Environmental Health Criteria 219


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

    First draft prepared by Professor W.F.O. Marasas (Medical Research
    Council, Tygerberg, South Africa), Professor J.D. Miller (Carlton
    University, Ottawa, Canada), Dr R.T. Riley (US Department of
    Agriculture, Athens, USA) and Dr A. Visconti (National Research
    Council, Bari, Italy)

    Published under the joint sponsorship of the United Nations
    Environment Programme, the International Labour Organisation, and the
    World Health Organization, and produced within the framework of the
    Inter-Organization Programme for the Sound Management of Chemicals.

    World Health Organization
    Geneva, 2000

         The International Programme on Chemical Safety (IPCS),
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    WHO Library Cataloguing in Publication Data

    Fumonisin B1.

         (Environmental health criteria; 219)

         1.Carboxylic acids - toxicity   2.Food contamination
         3.Environmental exposure        4.Risk assessment   I.Series

         ISBN 92 4 157219 1  (NLM Classification: QD 341.P5)
         ISSN 0250-863X

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         1.1. Summary
              1.1.1. Identity, physical and chemical properties, and
                     analytical methods
              1.1.2. Sources of human exposure
              1.1.3. Environmental transport, distribution and
              1.1.4. Environmental levels and human exposure
              1.1.5. Kinetics and metabolism in animals
              1.1.6. Effects on animals and  in vitro test systems
              1.1.7. Effects on humans
              1.1.8. Effects on other organisms in the laboratory
         1.2. Evaluation of human health risks
              1.2.1. Exposure
              1.2.2. Hazard identification
              1.2.3. Dose-response assessment
              1.2.4. Risk characterization
         1.3. Recommendations for protection of human health


         2.1. Identity
         2.2. Physical and chemical properties of the pure substance
         2.3. Analytical methods
              2.3.1. Sampling and preparation procedures
              2.3.2. Extraction
              2.3.3. Analysis





         6.1. Absorption
         6.2. Distribution
         6.3. Elimination, excretion and metabolic transformation
         6.4. Retention and turnover
         6.5. Reaction with body components


         7.1. Laboratory animals and  in vitro test systems
              7.1.1. Single exposure
              7.1.2. Repeated exposure
             Body weight loss
             Hepatocarcinogenicity and nephrotoxicity
              7.1.3. Skin and eye irritation
              7.1.4. Reproductive toxicity, embryotoxicity and
              7.1.5. Mutagenicity and related end-points
              7.1.6. Carcinogenicity
             Carcinogenicity bioassays
             Short-term assays for carcinogenicity
         7.2. Other mammals
              7.2.1. Equine leukoencephalomalacia
              7.2.2. Porcine pulmonary oedema syndrome
              7.2.3. Poultry toxicity
              7.2.4. Non-human primate toxicity
              7.2.5. Other species
         7.3. Mechanisms of toxicity - mode of action
              7.3.1. Disruption of sphingolipid metabolism
             Sphingolipids and their metabolism
             Fumonisin-induced disruption of sphingolipid
                              metabolism  in vitro 
             Fumonisin disruption of sphingolipid
                              metabolism  in vivo 
             Tissue and species specificity
             Fumonisin-induced sphingolipid alterations:
                              effects on growth, differentiation and cell
             Sphingolipid-mediated cellular deregulation
                              and fumonisin diseases
              7.3.2. Altered fatty acid metabolism in liver
              7.3.3. Other biochemical changes
         7.4. Factors modifying toxicity;  toxicity of metabolites


         8.1. Transkei, South Africa
         8.2. China
         8.3. Northern Italy


         9.1. Microorganisms
         9.2. Plants
              9.2.1. Duckweed and jimsonweed
              9.2.2. Tomato
              9.2.3. Maize









         Every effort has been made to present information in the criteria
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    Criteria monographs, readers are requested to communicate any errors
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    on Chemical Safety, World Health Organization, Geneva, Switzerland, in
    order that they may be included in corrigenda.

                              *     *     *

         A detailed data profile and a legal file can be obtained from the
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                              *     *     *

         This publication was made possible by grant number
    5 U01 ES02617-15 from the National Institute of Environmental Health
    Sciences, National Institutes of Health, USA, and by financial support
    from the European Commission.

    Environmental Health Criteria



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    Dr R.V. Bhat, Food and Drug Toxicology Research Centre, National
         Institute of Nutrition, Indian Council of Medical Research,
         Hyderabad, India

    Dr M. Hirose, Division of Pathology, Biological Research Centre,
         National Institute of Health Sciences, Tokyo, Japan

    Dr P.C. Howard, Division of Biochemical Toxicology, National Center
         for Toxicology Research, US Food and Drug Administration,
         Jefferson, Arkansas, USA

    Dr S. Humphreys, Center for Food Safety and Applied Nutrition, US Food
         and Drug Administration, Washington DC, USA

    Professor M. Kirsch-Volders, Laboratory for Cellular Genetics,
         Brussels, Belgium  (Chairman) 

    Professor W.F.O. Marasas, Medical Research Council, Tygerberg, South

    Professor J.D. Miller, Department of Chemistry, Carleton University,
         Ottawa, Ontario, Canada

    Dr J.H. Olsen, Institute of Cancer Epidemiology, Danish Cancer
         Society, Copenhagen, Denmark

    Dr R. Plestina, Toxicology Unit, Institute for Medical Research and
         Occupational Health, Zagreb, Croatia

    Dr R.T. Riley, Agricultural Research Service, US Department of
         Agriculture, Athens, USA

    Dr A. Visconti, Institute for Toxins and Mycotoxins of Plant
         Parasites, National Research Council, Bari, Italy


    Dr A. Aitio, International Programme on Chemical Safety, World Health
         Organization, Geneva, Switzerland  (Joint Secretary) 

    Mr Y. Hayashi, International Programme on Chemical Safety, World
         Health Organization, Geneva, Switzerland  (Joint Secretary) 

    Dr J.M. Rice, International Agency for Research on Cancer, Lyon,


         A WHO Task Group on Environmental Health Criteria for Fumonisin
    B1 met at the World Health Organization, Geneva, Switzerland from 10
    to 14 May 1999.  Dr M. Younes, Acting Coordinator, Programme for the
    Promotion of Chemical Safety, opened the meeting and welcomed the
    participants on behalf of the IPCS and its three cooperating
    organizations (UNEP/ILO/WHO).  The Task Group reviewed and revised the
    draft monograph and made an evaluation of the risks for human health
    and the environment from exposure to fumonisin B1.

         Professor W.F.O. Marasas, Professor J.D. Miller, Dr R.T. Riley
    and Dr A. Visconti prepared the first draft of this monograph.  The
    second draft incorporated comments received following the circulation
    of the first draft to the IPCS Contact Points for Environmental Health
    Criteria monographs.

         Dr A. Aitio, Mr Y. Hayashi and Dr P. Jenkins of the IPCS Central
    Unit were responsible for the overall scientific content and technical
    editing, respectively.

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

                                  *   *   *

         Financial support for this Task Group was provided by the US Food
    and Drug Administration as part of its contributions to the IPCS.


    2-AAF          2-acetylaminofluorene
    AAL-toxin       Alternaria alternata lycopersici toxin
    AMP            adenosine monophosphate
    AP             aminopentol
    CV             coefficient of variation
    CZE            capillary zone electrophoresis
    DEN            diethylnitrosamine
    DNA            deoxyribonucleic acid
    EDL            effective dose level
    EGF            epidermal growth factor
    ELEM           equine leukoencephalomalacia
    ELISA          enzyme-linked immunosorbent assay
    FA, FAK        fumonisin A, fumonisin AK
    FB             fumonisin B
    FC             fumonisin C
    FP             fumonisin P
    GC             gas chromatography
    GGT            gamma-glutamyltranspeptidase
    HPLC           high-performance liquid chromatography
    IC50           median inhibitory concentration
    LC50           median lethal concentration
    IFN-gamma      interferon-gamma
    LPS            lipopolysaccharide
    MAPK           mitogen-activated protein kinase
    MME            monomethyl ester
    MS             mass spectrometry
    NADH           reduced nicotinamide adenine dinucleotide
    NADPH          reduced nicotinamide adenine dinucleotide phosphate
    NCTR           National Center for Toxicological Research (USA)
    NMBA            N-methylbenzylnitrosamine
    NOEL           no-observed-effect level
    NTD            neural tube defect
    NTP            National Toxicology Program (USA)
    OPA             o-phthaldialdehyde
    PDI            probable daily intake
    PFC            plaque-forming cell
    PGST           placental glutathione  S-transferase
    PIM            pulmonary intravascular/interstitial macrophage
    PKC            protein kinase C 
    PPE            porcine pulmonary oedema
    PUFA           polyunsaturated fatty acid
    Sa/So          sphinganine/sphingosine
    TCA            tricarbalyllic acid moiety
    TLC            thin-layer chromatography
    TNF-alpha      tumour necrosis factor-alpha


         In this document, the fungus previously referred to as
     Fusarium moniliforme Sheldon, is referred to as  Fusarium 
     verticillioides (Sacc.) Nirenberg in accordance with a decision
    taken at the 8th International  Fusarium Workshop held at CABI
    BioScience, Egham, United Kingdom, 17-20 August 1998.

         This monograph focuses on fumonisin B1, the most abundant
    naturally occurring fumonisin. Some information is also given on
    fumonisins B2 and B3, which frequently occur with FB1, both in
    culture material and in naturally contaminated samples.


    1.1  Summary

    1.1.1  Identity, physical and chemical properties, and analytical 

         Fumonisin B1 (FB1) has the empirical formula C34H59NO15 and
    is the diester of propane-1,2,3-tricarboxylic acid and
    2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxyeicosane (relative
    molecular mass: 721). It is the most prevalent of fumonisins, a family
    of toxins with at least 15 identified members. The pure substance is a
    white hygroscopic powder, which is soluble in water,
    acetonitrile-water or methanol, is stable in acetonitrile-water (1:1),
    is unstable in methanol, and is stable at food processing temperature
    and to light.

         Several analytical methods have been reported, including
    thin-layer chromatography (TLC) and liquid chromatographic (LC), mass
    spectroscopic (MS), post-hydrolysis gas chromatographic and
    immunochemical methods, although the majority of studies have been
    performed using LC analysis of a fluorescent derivative.

    1.1.2  Sources of human exposure

         FB1 is produced by several  Fusarium species, mainly by
     Fusarium verticillioides (Sacc.) Nirenberg (=  Fusarium 
     moniliforme Sheldon), which is one of the most common fungi
    associated with maize worldwide. Significant accumulation of FB1 in
    maize occurs when weather conditions favour Fusarium kernel rot.

    1.1.3  Environmental transport, distribution and transformation

         There is evidence that fumonisins can be metabolized by some soil
    microorganisms. However, little is known about the environmental fate
    of fumonisins after they are either excreted or processed.

    1.1.4  Environmental levels and human exposure

         FB1 has been detected in maize and maize-based products
    worldwide at mg/kg levels, sometimes in combination with other
    mycotoxins. Concentrations at mg/kg levels have also been reported in
    food for human consumption. Dry milling of maize results in the
    distribution of fumonisin into the bran, germ and flour. In
    experimental wet milling, fumonisin was detected in steep water,
    gluten, fibre and germ, but not in the starch. FB1 is stable in maize
    and polenta, whereas it is hydrolysed in nixtamalized maize-based
    foods, i.e. foods processed with hot alkali solutions.

         FB1 is not present in milk, meat or eggs from animals fed grain
    containing FB1 at levels that would not affect the health of the
    animals. Human exposure estimates for the USA, Canada, Switzerland,

    the Netherlands and the Transkei (South Africa) ranged from 0.017 to
    440 µg/kg body weight per day. No data on occupational inhalation
    exposure are available.

    1.1.5  Kinetics and metabolism in animals

         There have been no reports on the kinetics or metabolism of FB1
    in humans. In experimental animals it is poorly absorbed when dosed
    orally, is rapidly eliminated from circulation and is recovered
    unmetabolized in faeces. Biliary excretion is important, and small
    amounts are excreted in urine. It can be degraded to partially
    hydrolysed FB1 in the gut of non-human primates and some ruminants. A
    small amount is retained in the liver and kidney.

    1.1.6  Effects on animals and in vitro test systems

         FB1 is hepatotoxic in all animal species tested including mice,
    rats, equids, rabbits, pigs and non-human primates. With the exception
    of Syrian hamsters, embryotoxicity or teratogenicity is only observed
    concurrent with or subsequent to maternal toxicity. Fumonisins are
    nephrotoxic in pigs, rats, sheep, mice and rabbits. In rats and
    rabbits, renal toxicity occurs at lower doses than hepatotoxicity.
    Fumonisins are known to be the cause of equine leukoencephalomalacia
    and porcine pulmonary oedema syndrome, both associated with the
    consumption of maize-based feeds. Limited information on immunological
    properties of FB1 is available. It was hepatocarcinogenic to male
    rats in one strain and nephrocarcinogenic in another strain at the
    same dose levels (50 mg/kg diet), and was hepatocarcinogenic at 50
    mg/kg diet in female mice. There appears to be a correlation between
    organ toxicity and cancer development.  FB1 was the first specific
    inhibitor of  de novo sphingolipid metabolism to be discovered and is
    currently widely used to study the role of sphingolipids in cellular
    regulation.  FB1 inhibits cell growth and causes accumulation of free
    sphingoid bases and alteration of lipid metabolism in animals, plants
    and some yeasts. It did not induce gene mutations in bacteria or
    unscheduled DNA synthesis in primary rat hepatocytes, but induced a
    dose-dependent increase in chromosomal aberrations at low
    concentration levels in one study on primary rat hepatocytes.

    1.1.7  Effects on humans

         There are no confirmed records of acute fumonisin toxicity in
    humans. Available correlation studies from the Transkei, South Africa,
    suggest a link between dietary fumonisin exposure and oesophageal
    cancer. This was observed where relatively high fumonisin exposure has
    been demonstrated and where environmental conditions promote fumonisin
    accumulation in maize, which is the staple diet. Correlation studies
    are also available from China. However, no clear picture on the
    relationship between either fumonisin or  F. verticillioides 
    contamination and oesophageal cancer emerged. Owing to the absence of
    fumonisin exposure data, no conclusion can be drawn from a case
    control study of males in Italy showing an association between maize

    intake and upper gastrointestinal tract cancer among subjects with
    high alcohol consumption.

         There are no validated biomarkers for human exposure to FB1.

    1.1.8  Effects on other organisms in the laboratory

         FB1 inhibits cell growth and causes accumulation of free
    sphingoid bases and alteration of lipid metabolism in  Saccharomyces 

         FB1 is phytotoxic, damages cell membranes and reduces
    chlorophyll synthesis. It also disrupts the biosynthesis of
    sphingolipids in plants and may play a role in the pathogenicity of
    maize by fumonisin-producing  Fusarium species.

    1.2  Evaluation of human health risks

    1.2.1  Exposure

         Human exposure as demonstrated by the occurrence of FB1 in maize
    intended for human consumption is common worldwide. There are
    considerable differences in the extent of human exposure between
    different maize-growing regions. This is most evident when comparing
    fully developed and developing countries. For example, although FB1
    can occur in maize products in the USA, Canada and western Europe,
    human consumption of those products is modest. In parts of Africa,
    South-Central America and Asia, some populations consume a high
    percentage of their calories as maize meal where FB1 contamination
    may be high (see Appendix 2). Maize contaminated naturally by FB1 can
    be simultaneously contaminated with other  F. verticillioides or
     F. proliferatum toxins or with other agriculturally important toxins
    including deoxynivalenol, zearalenone, aflatoxin and ochratoxin.

         FB1 is stable to food processing methods used in North America
    and western Europe. Treating maize with base and/or water washing
    effectively lowers the FB1 concentrations. However, its
    hepatotoxicity and/or nephrotoxicity in experimental animals are still
    evident. Little is known about how food processing techniques used in
    the developing world affect FB1 in maize products.

    1.2.2  Hazard identification

         The causal role of FB1 exposure in the disease equine
    leukoencephalomalacia has been established. Large-scale outbreaks of
    this fatal disease occurred in the USA during the 19th century and as
    recently as 1989-1990. The causal role of FB1 exposure in the fatal
    disease porcine pulmonary oedema has been established. As observed in
    pregnant females, low exposures to FB1 are fatal to rabbits. Exposure
    has been demonstrated to result in renal toxicity and causes
    hepatotoxicity in all animal species studied, including non-human
    primates. FB1 exposure causes hypercholesterolaemia in several animal

    species, including non-human primates. There is good evidence for
    altered lipid metabolism in the animal diseases associated with FB1
    exposure. Disruption of sphingolipid metabolism is evident either
    before or concurrent with  in vitro and  in vivo toxicity. The use
    of fumonisins as tools to study the function of sphingolipids has
    revealed that sphingolipids are required for cell growth and affect
    signalling molecules in several pathways, leading to apoptotic and
    necrotic cell death, cellular differentiation and altered immune
    responses. Altered lipid metabolism and changes in the activity and/or
    expression of key enzymes responsible for normal cell cycle progress
    appear to be common factors following exposure to FB1. FB1 is not a
    developmental toxin to rat, mouse or rabbit. It induces fetotoxicity
    in Syrian hamster at high doses without maternal toxicity.

