 1968                 B207
                                    S. kubensis Grossh.                             Khalilov & Telezhenetskaya, 1973a            B208
                                    S. lampsanoides                                 Khalilov & Damirov, 1974                     B210
                                    S. laricifolius H.B.K.                          Bohlmann et al. 1986                         A
                                    S. latifolius DC.                               Danilova et al. 1960                         B213
                                    S. longiflorus Sch. Bip.                        de Waal & van Twisk, 1964                    A
                                    S. longilobus Benth.                            Adams & Govindachari, 1949b; Adams &         B149,
                                                                                    Looker, 1951                                 151
                                    S. minimus Poir.                                White, 1969                                  B158
                                    S. multivenius Benth. in Oerst                  Bohlmann et al. 1986                         A
                                    S. othonnae Bieb.                               Zhdanovich & Men'shikov, 1941;               B220
                                                                                    Danilova et al. 1962                         B221
                                    S. palmatus Pall.                               Alekseev, 1960                               B226
                                    S. paludosus L.                                 Blackie, 1937; Dorosh & Alekseev,            B153,
                                                                                    1960;                                        227
                                                                                    Alekseev, 1961b                              B228
                                    S. pancicii Degen var arnautorum                Jizba et al. 1982                            C
                                    (Velen.) Stoj. Stef. et Kit.
                                    S. pancicii Degen var panicii                   Jizba et al. 1982                            C
                                    S. patagonicus Hook. and Arn.                   Villarroel et al. 1985                       A
                                    S. paucifolius S.G. Gmel.                       Alekseev & Ban'kovskii, 1965                 B231
                                    S. phillipicusRoegel et Koern                   Gonzalez et al. 1986a                        A
                                    S. platyphylloides Somm. et Lev.                Murav'eva, 1964b, 1965; Dauksha, 1970        B233, 234
                                                                                                                                 B235
                                    S. platyphyllus (Bieb.) DC.                     Orekhov, 1935; Konovalova &                  B236,
                                                                                    Orekhov, 1938;                               237
                                                                                    Konovalova, 1951                             B238
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    S. pojarkovae                                   Chernova & Murav'eva, 1974                   B243
                                    S. propinquus Ait.                              Khalilov et al. 1972                         B245
                                    S. pterophorus DC.                              de Waal, 1940b, 1941; Culvenor &             B192,
                                                                                    Smith, 1954                                  188
                                                                                                                                 B127
                                    S. quadridentatus Labill.                       Culvenor & Smith, 1955                       B247
                                    S. racemosus                                    Khmel, 1961                                  B248
                                    S. renardii Winkl.                              Danilova & Konovalova, 1950                  B249
                                    S. rhombifolius (Willd.) Sch. Bip.              Khalilov & Telezhenetskaya, 1973a            B208
                                    S. scandens                                     Batra & Rajagopalan, 1977                    B256
                                    S. spartioides Torr. et A. Gray                 Manske, 1939a; Adams & Gianturco,            B156,
                                                                                    1957b                                        262
                                    S. spathulatus                                  Benn et al. 1979                             B263
                                    S. stenocephalus Maxim.                         Konovalova & Orekhov, 1937b                  B266
                                    S. subalpinus C. Koch.                          Trivedi & Santavy, 1963; Klasek              B267,
                                                                                    et al. 1968b                                 147
                                    S. vernalis Walst. et Kit.                      Sener et al. 1986; Hartmann &                A, A
                                                                                    Zimmer, 1986
                                    S. vulgaris L.                                  Barger & Blackie, 1936; Konovalova &         B264,
                                                                                    Orenkhov, 1937a;                             276
                                                                                    Tschu Shun et al. 1960                       B277

    Senecivernine                   Senecio inaequidens                             Bicchi et al. 1985                           A
                                    S. seratophylloides Griseb.                     Pestchanker & Giordano, 1986                 A
                                    S. vernalis Waldst. et Kit.                     Roder et al. 1979b                           C

    Senkirkine                      Brachyglottis repanda Forst. et Forst.          Mortimer & White, 1967                       B117
                                    Crotalaria laburnifolia subsp. eldomae          Crout, 1972                                  B312
                                    Farfugium japonicum Kitam.                      Furuya et al. 1971                           B133
                                    Petasites albus L.                              Luthy et al. 1983                            C
                                    P. hybridus L.                                  Luthy et al. 1983                            C
                                    P. japonicus Maxim.                             Yamada et al. 1978a                          B140
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

                                    P. laevigatus (Willd.) Reichenb.                Massagetov & Kuzovkov, 1953                  B142
                                    Senecio antieuphorbium (L.) Sch. Bip.           Rodriguez & Gonzalez, 1969                   B152
                                    S. desfontainei Druce                           Rizk et al. 1983                             A
                                    S. grandifolius Less.                           Bohlmann et al. 1986                         A
                                    S. illinitus Phill.                             Gonzalez et al. 1986a                        A
                                    S. jacobaea L.                                  Akramov et al. 1968                          B51
                                    S. kirkii Hook. f. ex Kirk.                     Briggs et al. 1948; Briggs et al. 1965       B203, 204
                                    S. kleinia Sch. Bip.                            Rodriguez et al. 1967                        B206
                                    S. laricifolius H.B.K.                          Bohlmann et al. 1986                         A
                                    S. pierotii                                     Asada & Furuya, 1982                         C
                                    S. procerus L. var. procerus Stoj.              Jovceva et al. 1978                          B244
                                    Stef. et Kit.
                                    S. quebradensis Greenm.                         Bohlmann et al. 1986                         A
                                    S. renardii Winkl.                              Danilova & Konovalova, 1950; Briggs          B249,
                                                                                    et al. 1965                                  204
                                    S. tenuifolius Burm.                            Bhakuni & Gupta, 1982                        C
                                    S. vernalis Waldst. et Kit.                     Roder et al. 1979b                           C
                                    S. uintahensis                                  Roitman et al. 1979                          B420
                                    Tussilago farfara                               Culvenor et al. 1976b; Borka &               A, B425,
                                                                                    Onshuus, 1979; Luthy et al, 1980             A

    Sincamidine                     Amsinckia intermedia Fisch. et C. Mey.          Culvenor & Smith, 1966a                      B30

    Spartioidine                    Senecio spartioides Torr. et A. Gray            Manske, 1939a; Adams & Gianturco, 1957       B156,
                                                                                                                                 262
                                    S. vulgaris L.                                  Pieters & Vlietinck, 1986                    A

    Spectabiline                    Crotalaria spectabilis Roth                     Culvenor & Smith, 1957b                      B336

    Spiracine                       Parsonsia spiralis Wall.                        Edgar et al. 1980                            B418

    Spiraline                       Parsonsia spiralis Wall.                        Edgar et al. 1980                            B418
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Spiranine                       Parsonsia spiralis Wall.                        Edgar et al. 1980                            B418

    Supinine                        Borago officinale L.                            Luthy et al. 1984                            A
                                    Eupatorium cannabinum L.                        Pederson, 1975a                              B128
                                    E. serotinum Michx.                             Locock et al. 1966                           B131
                                    E. stoechadosmum Hanse                          Furuya & Hikichi, 1973                       B132
                                    Heliotropium europeum L.                        Culvenor, 1954                               B66
                                    H. indicum L.                                   Hoque et al. 1976                            B72
                                    H. supinum L.                                   Men'shikov & Gurevich, 1949; Crowley &       B81, 83
                                                                                    Culvenor, 1959
                                    Tournefortia sarmentosa Lam.                    Crowley & Culvenor, 1955                     B107
                                    Trichodesma zeylanicum (Burm. f.) R. Br.        O'Kelly & Sargeant, 1961                     B113

    Swazine                         Senecio barbellatus DC.                         Gordon-Gray & Wells, 1974                    B270
                                    S. swaziensis Compton                           Gordon-Gray et al. 1972; Laing &             B268,
                                                                                    Sommerville, 1972                            269

    Symlandine                      Symphytum asperum Lepech.                       Roitman, 1981                                A
                                    S. officinale Linn.                             Roder et al. 1982                            C
                                    S. tuberosum L.                                 Gray et al. 1983                             A
                                    S. x uplandicum Nyman                           Culvenor et al. 1980a, 1980b                 A, A

    Symphytine                      Myosotis scorpioides L.                         Resch et al. 1982                            C
                                    Symphytum aspera                                Roitman, 1981                                A
                                    S. officinale Linn.                             Furuya & Araki, 1968; Furuya &               B15, 16
                                                                                    Hikichi, 1971
                                    S. peregrinum Ledeb.                            Gadella et al. 1983                          A
                                    S. x uplandicum Nyman                           Culvenor et al. 1980a, 1980b                 A, A
    Syneilesine                     Syneilesis palmata Maxim.                       Hikichi & Furuya, 1974, 1976                 B278, 279
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Triangularine                   Alkanna tinctoria Tausch                        Roder et al. 1984b                           C
                                    Senecio triangularis Hook.                      Roitman, 1983b                               C