         The carcinogenicity of FB1 in rodents varies between species,
    strains and sex.  The only study with B6C3F1 mice indicated that FB1
    was hepatocarcinogenic to females at 50 mg/kg in the diet.  Primary
    hepatocellular carcinomas and cholangial carcinomas were induced in
    male BD IX rats fed diets at 50 mg FB1/kg for up to 26 months. Renal
    tubule adenomas and carcinomas were detected in male F344/N Nctr rats
    fed 50 mg FB1/kg. There appears to be a correlation between organ
    toxicity and cancer development.

         A limited number of genotoxicity studies are available. FB1 was
    not mutagenic in bacterial assays. In  in vitro mammalian cells,
    unscheduled DNA synthesis was not detected but FB1 caused chromosomal
    breaks in rat hepatocytes in one study. Other studies have shown that
    FB1 causes increased lipid peroxidation  in vivo and  in vitro. It
    is possible that chromosome-breaking effects and lipid peroxidation
    are causally related.

         FB1 levels above 100 mg/kg, which have been reported in maize
    consumed by humans in Africa and China, would probably cause
    leukoencephalomalacia, pulmonary oedema syndrome or cancer if fed to
    horses, pigs and rats or mice, respectively. Despite these cases of
    very high human exposure, there are no confirmed records of acute
    fumonisin toxicity in humans. Available correlation studies from the
    Transkei, South Africa, suggest a link between dietary fumonisin
    exposure and oesophageal cancer. Elevated rates of oesophageal cancer
    have been observed where relatively high fumonisin exposure has been
    demonstrated and where environmental conditions promote the
    accumulation of fumonisin in maize, which is the staple diet.

         One case-control study in males from Italy found an association
    between maize intake and cancers of the upper digestive tract,
    including oesophageal cancer, among subjects with high alcohol
    consumption. There were no data on fumonisin exposure.

    1.2.3  Dose-response assessment

         The lowest dose of FB1 that induced hepatocarcinomas in
    experimental animals was 50 mg/kg diet in male BD IX rats and female
    B6C3F1/Nctr mice; no cancer induction was observed at 25 or 15 mg/kg
    diet, respectively.  In each case, indications of hepatotoxicity or
    lipid alterations were noted at the same or lower doses in studies
    with these same rat and mouse strains. The lowest dose of FB1 that
    induced renal carcinomas in the male F344/N Nctr rats was 50 mg/kg
    diet; no cancer induction was observed at 15 mg/kg diet. Renal tubular
    apoptosis and cell proliferation, as well as tissue and urinary
    sphingolipid changes, occurred at lower doses than those required for
    the induction of cancer in these studies.

         No data are available to assess quantitatively the relationship
    between exposure to FB1 and possible effects in humans.

    1.2.4  Risk characterization

         FB1 is carcinogenic in mice and rats and induces fatal diseases
    in pigs and horses at levels of exposure that humans encounter. The
    Task Group was not in a position to perform a quantitative estimation
    of the human health risks, but considered that such an estimation is
    urgently needed.

    1.3  Recommendations for protection of human health

         a)   Limits for human dietary exposure should be established.
              Special consideration should be given to populations
              consuming a high percentage of their calories as maize meal.

         b)   Measures should be taken to limit fumonisin exposure and
              maize contamination by: 

              *    planting alternative crops in areas where maize is not
                   well adapted;
              *    developing maize resistant to Fusarium kernel rot;
              *    practising better crop management;
              *    segregating mouldy kernels.

         c)   Early awareness of potential food contamination should be
              increased by improving communication between veterinarians
              and public health officials on outbreaks of mycotoxicoses in
              domestic animals.

         d)   A robust, low-cost and simple screening method for the
              detection of fumonisin contamination in maize should be


    2.1  Identity

    Common name:             Fumonisin B1 (FB1)

    Chemical formula:        C34H59NO15

    Chemical structure:


    Relative molecular mass:      721

    CAS Name:                1,2,3-Propanetricarboxylic acid,

    IUPAC name:              None

    CAS registry number:     116355-83-0

    RTECS No.:               TZ 8350000

    Synonym:                 Macrofusine

         At least 15 different fumonisins have so far been reported and
    other minor metabolites have been identified, although most of them
    have not been shown to occur naturally. They have been grouped into
    four main categories (Plattner, 1995; Abbas & Shier, 1997; Musser &
    Plattner, 1997): FA1, FA2, FA3, FAK1; FB1, FB2, FB3, FB4;
    FC1, FC2, FC3, FC4; FP1, FP2 and FP3. FB2, FB3 and FB4
    differ from FB1 in that they lack hydroxyl groups present in FB1;
    FA1, FA2 and FA3 are like FB1, FB2 and FB3, but are

     N-acetylated; FAK1 is like FA1 but is 15-keto functionalized; FCs
    are like FBs but lack the methyl group adjacent to the amino group;
    FPs have a 3-hydroxypyridium group instead of the amine group in the
    FBs. This monograph will focus mainly on FB1, the most abundant of
    the naturally occurring fumonisins.

    2.2  Physical and chemical properties of the pure substance

    Physical state:     White hygroscopic powder

    Melting point:      Not known (has not been crystallized)

    Optical rotation:   Not known

    Spectroscopy:       Mass spectral and nuclear magnetic resonance data
                        are given in Bezuidenhout et al. (1988), Laurent
                        et al. (1989a) and Savard & Blackwell (1994)

    Solubility:         Soluble in water to at least to 20 mg/ml (US NTP,
                        1999); soluble in methanol, acetonitrile-water.

     n-Octanol/water     1.84 (Norred et al., 1997)
      (log P):

    Stability:          Stable in acetonitrile-water (1:1) for up to 6
                        months at 25°C; unstable in methanol (25% or 35%
                        concentration decrease after 3 or 6 weeks at 25°C,
                        respectively), giving rise to monomethyl or
                        dimethyl esters (Gelderblom et al., 1992a;
                        Visconti et al., 1994); stable in methanol up to 6
                        weeks at -18°C (Visconti et al., 1994); stable at
                        78°C for 16 h in buffer solutions at pH between
                        3.5 and 9 (Howard et al., 1998)

    2.3  Analytical methods

         Six general analytical methods have been reported: thin-layer
    chromatographic (TLC), liquid chromatographic (LC), mass spectrometric
    (MS), post-hydrolysis gas chromatographic, immunochemical and
    electrophoretic methods (Sydenham & Shephard, 1996; Shephard, 1998).
    The majority of studies have been performed using LC analysis of a
    fluorescent derivative.

    2.3.1  Sampling and preparation procedures

         In raw maize, FB1 is present in both visibly damaged and
    undamaged kernels (Bullerman & Tsai, 1994).  This means that the
    problem that occurs with the mycotoxin aflatoxin, i.e., a few highly
    contaminated kernels in otherwise aflatoxin-free kernels, is probably
    less of an issue.  However, it has been shown that higher levels of
    fumonisins are concentrated in visibly damaged kernels (Pascale et

    al., 1997).  Studies to determine the minimum representative sample in
    a lot of maize have not been reported.  However, homogeneous material
    (CV < 10%) for fumonisin analysis was obtained by grinding
    contaminated maize to a particle size less than 2 mm with test portion
    sizes of 25 and 10 g (Visconti & Boenke, 1995).

    2.3.2  Extraction

         Methanol-water (3:1) is the solvent of choice (e.g., Shephard et
    al., 1990; Stack & Eppley, 1992; Doko & Visconti, 1994; Scott &
    Lawrence, 1994) with a long shaking time or homogenization with a
    blender (Sydenham et al., 1992; Bennett & Richard, 1994; Visconti &
    Boenke, 1995; Visconti et al., 1995). The use of acetonitrile-water
    has also been reported, with conflicting data on its performance
    relative to methanol-water (Sydenham et al., 1992a; Bennett & Richard,
    1994; Visconti & Boenke, 1995). Use of an acidic extraction procedure
    may lead to higher extraction efficiencies (Zoller et al., 1994;
    Meister, 1998). However, remarkable variability in extraction
    efficiency has been reported by several authors, and more work needs
    to be done to establish the best extraction solvents for various food

         Clean-up involves the use of solid-phase extraction with strong
    anion exchange (Shephard et al., 1990) or C18 reversed-phase (Ross et
    al., 1990) or a combination of both (Miller et al., 1993). Improved
    recoveries can be achieved by using anion exchange instead of
    reversed-phase material for sample clean-up (Stockenström et al.,
    1994; Dawlatana et al., 1995). Immunoaffinity columns (Scott &
    Trucksess, 1997) have also been shown to be useful for clean-up of
    crude extracts of maize (Ware et al., 1994; Duncan et al., 1998),
    sweet corn (Trucksess et al., 1995), beer (Scott & Lawrence, 1995) and
    milk (Scott et al., 1994).

         Fumonisins are relatively stable compounds (Alberts et al., 1990;
    Dupuy et al., 1993a; Le Bars et al., 1994; Visconti et al., 1994;
    Pascale et al., 1995; Jackson et al., 1996a,b, 1997). A number of
    factors make them difficult to extract from processed food (Scott,
    1993; Bullerman & Tsai, 1994). Binding of FB1 to maize bran flour
    occurs at room temperature and above (Scott & Lawrence, 1994). Added
    iron may also affect recoveries of fumonisin (Scott & Lawrence, 1994).
    Unknown processing factors or ingredients can change the recovery of
    fumonisin from cereal products (Scott & Lawrence, 1994). Only 45% of
    FB1 present in spiked corn meal was recovered following baking at
    175-200°C for 20 min (Jackson et al., 1997). Fumonisins have been
    shown to react with reducing sugars at elevated temperatures (Murphy
    et al., 1996; Lu et al., 1997). The product of the reaction of FB1
    with reducing sugars was identified as  N-carboxymethyl-FB1 (Howard
    et al., 1998). This product was found in raw corn samples at 4% of the
    FB1 levels (Howard et al., 1998). Ammoniation and treatment with base
    reduces apparent fumonisin concentrations while increasing the
    concentration of hydrolysed fumonisins without eliminating the
    toxicity of the treated product, again suggesting analytical
    difficulties (Norred et al., 1991; Hendrich et al., 1993).

         Methods have been reported for the extraction of FB1 and FB2 in
    plasma and urine (Shephard et al., 1992c, 1995c; Shetty & Bhat, 1998),
    bile of rats and vervet monkeys (Shephard et al., 1994c, 1995a),
    faeces of vervet monkeys (Shephard et al., 1994b), liver, kidney and
    muscle of beef cattle (Smith & Thakur, 1996), and milk (Maragos &
    Richard, 1994; Scott et al., 1994; Prelusky et al., 1996a).

    2.3.3  Analysis

         Normal phase silica TLC can be used for analysis, with fumonisins
    being visualized by spraying with  p-anisaldehyde (Plattner et al.,
    1990; Sydenham et al., 1990a; Dupuy et al., 1993b). For C18 HPLC or
    TLC, visualization has been accomplished with fluorescamine
    (Rottinghaus et al., 1992; Miller et al., 1995) and vanillin (Pittet
    et al., 1992). The detection limit for fumonisins in maize by these
    methods is 1 mg/kg (Miller et al., 1995). Improved TLC methods with
    adequate sensitivity are needed, particularly to control maize
    contamination in developing countries.

         A number of fluorescent derivatives have been used for HPLC
    detection including fluorescamine (Ross et al., 1991a,b),
    naphthalene-2,3-dicarboxaldehyde/potassium cyanide (Ware et al., 1993;
    Bennett & Richard, 1994; Scott & Lawrence, 1994),
    4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole (Scott & Lawrence, 1992,
    1994), 6-aminoquinolyl  N-hydroxysuccinimidylcarbamate (Velázquez et
    al., 1995), 9-fluorenylmethyl chloroformate (Holcomb et al., 1993) and
     o-phthaldialdehyde (OPA) (Shephard et al., 1990; Sydenham et al.,
    1992). In most laboratories, these methods have reported limits of
    detection or limits of quantification ranging from 5 to 100 µg/kg. The
    OPA method is widely used and methodology using this derivative has
    been the subject of international collaborative trials (Thiel et al.,
    1993; Visconti et al., 1993; Sydenham et al., 1996). Particularly
    satisfactory results were achieved in the trial by Sydenham et al.
    (1996) with FB1 concentrations ranging from 0.5 to 8.0 mg/kg. 
    Relative standard deviations for within-laboratory repeatability
    ranged from 5.8% to 13.2% for FB1. Relative standard deviations for
    between-laboratory reproducibility were 13.9% to 22.2% for FB1.
    HORRAT ratios for 7 samples in the test varied from 0.75 to 1.73 for
    FB1 (Sydenham et al., 1996). Ratios of less than 2 are considered
    acceptable. This method has been adopted by the Association of
    Official Analytical Chemists International as an official method for
    the analysis of maize.

         There are no standardized methodologies for fumonisin analysis in
    different food products. A method for the extraction and analysis of
    FB1 in beer has been reported (Scott & Lawrence, 1994; Scott et al.,

         Hydrolysis of samples to the aminopentol chain followed by the GC
    analysis of the trimethylsilyl or trifluoroacetate derivative by flame
    ionization detection or mass spectrometry has been reported (Plattner
    et al., 1990, 1992; Plattner & Branham, 1994). Determination of

    hydrolysed FB1 in alkali-processed corn foods by HPLC with
    fluorescent derivatives has also been reported (Scott & Lawrence,

         Analyses of maize extracts with antibodies reactive with FB1
    (and FB2 plus FB3) by direct and indirect assays have been reported
    (Azcona-Olivera et al., 1992a,b; Usleber et al., 1994; Scott &
    Trucksess, 1997; Mullett et al., 1998). Detection limits using these
    methods have been reported to be 0.1-100 µg/litre. In one study, an
    ELISA method gave higher estimates of fumonisin concentrations
    compared to GC-MS and HPLC (Pestka et al., 1994).

         To a very limited extent, fumonisins have also been determined by
    capillary zone electrophoresis (CZE). In order to achieve resolution
    of the FB1 and FB2 analogues, samples were derivatized with either
    9-fluorenylmethyl chloroformate (Holcomb & Thompson, 1996) or
    fluorescein isothiocyanate (Maragos, 1995) prior to separation.

         As an analytical tool for the determination of fumonisins, MS was
    initially used as a detector after gas chromatographic separation of
    the hydrolysed fumonisins (Plattner et al., 1990). Although MS methods
    using fast-atom bombardment (Plattner & Branham, 1994) and particle
    beam interfaces (Young & Lafontaine, 1993) have been described, the
    application of the electrospray interface has led to the greatest
    advance in the use of MS for fumonisin determination. These methods
    rely on the LD separation of the underivatized fumonisins and
    detection of the different analogues as their protonated molecular
    ions (Doerge et al., 1994; Plattner, 1995; Lukacs et al., 1996;
    Churchwell et al., 1997). A combined on-line immunoaffinity capture,
    HPLC/MS method has also been described, and this permits analysis of
    non-derivatized fumonisins at sub µg/kg levels (Newkirk et al., 1998).


         FB1 was isolated in 1988 by Gelderblom et al. (1988). It was
    chemically characterized by Bezuidenhout et al. (1988), and shortly
    thereafter as "macrofusine" by Laurent et al. (1989a), from cultures
    of  Fusarium verticillioides (Sacc.) Nirenberg  (Fusarium 
     moniliforme Sheldon). A selection of FB1 occurrence data in maize
    and food products is given in Table 1 and Appendix 2. A worldwide
    survey of fumonisin contamination of maize and maize-based products
    was reported by Shephard et al. (1996a).

         FB1 is produced by isolates of  Fusarium verticillioides, 
     F. proliferatum, F. anthophilum, F. beomiforme, F. dlamini, 
     F. globosum, F. napiforme, F. nygamai, F. oxysporum, 
     F. polyphialidicum, F. subglutinans and  F. thapsinum isolated from
    Africa, the Americas, Oceania, Asia and Europe (Gelderblom et al.,
    1988; Ross et al., 1990; Thiel et al., 1991a; Nelson et al., 1991,
    1992; Chelkowski & Lew, 1992; Leslie et al., 1992, 1996; Rapior et
    al., 1993; Miller et al., 1993, 1995; Visconti & Doko, 1994;
    Desjardins et al., 1994; Abbas et al., 1995; Abbas & Ocamb, 1995;
    Logrieco et al., 1995; Klittich et al., 1997; Musser & Plattner, 1997;
    Sydenham et al., 1997). A species of  Alternaria (A. alternata f. sp.
     lycopersici) has also been demonstrated to synthesize B fumonisins
    (Abbas & Riley, 1996). Fumonisins can be produced by culturing strains
    of the  Fusarium species that produce these toxins on sterilized
    maize (Cawood et al., 1991), and yields of up to 17.9 g/kg have been
    obtained with  F. verticillioides strain MRC 826 (Alberts et al.,
    1990). Yields of 500-700 mg/litre for FB1 plus FB2 have been
    obtained in liquid fermentations and high recoveries of the toxins are
    possible (Miller et al., 1994). The most predominant toxin produced is
    FB1. FB1 frequently occurs together with FB2, which may comprise
    15-35% of FB1 (IARC, 1993; Diaz & Boermans, 1994; Visconti & Doko,

          Fusarium verticillioides and  F. proliferatum are amongst the
    most common fungi associated with maize. These fungi can be recovered
    from most maize kernels including those that appear healthy
    (Hesseltine et al., 1981; Bacon & Williamson, 1992; Pitt el al., 1993;
    Sanchis et al., 1995). The formation of fumonisins in maize in the
    field is positively correlated with the occurrence of these two fungal
    species, which are predominant during the late maturity stage (Chulze
    et al., 1996). These species can cause Fusarium kernel rot of maize,
    which is one of the most important ear diseases in hot maize-growing
    areas (King & Scott, 1981; Ochor et al., 1987; De León & Pandey, 1989)
    and is associated with warm, dry years and/or insect damage
    (Shurtleff, 1980).

        Table 1a  Worldwide occurrence of fumonisin B1 (FB1) in maize-based products

    Product                          Countries                                                 Detected / total     FB1 (mg/kg)

    North America
    Maize                            Canada, USA                                               324/729              0.08-37.9
    Maize flour, grits               Canada, USA                                               73/87                0.05-6.32
    Miscellaneous maize foodsb       USA                                                       66/162               0.004-1.21
    Maize feed                       USA                                                       586/684              0.1-330

    Latin America
    Maize                            Argentina, Uruguay, Brazil                                126/138              0.17-27.05
    Maize flour, alkali-treated 
      kernels, polenta               Peru, Venezuela, Uruguay                                  5/17                 0.07-0.66
    Miscellaneous maize foodsb       Uruguay, Texas-Mexico border                              63/77                0.15-0.31
    Maize feed                       Brazil, Uruguay                                           33/34                0.2-38.5

    Maize                            Austria, Croatia, Germany, Hungary, Italy, Poland,        248/714              0.007-250
                                     Portugal, Romania, Spain, United Kingdom

    Maize flour, maize grits, 
      polenta, semolina              Austria, Bulgaria, Czech Republic, France, Germany,       181/258              0.008-16
                                     Italy, Netherlands, Spain, Switzerland, United Kingdom

    Miscellaneous maize foodsb       Czech Republic, France, Germany, Italy, Netherlands,      167/437              0.008-6.10
                                     Spain, Sweden, Switzerland, United Kingdom

    Imported maize, grits and 
      flour                          Germany, Netherlands, Switzerland                         143/165              0.01-3.35

    Maize feed                       France, Italy, Spain, Switzerland, United Kingdom         271/344              0.02-70

    Table 1 (continued)

    Product                          Countries                                                 Detected / total     FB1 (mg/kg)

    Maize                            Benin, Kenya, Malawi, Mozambique, South Africa,           199/260              0.02-117.5
                                     Tanzania, Uganda, Zambia, Zimbabwe

    Maize flour, grits               Botswana, Egypt, Kenya, South Africa, Zambia,             73/90                0.05-3.63

    Miscellaneous maize foodsb       Botswana, South Africa                                    8/17                 0.03-0.35

    Maize feed                       South Africa                                              16/16                0.47-8.85

    Maize                            China, Indonesia, Nepal, Philippines, Thailand,           361/614              0.01-155

    Maize flour, grits, gluten       China, India, Japan, Thailand, Vietnam                    44/53                0.06-2.60

    Miscellaneous maize foodsb       Japan, Taiwan                                             52/199               0.07-2.39

    Maize feed                       Korea, Thailand                                           10/34                0.05-1.59

    Maize                            Australia                                                 67/70                0.3-40.6
    Maize flour                      New Zealand                                               0/12                 -

    a   This table is a summary of the information in Appendix 2
    b   Includes maize snacks, canned maize, frozen maize, extruded maize, bread, maize-extruded bread, biscuits, cereals, chips, 
        flakes, pastes, starch, sweet maize, infant foods, gruel, purée, noodles, popcorn, porridge, tortillas, tortilla chips, 
        masas, popped maize, soup, taco, tostada

         There is a strong relationship between insect damage and Fusarium
    kernel rot. A field survey demonstrated that the incidence of the
    European corn borer increased  F. verticillioides disease and
    fumonisin concentrations (Lew et al., 1991). Disease incidence was
    also shown to correlate to populations of thrips  (Frankliniella 
     occidentalis) (Farrar & Davis, 1991). Hybrids with a thin kernel
    pericarp were more susceptible to insect wounds, which allowed easier
    access to the fungus (Hoenisch & Davis, 1994). Hybrids with an
    increased propensity for kernel splitting had more disease (Odvody et
    al., 1990). Kernel splitting is worse under drought conditions. Ears
    infected by  F. graminearum may be predisposed to 
     F. verticillioides infection and fumonisin accumulation (Schaafsma
    et al., 1993). In maize ears inoculated one week after silk emergence
    with  F. verticillipodes fumonisins accumulated in the visibly
    damaged (mouldy) kernels (Pascale et al., 1997; Desjardins et al.
    1998). Sydenham et al. (1995) showed that in lightly contaminated
    kernels FB1 was concentrated in the pericarp of the maize kernel.

         A study of fumonisin occurrence in hybrids grown across the USA
    maize belt indicated that hybrids grown outside their range of
    adaptation had higher fumonisin concentrations (Shelby et al., 1994b),
    again suggesting the important role of temperature stress. Data from
    samples collected in Africa, Italy and Croatia also indicate fumonisin
    accumulation in lines grown outside their area of adaptation (Doko et
    al., 1995; Visconti, 1996). The occurrence of fumonisin in Ontario,
    Canada (a cool maize-growing region) was limited to drought-stressed
    fields (Miller et al., 1995).

         Significant fumonisin accumulation in maize occurs when weather
    conditions favour Fusarium kernel rot, and the severity of ear
    infection has been found to be a good indicator of fumonisin
    accumulation in maize ears artificially inoculated with
     F. verticillioides (Pascale et al., 1997). Since monitoring began in
    the USA, warm, dry years have greater concentrations than cooler years
    (Murphy et al., 1993). The direct influence of low moisture and dry
    weather on fumonisin accumulation could not be proven (Murphy et al.,
    1996; Pascale et al., 1997), although maize grown under normal
    conditions in cooler maize-growing areas is not significantly
    contaminated by fumonisin (Doko et al., 1995; Miller et al., 1995).

         Dry milling of maize results in the distribution of fumonisin
    into the bran, germ and flour (Bullerman & Tsai, 1994). Fumonisin may
    be present in beer where maize has been used as a wort additive (Scott
    et al., 1995). Little degradation of fumonisin occurs during
    fermentation and the fumonisins are found in the spent grain. No
    toxins can be detected in the distilled ethanol (Bothast et al., 1992;
    Scott et al., 1995; Bennett & Richard, 1996). Fumonisin is stable in
    polenta (Pascale et al., 1995), whereas it is hydrolysed, and the
    pericarp is removed, by nixtamalization, i.e. the treatment of
    maize-based foods with calcium hydroxide and heat (Hendrich et al.,
    1993). FB1 has been shown to form  N-(carboxymethyl)-FB1 when

    heated in the presence of reducing sugars (Howard et al. 1998), and
    the latter substance has been detected in raw corn (Howard et al.,

         FB1 is not significantly transferred into pork, chicken meat or
    eggs (Prelusky et al., 1994, 1996a; Vudathala et al., 1994), but a
    small amount accumulates in the liver and kidney of pigs as a function
    of exposure (Prelusky et al., 1996b; see also section 6.2). Fumonisin
    is not significantly transferred into milk from short-term dietary
    exposure (Scott et al., 1994; Prelusky et al., 1996a), and FB1 was
    found in only one of 165 samples of milk from Wisconsin, USA at a
    level close to 5 ng/ml (Maragos & Richard, 1994).


         Maize is the only commodity that contains significant amounts of
    fumonisins. It is consumed either directly or processed into products
    for human or animal consumption. Because fumonisins are known to be
    heat stable (Dupuy et al., 1993a; Howard et al., 1998), light stable
    (IARC, 1993), water soluble (US NTP, 1999), poorly absorbed, poorly
    metabolized and rapidly excreted by animals (see sections 6.1 to 6.5),
    most fumonisin will eventually end up being recycled into the
    environment in a manner that will concentrate its spatial
    distribution. The amount that enters the environment may be quite
    large. For example, in the USA, maize production exceeds 200 million
    tonnes per year. The concentration of FB1 and FB2 in field maize in
    the USA often exceeds 1 g/tonne of maize (Murphy et al., 1993 and
    Appendix 2). There is some evidence that fumonisins can be metabolized
    by some microorganisms (Duvick et al., 1994, 1998). However, little is
    known about the environmental fate of fumonisin after it is either
    excreted or processed.


         Table 1 summarizes the results of a number of surveys on the
    natural occurrence of FB1 in maize and maize-based foods and feeds
    (see Appendix 2 for more detail). The list is not exhaustive of the
    surveys carried out worldwide as there is continual production of
    similar data from every corner of the globe. Based on Table 1, 60% of
    the 5211 samples analysed have been found to be contaminated with
    FB1, the highest incidences of contamination being in Oceania (82% of
    82 samples) and Africa (77% of 383 samples), followed by Latin America
    (85% of 266 samples), North America (63% of 1662 samples), Europe (53%
    of 1918 samples) and Asia (52% of 900 samples).

         The data show that levels and incidence of contamination vary
    considerably in relation to the commodities tested and the source. The
    highest incidence was recorded in maize feeds (82% of 1112 samples),
    followed by ground maize products, such as flour, grits, polenta,
    semolina and gluten (73% of 517 samples), maize kernels (52% of 2525
    samples) and miscellaneous maize foods (40% of 892 samples).

         FB1 levels in animal feedstuffs can be exceptionally high, and
    reached maximum values of 330, 70, 38, 9 and 2 mg/kg in North America
    (USA), Europe (Italy), Latin America (Brazil), Africa (South Africa)
    and Asia (Thailand), respectively. The majority of the highly
    contaminated feeds were implicated in cases of equine
    leukoencephalomalacia, porcine pulmonary oedema and other

         In maize kernels available commercially or from experimental or
    breeding stations, FB1 has been detected in 96% (of 70 samples), 91%
    (of 138 samples), 76% (of 260 samples), 59% (of 614 samples), 44% (of
    729 samples) and 35% (of 714 samples) of samples from Oceania, Latin
    America, Africa, Asia, North America and Europe, respectively. Maximum
    FB1 levels were 40.6 mg/kg (Australia), 27 mg/kg (Argentina),
    117 mg/kg (South Africa), 155 mg/kg (China), 38 mg/kg (USA) and 250
    mg/kg (Italy).

         The list of commercial retail foods subject to fumonisin
    contamination (Table 1) includes maize flour, grits, polenta,
    semolina, maize snacks, cornflakes, sweet maize, canned maize, frozen
    maize, extruded maize, bread, maize-extruded bread, biscuits, cereals,
    chips, pastes, starch, infant foods, gruel, purée, noodles, popcorn,
    porridge, tortillas, tortilla chips, masas, popped maize, soup, taco
    and tostada.

         Of these samples, the global incidence of contamination in
    non-treated or minimally treated maize products (flour, grits,
    polenta, semolina) was 73% out of 517 samples analysed. The highest
    FB1 levels were recorded in Europe (16 mg/kg), followed by North
    America (6.3 mg/kg), Africa (3.6 mg/kg), Asia (2.6 mg/kg) and Latin
    America (0.7 mg/kg). In the remaining food products (892 samples) the
    incidence of contamination was 40%, the highest level (6.1 mg/kg FB1)

    being found in a sample of extruded maize from Italy. Generally
    processed maize foods have lower levels and incidence of contamination
    than non-treated maize. These differences might be the results of
    dilution of maize in food commodities, or may depend on the
    differences in maize cultivar or quality requirements for various

         Apart from maize and maize products, fumonisins have seldom been
    found in other food products, such as rice (Abbas et al., 1998),
    asparagus (Logrieco et al., 1998), beer (Torres et al., 1998) and
    sorghum (Shetty & Bhat, 1997). Surveys on other cereals, such as
    wheat, rye, barley and oats, did not show the occurrence of the toxin
    (Meister et al., 1996).

         Human exposure estimates have been made for fumonisins in several
    countries, including Switzerland, Canada, South Africa, USA and the
    Netherlands (Zoller et al., 1994; Contaminants Standards Monitoring
    and Programs Branch, 1996a,b; Gelderblom et al., 1996b; Kuiper-Goodman
    et al., 1996; Humphreys et al., 1997; Marasas, 1997; de Nijs, 1998).
    Human exposure estimates of 0.017-0.089 µg/kg body weight per day have
    been prepared for Canada for the period 1991 to early 1995
    (Kuiper-Goodman et al., 1996). For the USA, a preliminary estimate of
    human exposure to fumonisins for maize eaters was 0.08 µg/kg body
    weight per day (Humphreys et al., 1997). The mean daily intake of
    fumonisins in Switzerland is estimated to be 0.030 µg/kg body weight
    per day (Zoller et al., 1994).

         Based on the daily average intakes of maize and maize products of
    3 g (general population average), 42 g (regular maize product eaters)
    and 162 g (individuals with gluten intolerance) in the Netherlands,
    the respective population groups had an estimated daily intake of 4,
    57 and 220 µg FB1 per person, respectively, based on a mean FB1
    content of 1.36 mg/kg maize produce. De Nijs et al. (1998a) estimated
    conservatively that 97% of individuals with gluten intolerance had a
    daily exposure of at least 1 µg FB1 and 37% at least 100 µg, while
    the proportions of the general population exposed to these levels of
    FB1 were 49% and 1%, respectively (de Nijs, 1998; de Nijs et al.,

         Thiel et al. (1992) estimated that human exposures in the
    Transkei, South Africa, are 14 and 440 µg FB1/kg body weight per day
    for healthy and mouldy corn, respectively. More recent estimates of
    the probable daily intake (PDI) of South Africans are summarized in
    Table 2. These vary from 1.2 to 355 µg/kg body weight per day in rural
    blacks in Transkei consuming home-grown mouldy maize (Gelderblom et
    al., 1996b; Marasas, 1997).

         These exposure estimates will vary considerably according to the
    source and extent of maize in the diet as well as the extent of
    Fusarium kernel rot prevalent in the harvested crop.

        Table 2.  Probable daily intake of fumonisin in South Africaa

    Product              Country of          No. of       Mean FB1 + FB2           Probable daily intake
                         origin              samples      concentration            (µg/kg body weight per day)
                                                                              Rural population     Urban population

    Commercial maize     South Africa           68             400                   2.6                 1.6
    Commercial maize     South Africa          209             300                   2.0                 1.2
    Corn meal            South Africa           52             200                   1.3                 0.8
    Home-grown maize     South Africab          18            7100                  46.6                28.0
    Home-grown maize     South Africac          18          54 000                 354.9               212.9
    Imported maize       USAd                 1682            1100                   7.2                 4.3

    Maize consumption
     (g/70 kg body weight per day)                                                 460                 276

    a  From: Marasas (1997)
    b  Transkei, from individual farms in high oesophageal cancer area, healthy maize
    c  Transkei, from individual farms in high oesophageal cancer area, mouldy maize
    d  Imported in 1993

         Occupational inhalation exposure could be a problem. In addition
    to the presence of fumonisins in maize dust, FB1 is present in the
    spores and mycelia of  F. verticillioides (Tejada-Simon et al.,
    1995). No data have been collected on airborne levels of fumonisin
    during the harvesting, processing and handling of
    fumonisin-contaminated maize.


         There have been no reports on the kinetics and metabolism of
    fumonisins in humans. Because fumonisins are known to be consumed by
    farm animals and are the causative agent or a suspected contributing
    factor in farm animal diseases, an effort has been made to understand
    the kinetics and metabolism in cows, pigs and poultry. Thus, this
    chapter will summarize results of studies on both laboratory and farm

         To date, published studies with radiolabelled FB1 or FB2 have
    been conducted with either [21,22-14C]fumonisins, biosynthesized
    using L-[methyl-14C]methionine (Plattner & Shackelford, 1992; Alberts
    et al., 1993), or [U-14C]FB1 labelled using [1,2-14C]acetate
    (Blackwell et al., 1994). In these studies the final [14C]fumonisins
    had a specific activity of < 1 mCi/mmol and radiochemical purity of
    > 95%. Several studies have used unlabelled fumonisins with reported
    purities ranging from 70% (Hopmans et al., 1997) to 98% (Prelusky et
    al., 1996a).

         Briefly, FB1 is: poorly absorbed when dosed orally; it is
    rapidly eliminated from plasma or circulation and recovered in faeces;
    biliary excretion is important; enterohepatic cycling is clearly
    important in some animals; small amounts are excreted in urine; a
    small but persistent (and biologically active) pool of [14C]label
    appears to be retained in liver and kidney; and some is degraded to
    partially hydrolysed FB1 in the gut of vervet monkeys. In a study
    with FB2 in rats, the results were similar to those of FB1 (Shephard
    et al., 1995b).

    6.1  Absorption

         There are no reports available of fumonisin absorption through
    inhalation or dermal exposure. However, because fumonisins are present
    in  F. verticillioides cells (mycelia, spores and conidiophores)
    (Tejada-Simon et al., 1995), there is a potential for absorption
    through inhalation or buccal exposure. The risk from absorption due to
    dermal exposure would seem slight, since fumonisins are very water
    soluble and, typically, polar compounds do not easily penetrate the
    undamaged skin (Flynn, 1985).