    Trichodesmine                   Crotalaria globifera E. Mey.                    Brown et al. 1984                            C
                                    C. juncea L.                                    Adams & Gianturco, 1956a                     B305
                                    C. lunata Beddome ex Polhill                    Rothschild et al. 1979                       C
                                    C. recta Steud ex A. Rich.                      Crout, 1968a                                 B326
                                    C. wightiana Grah. ex Wight & Arn.              Atal et al. 1966b                            B329
                                    C. tetragona Roxb.                              Puri et al. 1974                             B314
                                    Heliotropium arguzioides Kar. et Kir.           Akramov et al. 1961aB54
                                    Trichodesma incanum Alph. DC.                   Men'shikov & Rubinstein, 1935;               B108
                                                                                      Men'shikov, 1936; Yunusov &                B109,
                                                                                      Plekhanova, 1957, 1959;                    B111, 112
                                                                                    Tashkhodzhaev et al. 1979                    C

    Uluganine                       Ulugbeckia tschimganica (B.Fedtsch.) Zak.       Khasanova et al. 1974                        B114

    Uplandicine                     Symphytum x uplandicum Nyman                    Culvenor et al. 1980a, 1980b                 A, A

    Usaramine                       Crotalaria brevidens Benth. var.                Suri et al. 1975b                            B304
                                    intermedia (Kotschy) Polhill
                                    C. brevifolia                                   Sawhney et al. 1967                          B291
                                    C. incana L.                                    Sawhney & Atal, 1970a                        B303
                                    C. pallida Ait.                                 Sawhney et al. 1967                          B291
                                    C. zanzibarica Benth. (C. usaramoensis)         Culvenor & Smith, 1966b                      B337
                                    Senecio glandulosus Don ex Hook. et Arn.        Pestchanker et al. 1985b                     A
                                    S. seratophylloides Griseb                      Pestchanker & Giordano, 1986                 A
                                    S. vulgaris L.                                  Pieters & Vlietinck, 1986                    A
                                                                                                                                                

                                                                                                                                                

    Alkaloid                        Plant Sources                                   Reference                                    Reference
                                                                                                                                 Location
                                                                                                                                                

    Uspallatine                     Senecio argentino Baker (vira-vira Hieron)      Pestchanker et al. 1985a                     A
                                    S. leucostachys Baker                           Pestchanker & Giordano, 1986                 A
                                    S. seratophiloides Griseb                       Pestchanker & Giordano, 1986                 A
                                    S. uspallatensis                                Pestchanker et al. 1985a                     A

    Yamataimine                     Cacalia yatabei Maxim.                          Hikichi et al. 1978                          B121
                                                                                                                                                

    A - References in this publication.
    B - References in Smith & Culvenor, J. Nat. Prod., 44, 129-152 (1981), with reference number.
    C - References in Mattocks, Chemistry and Toxicology of Pyrrolizidine Alkaloids, 1986.
            APPENDIX II
    Table 1.  Plants containing hepatotoxic pyrrolizidine alkaloids 
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Apocynaceae

    Fernaldia pandurata           loroquin                root           B  29
    (syn. Urechites karwinsky
    Mueller)

    Parsonsia eucalyptophylla     lycopsamine             aeriel         B  28
    (F. Muell.)

    Parsonsia heterophylla        parsonsine              whole          B  417
    A. Cunn.                      heterophylline                         B  418

    Parsonsia spiralis Wall.      heterophylline          leaf           B  418
                                  parsonsine
                                  spiracine
                                  spiranine
                                  spiraline

    Parsonsia straminea (R. Br.)  lycopsamine             aerial         B  28
    F. Muell.

    Parsonsia estonia sp.         echinatine                             A  Edgar (1985)

    Boraginaceae

    Alkanna tinctoria Tausch      7-angelylretronecine                   C  Roder et al. 
                                  dihydroxytriangularine                    (1984b)                 
                                  triangularine

    Amsinckia hispida (Ruiz       intermedine             whole          B  30
    et Pav.) M. Johnston          lycopsamine
                                  echiumine

    Amsinckia intermedia Fisch    intermedine             whole          B  30
    et C. Mey                     lycopsamine
                                  echiumine
                                  sincamidine
                                  echimidine

    Amsinckia lycopsoides Lehm.   intermedine             whole          B  30
                                  lycopsamine
                                  echiumine

    Amsinckia menziesii (Lehm.)   7-acetyllycopsamine     aerial         C  Roitman (1983a)
    Nels and Macbr.               3'-acetyllycopsamine                                       
                                  diacetyllycopsamine
                                  lycopsamine
                                  intermedine
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Boraginaceae (contd.)

    Anchusa arvensis ( L.) Bieb.  echinatine              whole          B  31
                                  (or diastereoisomer)

    Anchusa officinalis L.        7-acetyllycopsamine     whole          B  31
                                  (or diastereoisomer)
                                  lycopsamine                            B  32

    Asperugo procumbens L.        supinine (or            whole          B  31
                                  diastereoisomer)
                                  lycopsamine (or 
                                  diastereoisomer)   


    Borago officinalis L.         lycopsamine             aerial, root   C  Larson et al. 
                                  amabiline                                 (1984)
                                  supinine                               A  Luthy et al. 
                                  intermedine                               (1984)
                                  acetylintermedine
                                  acetyllycopsamine
                                  thesinine               seed, flower

    Cynoglossum amabile  Stapf    amabiline               whole          B  33, 34
    & Drummond                    echinatine

    Cynoglossum australe R. Br.   cynaustine              whole          B  33
                                  cynaustraline
                                  heliosupine

    Cynoglossum creticum          heliosupine             aerial         B  419
                                  echinatine

    Cynoglossum glochidiatum      amabiline               whole          B  36
    Wall. ex Lindl.

    Cynoglossum lanceolatum       cynaustraline           whole          B  36
    Forsk                         cynaustine

    Cynoglossum latifolium        latifoline              aerial         B  37
    R. Br.                        7-angelylretronecine

    Cynoglossum officinale L.     heliosupine             aerial         B  38, 39, 40
                                  echinatine              root, aerial   B  41, 42
                                  acetylheliosupine       aerial         B  43        
                                  7-angelylheliotridine

    Cynoglossum pictum Ait.       heliosupine             root, aerial   B  44, 45
                                  echinatine              
                                  pictumine               aerial         B  46
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Boraginaceae (contd.)

    Cynoglossum viridiflorum      viridiflorine           root           B  47
    Pallas ex Lehm.               heliosupine                            B  34

    Echium plantagineum L.        echiumine               aerial         B  48
    (Echium lycopsis  L.)         echimidine

    Echium vulgare L.             heliosupine             aerial         B  49
                                  asperumine              aerial         B  50
                                  echinatine (or          whole          B  31
                                  diastereoisomer)

    Hackelia floribunda           latifoline                             A  Hagglund et al.
                                                                            (1985)                     

    Heliotropium acutiflorum      heliotrine              aerial         B  51

    Heliotropium arbainense       heliotrine              aerial         B  419
                                  europine
                                  lasiocarpine

    Heliotropium arborescens L.   lasiocarpine            aerial         B  52
    (Heliotropium peruvianum L.)

    Heliotropium arguzioides      heliotrine              aerial         B  53
    Kar. et Kir.                  trichodesmine           aerial, root   B  54, 55

    Heliotropium curassavicum     heliotrine              whole          B  56
    Linn.                         lasiocarpine
                                  angelylheliotridine
                                  curassavine             aerial         B  57
                                  heliovicine
                                  trachelanthamidine                     B  58
                                  acetylcurassavine       aerial         C  Mohanraj et al.
                                  heliocurassavinine                        (1982)
                                  heliocurassavine
                                  heliocoromandaline
                                  heliocurassavicine
                                  curassanecine
                                  curassavinine
                                  coromandalinine
                                  heliovinine

    Heliotropium dasycarpum       heliotrine              aerial, root   B  54
    Ledeb.                                                seed           B  59
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Boraginaceae (contd.)

    Heliotropium digynum          heliotrine                             A  Hammouda et al.
    (Heliotropium luteum)         lasiocarpine                              (1984)                  
                                  europine
                                  angelylheliotrine

    Heliotropium eichwaldi        heliotrine              whole          B  60, 61
    Steud. ex DC.                 lasiocarpine            aerial         B  62
                                  7-angelylheliotrine     aerial         B  63

    Heliotropium europaeum        heliotrine              whole          B  64, 65
                                  lasiocarpine
                                  europine                whole          B  66, 67
                                  supinine
                                  heleurine
                                  N-dihydropyrrolizino-   whole          B  68
                                   methyl-
                                  heliotrine chloride
                                  acetyllasiocarpine      whole          B  69

    Heliotropium indicum L.       indicine                aerial         B  70
                                  acetylindicine          aerial         B  71
                                  indicinine
                                  echinatine              aerial         B  72
                                  supinine
                                  heleurine
                                  lasiocarpine

    Heliotropium lasiocarpum      heliotrine              aerial         B  73
    Fisch. et Mey.                lasiocarpine
                                  europine                               A  Culvenor et al.
                                  heleurine                                 (1986)

    Heliotropium maris-mortui     europine                aerial         B  74
                                  lasiocarpine            aerial         B  419

    Heliotropium olgae            heliotrine              aerial, root   B  75

    Heliotropium popovii H.       heliotrine              seed           B  77
    Riedl. subsp. gillianum 
    H. Riedl.

    Heliotropium ramosissimum     heliotrine              aerial         B  78, 79
    (Lehm.) Dec. (syn.            heleurine
    Heliotropium persicum L.,     supinine
    Heliotropium undulatum)       lasiocarpine

    Heliotropium rotundifolium    europine                aerial         B  74
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Boraginaceae (contd.)

    Heliotropium steudneri Vatke  lycopsamine             leaf           B  80

    Heliotropium suaveolens Bieb. heliotrine              aerial         A  Guner (1986)
                                  lasiocarpine
                                  europine
                                  echinatine

    Heliotropium supinum L.       supinine                root           B  81
                                  heliosupine             root           B  82
                                  echinatine              whole          B  83
                                  7-angelylheliotridine
                                  7-angelylheliotridine 
                                   viridiflorate
                                  7-angelylheliotridine 
                                   trachelanthate
                                  heliotrine              seed, leaf     C  Pandey et al. 
                                  lasiocarpine                              (1983)        

    Heliotropium transoxanum      heliotrine              aerial         B  51

    Lappula glochidiata           echinatine              aerial         B  84

    Lappula intermedia            lasiocarpine            aerial         B  85

    Lindelofia angustifolia       echinatine              aerial         B  62, 36
    (Schrenk) Brand.              amabiline

    Lindelofia spectabilis        echinatine              aerial         B  62, 63
    Lehm.                         7-acetylechinatine
                                  monocrotaline

    Lindelofia stylosa            viridiflorine           aerial         B  86
    (Kar. et Kir.) Brand          echinatine              seed           B  75
                                  lindelofine

    Lindelofia tschimganica       carategine              aerial         B  87
                                  echinatine
                                  viridiflorine

    Lithospermum officinale L.    acetylechimidinyl-      whole          B  31
                                   retronecine
                                  (or diastereoisomer)

    Messerschmidia sibirica       lycopsamine             whole          C  Hikichi et al.
                                  angelylretronecine                        (1980)
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Boraginaceae (contd.)

    Myosotis scorpioides L.       7-acetylscorpioidine    aerial         C  Resch et al. 
    (syn. Myosotis                scorpioidine                              (1982)   
    palustris L.)                 symphytine
                                  myoscorpine

    Paracynoglossum imeritinum    heliosupine             aerial, root   B  88
    (Kusn.) M. Pop.               echinatine                             B  89, 90

    Rindera austroechinata        echinatine              whole, seed    B  87
    M. Pop.

    Rindera baldshuanica          rinderine               aerial         B  91
    Kusnezov                      echinatine              aerial         B  87
                                  trachelanthamine
                                  turkestanine

    Rindera cyclodonta Bge.       echinatine              aerial         B  59

    Rindera echinata Regel        echinatine              aerial         B  92
                                  trachelanthamine        aerial         B  59

    Rindera oblongifolia          carategine              aerial         B  87
    M. Pop.                       echinatine
                                  turkestanine

    Solenanthus circinnatus       echinatine              seed, aerial,  B  93
    Ledeb.                                                root

    Solenanthus coronatus         echinatine              aerial         B  75

    Solenanthus karateginius      carategine              aerial         B  93
    Lipsky                        echinatine

    Solenanthus turkestanicus     rinderine               aerial         B  94
                                  turkestanine

    Symphytum asperum Lepech.     asperumine              aerial, root   B  95, 96
                                  echinatine              aerial, root   B  97
                                  acetylheliosupine       whole          B  31
                                  (or diastereoisomer)
                                  7-acetyllycopsamine     leaf, root     C  Roitman (1981)
                                  intermedine
                                  symlandine
                                  7-acetylintermedine
                                  symphytine
                                  lycopsamine
                                  echimidine
                                  angelylechimidine       root           A  Gadella et al.
                                  (or diastereoisomer)                      (1983)
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Boraginaceae (contd.)

    Symphytum caucasicum Bieb.    lasiocarpine            aerial, root   B  95
                                  asperumine              aerial, root   B  98, 99
                                  echinatine
                                  echimidine

    Symphytum officinale Linn.    symphytine              root           B  15, 16
                                  echimidine                             B  100
                                  lasiocarpine            aerial, root   B  95
                                  heliosupine             aerial, root   B  101
                                  viridiflorine           root
                                  echinatine              root
                                  acetylechimidine                       B  31
                                  (or diastereoisomer)
                                  7-acetyllycopsamine     aerial, root   A  Huizing et al.
                                  lycopsamine                               (1981)
                                  intermedine
                                  7-acetylintermedine
                                  symlandine                             C  Roder et al.
                                  myoscorpine             root              (1982a)
                                                                         C  Resch et al.
                                                                            (1982)

    Symphytum orientale           anadoline               whole          B  102, 103, 104
                                  symphytine
                                  echimidine

    Symphytum peregrinum Ledeb.   echimidine              root           A  Gadella et al.
                                  symphytine                                (1983)

    Symphytum tuberosum L.        echimidine              whole          B  105
                                  anadoline
                                  symlandine              root           A  Gray et al.
                                                                            (1983)

    Symphytum x uplandicum Nyman  lycopsamine             aerial         B  31, 17, 106
                                  intermedine
                                  uplandicine
                                  7-acetyllycopsamine
                                  7-acetylintermedine
                                  echimidine
                                  symphytine
                                  symlandine
                                  angelylechimidine       root           A  Gadella et al.
                                  (or diastereoisomer)                      (1983)

    Tournefortia sarmentosa Lam.  supinine                leaf, stem     B  107
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Boraginaceae (contd.)

    Trichodesma africana          intermedine             aerial         B  419
                                  europine

    Trichodesma incanum Alph.     trichodesmine           aerial         B  108, 109
    DC.                           incanine                seed, aerial,  B  110, 111, 112
                                                          root

    Trichodesma zeylanicum        supinine                seed           B  113
    (Burm. f) R. Br.

    Ulugbekia tschimganica        uluganine                              B  114

    Compositae

    Adenostyles alliariae         platyphylline           root           B  115
                                  seneciphylline

    Adenostyles glabra            seneciphylline                         C  Wiedenfeld et al.
                                                                            (1984)

    Adenostyles rhombifolius      platyphylline           aerial         B  116
    (Willd.) M. Pimen. ssp.       seneciphylline
    platyphylloides

    Brachyglottis repanda         senecionine             aerial         B  117
    Forst. et Forst.              senkirkine
                                  brachyglottine                         B  118

    Cacalia floridana (= Senecio  otosenine               aerial         B  119
    floridanus Sch. Bip.)         florosenine
                                  floridanine
                                  floricaline

    Cacalia hastata L. subsp.     integerrimine           root           B  120
    orientalis Kitamura

    Cacalia yatabei Maxim         yamataimine             root           B  121

    Conoclinium coelestinium      intermedine                            C  Herz et al. (1981)
    (L.) DC                                                                                   

    Crassocephalum crepidioides   jacobine                aerial         A  Asada et al.
                                  jacoline                                  (1985)

    Doronicum macrophyllum        otosenine               root           B  122
                                  floridanine
                                  doronine
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Doronicum pardalianches       otosenine               root           A  Rajagopalan & Negi
    Linn.                                                                   (1985)

    Echinacea angustifolia DC.    tussilagine             whole          C  
                                  isotussilagine

    Echinacea purpurea M.         tussilagine             whole          C  
                                  isotussilagine

    Emilia flammea Cass.          otosenine               aerial, root   B  123, 124, 125
                                  emiline

    Erechtites hieracifolia (L.)  senecionine             aerial         B  126, 127
    Raf. ex DC.                   seneciphylline