         The quantity of FB1 detected in plasma after oral dosing in
    pigs, laying hens, vervet monkeys, dairy cows and rats is very low. In
    rats (BD IX, Sprague-Dawley or Wistar) administered [14C]FB1 orally,
    accumulation of 14C-labelled compounds in tissues is also very low,
    suggesting that absorption is very poor (negligible to < 4% of dose)
    (Shephard et al., 1992a,b, 1994c; Norred et al., 1993). Similar
    results indicating that fumonisins are poorly absorbed (2 to < 6% of
    dose) have been reported in vervet monkeys, dairy cows and pigs
    (Prelusky et al., 1994, 1995, 1996a,b; Shephard et al., 1994a,b). In
    orally dosed laying hens and dairy cows, systemic absorption based on
    plasma levels and accumulation of 14C-labelled compounds in tissues

    has been estimated to be less than 1% of dose (Scott et al., 1994;
    Vudathala et al., 1994; Prelusky et al., 1996a). A study using beef
    cattle fed  F. verticillioides culture material (corn grits)
    containing FB1 plus FB2 (530 mg/kg) found that the majority of the
    fumonisin dose was recovered unmetabolized in faeces, and only traces
    were detected in blood and urine (Smith & Thakur, 1996). Following
    single gavage doses of 1 or 5 mg/kg body weight to cows, no FB1 or
    known metabolites could be found in the plasma, indicating no or very
    limited bioavailability in ruminants (Prelusky et al., 1995). Rumen
    metabolism may reduce the bioavailability of FB1 as the hydrolysed
    form of FB1 comprised 60-90% of the total amount of FB1 found in
    faeces. In non-ruminants the parent compound was the dominant species
    present (Rice & Ross, 1994).

    6.2  Distribution

         In rats and pigs orally dosed with [14C]FB1, the 14C label is
    distributed to most tissues, with the liver and kidney containing the
    highest concentration of radiolabel (Shephard et. al., 1992b; Norred
    et al., 1993; Prelusky et al., 1994, 1996a,b; Haschek et al., 1996).
    Typically, the liver contains more 14C label than the kidney,
    although in the study by Norred et al. (1993) the measured
    radioactivity in the kidney was greater than in the liver. In chickens
    and dairy cows the poor absorption of [14C]FB1 (< 1% of oral dose)
    was reflected in the fact that only trace amounts of radioactivity
    were recovered in tissues (Prelusky et al., 1996a), no residues were
    recovered in eggs of laying hens (Vudathala et al., 1994) and no FB1
    or aminopentol hydrolysis products were recovered in milk (Scott et
    al., 1994; Prelusky et al., 1996a). In pregnant rats dosed
    intravenously with [14C]fumonisin, approximately 14.5% and 4% of the
    dose were recovered in the liver and kidney, respectively, after 1 h
    (Voss et al., 1996a). Based on the known pharmacokinetics (Norred et
    al., 1993) in the rat, 1-h exposure and intravenous injection were
    chosen so as to optimize the presentation in blood of the [14C]FB1
    to the placentae. In contrast to liver and kidney, the uteri contained
    0.24 to 0.44%, individual placentae contained 0 to 0.04%, and total
    fetal recovery was < 0.015% of dose/dam (Voss et al., 1996a).
    Recent studies have confirmed the lack of placental transfer of FB1
    in rats (Collins et al., 1998a,b) and rabbits (LaBorde et al., 1997).

         FB1 inhibition of the enzyme sphinganine  N-acyltransferase
    results in a large increase in intercellular free sphinganine (Wang et
    al., 1991; Yoo et al., 1992). In animal tissues the fumonisin-induced
    increase in free sphinganine tends to parallel the distribution of
    14C label reported in the studies cited above using [14C]FB1. For
    example, relative to other tissues examined, liver and kidney in
    rabbits, pigs and catfish showed the greatest increases in free
    sphinganine following exposure of animals to fumonisins or consumption
    of diets containing fumonisins (Goel et al., 1994; Gumprecht et al.,
    1995). The free sphinganine concentration in tissues has been shown to
    be an easily detectable biomarker for exposure to fumonisins (Riley et
    al., 1994c), although it has not been validated as a biomarker in

    6.3  Elimination, excretion and metabolic transformation

         When [14C]FB1 is dosed by intraperitoneal or intravenous
    injection in rats (BD IX, Sprague-Dawley or Wistar), initial
    elimination (subsequent to the distribution phase) is rapid (half-life
    of approximately 10-20 min) with little evidence of metabolism
    (Shephard et al., 1992a,b, 1994c; Norred et al., 1993). In rats the
    elimination kinetics based on intraperitoneal or intravenous dosing
    are consistent with a one- (Shephard et al., 1992b) or two-compartment
    model (Norred et al., 1993). Because FB1 is poorly absorbed from the
    rat gastrointestinal tract and extensively distributed in rat tissues
    (Norred et al., 1993), the tissue elimination kinetics following oral
    dosing is not as easily described. In vervet monkeys, as in rats, the
    14C label is widely distributed and rapidly eliminated (half-life of
    40 min) after intravenous injection (Shephard et al., 1994a,b). The
    elimination kinetics following oral dosing in a non-human primate has
    not been determined. Following single intravenous injection of 0.05 or
    0.20 mg FB1/kg body weight to cows, the toxin is cleared rapidly from
    the blood. A two-compartment model (half-lives of < 2 and 15-18 min,
    respectively) satisfactorily described the plasma kinetics. No toxin
    could be detected 120 min after dosing. No known metabolites were
    detected in the plasma (Prelusky et al., 1995).

         In pigs, clearance of [14C]FB1 from blood following an
    intravenous injection was best described by a 3-compartment model
    (half-lives of 2.5, 10.5 and 183 min, respectively), and cannulation
    of the bile duct (bile removed) resulted in a much more rapid
    clearance (best described by a 2-compartment model). The effect of
    bile removal was observed whether dosing was intravenous or
    intragastric (Prelusky et al., 1994, 1996a). The half-life in pigs
    dosed intragastrically without bile removal was determined to be 96
    min (Prelusky et al., 1996a). The studies with pigs strongly support
    the importance of enterohepatic circulation of FB1 in pigs. As in the
    study with rats, the majority of 14C label dosed orally was recovered
    in faeces (approximately 90%) with less than 1% recovered in urine
    (Prelusky et al., 1994, 1996a). In the LLC-PK1 renal cell line,
    uptake of [14C]FB1 reached an equilibrium concentration with the
    extracellular [14C]FB1 concentration after 4 to 16 h, and kinetics
    were indicative of a simple diffusion process (Riley & Yoo, 1995).
    Efflux was rapid with a half-life of less than 5 min.

         Following intravenous injection into rats, FB1 is excreted
    unchanged in bile (Norred et al., 1993; Shephard et al., 1994c). In
    vervet monkeys there is evidence of metabolism to partially hydrolysed
    (one propane tricarboxylic acid residue removed) FB1, and to a much
    lesser extent the fully hydrolysed (both propane tricarboxylic acid
    residues removed) aminopentol backbone, in faeces while in urine 96%
    of the 14C label was recovered as FB1 (Shephard et al., 1994a,b).
    Metabolism was most likely occurring in the gut since partially
    hydrolysed and fully hydrolysed FB1 were recovered in the faeces but
    not in the bile of vervet monkeys (Shephard et al., 1995a). Because
    hydrolysed FB1 and FB1-fructose adduct can be formed during
    processing, Hopmans et al. (1997) evaluated the excretion of these

    products and FB1 in Fischer-344 rats. Based on the amount of each
    FB1-related compound recovered in urine and faeces, it was concluded
    that hydrolysed FB1 and the FB1-fructose adduct were better absorbed
    than FB1 (Hopmans et al., 1997).

         Dairy cows dosed with pure FB1 either orally (1.0 and 5.0 mg
    FB1/kg body weight) or by intravenous injection (0.05 and 0.20 mg
    FB1/kg body weight) showed no detectable residues of FB1, AP1 (the
    aminopentol hydrolysis product of FB1) or their conjugates in the
    milk (Scott et al. 1994). FB1 does not react with monoamine or
    diamine oxidase (Murphy et al., 1996).  In vitro studies using rat
    primary hepatocytes and microsomal preparations (Cawood et al., 1994)
    or studies with the LLC-PK1 renal epithelial cell line (Riley & Yoo,
    1995) indicated that there was no metabolism of FB1 in these systems.

         Repeated intraperitoneal injection of FB1 resulted in induction
    of cytochrome P-4501A1 and P-4504A1 activities (Martinez-Larrañaga et
    al., 1996). However there is no evidence that fumonisin is metabolized
    by P-450 enzymes. Whether or not the induction was due to a direct
    interaction between fumonisins and the metabolizing systems could not
    be determined. However, it has been shown that some of the same
    sphingolipid metabolites that are altered in fumonisin-treated animals
    also mediate the cytokine-induced alterations in P-4502C11 in rat
    hepatocytes (Nikolova-Karakashian et al., 1997).

    6.4  Retention and turnover

         [14C]FB1 is widely distributed in tissues of the rat and pig.
    However, only the liver and kidney retain small but persistent amounts
    of 14C label based on measured radioactivity (Norred et al., 1993;
    Prelusky et al., 1994, 1996b). In rats given three repeated oral
    doses, once accumulated, the measured radioactivity in liver and
    kidney remained unchanged for at least 72 h after the last
    intragastric dose (Norred et al., 1993). In pigs, it was estimated
    that exposure to dietary FB1 at 2-3 mg/kg in feed would require a
    withdrawal period of at least 2 weeks for the 14C label to be
    eliminated from the liver and kidney (Prelusky et al., 1996b). The
    chemical nature of the 14C-labelled material retained in liver and
    kidney was primarily FB1.

          In vitro studies with rat primary hepatocytes and the cultured
    kidney cell line LLC-PK1 also indicate that a low but persistent pool
    of 14C-labelled material is retained inside cells long after the
    rapidly diffusible pool of [14C]fumonisin has exited the cells
    (Cawood et al., 1994; Riley et al., 1998). This retained pool appears
    to be capable of maintaining the elevation of cellular (LLC-PK1
    cells) and urinary (in rats) free sphingoid base concentration, a
    biomarker of fumonisin exposure (Solfrizzo et al. 1997b; Riley et al.,
    1998; Wang et al., 1999).

    6.5  Reaction with body components

         Fumonisins are potent inhibitors of the enzyme sphinganine
    (sphingosine)  N-acyltransferase in the  de novo sphingolipid
    biosynthesis and sphingolipid turnover pathways (Wang et al., 1991).
    The consequences of this reaction will be discussed in sections 7.8
    and 7.9. FB1 may also interact directly with protein kinase C (Yeung
    et al., 1996) and/or with mitogen-activated protein kinases
    (Wattenberg et al., 1996). The only other information concerning
    reaction with body components is that FB1 does not bind strongly to
    chicken plasma proteins (Vudathala et al., 1994).

         Cytotoxicity studies in primary rat hepatocytes and binding
    studies using subcellular fractions indicated that 14C-labelled FB1
    binds tightly to hepatocytes and microsomal and plasma membrane
    fractions (Cawood et al. 1994). FB1 has been shown to interact
    directly with liposomes (Yin et al., 1996). Since fumonisins are water
    soluble, are not accumulated and are rapidly eliminated, the
    toxicological significance of this finding is unclear.


    7.1  Laboratory animals and in vitro test systems

         The studies described below used either purified FB1, naturally
    contaminated corn or cultures of  Fusarium. It is generally accepted
    that the  in vivo toxicity of  Fusarium verticillioides MRC 826
    culture material is the result of its high FB1 content. Culture
    materials other than MRC 826 may contain several other products such
    as other fumonisins, fusarins, moniliformin and beauvericin.

    7.1.1  Single exposure

         In the male Sprague-Dawley rat, intravenous injection of FB1
    (95% purity) at 1.25 mg/kg body weight resulted in renal lesions
    localized to the tubules in the outer medulla and consisted of both
    proliferation and death of cells. An increased number of mitotic
    figures, stained with 5-bromo-2'-deoxyuridine (not quantified), and
    apoptosis followed by severe nephrosis were observed (Lim et al.,
    1996). Cell proliferation was also detected in the liver 24 h after
    dosing, but was not significantly different from control values at
    later times (day 2 to day 5). In the oesophagus, increased cell
    proliferation was measured on day 3, but this returned to the control
    level on day 5. While kidney lesions were reported as severe, the
    increased mitotic activity in the liver and oesophagus occurred in the
    absence of morphological injury (Lim et al., 1996).

         No information is available on the toxicological effects of
    single exposure to FB1 by the inhalation or dermal route.

    7.1.2  Repeated exposure  Body weight loss

         In male BD IX rats consuming a diet containing 1 g FB1/kg during
    a 4-week promotion treatment, the mean body weights were 50% lower
    than those of non-treated rats ( P < 0.0001), both with and without
    initiation with diethylnitrosamine (DEN) (Gelderblom et al., 1988).
    Similarly, the body weight gains of male Fischer rats fed the same
    concentration of FB1 over a 26-day initiation period were 80% lower
    than those of the controls ( P < 0.0025) (Gelderblom et al., 1992b).

         Male Fischer rats fed diets containing 1 g FB1 (and FB2 plus
    FB3) per kg over a 21-day initiating period started to lose weight
    within the first week, and the level of the FB compounds had to be
    reduced by half (Gelderblom et al., 1993). Body weight losses were
    first observed in rats fed FB2, where a significant ( P = 0.008)
    reduction compared to the controls was recorded after 4-5 days. In the
    case of FB1 and FB3, significant ( P = 0.01) reductions in body
    weight occurred after 7-8 days. Body weight loss induced by FB1 and
    FB2 was significantly ( P = 0.001) higher than that induced by FB3
    (Gelderblom et al., 1993). In female Sprague-Dawley rats administered

    purified FB1 at gavage doses of 0, 1, 5, 15, 35 or 75 mg FB1/kg body
    weight per day for 11 consecutive days, significant depression of body
    weight and food consumption was observed at 35 and 75 mg FB1/kg body
    weight per day (Bondy et al., 1998).

         The reduction in body weight gain of male Fischer rats induced by
    FB1 is apparently due to a feed refusal effect (Gelderblom et al.,
    1994). During a feeding study over 21 days, the body weight gains of
    rats receiving 750, 500, 250 and 100 mg FB1/kg diet were
    significantly (0.01 <  P < 0.05) lower than those of the controls
    as well as those of rats receiving 50 and 25 mg/kg. Based on the
    weekly feed intake profiles, the reduction in body weight gain was
    accompanied by a concomitant reduction in feed intake. The reduction
    in feed intake was overcome after the second week, resulting in a feed
    intake similar to that of the controls at the end of the 21-day
    initiating treatment (Gelderblom et al., 1994).

         In male Fischer-344/N Nctr BR rats, exposure to 234 and 484 mg
    FB1/kg diet resulted in 10% and 17%, respectively, less gain in body
    weight after 28 days of feeding in the range-finding study by the US
    National Toxicology Program (US NTP, 1999). Female rats had decreased
    body weight only at 484 mg FB1/kg diet.

         In the NTP 2-year carcinogenicity study (US NTP, 1999) (see
    section, there was no difference in body weight or feed
    consumption in male or female Fischer-344/N Nctr BR rats or
    B6C3F1/Nctr BR mice fed FB1 when compared to rats or mice on control

         The characteristic reduction in the body weight of rats induced
    by FB1, was also induced by FB2, FB3 and the monomethyl esters of
    FB1 (MME, an artefact of the isolation procedure of FB1 and a minor
    contaminant of FB1 preparations), and to a much lesser extent by the
     N-acetylated analogue FA1, but not by the aminopolyol hydrolysis
    products AP1 and AP2 or the tricarbalyllic acid moiety (TCA)
    (Gelderblom et al., 1993).  Hepatotoxicity and nephrotoxicity

         The acute toxicity of FB1 was tested by dosing four male BD IX
    rats orally with 240 mg FB1/kg body weight per day (Gelderblom et
    al., 1988). Three of the four rats died within 3 days and exhibited
    toxic hepatosis characterized by scattered single-cell necrosis
    accompanied by mild fatty changes, hydropic (i.e., the abnormal
    accumulation of serous fluid in the cellular tissue or in a body
    cavity) degeneration and hyaline droplet degeneration. Hepatocellular
    nuclei varied in size and some were markedly enlarged. In addition to
    the hepatotoxic changes, fatty changes and scant necrosis were present
    in the proximal convoluted tubules of the kidney, prominent lymphoid
    necrosis was observed in Peyer's patches, and severe disseminated
    acute myocardial necrosis and severe pulmonary oedema were observed in
    two of the rats (Gelderblom et al., 1988).

         In a separate experiment, male BD IX rats were dosed orally with
    48 mg FB1/kg body weight per day for 12 days, followed by 70 mg
    FB1/kg body weight per day for the remaining 9 days of the experiment
    (Gelderblom et al., 1988). In the rats killed after 21 days, chronic
    toxic hepatosis was present and characterized by marked hydropic
    degeneration, single-cell necrosis and a few hyaline droplets, early
    bile duct proliferation and fibrosis, and enlargement of
    hepatocellular nuclei (Gelderblom et al., 1988).

         In the livers of rats killed after 33 days on a diet containing
    1 g FB1/kg, the hepatic changes were similar to those described
    above, but more advanced (Gelderblom et al., 1988). The proliferation
    of bile ducts and fibrosis caused distortion of the lobular structure
    of the liver and, together with the development of hyperplastic
    nodules, gave the liver a distinctly nodular appearance. The authors
    reported that many nuclei were enlarged in hepatic cells and numerous
    mitotic figures, some of which were abnormal, were present. The
    lesions in the kidneys were similar, but less severe, than those seen
    in the rats that died within 3 days (Gelderblom et al., 1988).