    Eupatorium altissimum L.      rinderine                              C  Herz et al. (1981)
                                  angelylheliotridine                         

    Eupatorium cannabinum L.      echinatine              aerial         B  128
                                  supinine
                                  amabiline                              A  Luthy et al.
                                                                            (1984)

    Eupatorium compositifolium    intermedine                            C  Herz et al. (1981)
    Walt.                         lycopsamine                                   

    Eupatorium maculatum L.       echinatine              root           B  129
                                  trachelanthamidine

    Eupatorium purpureum          probably echinatine     aerial         B  130

    Eupatorium serotinum Michx.   supinine                aerial         B  131
                                  rinderine

    Eupatorium stoechadosmum      lindelofine             root           B  132
    Hance                         supinine

    Farfugium japonicum Kitam.    senkirkine              root, leaf     B  133
                                  farfugine               whole          C  Niwa et al. (1983)
                                  petasitenine            whole          A  Niwa et al. (1985)    

    Gynura scandens O. Hoffm.     gynuramine                             C  Wiedenfeld (1982)
                                  acetylgynuramine                                           

    Gynura segetum (Lour.) Merr.  senecionine                            C  Liang & Roder
                                                                            (1984)
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Ligularia brachyphylla        clivorine               aerial         B  134
    Hand.-Mazz.                   ligularine
                                  ligudentine

    Ligularia clivorum            clivorine               aerial         B  135, 136,
                                                                            137, 138

    Ligularia dentata             clivorine               aerial         B  134
    (A. Gray) Hara                ligularine
                                  ligudentine
                                  ligularidine            whole          B  436
                                  ligularinine            aerial, root   C  Asada & Furuya
                                  ligularizine                              (1984a)
                                  neoligularidine

    Ligularia elegans (Cass.)     clivorine               aerial         B  134
    [syn. Ligularia macrophylla   ligularine
    (Ledeb. DC.)]

    Ligularia japonica            senecionine             root           A  Asada et al.
                                  neopetasitenine                           (1981)
                                  platyphylline

    Petasites albus L.            senkirkine              aerial         C  Luthy et al.
                                                                            (1983)

    Petasites hybridus L.         senecionine             aerial         C  Luthy et al.
                                  integerrimine                             (1983)
                                  senkirkine

    Petasites japonicus Maxim.    petasitenine            aerial         B  20, 19, 139
                                   (fukinotoxin)
                                  neopetasitenine
                                  senkirkine              stem           B  140
                                  petasinine              aerial         B  141
                                  petasinoside

    Petasites laevigatus (Willd.) platyphylline           aerial         B  142
    Reichenb. [syn. Nardosmia     senkirkine (renardine)  aerial         B  143, 144
    laevigata (Willd.) DC.]       senecionine

    Senecio abrotanifolius        doronine                aerial         A  Roder et al.
    ssp. abrotanifolius           doronenine                                (1984a)
                                  bulgarsenine

    Senecio abrotanifolius        doronine                aerial         A  Roder et al.
    ssp. abrotanifolius var.      doronenine                                (1984a)
    tiroliensis                   bulgarsenine
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                         Constituent alkaloids   Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Senecio aegypticus L.         senecionine             whole          B  145, 146
                                  otosenine               aerial
                                  riddelliine
                                  erucifoline

    Senecio alpinus (L.) Scop.    seneciphylline          aerial         B  147
                                  jacozine
                                  jacobine                whole          A  Luthy et al. 
                                  integerrimine                             (1981) 
                                  jacoline
                                  senecionine
                                  jaconine

    Senecio ambrosioides          retrorsine              whole          B  148
    (= Senecio brasiliensis       seneciphylline
    Less.)                        senecionine
                                  riddelliine             whole          B  420

    Senecio ampullaceus Hook.     senecionine             whole          B  149, 150, 151
                                  seneciphylline
                                  retrorsine

    Senecio antieuphorbium        integerrimine           aerial         B  152
    (L.) Sch. Bip.                senkirkine

    Senecio aquaticus Hill        seneciphylline          aerial         B  153, 154

    Senecio aureus L.             senecionine             aerial         B  155, 156
                                  otosenine               aerial         C  Resch et al.
                                  floridanine                               (1983)
                                  florosenine                            C  Roder et al.
                                                                            (1983)

    Senecio auricola Bourg.       neosenkirkine           aerial         B  157

    Senecio barbellatus DC.       swazine                                B  270
                                  retrorsine

    Senecio bipinnatisectus       retrorsine              aerial, root   B  158
    Belcher (syn.
    Erechtites atkinsoniae)

    Senecio borysthenicus         seneciphylline          aerial, root   B  159, 160
    (= Senecio prealtus
    Berthol.)

    Senecio brasiliensis          senecionine             leaf           B  161, 162
    DC. (syn.                     seneciphylline                         B  148, 163
    Senecio ambrosioides)         jacobine
                                  integerrimine
                                  retrorsine
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Senecio brasiliensis Less      integerrimine                         A  Nardi et al.
    var tripartitus                                                         (1980)

    Senecio bupleuroides DC.       retrorsine             aerial         B  164

    Senecio cacaliaster (Lam.)     sencalenine                           A  Roder et al.
                                   bulgarsenine                             (1984b)
                                   7-senecioylretrone-
                                    cine
                                   7-senecioyl-9-sarra-
                                    cinoylretronecine

    Senecio cannabifolius Less.    seneciphylline         aerial         B  165
                                   senecicannabine        aerial, root   C  Asada et al.
                                   jacozine                                 (1982a)

    Senecio carthamoides Greene    senecionine            whole          B  149, 151
                                   seneciphylline

    Senecio caudatus DC.           7-senecioylretrone-    aerial         A  Bohlmann et al.
                                    cine                                    (1986)
                                   9-senecioylretrone-                                      
                                    cine
                                   7-senecioyl-9-sarra-
                                    cinylretronecine
                                   7-senecioyl-9-(2-
                                    methyl-2,3-dihyroxy-
                                    butyryl)retronecine
                                   7-senecioyl-9-(2-
                                    methy-2-hydroxy-3-
                                    acetoxybutyryl)
                                    retronecine
                                   retronecine
                                   2-senecioyl-(-)-
                                    macronecine
                                   9-senecioyl-(-)-
                                    macronecine
                                   senecicaudatine-9-
                                    senecioate
                                   senecidaudatine-9-
                                    isovalerate
                                   norsenecicaudatine-
                                    9-sencioate
                                   senecicaudatinal 
                                    semiacetal

    Senecio chrysanthemoides       seneciphylline                        B  166
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Senecio cineraria DC.          jacobine               aerial         B  153, 167
                                   senecionine            seed           B  168
                                   seneciphylline         aerial         B  169
                                   otosenine              aerial         B  170
                                   retrorsine             aerial         B  171

    Senecio congestus (R. Br.)     senecionine                           C  Roder et al.
    DC. [syn. Senecio palustris    neoplatyphylline                         (1982b)
    (L.) Hooker, Senecio           platyphylline
    tubicaulis Mansfeld)

    Senecio cruentus DC.           senecionine                           C  Asada et al.
                                   seneciphylline                           (1982b)
                                   retrorsine
                                   riddelliine

    Senecio cymbaroides            senecionine            whole          B  420
                                   seneciphylline
                                   riddelliine
                                   retrorsine

    Senecio desfonainei Druce      senecionine            aerial         B  173, 145
                                   otosenine
                                   riddelliine
                                   seneciphylline         aerial         B  146
                                   retrorsine                            A  Rizk et al. (1983)
                                   senkirkine                               
                                   angelylretronecine

    Senecio discolor DC.           retrorsine             leaf           B  174
                                   senecionine            aerial         B  175

    Senecio dolichodoryius         15,20-dihyroxyeruci-   aerial         A  Bohlmann et al.           
    Cuatr.                          foline                                  (1986)                         

    Senecio doronicum  L.          bulgarsenine           leaf           B  176
                                   doronenine

    Senecio douglasii  DC.         retrorsine             whole          B  149, 150, 151
                                   riddelliine
                                   seneciphylline
                                   senecionine

    Senecio durieui Gay            integerrimine          whole          B  157

    Senecio eremophilus            senecionine            aerial         B  149, 150, 151
    Richards                       seneciphylline
                                   retrorsine
                                   riddelliine
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Senecio erraticus Berthol.     senecionine            aerial         B  177
                                   otosenine
                                   floridanine

    Senecio erraticus Berthol.     senecionine                           B  178, 179, 180
    subsp. barbaraeifolius Krock   otosenine
                                   erucifoline
                                   seneciphylline         leaf           B  181
                                   integerrimine          aerial         B  182

    Senecio erucifolius L.         senecionine            aerial         B  153
                                   seneciphylline         aerial         B  183, 180
                                   erucifoline
                                   retrorsine             aerial         B  184

    Senecio faberi Hemsl.          integerrimine                         C  Wei et al. (1982)

    Senecio filaginoides           senecionine            root           A  Pestchanker &
    (H. et A.) DC.                 retrorsine                               Giordano (1986)


    Senecio fistulosus             senecionine                           A  Gonzalez et al.
    Poepp. ex Less.                                                         (1986b)

    Senecio fluviatilis Wallr.     seneciphylline         aerial         B  185
                                   otosenine
                                   florosenine

    Senecio formosus               integerrimine          aerial         B  186
                                   retrorsine

    Senecio fremontii Torr.        seneciphylline         whole          B  148
    et A. Gray                     senecionine

    Senecio gilliesiano            senecionine            root           A  Guidugli et al.
                                   retrorsine                               (1986)

    Senecio glabellus              senecionine            whole          B  187
    (Turcz.) DC.

    Senecio glaberrimus DC.        retrorsine             aerial         B  153

    Senecio glandulosus            integerrimine          root           A  Pestchanker et al.
    Don ex Hook. et Arn.           retrorsine                               (1985b)
                                   usaramine

    Senecio graminifolius          retrorsine             aerial         B  188
    N.J. Jacq.                     graminifoline
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Senecio grandifolia Jaqu.      platyphylline          root, leaf,    B  189
                                   seneciphylline         stem

    Senecio grandifolius Less.     senkirkine             aerial         A  Bohlmann et al.
                                   neosenkirkine                            (1986)

    Senecio griesbachii Baker      retrorsine             aerial         B  190

    Senecio ilicifolius  Thunb.    senecionine            aerial         B  191, 192, 188, 127
                                   seneciphylline                           
                                   retrorsine

    Senecio illinitus Phill.       senkirkine             aerial         A  Gonzalez et al.
                                   O-acetylsenkirkine                       (1986a)
                                   senecionine

    Senecio inaequidens DC.        retrorsine                            A  Roder et al.
                                   senecionine                              (1981)
                                   senecivernine          aerial         A  Bicchi et al.
                                   integerrimine                            (1985)

    Senecio incanus L.             seneciphylline         aerial         B  147
    subsp. carniolicus             integerrimine
    (Willd.) Br.-Bl.

    Senecio integerrimus Nutt.     integerrimine          aerial         B  156
                                   senecionine
                                   neoplatyphylline       whole          B  420
                                   platyphylline

    Senecio isatideus DC.          retrorsine             aerial         B  153, 193

    Senecio jacobaea L.            seneciphylline         aerial         B  194, 195
                                   senecionine                           B  196, 197
                                   jacobine                              B  153, 197, 198
                                   jaconine                              B  199, 200, 201
                                   jacozine
                                   otosenine              aerial         B  51
                                   senkirkine
                                   retrorsine             aerial         B  184

    Senecio kirkii Hook. f.        senkirkine             bark, leaf     B  203
    ex Kirk                        O-acetylsenkirkine     leaf           B  204

    Senecio kleinia  Sch. Bip.     integerrimine          stem           B  205
                                   senkirkine             stem           B  206

    Senecio krylovii               seneciphylline         aerial         B  207
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Senecio kubensis Grossh.       seneciphylline         aerial         B  208

    Senecio lampsanoides           seneciphylline         aerial, root   B  209, 210

    Senecio laricifolius           senecionine            aerial         A  Bohlmann et al.
    H.B.K.                         seneciphylline                           (1986)
                                   senkirkine
                                   19-hydroxysenkirkine
                                   19-acetoxysenkirkine

    Senecio latifolius DC.         retrorsine             aerial         B  211, 212
    (syn. Senecio sceleratus       seneciphylline         aerial         B  213
    Schweikerdt)                   platyphylline
                                   sceleratine            aerial         B  260
                                   chlorodeoxysceleratine aerial         B  261;
                                    (merenskine)                         C  Bredenkamp et al.
                                                                            (1985)

    Senecio leucostachys Baker     senecionine            root           A  Pestchanker &
                                                                            Giordano (1986)

    Senecio longilobus Benth.      seneciphylline         whole          B  156, 214, 150
                                   retrorsine                            B  151
                                   riddelliine            whole          B  149

    Senecio magnificus F. Muell.   senecionine            aerial         B  215
                                   integerrimine                         B  216

    Senecio megaphyllus Green.     13,19-epoxyseneci-     aerial         A  Bohlmann et al.
                                    phylline                                (1986)
                                   13,19-epoxysparti-                     
                                    oidine

    Senecio minimus Poir           seneciphylline         aerial         B  158


    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Senecio morrisonensis Hayata   integerrimine          whole          B  217

    Senecio multilobatus           senecionine                           A  McCoy et al.
                                                                            (1983)

    Senecio multivenius            seneciphylline         aerial         A  Bohlmann et al.
    Benth. in Oerst.               senecionine                              (1986)

    Senecio nebrodensis L.         integerrimine          whole          B  218
    var sicula                     senecionine

    Senecio nemorensis L.          bulgarsenine           leaf           B  219
    var bulgaricus                 retrosisosenine
    (Vel) Stoj.                    nemorensine

    Senecio nemorensis L. ssp.     fuchsisenecionine                     B  362, 363, 364, 365
    fuchsii Gmel.                                                             
                                   senecionine                           B  423

    Senecio nemorensis L.          nemorensine            aerial, root   B  371
    subdecurrens  Griseb.          retroisosenine         aerial         B  422
                                   bulgarsenine

    Senecio othonnae Bieb.         otosenine              aerial, root   B  220
                                   onetine                root           B  221
                                   seneciphylline
                                   floridanine            aerial, root   B  222
                                   doronine               aerial         B  223

    Senecio othonniformis          bisline                aerial         B  22, 225
    Fourcade                       isoline

    Senecio palmatus Pall.         seneciphylline         root           B  226

    Senecio paludosus L.           seneciphylline         root, aerial   B  153, 227, 228

    Senecio pampeanus Cabrera      senecionine            aerial         B  229

    Senecio pancicii Degen         senecionine            whole          C  Jizba et al.
    var arnautorum (Velen.)        seneciphylline                           (1982)
    Stoj., Stef. et Kit.

    Senecio pancicii Degen         senecionine            whole          C  Jizba et al.
    var pancicii                                                            (1982)

    Senecio patagonicus            senecionine            aerial         A  Villaroel et al.
    Hook. and Arn.                 seneciphylline                           (1985)
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part     Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Senecio paucicalyculatus       paucicaline            whole           B  230
    (Klatt.)                       retrorsine

    Senecio paucifolius            seneciphylline                         B  231
    S.G. Gmel.

    Senecio petasitis DC.          senecionine            leaf            B  146
                                   bisline (?)            aerial          B  232

    Senecio phillipicus            retrorsine             aerial          A  Gonzalez et al.
    Rogel et Koern.                                                          (1986a)

    Senecio pierotii               neosenkirkine          aerial, root    C  Asada & Furuya
                                   senkirkine                                (1982)

    Senecio pimpinellifolius       senecionine            aerial          A  Bohlmann et al.
    H.B.K.                                                                   (1986)

    Senecio platyphylloides        platyphylline          root            B  233, 234, 235
    Somm. et Lev.                  seneciphylline

    Senecio platyphyllus           platyphylline          root, aerial    B  236, 237, 238
    (Bieb.) DC.                    seneciphylline         leaf            B  239
                                   neoplatyphylline       root            B  240, 241
                                   sarracine              root            B  242

    Senecio pojarkovae             sarracine              root            B  243
                                   seneciphylline

    Senecio procerus L.            senkirkine             aerial, root    B  244
    var procerus                   procerine
    Stoj. Stef. et Kit.