         In male Fischer rats fed a diet containing 1 g FB1 (90-95% pure)
    per kg during an initiating period of 26 days, followed by partial
    hepatectomy and a promoting regimen of 2-acetyl-aminofluorene (2-AAF)
    and carbon tetrachloride, early pathological changes in the liver were
    very similar to those described above (Gelderblom et al., 1992b).
    Early hepatocyte nodules were evident as discrete focal changes in
    hepatocytes characterized by somewhat bigger cells that displayed more
    mitotic figures than the cells in the surrounding liver and also
    showed vacuolization. Another prominent pathological feature was the
    mild-to-moderate proliferation of bile ducts (Gelderblom et al.,
    1992b). Similar hepatic changes have been described in male Fischer
    rats fed diets containing, at a level of 0.5-1 g/kg, FB1, FB2, FB3
    and MME during an initiating period of 21 days followed by a promoting
    treatment of 2-AAF and partial hepatectomy (Gelderblom et al., 1993).
    The short-term toxicological effects in rats of FB2 and FB3 are
    similar to those of FB1 (Gelderblom et al., 1992a).

         Changes including hydropic swelling, hyaline droplet
    accumulation, single-cell necrosis, increased mitotic figures, lipid
    accumulation, fibrosis, and bile duct proliferation were also observed
    in the liver of male Fischer rats that died after gavage treatment
    with 50 mg FB1/kg body weight in 6 dosages over 11 days (Gelderblom
    et al., 1994).

         A 4-week exposure of Sprague-Dawley rats to aqueous extracts of
     Fusarium verticillioides (MRC 826) cultures (containing fumonisins)
    resulted in decreased body weights, increased serum alanine and
    aspartate aminotransferase and alkaline phosphatase activities,
    decreased relative liver weights and microscopic liver lesions in rats
    (Voss et al., 1990).

         Male and female Sprague-Dawley rats (3 of each sex per group)
    were fed diets containing 0, 15, 50 and 150 mg/kg of FB1 (> 99%
    pure) for 4 weeks (Voss et al., 1993). No significant differences in
    weight gain or food consumption were found, but significant increases
    in serum triglycerides, cholesterol and alkaline phosphatase confirmed
    that a dietary level of 150 mg/kg was hepatotoxic to both sexes.
    Histopathological changes in the liver of these rats were
    characterized by scattered single-cell hepatocellular necrosis,
    variability in nuclear size and staining and hepatocellular
    cytoplasmic vacuolation. Nephrosis, consisting of focal cortical
    proximal tubular epithelial basophilia, hyperplasia and single cell
    necrosis or pyknosis, was found in males fed > 15 mg/kg and in
    females fed > 50 mg/kg (Voss et al., 1993). The incidence and
    severity of ultrastructural alterations in kidney and liver were
    closely correlated with increased sphinganine concentration in
    tissues, serum and urine (Riley et al., 1994a).

         The apparent no-observed-effect level (NOEL) for renal toxicity
    in FB1-fed rats was less than the NOEL for hepatic effects (4.1
    < NOEL < 13.6 mg/kg diet for 28 days), and renal toxicity was more
    severe in males (NOEL < 1.4 mg/kg diet for 28 days) than females
    (1.4 < NOEL < 4.1 mg/kg diet for 28 days). Furthermore, liver
    lesions found in females appeared (subjectively) more advanced than
    those found in males. The results of subacute toxicity studies (7.5
    and 10 mg/kg body weight per day for 4 days) (Bondy et al., 1995;
    Suzuki et al., 1995) and of an independent (Tolleson et al., 1996a)
    4-week study in Fischer-344 rats fed 0, 99, 163, 234 or 484 mg FB1/kg
    diet corroborated the findings of nephrotoxicity by Voss et al.
    (1993). Hepatopathy of the same type was found in males fed > 234
    mg/kg diet and females fed > 163 mg/kg diet. Nephropathy was found
    in males from all FB1-fed groups and in females fed > 163 mg/kg
    diet (Tolleson et al., 1996a). Apoptotic hepatocytes and renal
    proximal tubule epithelial cells were accompanied by cell
    proliferation in Fischer-344 rats, suggesting that fumonisin induces
    or accelerates programmed cell death in both liver and kidney
    (Tolleson et al. 1996a; US NTP, 1999).

         In male and female B6C3F1 mice administered FB1 at gavage doses
    ranging from 1 to 75 mg FB1/kg body weight per day for 14 days,
    effects on liver, bone marrow, adrenals and kidneys were observed. In
    general, however, the degree of change observed indicates that mice
    are not as sensitive to FB1 toxicity as rats (Bondy et al., 1995,

         In B6C3F1 mice fed 99 to 484 mg FB1/kg diet for 4 weeks, the
    liver, not the kidney, was the target organ (US NTP, 1999). As for
    rats, the NOEL was lower in females as liver lesions were found in the
    females of all FB1-fed groups, while in males hepatopathy was
    confined to the highest dose group. In male BALB/c mice dosed
    subcutaneously (0.25 to 6.25 mg FB1/kg body weight per day), a
    dose-dependent increase in apoptosis was observed in both liver and
    kidney (Sharma et al., 1997).

         To obtain dose-response data under longer-term exposure
    conditions, Fischer-344 rats and B6C3F1 mice were fed diets
    containing 0, 1, 3, 9, 27 or 81 mg FB1/kg diet for 13 weeks (Voss et
    al., 1995). In rats, toxicity was confined to the kidneys. Lesions of
    the proximal tubule located in the outer medulla (sometimes referred
    to as the corticomedullary junction) were found in males fed > 9
    mg/kg diet and in females fed 81 mg/kg diet. Qualitatively these
    lesions were of the same type as those found in the 4-week study (Voss
    et al., 1993). No differences in the incidence or severity of
    nephropathy between rats examined after 4 (n = 5 rats/group) or 13
    (n = 10/group) weeks were found.

         Renal lesions were accompanied by decreased relative kidney
    weight (as a percentage of body weight), which was found in males fed
    > 27 mg/kg diet for 4 weeks and in both sexes fed > 9 mg/kg diet
    for 13 weeks. Serum creatinine was increased after 13, but not 4,
    weeks in males fed > 27 mg/kg diet and in females fed 81 mg/kg diet
    (Voss et al. 1995).

         In mice, hepatopathy and serum chemical evidence of liver
    dysfunction were found after 13 weeks in females fed 81 mg FB1/kg
    diet (Voss et al., 1995). Liver lesions in female mice were primarily
    centrilobular, although some midzonal involvement and apparent
    "bridging" between adjacent central areas were evident. Single cell
    hepatocyte necrosis, cytomegaly, increased numbers of mitotic figures,
    some mixed infiltration of neutrophils and macrophages were present
    and, in more advanced lesions, the loss of hepatocytes caused an
    apparent collapse around the central vein. Hepatopathy was not found
    in male mice and FB1-related kidney lesions did not occur in either
    sex. A few macrophages containing minimal to mild amounts of
    cytoplasmic pigment, presumably ceroid, were also found in the adrenal
    cortex of high-dose (81 mg/kg diet) females only.

         Taken together, the findings from 4-week and 90-day toxicity
    studies in rats and mice (Voss et al., 1993, 1995; Tolleson et al.,
    1996a; US NTP, 1999) indicate that the liver is a target organ in both
    species, and the data seem to indicate that females exhibit hepatic
    effects at lower doses than males. In rats, however, the kidney is
    also an important target organ and, in contrast to liver, the males
    were affected at lower doses.  Immunotoxicity

         There have been very few studies that address directly the
    potential for fumonisins to modify immune response  in vivo. 
    Nonetheless, there are many studies with fumonisins or
    fumonisin-containing diets that show either altered function of blood
    cells  in vitro or changes in haematological parameters  in vivo. 
    Fumonisins are inhibitors of ceramide synthase (see section 7.3) and
    ceramide and glycosphingolipids are important signalling molecules and
    recognition sites in the cellular immune response and attachment sites
    for many infectious agents and microbial toxins (Ballou et al., 1996;
    Merrill et al., 1997a).

         In a study with pure FB1, changes in selected haematological
    parameters in pigs were reported at dietary levels as low as 1 mg/kg
    (Rotter et al., 1996). Consumption of culture-material diets (MRC 826)
    containing fumonisins decreased the ability to clear  Pseudomonas 
     aeruginosa and inhibited pulmonary interstitial macrophage function
    (Smith et al., 1996c). It was hypothesized that pulmonary
    intravascular macrophage (PIM) dysfunction could contribute to
    increase susceptibility to microbial diseases (Smith et al., 1996c).

         Cytokine production has been shown to be modified by exposure to
    fumonisin. For example, serum tumour necrosis factor-alpha
    (TNF-alpha)-like activity was increased in pigs fed culture material
    (M 1325 = MRC 826) containing 150 mg/kg fumonisins (Guzman et al.,
    1997). Fumonisin-induced changes in the TNF pathway have also been
    seen in lipopolysaccharide (LPS)-stimulated macrophages collected from
    BALB/c mice dosed with pure FB1 (Dugyala et al., 1998).

         Immunosuppression in chickens was produced in birds fed maize
    cultured with  F. verticillioides (MRC 826) (Marijanovic et al.,
    1991). Broiler chicks fed diets containing 10 mg pure FB1/kg diet, or
    diets formulated from  Fusarium verticillioides (MRC 826) culture
    material to contain 30 to 300 mg FB1/kg diet, had reduced spleen
    and/or bursa weights and altered haematological parameters (Espada et
    al., 1994, 1997).

         In male and female rats (10 rats/group) gavaged daily for 14 days
    with doses of 0, 5, 15 or 25 mg FB1/kg body weight per day, a
    significant dose-related linear trend toward decreased plaque-forming
    cell number per 106 spleen mononuclear leukocytes (PFC per 106
    splenocytes) ( P = 0.003) and PFC per spleen cells ( P = 0.001) was
    observed in the male rats. However, the PFC numbers in female rats
    were not affected significantly by treatment ( P > 0.05) (Tryphonas
    et al., 1997).

    7.1.3  Skin and eye irritation

         No information is available on the effects of FB1 on skin and
    eye irritation and/or sensitization.

    7.1.4  Reproductive toxicity, embryotoxicity and teratogenicity

         Concern about the reproductive and developmental effects of
    fumonisins originated with: (a) the observation of abortions in
    pregnant sows fed fumonisin-contaminated diets (Harrison et al.,
    1990); (b) the suggestion that a cluster of birth defects among
    residents in Brownsville, Texas, USA (Hendricks, 1999) might be
    associated with consumption of maize from the 1989 maize crop; (c) the
    association of "mystery swine disease" with fumonisin-contaminated
    maize (Bane et al., 1992); and (d) the discovery that fumonisins are
    inhibitors of sphingolipid biosynthesis (Wang et al., 1991). Currently
    there are no data to support the conclusion that consumption of
    fumonisins is a developmental or reproductive toxicant in farm animals

    or humans. There are also no data demonstrating that fumonisin
    consumption results in transfer to chicken eggs (Vudathala et al.,
    1994; Prelusky et al., 1996a) or that it crosses the placenta in rats
    (Voss et al., 1996a; Collins et al., 1998a,b), mice (Reddy et al.,
    1996) or rabbits (LaBorde et al., 1997).

         Injection of purified FB1 into fertile chicken eggs resulted in
    time- and dose-dependent embryopathic and embryocidal effects (Javed
    et al., 1993b). Embryonic changes included hydrocephalus, enlarged
    beaks and elongated necks. Pathological changes were noted in most
    organ systems. At the low FB1 dose (1 µM = 0.72 µg/ml), stimulation
    of chick embryo development was observed. Stimulated embryo
    development  in vitro in pre-somite rat embryos exposed to
    0.5-1 µg/ml of hydrolysed FB1 has been reported in an abstract (Flynn
    et al., 1994). Higher concentrations of fully hydrolysed FB1 (Flynn
    et al., 1997) and all concentrations of FB1 > 0.2 µg/ml inhibited
    growth and development of pre-somite rat embryos  in vitro (Flynn et
    al., 1994, 1996). Johnson et al. (1993) reported that FB1 was a weak
    developmental toxin to organogenesis stage rat embryos (day 10.5;
    lowest-observed-effect level = 0.5 mM). FB1 (> 2.5 mM = 1.8 µg/ml)
    inhibited reaggregation and growth of chicken embryo neural retina
    cells, a commonly used  in vitro assay for screening potential
    developmental toxins (Bradlaw et al., 1994). Bacon et al. (1995) found
    effects of FB1 in fertile chicken eggs similar to those reported by
    Javed et al. (1993b). In addition it was found that co-injection of
    fusaric acid and FB1 resulted in a synergistic toxic response (Bacon
    et al., 1995). Zacharias et al. (1996) found that morphological
    changes, due to direct administration of FB1 to chick embryos, were
    correlated with inhibition of glycosphingolipid biosynthesis.

         Syrian hamsters orally gavaged with aqueous extracts of
     F. verticillioides (M 1325 = MRC 826) culture material containing
    fumonisins (0.25-18 mg FB1/kg body weight) or pure FB1 (12 mg/kg and
    18 mg/kg) did not exhibit maternal toxicity based on weight gain,
    serum aspartate aminotransferase activity or total bilirubin.
    Histological examination of liver, kidney and placenta did not reveal
    important changes, although mild karyomegalic changes in liver were
    observed in the hamsters dosed with either aqueous extracts or pure
    FB1 at > 6 mg FB1/kg body weight (Floss et al., 1994a,b). When
    aqueous extracts were given by oral gavage from day 8 to day 10 or 12
    of gestation, there appeared to be an increase in the number of fetal
    deaths, but statistical significance was not achieved (Floss et al.,
    1994a). Relative to controls, statistically significant increases in
    fetal deaths occurred only in the hamsters given 18 mg FB1/kg body
    weight (aqueous culture extracts and pure material) (Floss et al.,
    1994b). Prenatal exposure to aqueous culture extracts containing
    fumonisins or to pure FB1 were detrimental to fetal hamster
    survivability in the absence of maternal toxicity (Floss et al.,
    1994a,b; Penner et al. 1998).

         In Fischer-344/N rats dosed orally from day 8 to 12 of gestation
    with 30 or 60 mg purified FB1/kg body weight, the high dose
    significantly suppressed growth and fetal bone development while an
    extract of  F. proliferatum (M 5991) in corn culture did not
    (Lebepe-Mazur et al., 1995a). Voss et al. (1996a) formulated diets
    using  F. verticillioides (MRC 826) culture material to provide 0, 1,
    10 or 55 mg FB1/kg diet. Based on consumption, the diet containing
    55 mg/kg provided about 3 to 4 mg FB1/kg body weight per day to the
    dams. The diets were fed to male and female Sprague-Dawley rats prior
    to and during the mating, gestational and lactational phases of the
    study. Nephropathy was observed in males and females fed diets
    containing > 10 mg/kg and 55 mg/kg, respectively. No statistically
    significant reproductive effects were observed in any of the males or
    females, and no developmental effects were found in fetuses during any
    phase of the study. Litter weight gains in the 10 and 55 mg/kg diet
    groups were slightly decreased. Increased levels of free sphinganine,
    a biomarker for fumonisin exposure, were demonstrated in the livers of
    dams in the 55 mg/kg diet group on gestation day 15. In contrast, no
    increase in the sphinganine/sphingosine (Sa/So) ratio was observed in
    fetuses at that time, suggesting that fetuses were not exposed
     in utero to FB1. This finding was supported by the study in which
    an intravenous injection of [14C]FB1 was given to dams on gestation
    day 15. Radiolabel was easily detected in tissues of pregnant females
    but was not detected in their fetuses. Culture material containing
    fumonisins, and by inference FB1, did not have reproductive effects
    at doses that were minimally toxic (Voss et al., 1996a). These
    findings have been recently confirmed (Sa/So ratios in fetuses were
    not affected and FB1 was not teratogenic at the doses tested) in a
    Charles River CD rats (Collins et al., 1998a,b).

         Gross et al. (1994) gavaged pregnant CD1 mice daily between
    gestation days 7 and 15 with a diet containing partially purified FB1
    extracted from  F. verticillioides (M 1325 = MRC 826) culture
    material. Maternal toxicity and fetal developmental abnormalities
    (e.g., hydrocephalus, digital and sternal ossification) occurred at
    FB1 dosages greater than 12.5 mg/kg body weight per day. Similar
    results were obtained in a second study using purified FB1 (Reddy et
    al., 1996). As in the study by Voss et al. (1996b), the Sa/So ratio
    was significantly increased in maternal liver but not in fetal liver,
    suggesting that developmental effects were mediated through maternal
    toxicity (Reddy et al., 1996).

         Unlike CD1 mice and Syrian hamsters, pregnant New Zealand white
    rabbits are very sensitive to the toxic effects of FB1 (LaBorde et
    al., 1997). Maternal toxicity was observed at daily gavage dosages (in
    water) as low as 0.25 mg/kg body weight from gestational day 3 to
    gestational day 19. Compared to controls there was no increase in
    fetal loss or in gross visceral or skeletal abnormalities, and no
    decrease in fetal weight or fetal organ weight at any dosage (0 to
    1.75 mg/kg body weight) (LaBorde et al., 1997). The maternal kidney,
    serum and urine Sa/So ratios were increased, but there were no
    increases in these ratios in fetal liver, brain or kidney (LaBorde et

    al., 1997). While FB1 is toxic in the pregnant dam, it is not a
    developmental toxin but is maternally toxic in rabbits (LaBorde et
    al., 1997). However, the lowest-observed-effect level for maternal
    toxicity was 0.1 mg FB1/kg body weight, which is equivalent to a
    calculated dietary fumonisin level of 2.3 mg/kg diet (LaBorde et al.,
    1997). Thus, in sensitive species, maternal toxicity and consequent
    fetal toxicity could occur at low dosages of FB1.

         There is currently no evidence of neonatal toxicity. However,
    average mean litter weights were reduced in litters from
    Sprague-Dawley dams fed  F. verticillioides (MRC 826) culture
    material containing 10 or 55 mg FB1/kg (Voss et al., 1996a). The
    Sa/So ratio was increased in litters at lactation day 21. However,
    given the likelihood that offspring had consumed the contaminated
    diets (Voss et al., 1996a), the authors could not ascertain the route
    of exposure (via milk or diet). Reduced weights and several
    alterations in haematological parameters were reported in mink kits
    lactationally exposed to fumonisins (Powell et al., 1996).

         No FB1 was detected in the milk of lactating sows fed diets
    containing non-lethal levels of FB1 and there was no evidence of
    toxicosis in their suckling pigs (Becker et al., 1995). However, in a
    study with lactating cows administered FB1 intravenously, the
    carry-over rate of FB1 into the milk reached a maximum of 0.11%
    (Hammer et al., 1996), while in other studies no fumonisins were
    detected in cow's milk (Scott et al., 1994; Richard et al., 1996). In
    a reproductive study with mink, fumonisins were detected in the milk
    at 0.7% of the dietary fumonisin concentrations (Powell et al., 1996).

         The question of neonatal toxicity is of concern since neonates
    may be more sensitive to fumonisins than adults. For example, a recent
    report by Kwon et al. (1997b) indicated that subcutaneous injection of
    FB1 in neonatal rats caused elevation in the Sa/So ratio in brain
    tissue and reduced myelin deposition. The elevated sphinganine level
    was determined to be the result of a direct effect on the neonate
    brain, indicating that FB1 can cross the blood-brain barrier (Kwon et
    al., 1997a). When maternal toxicity was minimal, there was little or
    no evidence of neonatal toxicity in rats (Ferguson et al., 1997).

    7.1.5  Mutagenicity and related end-points

         The fumonisins FB1, FB2 and FB3 (98, 98, 90% pure,
    respectively) were non-mutagenic in the  Salmonella assay against the
    tester strains TA97a, TA98, TA100 and TA102, in both the presence and
    absence of the S-9 microsomal preparation (Gelderblom & Snyman, 1991).
    The non-mutagenicity of FB1 (approximately 90% pure) to  Salmonella 
    tester strain TA100 at concentrations up to 100 mg/plate was confirmed
    by Park et al. (1992). Similarly negative results were reported with
    FB1 in  Salmonella TA98 and TA100, as well as in SOS chromotest in
     E. coli PQ37 and differential DNA repair assays with  E. coli K12
    strains (343/753,  uvrB/ recA and 343/765,  uvr+ rec+)
    (Knasmüller et al., 1997). In contrast, Sun & Stahr (1993), using a

    commercial bioluminescent bacterial  (Vibrio fischeri) genotoxicity
    test, reported that FB1 showed in the concentration range 5-20 µg/ml
    genotoxic activity without the metabolic activation of S-9 fraction.

         FB1 (and FB2) were non-genotoxic in the  in vitro rat
    hepatocyte DNA repair assay at concentrations ranging from 0.04 to 80
    µM (and FB2 from 0.04 to 40 µM) as well as in the  in vivo assay at
    a dose of 100 mg/kg body weight administered by gavage (FB1 or FB2)
    (Gelderblom et al., 1989, 1992b). The finding that FB1 does not
    induce unscheduled DNA synthesis was confirmed in the  in vitro assay
    in primary rat hepatocytes at concentrations ranging from 0.5 to 250
    µM (Norred et al., 1992a).

         FB1 induced DNA strand breaks in isolated rat liver nuclei (Sahu
    et al., 1998).

         FB1 induced a moderate increase in the micronucleus frequency in
    primary rat hepatocytes at concentrations ranging from 0.01 to
    1 µg/ml. No concentration-dependent increase of micronuclei occurred.
    A significant concentration-dependent increase in chromosomal
    aberrations was observed in isolated hepatocytes exposed to FB1 at
    concentrations ranging from 1 to 100 µg/ml (Knasmüller et al., 1997).

         FB1 induced lipid peroxidation in isolated rat liver nuclei at
    concentrations ranging from 40 to 300 µM (Sahu et al., 1998). It also
    increased significantly the level of thiobarbituric-acid-reactive
    substances  in vitro in primary rat hepatocytes at concentrations of
    75 and 150 µM and  in vivo in the liver of rats fed a dietary FB1
    level of 250 or 500 mg/kg for 21 days (Abel & Gelderblom, 1998).

         Single gavage doses of 50, 100 and 200 mg FB1/kg body weight
    significantly ( P = 0.05) inhibited hepatocyte proliferation as
    measured by the incorporation of radiolabelled thymidine into DNA in
    partially hepatectomized male Fischer rats (Gelderblom et al., 1994).
    Inhibition of hepatocyte proliferation was also observed after dietary
    exposure to FB1 (> 50 mg/kg diet) (Gelderblom et al., 1996c). FB1
    also inhibited DNA synthesis induced by epidermal growth factor in
    primary rat hepatocytes (Gelderblom et al., 1995).

         In BALB/3T3 A31-1-1 mouse embryo cells, FB1 (90% pure) treatment
    produced transforming activity at 500 µg/ml but not at lower or higher
    concentrations (Sheu et al., 1996).

    7.1.6  Carcinogenicity  Carcinogenicity bioassays

         When inbred BD IX rats were fed commercial diet containing
    freeze-dried or oven-dried culture material inoculated with
     F. verticillioides MRC 826 for 2 years, the incidence of liver
    tumours (hepatocellular and cholangiocellular carcinomas combined) was
    increased (control: 0/20, freeze-dried 13/20, oven-dried, 16/20)

    (Marasas et al, 1984b). When 30 rats of the same inbred strain were
    given  F. verticillioides MR 826 (containing fusarin C and later
    found to produce FB1 and FB2) for 23-27 months, two hepatocellular
    and eight cholangiocellular cancers were observed. In addition,
    neoplastic hepatic nodules were observed in all surviving 21 animals,
    but none among the controls. However, in rats similarly administered
     F. verticillioides MRC 1069 culture material containing 104 mg/kg
    fusarin C (but suspected of being low in FB1), no increase in hepatic
    carcinomas was observed (Jaskiewicz et al., 1987b).

         Maize from a field outbreak of equine leukoencephalomalacia in
    the USA, shown to be naturally contaminated with
     F. erticillioides, was fed to 12 male Fischer-344 rats and
    commercial rodent feed to 12 controls by Wilson et al. (1985). All
    treated rats necropsied from 123 to 176 days had multiple hepatic
    neoplastic nodules, adenofibrosis and cholangiocarcinoma, whereas no
    such lesions were found in the controls. The authors considered these
    lesions in the livers of male Fischer-344 rats to be similar to those
    described in male BD IX rats by Marasas et al. (1984b). The fact that
    the lesions observed by Wilson et al. (1985) developed more rapidly
    (as early as 123 days) than those described by Marasas et al. (1984b)
    (more than 450 days) was attributed by Wilson et al. (1985) to the
    dietary deficiencies, which included choline and methionine, in the
    maize-only diet used in their study.

         Hendrich et al. (1993) reported that in Fischer-344/N rats fed
    diets containing  Fusarium proliferatum maize culture material (with
    known amounts of FB1) there was an increased incidence of
    hepatocellular adenomas, relative to rats fed the control diets. When
    rats were fed nixtamalized  Fusarium proliferatum (M 5991) maize
    culture material diets (converting FB1 to hydrolysed FB1) the
    incidence of hepatic adenomas and cholangiomas was reduced relative to
    rats fed the diets containing FB1. The frequency of
    hepatic/cholangiocellular adenomas in rats given the nixtamalized diet
    was higher in rats receiving nutrient-supplemented diet (equivalent to
    AIN-76) than in rats given a diet not supplemented with nutrients
    (nutritionally deficient relative to AIN-76). In a 4-week feeding
    study with male Sprague-Dawley rats, Voss et al. (1996c) found that
    hydrolysed FB1 (58 mg/kg diet) from nixtamalized
     F. verticillioides (MRC 826) was both hepatotoxic and nephrotoxic.
    However, the extent and severity of the hepatotoxicity was
    significantly less than that caused by FB1 (71 mg/kg diet) from
     F. verticillioides (MRC 826), whereas the kidney toxicity was
    similar (Voss et al., 1996c).

         In male BD IV rats treated with the known oesophageal carcinogen
     N-methylbenzylnitrosamine (NMBA) (2.5 mg/kg body weight) and FB1
    (5 mg/kg body weight), there was no synergistic interaction between
    NMBA and FB1 in the rat oesophagus when the two compounds were
    administered together (Wild et al., 1997).

         A semi-purified maize-based diet containing FB1 (not less than
    90% pure) at 50 mg/kg diet was fed to 25 inbred male BD IX rats over a
    period of 26 months (Gelderblom et al., 1991). A control group
    received the same diet without FB1 (the FB1 content of the control
    diet was approximately 0.5 mg/kg and no aflatoxin B1 could be
    detected). Five rats from each group were killed at 6, 12, 20 and 26
    months. All FB1-treated rats (50 mg/kg diet) that died or were killed
    from 18 months onward suffered from a micro- and macronodular
    cirrhosis and had large expansile nodules of cholangiofibrosis at the
    hilus of the liver. The pathological changes terminating in cirrhosis
    and cholangiofibrosis were already present in the liver of rats killed
    6 months after the initiation of the experiment and included fibrosis,
    bile duct hyperplasia and lobular distortion. The severity of the
    hepatic lesions increased with time and the histological changes were
    consistent with those of a chronic toxic hepatosis progressing to
    cirrhosis. Ten out of 15 FB1-treated (50 mg/kg diet) rats (66%) --
    but none in the controls -- that were killed or died between 18 and 26
    months developed primary hepatocellular carcinoma. Metastases to the
    heart, lungs or kidneys were present in four of the rats with
    hepatocellular carcinoma. Apart from the hepatocellular carcinoma,
    FB1 also induced cholangiofibrosis consistently from 6 months onward,
    and toward the end of the experiment, cholangiocarcinoma. However, the
    authors noted that the experiment was performed under nutritionally
    compromised conditions, using diets deficient in vitamins, methionine
    and choline, that may have had an enhancing effect on the action of
    FB1 in the liver (Table 3).

         The detailed results of a second long-term experiment in rats fed
    diets containing 0, 1, 10 and 25 mg FB1/kg diet over a period of
    24 months have not yet been published (Gelderblom et al., 1996b).
    However, in preliminary reports, Gelderblom et al. (1996b, 1997) noted
    that no cancers were observed in these rats, including those fed 25 mg
    FB1/kg diet.

         Male and female Fischer-344/N Nctr BR rats and B6C3F1/Nctr BR
    mice were given diets containing FB1 for 2 years as part of the US
    National Toxicology Program (NTP) tumorigenesis studies on FB1
    (US NTP, 1999). The FB1 that was used in these studies was > 96%
    pure. Dietary levels of FB1 were 0, 5, 15, 50 and 150 mg/kg diet for
    the male rats, resulting in average daily FB1 doses of 0, 0.26, 0.76, 2.5 
    and 7.5 mg/kg body weight. The dietary levels for female rats were 0, 5, 15, 50
    and 100 mg/kg diet, resulting in average daily FB1 doses of 0.31, 0.91, 3.0 
    and 6.1 mg/kg body weight.

         There was no difference in body weight, survival or feed
    consumption in male rats fed FB1 when compared to rats on control
    diets. The only compound-related change in tumour incidence was the
    induction of renal adenomas and carcinomas in male rats. The overall
    incidence of renal tubule tumours in male rats receiving 0, 5, 15, 50
    and 150 mg FB1/kg diet were 0/48, 0/40, 0/48, 2/48 and 5/48 for
    adenomas and 0/48, 0/40, 0/48, 7/48 and 10/48 for carcinomas. Renal
    tubule adenomas were characterized as an expansive proliferation of

        Table 3.  Summary of the induction of neoplasia in long-term feeding studiesa

    Neoplasia                    Species and strain     Sex               Fumonisin concentration (mg/kg feed)              Reference
                                                                  0       5       15      50      80      100     150

    Hepatocellular carcinoma     BD IX rats             male      0/15                    10/15                             Gelderblom
    Cholangiofibrosisb           BD IX rats             male      0/15                    15/15                             et al. (1991)

    Renal tubule adenoma         F-344/N Nctr rats      male      0/48    0/40    0/48    2/48                    5/48      US NTP (1999)
    Renal tubule carcinoma       F-344/N Nctr rats      male      0/48    0/40    0/48    7/48                    10/48     US NTP (1999)
    Renal tubule adenoma         F-344/N Nctr rats      female    0/48    0/40    1/48    0/48            0/48              US NTP (1999)
    Renal tubule carcinoma       F-344/N Nctr rats      female    0/48    0/40    0/48    0/48            1/48              US NTP (1999)

    Hepatocellular adenoma       B6C3F1/Nctr mice       female    5/47    3/48    1/48    16/47   31/45                     US NTP (1999)
    Hepatocellular carcinoma     B6C3F1/Nctr mice       female    0/47    0/48    0/48    10/47   9/45                      US NTP (1999)
    Hepatocellular adenoma       B6C3F1/Nctr mice       male      9/47    7/47    7/48            6/48            8/48      US NTP (1999)
    Hepatocellular carcinoma     B6C3F1/Nctr mice       male      4/47    3/47    4/48            3/48            2/48      US NTP (1999)

    a  This summarizes the data for tissues where fumonisin dose-dependent induction of tumours was detected (US NTP, 1999)
    b  In Gelderblom et al. (1991), cholangiofibrosis was considered to have progressed to cholangiocarcinoma

    renal tubule epithelial cells that tended to be separated into lobules
    by a delicate fibrous stroma. The neoplastic cells had nuclei that
    were slightly larger with increased cytoplasmic volume. The
    cytoplasmic changes were uniform within individual lesions and varied
    from clear to basophilic. Renal tubule carcinomas were characterized
    by cellular atypia, necrosis within a lesion, invasion of the adjacent
    normal renal parenchyma, or metastasis to distant organs.
    Historically, renal tubule carcinomas have not occurred in
    Fischer-344/N Nctr male rats on control diets. There was no apparent
    involvement of alpha-2 microglobulin in the tumorigenicity in male rat
    kidneys. The incidence in renal tumours was accompanied by an
    increased incidence in renal tubule epithelial cell hyperplasia at 50
    and 150 mg FB1/kg diet at 2 years (2/48, 1/40, 4/48, 14/48 and 8/48
    of the male rats receiving 0, 5, 15, 50 and 150 mg FB1/kg diet).
    Similarly, increased renal tubule epithelial cell apoptosis and
    proliferation were detected at 50 and 150 mg FB1/kg diet in male rats
    sacrificed following 6, 10, 14 and 26 weeks on FB1-containing diets.
    As in the 28-day range-finding studies, the apoptosis was confined to
    tubules of the inner cortex and was characterized by cellular
    shrinkage from adjacent cells, cytoplasmic eosinophilia, and chromatin
    condensation and margination in the nucleus. Apoptotic cells were
    additionally detected using an  in situ method for detection of DNA
    fragmentation. In serum removed from male rats killed at 6, 10, 14 or
    26 weeks, no FB1-dependent changes were noted (e.g., in cholesterol,
    triglycerides or serum alanine aminotransferase levels). The urinary
    Sa/So ratio was increased in the urine of male rats fed 5, 15, 50 or
    150 mg FB1/kg diet, while kidney tissue Sa/So ratios were increased
    at 15, 50 or 150 mg FB1/kg diet. Increased tissue sphingoid base
    changes, and renal tubule tumour incidence and hyperplasia were
    detected at 50 and 150 mg FB1/kg diet, while urinary sphingolipid
    changes were detected at 15, 50 or 150 mg FB1/kg diet. In the livers
    of the male rats, an increase in basophilic foci was detected at 150
    mg FB1/kg diet, but liver tissue Sa/So ratios were not affected.

         FB1 at 5, 15, 50 and 100 mg/kg diet did not affect body weight,
    survival, feed consumption, serum analytes or tumour incidence in the
    female rats. Significant increases in urinary and kidney tissue Sa/So
    ratios were detected at 50 and 100 mg FB1/kg diet, but the extent of
    induction was not as high as in the male rats.

         In the female mice, dietary levels of FB1 were 0, 5, 15, 50 and
    80 mg/kg diet, resulting in average daily FB1 consumption of 0.7,
    2.1, 7.0 and 12.5 mg/kg body weight, respectively. In the male mice,
    dose levels of 0, 5, 15, 80 and 150 mg FB1/kg diet resulted in
    average daily FB1 doses of 0, 0.6, 1.7, 9.5 and 17 mg/kg body weight,

         In female B6C3F1/Nctr mice, there were essentially no
    differences in the mean body weights and diet consumption. The body
    weights of the mice on this study were less than those reported for
    the NTP and control studies at the National Center for Toxicological
    Research (NCTR). This was attributed to an unintended restriction of

    the powdered feed in the individual feeders. The female mice consuming
    diets containing 80 mg FB1/kg diet had a significantly reduced
    survival compared to the female mice on control diets.