    Senecio propinquus Ait.        seneciphylline         aerial, root    B  209, 245

    Senecio pseudo-arnica Less.    senecionine            aerial          B  156

    Senecio pterophorus            senecionine            aerial          B  192, 188, 127
                                   seneciphylline
                                   retrorsine
                                   rosmarinine
                                   acetylseneciphylline

    Senecio quadridentatus         senecionine            aerial          B  247
    Labill. (syn. Erechtites       seneciphylline
    quadridentata DC.)             retrorsine
    ------------------------------------------------------------------------------------------

    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Senecio quebradensis           senkirkine             aerial          A  Bohlmann et al.
    Greenm.                        florosenine                               (1986)

    Senecio racemosus DC.          seneciphylline         root            B  248

    Senecio renardii  Winkl.       seneciphylline         aerial          B  249, 250
                                   senkirkine (renardine)
                                   otosenine

    Senecio retrorsus DC.          retrorsine             aerial          B  194, 193

    Senecio rhombifolius           sarracine              root            B  251, 233
    (Willd.) Sch. Bip.             platyphylline          aerial, root    B  208
                                   seneciphylline
                                   neoplatyphylline

    Senecio riddellii              riddelliine            aerial          B  156
    Torr. et A. Gray                                      whole           B  252, 253
                                   retrorsine                             B  420

    Senecio riddellii              retrorsine             whole           B  149
    Torr. et A. Gray               riddelliine
    var. parksii (Cory)

    Senecio ruderalis Harvey       retrorsine             aerial          B  254

    Senecio ruwenzoriensis         ruwenine               whole           B  255
    S. Moore                       ruzorine

    Senecio sandrasicus            senecionine                            A  Temizer et al.
                                                                             (1985)

    Senecio scandens               senecionine            whole           B  256
                                   seneciphylline

    Senecio seratophiloides        senecionine            root            A  Pestchanker &
    Griseb.                        senecivernine                             Giordano (1986)
                                   usaramine                                  
                                   retrorsine
                                   uspallatine

    Senecio spartioides            seneciphylline         aerial          B  156, 262
    Torr. et A. Gray               senecionine
                                   spartiodine
                                   riddelliine            whole           B  420
                                   retrorsine
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Senecio spathulatus            senecionine            aerial, root    B  158
    A. Rich.                       integerrimine
                                   seneciphylline                         B  263

    Senecio squalidus L.           senecionine            aerial          B  153, 264, 265
                                   integerrimine                          B  205

    Senecio stenocephalus          seneciphylline         aerial          B  266
    Maxim.

    Senecio subalpinus             senecionine            leaf            B  267
    C. Koch.                       seneciphylline         aerial          B  147
                                   integerrimine

    Senecio subulatus              dihydroretrosine       root            A  Pestchanker et
    Don ex Hook.                   retrorsine                                al. (1985b)
    et Arn. var. erectus           senecionine

    Senecio swaziensis             retrorsine             aerial          B  268, 269, 270
    Compton                        swazine

    Senecio tenuifolius            senecionine            aerial          C  Bhakuni & Gupta
    Burm.                          integerrimine                             (1982)
                                   senkirkine
                                   o-acetylsenkirkine

    Senecio tomentosus             senecionine            aerial          B  271, 179
                                   otosenine 
                                    (tomentosine)

    Senecio triangularis           senecionine            aerial          B  272
    Hook.                          integerrimine                          C  Roitman (1983b)
                                   platyphylline                             
                                   rosmarinine
                                   retrorsine
                                   triangularine
                                   neotriangularine
                                   7-angelylretronecine   whole           C  Rueger & Benn
                                   7-senecioylretrone-                       (1973)
                                    cine
                                   7-angelyl-9-sarra-
                                    cinylretronecine
                                   7-senecioyl-9-sarra-
                                    cinylretronecine

    Senecio uintahensis            senkirkine             whole           B  420
                                   senecionine
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Senecio umgeniensis            7-senecioyl-9-sarra-   aerial          A  Bohlmann et al.
    Thell.                          cinylretronecine                         (1986)

    Senecio usgorensis             13,19-epoxyseneci-     aerial          A  Bohlmann et al.
    Cuatr.                          phylline                                 (1986)

    Senecio uspallatensis          retrorsine             root            A  Pestchanker et 
                                   uspallatine                               al. (1985a)

    Senecio variabilis Sch.        7-senecioylretrone-    aerial, root    A  Bohlmann et al.
    Bip.                            cine                                     (1986)
                                   9-senecioylretrone-                     
                                    cine
                                   7-senecioyl-9-sarra-
                                    cinylretronecine

    Senecio venosus Harvey         retrorsine             aerial          B  153

    Senecio vernalis Walst.        retrorsine             aerial          B  273, 274
    et Kit.                        senecionine
                                   senkirkine
                                   senecivernine
                                   integerrimine                          A  Sener et al.
                                   seneciphylline                            (1986)
                                   riddelliine
                                   retronecine

    Senecio viscosus L.            senecionine            aerial          B  264, 153
                                   integerrimine          aerial          B  182

    Senecio vulgaris L.            senecionine            aerial          B  275, 264, 153
                                   seneciphylline         aerial          B  155, 276, 277
                                   retrorsine
                                   riddelliine            whole           B  420
                                   integerrimine                          A  Pieters &
                                   spartioidine                              Vlietinck (1986)
                                   usaramine                                

    Senecio                        senecionine            whole           B  420
    werneriaefolius                retrorsine

    Syneilesis palmata             syneilesine            aerial, root    B  278, 279
    Maxim.                         acetylsyneilesine
                                   senecionine
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Compositae (contd.)

    Tussilago farfara L.           senkirkine             flower          B  18, 425
                                                          leaf, stem      C  Rosberger et al.
                                                                             (1981)
                                   senecionine            leaf, stem      A  Luthy et al.
                                                                             (1980)
                                   tussilagine                            C  Roder et al.
                                                                             (1981b)

    Leguminosae

    Crotalaria aegyptica           crosemperine                           B  419
    Benth.                         monocrotaline
                                   7beta-hydroxy-1-                       B  426
                                    methylene-8alpha-
                                    pyrrolizidine

    Crotalaria agatiflora         maduraensine            aerial          B  280
    Schweinf.                     anacrotine              aerial          B  281
                                  7-acetylmadurensine
                                  6-acetylanacrotine
                                  7-acetyl-cis-
                                   madurensine
                                  6-acetyl-trans-
                                   anacrotine
                                  crotaflorine
                                  6-angelyl-trans-
                                   anacrotine

    Crotalaria assamica Benth.    monocrotaline                           B  285, 286

    Crotalaria axillaris Ait.     axillarine              seed            B  287, 288
                                  axillaridine

    Crotalaria barbata R. Graham  crobarbatine            seed            B  289
    ex R. Wight et Walk.-Arn.

    Crotalaria berteroana DC.     fulvine                 aerial          B  297
    (syn. Crotalaria fulva Roxb.)

    Crotalaria brevidens          integerrimine           seed            B  304
    Benth. var. intermedia        usaramine
    (Kotschy) Polhill(syn.
    Crotalaria intermedia
    Kotschy)

    Crotalaria breviflora DC.     integerrimine           seed            B  290, 291
                                  usaramine
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Leguminosae (contd.)

    Crotalaria burhia Ham.         crotalarine            aerial          B  292, 293
    ex Benth.                      monocrotaline

    Crotalaria candicans           crocandine             seed            B  380
    W. and A.                      isocorcandine
                                   isocromadurine         seed            C  Suri et al.
                                   crispatine                                (1982)
                                   turneforcidine
                                   cropodine              seed            C  Haksar et al.
                                                                             (1982)

    Crotalaria cephalotes Steud.   monocrotaline          seed            C  Pilbeam et al.
    ex A. Rich                                                               (1983)

    Crotalaria crispata            monocrotaline          whole           B  294
    F. Muell. ex Benth.            fulvine
                                   crispatine

    Crotalaria cunninghamii        monocrotaline          seed            C  Pilbeam et al.
    R. Br.                                                                   (1983)

    Crotalaria dura                dicrotaline            aerial          B  295, 296
    J.M. Wood et Evans

    Crotalaria fulva Roxb.
    (see Crotalaria
    berteroana DC.)

    Crotalaria globifera E. Mey    dicrotaline            aerial          B  295, 296
                                   globiferine            seed            C  Brown et al.
                                   grantianine                               (1984)
                                   grantaline
                                   trichodesmine

    Crotalaria grahamiana          monocrotaline          seed            B  298
    Wight & Arn.                   grahamine              seed            B  299
                                   monocrotalinine        whole           B  300

    Crotalaria incana L.           integerrimine          seed            B  187
                                   anacrotine             aerial          B  302
                                   usaramine              seed            B  303

    Crotalaria juncea  L.          senecionine            seed            B  305, 306, 307
                                   seneciphylline
                                   riddelliine
                                   trichodesmine
                                   junceine
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Leguminosae (contd.)

    Crotalaria laburnifolia L.     anacrotine             seed            B  308, 309, 310,
                                    (crotalaburnine)                         291, 311

    Crotalaria laburnifolia L.     madurensine            aerial          B  312
    subsp. eldomae                 anacrotine
                                   senkirkine
                                   hydroxysenkirkine
                                   crotafoline

    Crotalaria leschenaultii       monocrotaline          seed            B  313

    Crotalaria leiloba Bartl.      monocrotaline          seed            B  314
    (syn. Crotalaria
    ferruginea Wall.)

    Crotalaria madurensis          madurensine            seed, flower,   B  280
    R. Wight                                              leaf
                                   crispatine             aerial          B  315
                                   fulvine
                                   cromadurine            seed            B  62, 316
                                   isocromadurine         seed            B  317

    Crotalaria micans Link.        1-methylenepyrroliz-   seed            B  282, 283
    (syn. Crotalaria anagyroides    idine                 seed            B  284
    Humb. et al.)                  senecionine                            B  280
                                   anacrotine 

    Crotalaria mitchellii Benth.   monocrotaline          whole           B  318
                                   retusamine             whole

    Crotalaria mitchellii Benth.   retusamine             whole           B  318
    subsp. laevis A. Lee,
    published as
    "sp. aff. mitchellii")

    Crotalaria mysorensis Roth.    monocrotaline          seed            B  319

    Crotalaria nana Burm.          crotananine            seed            B  320
                                   cronaburmine           seed            B  427

    Crotalaria nitens Kunth.       monocrotaline                          A  Hoet et al.
                                                                             (1981)

    Crotalaria novae-hollandiae    monocrotaline          whole           B  318
    DC. subsp. lasiophylla         retusamine
    (Benth) A. Lee
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Leguminosae (contd.)

    Crotalaria novae-hollandiae    retusamine             seed            B  318
    DC. subsp. novae-hollandiae
    (syn. Crotalaria
    crassipes Hook.)

    Crotalaria pallida Ait.        usaramine              seed            B  291
    (syn. Crotalaria mucronata,    nilgirine              seed            B  322
    Crotalaria striata)            crotastriatine         seed            B  323, 324

    Crotalaria paniculata Willd.   fulvine                seed            B  325

    Crotalaria paulina Schrank     monocrotaline          seed            C  Pilbeam et al.
                                                                             (1983)

    Crotalaria quinquefolia L.     monocrotaline          seed            C  Pilbeam et al.
                                                                             (1983)

    Crotalaria recta Steud.        monocrotaline          aerial          B  326
    ex A. Rich

    Crotalaria retusa L.           monocrotaline          seed            B  327
                                   retusine               seed, aerial    B  328
                                   retusamine
                                   retronecine

    Crotalaria sagittalis L.       monocrotaline          seed            B  330

    Crotalaria scassellatii        axillaridine           seed            A  Wiedenfeld et
    Chiov.                         axillarine                                al. (1985)
                                   deoxyaxillarine

    Crotalaria semperflorens       crosemperine           seed            B  331
    Vent.

    Crotalaria spartioides DC.     retrorsine             aerial          B  334

    Crotalaria spectabilis Roth.   monocrotaline          seed            B  335, 227
    (syn. Crotalaria sericea       spectabiline           seed, whole     B  336
    Retz)

    Crotalaria stipularia Desv.    monocrotaline          seed            B  314

    Crotalaria tetragona Roxb.     integerrimine          seed            B  314
                                   trichodesmine

    Crotalaria verrucosa L.        anacrotine             seed            B  338
                                   crotaverrine           seed            B  339
                                   acetylcrotaverrine
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 1.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Leguminosae (contd.)

    Crotalaria virgulata           grantianine            seed            B  301, 259
    subsp. grantiana (Harv.)       grantaline                             C  Smith & Culvenor
    Polhill (syn. Crotalaria       1-hydroxymethyl-                          (1984) 
    grantiana Harv.)                1beta,2beta-epoxy-                                            
                                    pyrrolizidine

    Crotalaria walkeri  Arn.       crotaverrine           seed            B  340
                                   acetylcrotaverrine

    Crotalaria wightiana Grah.     junceine               seed            B  329
    ex Wight & Arn. (syn.          trichodesmine
    Crotalaria rubiginosa Willd.
    var. wightiana J.G. Baker)

    Crotalaria zanzibarica         integerrimine          seed            B  187
    Benth. (syn. Crotalaria        usaramine              seed            B  337
    usaramoensis E.G. Baker)       senecionine
                                   retrorsine

    Ranunculaceae

    Caltha biflora DC.             senecionine            aerial          B  341

    Caltha leptosepala DC.         senecionine            aerial, root    B  341

    Scrophulariaceae

    Castilleja rhexifolia          senecionine                            B  342
    Rydb.                          sarracine                              C  Roby & Stermitz
                                   indicine or isomer                        (1984)
    ------------------------------------------------------------------------------------------
    a A = References in the reference list of this document.
      B = References in Smith & Culvenor (1981), J. nat. Prod., 44: 129-152 (with reference 
          number).
      C = References in Mattocks (1986),  Chemistry and toxicology of pyrrolizidine alkaloids.

    APPENDIX II
    Table 2.  Plants containing known alkaloids that are non-hepatotoxic (aminoalcohols and esters)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    A.  Families in which hepatotoxic alkaloids also occur

    Apocynaceae

    Alafia multiflora              alafine                seed            B  343

    Anodendron affine Druce        alloanodendrine        aerial          B  344, 345
                                   anodendrin

    Boraginaceae

    Caccinea glauca Savi           7,9-dibenzoylretrone-  fl              B  346
                                    cine

    Ehretia aspera Willd.          ehretinine             leaf            A  Suri et al. 
                                                                             (1980)
    Heliotropium angiospermum      1-hydroxymethyl-       whole           C  Birecka et al.
    Murray                          1beta,2beta-epoxy-                       (1983)
                                    pyrrolizidine

    Heliotropium ovalifolium       heliofoline            whole           C  Mohanraj et al.
    Forsk                          retronecine                               (1981)

    Heliotropium spathulatum       acetylcurassavine      aerial          A  Birecka et al.
    Rydb.                          curassavine                               (1980)

    Heliotropium strigosum Willd.  strigosine             aerial          B  347

    Lindelofia macrostyla (Bunge)  lindelofine            aerial          B  348
    M. Pop. (syn. Lindelofia       lindelofamine
    anchusoides,Paracaryum  
    heliocarpum Kern.)

    Lindelofia olgae (Regel et     viridiflorine          aerial          B  94
    Smirnov) Brand

    Lindelofia pterocarpa (Rupr.)  viridiflorine          aerial          B  93
    M. Pop.

    Macrotomia echioides Boiss.    macrotomine            aerial          B  350

    Paracaryum himalayense         viridiflorine          aerial          B  93
    (Klotsch) C.B. Clark

    Tournefortia sibirica L.       turneforcine           aerial          B  351

    Trachelanthus hissoricus       viridiflorine          leaf            B  352
    Lipsky                         trachelanthamine
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 2.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Boraginaceae  (contd.)

    Trachelanthus korolkovii       trachelanthamine       aerial          B  353, 354, 355, 94
    (Lipsky) B. Fedtsch.

    Celastraceae

    Bhesa archboldiana             9-angelylretronecine   bk              B  356
    (Merr. & Perry) Ding Hou 
    [syn. Kurrimia archboldiana 
    (Merr. & Perry)]

    Compositae

    Adenostyles rhombifolius       sarracine              aerial          B  116
    (Willd.) M. Pimen. ssp. 
    rhombifolia chemovar. 
    sarracinifera

    Adenostyles rhombifolius       platyphylline          aerial          B  116
    (Willd.) M. Pimen. ssp. 
    rhombifolia chemovar. 
    platyphyllinifera
    Cacalia hastata L.             hastacine              root            B  357, 241

    Cacalia robusta                hastacine                              B  358

    Senecio amphibolus             macrophylline          aerial          B  359

    Senecio angulatus L.           angularine             whole           B  360
                                   rosmarinine

    Senecio aronicoides            hygrophylline          whole           B  420

    Senecio brachypodus DC.        rosmarinine            aerial, root    B  361

    Senecio francheti Winkl.       sarracine              aerial          B  352
                                   franchetine

    Senecio glastifolius           sarracine                              A  Mortimer & White
                                                                             (1975)

    Senecio hygrophyllus R.A.      platyphylline          aerial          B  366, 361
    Dyer et C.A. Smith (syn.       rosmarinine
    Senecio adnatus DC.)           hygrophylline

    Senecio macrophyllus Bieb.     macrophylline          aerial          B  368

    Senecio mikanioides Otto. ex   sarracine              aerial          B  155, 369, 370
    Walp.
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 2.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Compositae  (contd.)

    Senecio nemorensis L. ssp.     nemorensine            aerial          B  371
    fuchsii var. nova (Zlatnik)

    Senecio nemorensis L. ssp.     nemorensine            aerial          B  371
    jaquinianus (Rchb.) Durand

    Senicio ovirensis ssp.         angelylheliotridine                    A  Roder et al. 
    gaudinil                                                                 (1980)

    Senecio pauciligulatus         rosmarinine            aerial          B  361
    Dyer et Sm.