         The only tissue that demonstrated FB1-dependent changes in
    tumour incidence was the liver in the female mice. Hepatocellular
    adenomas were present in 5/47, 3/48, 1/48, 16/47 and 31/45 female mice
    and hepatocellular carcinomas were present in 0/47, 0/48, 0/48, 10/47
    and 9/45 female mice consuming 0, 5, 15, 50 and 80 mg FB1/kg diet,
    respectively. Hepatocellular adenomas were characterized as discrete
    lesions with compression of adjacent normal tissues. The normal
    hepatic lobular structure was absent with uneven growth patterns. The
    cells in the adenoma appeared to be well differentiated and either
    eosinophilic, basophilic or vacuolated. Hepatocellular carcinomas were
    characterized as foci of cells with distinct trabecular or adenoid
    structure. Histological evidence of local invasiveness or metastasis
    was usually evident. The cells within the carcinoma were poorly
    differentiated or anaplastic. Some of the carcinomas appeared to arise
    within adenomas. The incidence of hepatocellular adenomas and
    carcinomas in the female mice was within the range of historical
    occurrence in B6C3F1/Nctr mice. The increased tumour incidence was
    accompanied by increased hepatocellular hypertrophy (0/47, 0/48, 0/48,
    27/47, 31/45) and hepatocellular apoptosis (0/47, 0/48, 0/48, 7/47,
    14/45) in the female mice. The mean liver weights (relative to body
    weight) of the mice after 2 years were increased at 50 and 80 mg
    FB1/kg diet. There were no consistent increases in serum analytes in
    mice killed at 3, 7, 9 and 24 weeks. Liver sphingoid bases were
    increased at 80 mg FB1/kg diet at weeks 3, 7 and 9 but not week 24.
    Levels of urinary sphingoid bases were not determined.

         No FB1-dependent changes in tumour incidences in the male mice
    receiving diets of 0, 5, 15, 80 and 150 mg FB1/kg diet were
    identified. The body weights, organ weights, survival, feed
    consumption and serum analytes were not affected by FB1 dose. The
    lack of demonstration of statistically significant sphingoid base
    increases in livers at intermediate sacrifices (3, 7 and 9 weeks) were
    probably due to the low sample number (n = 4) (US NTP, 1999).  Short-term assays for carcinogenicity

         The short-term assays have mainly used rat liver nodules as the
    end-point, assessed either using traditional microscopy or
    histochemical analysis of different enzyme activities such as
    gamma-glutamyl transferase (GGT) or placental glutathione
     S-transferase (PGST). In many of these assays, different stages
    (initiation, promotion) of the multistage carcinogenesis model have
    been investigated by combining the FB1 treatment with classical
    promotion assays. In these studies FB1 (or a  Fusarium extract) was
    administered alone, or before a promoting treatment, or after an
    initiating treatment.

    a)    Initiation studies 

         In male BD IX rats fed a diet containing 0.1% FB1 during 4
    weeks, GGT-positive (GGT+) foci were induced in the liver
    (Gelderblom et al., 1988).

         GGT+ foci were induced in the liver of male Fischer rats fed a
    diet containing 0.5-1 g/kg of FB1 (90-95% pure) for 21-26 days,
    followed by partial hepatectomy and treatment with 2-AAF and carbon
    tetrachloride. However, foci were not induced when single or multiple
    doses (50-200 mg/kg) of FB1 (and FB2) were administered by gavage to
    hepatectomized rats (Gelderblom et al., 1992b, 1993).

         In subsequent dose-response studies in male Fischer rats using
    the same experimental approach, Gelderblom et al. (1994) reported that
    the lowest dietary level to produce cancer initiation (GGT+-foci)
    over 21 days was 250 mg FB1/kg diet. The lowest levels to cause
    cancer initiation over 14 and 7 days were 500 mg and 750 mg FB1/kg
    diet, respectively. Based on the feed intake values, the effective
    dosage level (EDL) for cancer initiation over a period of 21 days was
    142 < EDL < 308 mg FB1/kg body weight and over 14 days the amount
    required for cancer initiation was 233 < EDL < 335 mg FB1/kg body
    weight. The dietary level of FB1 required for cancer initiation is
    dependent on the duration of exposure since a dose of 293 mg FB1/kg
    body weight over 7 days did not initiate cancer whereas a similar dose
    (308 mg FB1/kg body weight) over 21 days did.

         Lebepe-Mazur et al. (1995b) fed female Fischer-344/N rats for one
    week with a semipurified diet with or without an aqueous extract of a
     Fusarium verticillioides (M 1325 = MRC 826) culture, providing 20 mg
    FB1/kg diet, and administered a single dose of 30 mg/kg body weight
    diethylnitrosamine (DEN) thereafter. Rats fed the  Fusarium culture
    showed more PGST+ hepatocytes than those treated with DEN alone.
    Continued dietary treatment with the  Fusarium moniliforme culture
    for 12 weeks after the DEN administration did not further increase the
    number of PGST foci. When the FB1 diet (one week) was followed by the
    DEN administration and a 7-day-non-treatment interval, no increase in
    PGST foci by FB1 was observed.

         In another study (Lebepe-Mazur et al., 1995c), female
    Sprague-Dawley rats were fed a diet supplemented with corn
    contaminated with  Fusarium proliferatum (containing 20 or 50 mg/kg
    FB1) for six months; GGT-foci were not observed but the number of
    PGST+ altered hepatic foci was increased in treated rats in
    comparison to rats fed a semipurified diet without supplementation. In
    a similar study (Lebepe-Mazur et al., 1995b), feeding diet containing
    20 mg/kg FB1 for one or 13 weeks failed to induce a statistically
    significant increase in PGST-altered foci.

         In male Sprague-Dawley rats administered purified FB1
    intraperitoneally at 10 mg/kg body weight per day for 4 days, as well
    as in male and female Sprague-Dawley rats given 35 and 75 mg/kg body

    weight per day orally for 11 days, significant increases in PGST+
    hepatocytes were observed (Mehta et al., 1998).

    b)    Promotion studies 

         In a study on the promotion activity of FB1, it was administered
    to male Fischer-344 rats (10, 50, 100, 250 or 500 mg/kg diet for 21
    days) after a dose of DEN (200 mg/kg body weight) (Gelderblom et al.,
    1996c). Dietary levels of 50 mg/kg or more markedly increased the
    number and size of the PGST+ foci in the liver. It was thus
    concluded (Gelderblom et al., 1996b,c) that the dose of FB1 required
    for cancer initiation was markedly higher than that required for
    cancer promotion.

         Female Sprague-Dawley rats were fed a semipurified diet with or
    without an aqueous extract of a  Fusarium verticillioides (M 1325 =
    MRC 826) culture, providing 20 or 50 mg FB1/kg for 6 months, after a
    single dose of 30 mg DEN/kg. The number of PGST-altered foci was
    increased at 20 mg/kg but not in the high-dose group, as compared to
    rats treated with DEN alone. GGT-altered foci were not observed
    (Lebepe-Mazur et al., 1995c).

         Gelderblom et al. (1996b) suggested that FB1-induced
    hepatocarcinogenesis in male BD IX rats developed against a background
    of chronic toxic hepatosis culminating in cirrhosis. Chronic
    hepatotoxicity appears to be a prerequisite for the development of
    liver cancer in the BD IX rat (Gelderblom et al., 1996b).

    7.2  Other mammals

    7.2.1  Equine leukoencephalomalacia

         Equine leukoencephalomalacia (ELEM) syndrome is characterized by
    the presence of liquefactive necrotic lesions in the white matter of
    the cerebrum. The name is somewhat misleading since the gray matter
    may also be involved (Marasas et al., 1988a). This fatal disease
    apparently occurs only in equids, although there has been one
    unconfirmed report of fumonisin-induced brain lesions and haemorrhage
    in rabbits gavaged with FB1 (Bucci et al., 1996) and there is some
    evidence that FB1 can cross the blood-brain barrier and disrupt brain
    sphingolipid metabolism in neonatal rats (Kwon et al., 1997b). In
    equids, the ELEM syndrome has been recognized since the 19th century
    as a sporadically occurring condition. ELEM was experimentally
    produced by feeding mouldy maize obtained from a field case in Kansas
    by Butler (1902). The disease was known as "mouldy maize poisoning"
    but attempts to identify the responsible fungus failed.

         Wilson & Maronpot (1971) succeeded in establishing the causative
    agent when they isolated  F. verticillioides as the predominant
    contaminant of mouldy maize that had caused cases of ELEM in Egypt and
    reproduced ELEM by feeding culture material of the fungus on maize to
    two donkeys. Subsequently investigators in South Africa confirmed the

    ability of  F. verticillioides (MRC 826) culture material to induce
    the characteristic clinical signs and pathological changes of ELEM as
    well as hepatosis in horses and donkeys (Kellerman et al., 1972;
    Marasas et al., 1976, 1988a; Kriek et al., 1981).

         The first symptoms of the syndrome are lethargy, head pressing
    and inappetence, followed by convulsions and death after several days.
    Elevated serum enzyme levels indicative of liver damage (Wilson et
    al., 1992) are preceded by elevation in the serum Sa/So ratio (Wang et
    al., 1992; Riley et al., 1997). Serum enzyme levels often return to
    near normal concentrations (Wang et al., 1992; Wilson et al., 1992;
    Ross et al., 1993; Riley et al., 1997) but usually increase markedly
    immediately prior to or at the onset of behavioral changes (Kellerman
    et al., 1990; Wang et al., 1992; Ross et al., 1993; Riley et al.,

         In addition to the brain lesions, histopathological abnormalities
    in liver and kidney have been reported in horses orally dosed with
    pure fumonisins, maize screenings naturally contaminated with
    fumonisins, or culture material containing known amounts of fumonisins
    (Kellerman et al., 1990; Wilson et al., 1992; Ross et al., 1993;
    Caramelli et al., 1993).

         Shortly after the isolation and structure elucidation of
    fumonisins in 1988 (Bezuidenhout et al., 1988; Gelderblom et al.,
    1988), Marasas et al. (1988a) successfully produced ELEM in a horse by
    the intravenous administration of pure FB1. This was done by avoiding
    as much as possible hepatotoxicity using serum enzymes indicative of
    it. ELEM has also been produced in horses given pure FB1 by stomach
    tube, again monitoring for liver toxicity (Kellerman et al., 1990).

         Fatal liver disease in the absence of any brain lesions has been
    induced by intravenous injection of FB1 (Laurent et al., 1989b). ELEM
    concurrent with significant liver disease has been observed in horses
    and ponies fed feeds naturally contaminated with fumonisins at low
    concentrations (Wilson et al., 1992; Ross et al., 1993). The
    development of brain lesions in the absence of major liver lesions
    does not preclude biochemical dysfunction in non-brain tissue from
    contributing to the brain lesions. Ross et al. (1993) concluded that
    length of exposure, level of contamination, individual animal
    differences, previous exposure, or pre-existing liver impairment may
    all contribute to the appearance of the clinical disease.

         To date, the lowest FB1 dose that has resulted in ELEM, in a
    controlled experiment, is 22 mg/kg in diets formulated with naturally
    contaminated maize screenings (Wilson et al., 1992). Analysis of feeds
    from confirmed cases of ELEM indicated that consumption of feed with a
    FB1 concentration greater than 10 mg/kg diet is associated with
    increased risk of development of ELEM, whereas, a concentration less
    than 6 mg/kg diet is not (Ross, 1994).

         A study by the National Veterinary Services Laboratory of the US
    Animal and Plant Health Inspection Agency (National Veterinary
    Services Laboratory, 1995) showed that horses fed 15 mg FB1/kg in
    diets formulated from  F. proliferatum (M 5991) culture material did
    not exhibit any clinical signs or altered serum biochemical parameters
    (including changes in the Sa/So ratio) after 150 days. A similar
    result was found with a pony fed a diet containing maize screenings
    naturally contaminated with 15 mg FB1/kg (Wang et al., 1992). Thus,
    the minimum toxic dose in equids appears to be < 22 mg/kg > 15 mg/kg
    based on studies with naturally contaminated maize screenings or
    culture material  (F. proliferatum) containing fumonisins. The
    minimum toxic dose of pure fumonisins is unknown.

         In a study using culture material containing primarily FB2 or
    FB3, Ross et al. (1994) found that a diet formulated from
     F. proliferatum (M 6290 and M 6104) culture material containing
    primarily FB2 at 75 mg/kg was capable of inducing ELEM with hepatic
    involvement in ponies after 150 days. In contrast, diets containing
    primarily FB3 (75 mg/kg) were without any effect (serum enzymes,
    clinical signs and histology were all normal relative to control
    ponies) after 57 to 65 days. It was concluded that FB3 was less toxic
    than FB2 or FB1 (Ross et al., 1994). However, analysis of serum and
    tissues from ponies fed the FB3 diets revealed that the FB3 diets
    significantly increased concentrations of free sphingoid bases
    relative to controls and that serum enzymes were elevated but within
    the normal range for ponies (Riley et al., 1997).

    7.2.2  Porcine pulmonary oedema syndrome

         The first report of the disease now known as porcine pulmonary
    oedema (PPE) was by Kriek et al. (1981). In experimental trials,
    culture material of  F. verticillioides (MRC 826) was fed to horses,
    pigs, sheep, rats and baboons (Kriek et al.,1981). Lung oedema
    occurred only in pigs. Clinical signs of PPE typically occur soon
    (2-7 days) after pigs consume diets (culture material or contaminated
    maize screenings) containing large amounts of fumonisins over a short
    period of time. Clinical signs usually include dyspnoea, weakness,
    cyanosis and death (Osweiler et al., 1992). At necropsy, the animals
    exhibit varying degrees of interstitial and interlobular oedema, with
    pulmonary oedema and hydrothorax (Colvin & Harrison, 1992; Colvin et
    al., 1993). Varying amounts of clear yellow fluid accumulate in the
    pleural cavity.

         Toxic hepatosis occurs concurrently with PPE (Osweiler et al.,
    1992; Colvin et al., 1993) and is also observed in animals that
    consume high levels of fumonisins but do not develop PPE (Haschek et
    al., 1996). Typically, the liver contains multiple foci of coagulative
    necrosis that do not show zonal distribution across the three zones of
    the liver (Osweiler et al., 1992; Colvin et al., 1993). Two studies
    have reported nodular hyperplasia in the pig liver (Casteel et al.,
    1993, 1994).

         The physiological alteration that results in the inability of the
    lung to maintain fluid equilibrium is unknown. However, several
    hypotheses have been proposed that are supported by experimentation.
    Casteel et al. (1994) found that feeding culture material diets
    (M 1325 = MRC 826) containing 150 to 170 mg FB1/kg for 210 days
    resulted in right ventricular hypertrophy and medial hypertrophy of
    the pulmonary arterioles. It was suggested that this cardiotoxic
    effect was an indirect consequence of fumonisin-induced
    hepatotoxicity. Cardiac failure is a well-known physiological
    mechanism inducing altered pulmonary haemodynamics which can result in
    pulmonary oedema (Colvin et al., 1993). Significant changes in oxygen
    consumption and several haemodynamic parameters in pigs fed diets
    containing fumonisins suggest that pulmonary hypertension caused by
    hypoxic vasoconstriction may contribute to PPE (Smith et al.,
    1996a,b). It has been hypothesized that the cardiovascular alterations
    are a consequence of sphingoid-base-induced inhibition of L-type
    calcium channels (Smith et al., 1996b).

         Haschek et al. (1992) hypothesized that PPE might be induced by
    dysfunction of pulmonary interstitial macrophages (PIM) resulting in
    release of vasoactive mediators. The accumulation of membranous
    materials in PIM, secondary to hepatotoxicity, was postulated as the
    possible basis for PIM dysfunction (Haschek et al., 1992). A similar
    phenomena has been observed in alveolar endothelial cells (Gumprecht
    et al., 1998). It has been shown that consumption of culture material
    diets (MRC 826) containing fumonisins does in fact alter PIM function
    (Smith et al., 1996c). How this might contribute to pulmonary oedema
    is not clear. However, it has been hypothesized that PIM dysfunction
    could contribute to increased susceptibility to microbial diseases
    (Smith et al., 1996c). It has been shown that serum tumour necrosis
    factor-alpha (TNF-alpha)-like activity was increased in pigs fed
    culture material (M 1325 = MRC 826) containing 150 mg FB1/kg (Guzman
    et al., 1997). Fumonisin-induced changes in the TNF pathway have also
    been seen in lipopolysaccharide-stimulated macrophages collected from
    BALB/c mice dosed with pure FB1 (Dugyala et al., 1998).