    Senecio rivularis DC.          7-angelylheliotridine  aerial          B  182, 135

    Senecio rosmarinifolius Linn.  rosmarinine            aerial          B  191, 192, 188

    Senecio salignus DC.           7-angelylheliotridine  aerial          A  Bohlmann et al.
                                                                             (1986)

    Senecio sarracenius L.         sarracine              aerial          B  153, 372, 373

    Senecio schvetsovii Korsh      macrophylline          aerial          B  231

    Senecio sylvaticus L.          silvasenecine          aerial          B  362, 153
                                   sarracine              aerial          B  374

    Senecio taiwanensis Hayata     rosmarinine            aerial          B  217

    Senecio tournefortii Lap.      platyphylline          aerial          B  375

    Leguminosae

    Crotalaria albida Heyne ex     croalbidine            aerial          B  376, 377
    Roth (syn. Crotalaria montana 
    Roxb.)

    Crotalaria aridicola Domin.    1-methoxymethyl-1,2-   aerial          B  378
                                    dehydropyrrolizidine
                                   7beta-hydroxy-1-
                                    methoxymethyl-1,2-
                                    dehydropyrrolizidine
                                   7beta-acetoxy-1-       whole           B  379
                                    methoxymethyl-1,2-
                                    dehydropyrrolizidine

    Crotalaria damarensis Engl.    1-methylenepyrrolizi-  whole, seed     B  381, 283
                                    dine
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 2.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Leguminosae  (contd.)

    Crotalaria goreensis Guill.    7beta-hydroxy-1-       aerial, seed    B  382
    et Perr.                        methylene-8beta-
                                    pyrrolizidine
                                   7beta-hydroxy-1-
                                    methylene-8alpha-
                                    pyrrolizidine

    Crotalaria grandistipulata     1-methylenepyrrolizi-  seed            B  434
    Harms                           dine

    Crotalaria lachnophora         1-methylenepyrrolizi-  seed            B  434
    A. Rich                         dine

    Crotalaria maypurensis Humb    7beta-hydroxy-1-       aerial          B  383
    et al.                          methylene-8beta- 
                                    pyrrolizidine
                                   7beta-hydroxy-1-
                                    methylene-8alpha-          
                                    pyrrolizidine

    Crotalaria medicaginea Lam.    1-methoxymethyl-1,2-   whole, seed     B  378
                                    dehydropyrrolizidine
                                   7beta-hydroxy-1-
                                    methoxymethyl-1,2-
                                    dehydropyrrolizidine
                                   1alpha-methoxymethyl-  whole, seed
                                    1beta,2beta-
                                    epoxypyrrolizidine
                                   7alpha-hydroxy-1-      seed
                                    methoxymethyl-1,2-
                                    dehydropyrrolizidine
                                   1alpha-hydroxymethyl-  aerial
                                    1beta,2beta-
                                    epoxypyrrolizidine

    Crotalaria natalitia Meissner  1-methylenepyrrolizi-  seed            B  434
                                    dine
    Crotalaria podocarpa DC.       7-hydroxy-1-methylene- seed            B   435
                                    pyrrolizidine

    Crotalaria stolzii (Bak. f.)   1-methylenepyrrolizi-  seed            B  434
    Milne-Redh. ex Polhill          dine

    Ranunculacae laburnum L.       laburnine              seed            B  387, 388, 389
                                   1-hydroxymethyl-7-e                    B  390
                                   hydroxypyrrolizidin          
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 2.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Scrophulariacea

    Castilleja "rhexifolia aff.    sarracine                              C  Roby & Stermitz
    miniata"                       7-angelylplatynecine                         (1984)
                                   8-angelylplatynecine

    B.  Families in which hepatotoxic alkaloids are not known to occur

    Orchidaceae

    Chysis bractescens Lindl.      1alpha-methoxycar-     whole           B  391, 392
                                    bonyl-8beta-
                                    pyrrolizidine
                                   1alpha-ethoxycar-
                                    bonyl-8beta-
                                    pyrrolizidine

    Doritis pulcherrima            phalaenopsine La or T  whole           B  393
    (syn. Phalaenopsis esmerelda)

    Hammarbya paludosa (L.) O.K.   paludosine             whole           B  394
                                   hammarbine             whole           B  395

    Kingiella taenialis (Lindl.)   phalaenopsine La       whole           B  396
    Rolfe
    Liparis auriculata (Blume)     auriculine             whole           B  397

    Liparis bicallosa Schltr.      laburnine              whole           B  397
                                   malaxine               whole           B  398, 399

    Liparis hachijoensis Nakai     laburnine              whole           B  397
                                   malaxine               whole           B  398

    Liparis keitaoensis Hay.       keitaoine              whole           B  395
                                   keitine

    Liparis kumokiri F. Maekawa    kumokirine             whole           B  400, 398

    Liparis loeselii (L.) L.C.     auriculine             whole           B  394
    Rich

    Liparis nervosa Lindl.         nervosine              whole           B  398, 401

    Malaxis congesta comb. nov.    malaxin                whole           B  402
    (Rchb. f.)

    Malaxis grandifolia Schltr.    grandifoline           whole           B  403

    Phalaenopsis amabilis Bl.      phalaenopsine T        whole           B  404, 405, 393

    Phalaenopsis amboinensis       phalaenopsine La       whole           B  393
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 2.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Orchidaceae  (contd.)

    Phalaenopsis aphrodite         phalaenopsine T        whole           B  393

    Phalaenopsis cornu-cervi       cornucervine           whole           B  404, 393
    Rchb. f.

    Phalaenopsis equestris         phalaenopsin ls        whole           B  393
    Rchb. f.                       phalaenopsin T

    Phalaenopsis fimbriata         phalaenopsine T        whole           B  393

    Phalaenopsis hieroglyfica      phalaenopsine T or La  whole           B  393

    Phalaenopsis lueddemanniana    phalaenopsine T or La  whole           B  393

    Phalaenopsis mannii Rchb. f.   phalaenopsine La       whole           B  393

    Phalaenopsis sanderiana        phalaenopsine La       whole           B  393
    Rchb. f.                       phalaenopsine T

    Phalaenopsis schilleriana      phalaenopsine La       whole           B  393

    Phalaenopsis stuartiana        phalaenopsine La       whole           B  393
    Rchb. f.                       phalaenopsine T

    Phalaenopsis sumatrana         phalaenopsis La        whole           B  393

    Phalaenopsis violacea          phalaenopsis La or T   whole           B  393

    Vanda cristata Lindl.          acetyllaburnine        whole           B  407

    Vanda helvola Bl.              laburnine              whole           B  408
                                   acetyllaburnine

    Vanda hindsii Lindl.           acetyllaburnine        whole           B  408

    Vanda luzonica Loher           acetyllaburnine        whole           B  408

    Vandopsis gigantea Pfitz.      laburnine              whole           B  408
                                   lindelofidine
                                   acetyllaburnine
                                   acetyllindelofidine

    Vandopsis lissochiloides       laburnine              whole           B  408
    Pfitz.                         lindelofidine
                                   acetyllaburnine
                                   acetyllindelofidine
    ------------------------------------------------------------------------------------------


    APPENDIX II  Table 2.  (contd.)
    ------------------------------------------------------------------------------------------
    Plant                          Constituent alkaloids  Plant part      Referencea
    ------------------------------------------------------------------------------------------
    Rhizophoraceae

    Cassipourea gummiflua Tulasne  cassipourine           stem, leaf      B  409
    var. verticellata Lewis                               bk              B  410

    Santalaceae

    Thesium minkwitzianum          thesine                aerial          B  411
    B. Fedtsch.                    thesinine                              B  412, 413
                                   thesinicine
                                   isoretronecanol        root

    Sapotaceae

    Mimusops elengi L.             1-hydroxymethyl-                       B  414
                                   pyrrolizidine
                                   tiglate

    Planchonella anteridifera      planchonelline         leaf            B  415
    (C.T. White et W.D. Francis)   tiglyllaburnine
    H.J. Lamb                      benzoyllaburnine

    Planchonella thyrsoidea C.T.   planchonelline         leaf            B  415
    White ex F.S. Walker           tiglyllaburnine
                                   benzoyllaburnine

    Planchonella sp. (NGF 24722)   trans-beta-thio-       leaf            B  416
                                    acrylyl-(-)-iso-
                                    retronecanol
                                   tiglylisoretronecanol
    ------------------------------------------------------------------------------------------
    a A = References in the reference list of this document.
      B = References in Smith & Culvenor (1981), J. nat. Prod., 44: 129-152 (with reference 
          number).
      C = References in Mattocks (1986),  Chemistry and toxicology of pyrrolizidine alkaloids.