         In 1989-1990 outbreaks of this disease were reported in different
    parts of the USA (Harrison et al., 1990; Osweiler et al., 1992; Ross
    et al., 1992). Maize screenings obtained from farms (Harrison et al.,
    1990; Osweiler et al., 1992) where pigs died of PPE were predominantly
    contaminated with  F. verticillioides. Feeding (Kriek et al., 1981;
    Osweiler et al., 1992; Fazekas et al., 1998) or intubation (Colvin et
    al., 1993) of  F. verticillioides culture material (MRC 826) produces
    PPE. Also, PPE and hepatotoxicity have been produced by feeding diets
    containing maize screenings naturally contaminated with fumonisins
    (Osweiler et al., 1992; Motelin et al., 1994). Purified FB1 has been
    shown to produce the disease when administered intravenously (Harrison
    et al., 1990; Haschek et al., 1992; Osweiler et al., 1992). However,
    PPE has not yet been produced by oral administration of pure

         As with ELEM, there is a strong correlation between fumonisin
    content of maize screenings obtained from different farms and
    outbreaks of PPE (Osweiler et al., 1992; Ross et al., 1992; Ross,
    1994). The highest concentration of FB1 ever reported was from maize
    screenings (330 mg/kg) associated with an outbreak of PPE (Ross et
    al., 1992). The minimum toxic dose has not been clearly established.
    Osweiler et al. (1992) induced PPE by feeding  F. verticillioides 
    (MRC-3033) maize culture material reportedly containing 17 mg FB1/kg
    for 5 days. In the same study, maize screenings containing fumonisins
    at 92 mg/kg induced PPE in several pigs after 5-7 days; similar
    results were obtained in studies by Harrison et al. (1990), Haschek et
    al. (1992) and Motelin et al. (1994). Based on feeding studies with
    maize screenings naturally contaminated with fumonisins, FB1
    concentrations of 92 to 166 mg/kg have induced PPE in 4-7 days.

         Pigs fed diets containing fumonisins (formulated with culture
    material or naturally contaminated maize screenings) often do not die
    of PPE, even when fumonisins are reported to be present at very high
    concentrations in the diet. Concentrations of FB1 as low as 17 mg/kg
    in culture material diets (MRC-3033) induced PPE in 5 days (Osweiler
    et al.,1992). In contrast, culture-material-formulated (M 1325 = MRC
    826) diets containing as much as 190 mg/kg have been fed for 83 days
    with no reported evidence of respiratory distress (Casteel et al.,
    1993) and a dose of 150 to 170 mg/kg diet for up to 210 days caused
    liver effects early on but no evidence of pulmonary oedema (Casteel et
    al., 1994).

         Colvin et al. (1993) concluded that the primary determinant of
    whether pulmonary oedema or liver failure caused death was the
    quantity of fumonisins fed or intubated per kg body weight per day.
    They proposed that > 16 mg/kg body weight per day induced PPE and
    < 16 mg/kg body weight per day induced liver failure. However, daily
    oral intake levels of FB1 plus FB2 (maize screenings) from 4.5 to
    6.3 mg/kg body weight have induced PPE (Haschek et al., 1992; Motelin
    et al., 1994). The FB1 concentration in these diets was 166 mg/kg and
    129 mg/kg, respectively. Liver lesions have been induced with maize
    screenings at 1.1 mg/kg body weight per day (17 mg FB1/kg diet)
    (Motelin et al., 1994).

         In pigs, tissues other than liver and lung have been reported to
    be targets for fumonisins, e.g., pancreas (Harrison et al., 1990),
    heart (Casteel et al., 1994), kidney (Colvin et al., 1993; Harvey et
    al., 1995, 1996), pulmonary intravascular macrophages (Haschek et al.,
    1992), and oesophagus (Casteel et al., 1993). None of these studies
    were conducted with pure fumonisins. In a recent study with pure FB1,
    altered growth and changes in selected haematological parameters in
    pigs were reported at dietary levels as low as 1 mg/kg (Rotter et al.,

    7.2.3  Poultry toxicity

         Several reports have been published implicating
     F. verticillioides contamination of feed in diseases of poultry
    (Marasas et al., 1984a; Bryden et al., 1987; Jeschke et al., 1987;
    Prathapkumar et al., 1997). The clinical features of the disease often
    include diarrhoea, weight loss, increased liver weight and poor
    performance. Immunosuppression in chickens was also produced in birds
    fed maize cultured with several different isolates of the fungus
    (Marijanovic et al., 1991). Functional and morphological changes were
    observed in chicken exposed to FB1 (Qureshi & Hagler, 1992). Several
    studies have confirmed that  F. verticillioides, F. proliferatum, 
    FB1 and moniliformin are toxic to poultry (broiler chicks, turkeys,
    ducklings) (Ledoux et al., 1992, 1996; Brown et al., 1992;
    Dombrink-Kurtzman et al., 1993; Javed et al., 1993a, 1995; Weibking et
    al., 1993a,b, 1995; Kubena et al., 1995a,b; Hall et al., 1995;
    Bermudez et al., 1996; Vesonder & Wu, 1998) and chicken embryos (Javed
    et al., 1993b; Bacon et al., 1995). The levels of fumonisins used in
    these studies were 75-644 mg/kg diet. Culture materials and naturally
    contaminated maize containing  F. proliferatum may contain, in
    addition to fumonisins, moniliformin and beauvericin (Kriek et al.,
    1977; Logrieco et al., 1993; Plattner & Nelson, 1994). Espada et al.
    (1994) reported toxicity and altered haematological parameters (Espada
    et al., 1997) in broiler chicks fed diets containing pure FB1
    (10 mg/kg) and FB1 (30 mg/kg) from  Fusarium verticillioides 
    (MRC 826) culture material.

    7.2.4  Non-human primate toxicity

         Kriek et al. (1981) fed three baboons  F. verticillioides 
    culture material (MRC 826). Baboon 1 and baboon 2 died of acute
    congestive heart failure after 248 and 143 days, respectively. The
    remaining baboon continued on feed for 720 days, at which time it was
    killed. Autopsy of baboon 3 revealed that the principle lesion was
    cirrhosis of the liver. Vervet monkeys fed  F. verticillioides 
    culture material (MRC 826) for 180 days exhibited various degrees of
    toxic hepatosis (Jaskiewicz et al., 1987a). Subsequent long-term
    studies (Fincham et al., 1992) with vervet monkeys fed MRC 826 culture
    material shown to contain fumonisins revealed an increase in serum
    cholesterol, plasma fibrinogen and blood coagulation factor VII
    (factors known to promote atherosclerosis). These changes occurred
    secondary to chronic hepatotoxicity at a dose calculated to average
    0.3 mg total fumonisins/kg body weight per day (low-dose diet) based
    on a retrospective analysis of the diets (Fincham et al., 1992) and a
    high-dose diet averaging approximately 0.8 mg total fumonisins/kg body
    weight per day (Shephard et al., 1996b). Analysis of the free
    sphingoid bases in serum from some of the animals used in the study by
    Fincham et al. (1992) showed that in serum the free sphinganine
    concentration and Sa/So ratio were significantly elevated in both the
    low-dose and high-dose animals (Shephard et al., 1996b). Free
    sphinganine and the Sa/So ratio were also elevated in urine at both
    dose levels, but not significantly (Shephard et al., 1996b).

    7.2.5  Other species

         Other species that have been studied using pure fumonisins,
    contaminated maize screenings or maize culture material of
     F. verticillioides include the following: catfish (Brown et al.,
    1994; Goel et al., 1994); cattle (Osweiler et al., 1993); hamsters
    (Floss et al., 1994a,b); lambs (Edrington et al., 1995); mink (Restum
    et al., 1995); and rabbits (Gumprecht et al., 1995; Bucci et al.,
    1996; LaBorde et al., 1997). In all cases where toxicity was evident
    it involved liver and/or kidney or homologous organs.

    7.3  Mechanisms of toxicity -- mode of action

         Several biochemical modes of action have been proposed to explain
    all or some of the fumonisin-induced animal diseases. Two of these
    invoke disruption of lipid metabolism as initial site of action. There
    are also several studies that hypothesize fumonisin-induced changes in
    key enzymes involved in cell cycle regulation, differentiation and/or
    apoptosis as initial or secondary sites of action.

    7.3.1  Disruption of sphingolipid metabolism

         The structural similarity between sphinganine and FB1 led Wang
    et al. (1991) to hypothesize that the mechanism of action of this
    mycotoxin might be via disruption of sphingolipid metabolism or a
    function of sphingolipids. At the moment, there are considerable data
    supporting the hypothesis that fumonisin-induced disruption of
    sphingolipid metabolism is an important event in the cascade of events
    leading to altered cell growth, differentiation and cell injury
    observed both  in vitro and  in vivo.  Sphingolipids and their metabolism

         The pathways of biosynthesis and turnover (Fig. 1) have not been
    as well studied in sphingolipids as in other lipid classes. In order
    to understand how disruption of sphingolipid metabolism might
    contribute to the farm animal and laboratory animal diseases
    associated with consumption of fumonisins, it is necessary to
    understand how sphingolipids are biosynthesized. Eukaryotic cells
    synthesize a diverse array (over 400 distinct molecules) of
    sphingolipids which serve as important structural molecules in
    membranes and as regulators of many cell functions (Bell et al.,
    1993). While sphingolipids have also been found in procaryotes
    (Karlsson, 1970), their biosynthesis and role in cellular regulation
    is poorly understood.

         Typically,  de novo sphingolipid biosynthesis proceeds via the
    reactions described below (Merrill & Jones, 1990; Sweeley, 1991; Bell
    et al., 1993). The first is the condensation of serine with
    palmitoyl-CoA by serine palmitoyltransferase, a pyridoxal
    5'-phosphate-dependent enzyme, and the resulting 3-ketosphinganine is
    reduced to sphinganine using NADPH. Sphinganine is acylated to

    dihydroceramides (also called  N-acylsphinganines) by ceramide
    synthase using various fatty acyl-CoAs. Headgroups (e.g.,
    phosphorylcholine, glucose, etc.) are subsequently added to the
    1-hydroxyl group. The 4,5-trans-double bond of the sphingosine
    backbone is added after acylation of the amino group of sphinganine by
    the enzyme dihydroceramide desaturase (Michel et al., 1997). Both
    dihydroceramide and dihydrosphingomyelin are substrates for the
    enzyme. Thus, free sphingosine is not an intermediate of  de novo 
    sphingolipid biosynthesis (Merrill, 1991; Rother et al., 1992).
    Sphingolipid turnover is thought to involve the hydrolysis of complex
    sphingolipids to ceramides, then to sphingosine. Sphingosine is either
    reacylated or phosphorylated and cleaved to a fatty aldehyde and
    ethanolamine phosphate. The fatty aldehyde and ethanolamine phosphate
    can be redirected into the biosynthesis of glycerophospholipids and
    other fats (Van Veldhoven & Mannaerts, 1993).

         In animal cells the initial steps from the condensation of serine
    and palmitoyl-CoA to the formation of ceramide take place in the
    endoplasmic reticulum. Subsequent processing of ceramide into
    glycosphingolipids and sphingomyelin takes place in the endoplasmic
    reticulum and Golgi apparatus. Degradation of complex sphingolipids
    occurs in the lysosomes, endosomes and the plasma membrane with
    degradation of free sphingoid bases occurring in the cytosol. For
    reviews of sphingolipid metabolism, see Merrill & Jones (1990),
    Merrill (1991), Sweeley (1991) and the volumes edited by Bell et al.
    (1993).  Fumonisin-induced disruption of sphingolipid metabolism in

         The term "fumonisin disruption of sphingolipid metabolism"
    includes inhibition of sphingosine and ceramide biosynthesis,
    depletion of more complex sphingolipids, increase in free sphinganine,
    decrease in reacylation of sphingosine derived from complex
    sphingolipid turnover and degradation of dietary sphingolipids,
    increase in sphingoid base degradation products (i.e. sphingosine
    (sphinganine) 1-phosphate, ethanolamine phosphate and fatty
    aldehydes), and increase in lipid products derived from the increase
    in the sphingoid base degradation products. FB1 is now widely used to
    reveal the function of sphingolipids and sphingolipid metabolism in
    cells (Merrill et al., 1996a).

         Fumonisins potently inhibit the acylation of sphinganine and
    sphingosine (Wang et al., 1991; Yoo et al., 1992; Merrill et al.,
    1993c). In primary rat hepatocytes, the IC50 for inhibition of serine
    incorporation into sphingosine is approximately 0.1 µM for FB1 and
    FB2 (Wang et al., 1991; Merrill et al., 1993c). In P388 murine
    macrophages, the IC50 is less than 0.5 µM (Balsinde et al., 1997). In
    cultured pig renal cells (LLC-PK1) the IC50 for inhibition of
     de novo sphingosine biosynthesis is approximately 20 µM FB1 (Yoo et
    al., 1992). The basis for this difference in sensitivity is unknown.

    .FIGURE 1a and 1b;V219EH03.BMP

    Fig. 1a. The pathway of  de novo sphingolipid biosynthesis and
    turnover in a mammalian cell. Large solid arrows indicate the
    enzymatic steps leading to biosynthesis of ceramide, a known effector
    of cell death, and large broken arrows show the enzymatic steps
    leading to the production of sphingosine 1-phosphate, an effector of
    cell survival. Also shown is the proposed role of mitochondrial
    perturbations triggering a redirection of palmitate from
    beta-oxidation into the  de novo pathway, resulting in increased
    biosynthesis of ceramide under conditions of oxidative stress.

    Fig. 1b. The sites of action of serine palmitoyltransferase (SPTase)
    inhibitors (SPTI) such as ISP-I = myriocin = thermozymozydin, and
    ceramide synthase (CER synthase) inhibitors (FB) such as fumonisins.
    The block on the ceramide synthase responsible for reacylation of
    sphingosine results in an increase in free sphingosine and possibly
    sphingosine 1-phosphate. However, it has been reported that unlike
    sphingosine 1-phosphate, sphinganine 1-phosphate does not exert a
    marked cytoprotective effect, but does bind to and signal via the G
    protein-coupled receptor encoded by endothelial differentiation gene 1
    (EDG1) (Spiegel, 1999).

    In both Fig 1a and Fig 1b, the biochemicals shown in all capital
    letters are those known or suspected to be lipid secondary messengers.
    Also shown in Fig 1a is the generation of ceramide by ligand-induced
    sphingomyelin hydrolysis.

    Abbreviations: DHC-desaturase (dihydroceramide desaturase), A-SMase
    (acidic sphingomyelinase), So-kinase and -lyase (sphingosine kinase
    and lyase).

         Inhibition of sphinganine (sphingosine)  N-acyltransferase
    (ceramide synthase) in cells leads to a concentration-dependent
    reduction in total complex sphingolipids, including sphingomyelin and
    glycosphingolipids (Wang et al., 1991; Merrill et al., 1993b; Yoo et
    al., 1996). In LLC-PK1 cells, the decrease in complex sphingolipids
    does not become apparent until 24 to 48 h after cells have been
    exposed to FB1 but before inhibition of cell growth and increased
    cell death (Yoo et al., 1996). In microsomal preparations from
    cultured mouse cerebellar neurons, inhibition of ceramide synthase was
    competitive with respect to both the long-chain (sphingoid) base and
    fatty acyl-CoA (Merrill et al., 1993b). This observation suggests that
    ceramide synthase recognizes both the amino group (sphingoid binding
    domain) and the tricarballylic acid side-chains (fatty acyl-CoA
    domain) of FB1 (Merrill et al., 1996b). The reduced inhibition by the
    hydrolysed derivatives of the fumonisin B series (Norred et al., 1997;
    van der Westhuizen et al., 1998) supports this hypothesis. Ceramide
    synthase has recently been shown to acylate hydrolysed FB1, AP1, to
    form  N-palmitoyl-AP1. The product was also found to be an inhibitor
    of ceramide synthase and to be 10 times more toxic than FB1 in a
    human colonic cell line, HT29 (Humpf et al., 1998). Sphingomyelin
    biosynthesis is approximately 10-fold more sensitive to inhibition by
    FB1 than glycosphingolipids. This is true for other inhibitors of
    ceramide synthesis, such as ß-fluoroalanine (Medlock & Merrill, 1988;
    Merrill et al.,1993b).

         The complete inhibition of ceramide synthase by fumonisins causes
    the intracellular sphinganine concentration to increase rapidly (Wang
    et al., 1991; Yoo et al., 1992). The amount of free sphinganine that
    accumulates in cells is a function of several factors. These include
    the extent of inhibition of ceramide synthase, the concentration of
    essential precursors (serine, palmitoyl-CoA), the growth rate of the
    cells, the rate of sphinganine degradation, and the rate of
    elimination from the cells. For example, in rat primary hepatocytes
    treated with a concentration of 1 µM FB1, which inhibits serine
    incorporation into sphingosine by > 90%, there was a significant
    increase in free sphinganine after 1 h, which increased to 110-fold
    over controls after 4 days (Wang et al., 1991). In LLC-PK1 cells
    approximately 50-fold and 128-fold increases were measured after
    exposure to FB1 (35 µM, 50 to 60% inhibition) for 6 h and 24 h,
    respectively (Yoo et al., 1992). Proliferating LLC-PK1 cells
    accumulate much higher levels of free sphingoid bases than confluent
    monolayers and cytotoxicity is only observed in proliferating cells
    (Yoo et al., 1996).

         While the level of free sphingosine does not increase in primary
    rat hepatocytes (Wang et al., 1991; Gelderblom et al., 1995), it does
    in LLC-PK1 cells, presumably due to inhibition of reacylation of
    sphingosine derived from sphingolipid turnover or from the growth
    medium. When cells begin to die, sphingosine levels will increase due
    to the breakdown of membrane lipids. However, in LLC-PK1 cells the
    increase in free sphingosine occurs before any evidence of increased
    cell death or inhibition of cell proliferation (Yoo et al., 1992).

    Nonetheless, approximately 95% of the increase in the levels of free
    sphingoid bases in LLC-PK1 cells was found to be due to the increase
    in free sphinganine level (Yoo et al., 1992). The fate of the
    accumulated sphinganine is unclear. While sphingosine has little
    difficulty in crossing cell membranes (Hannun et al., 1991), the
    half-life of sphinganine inside LLC-PK1 cells is much longer than the
    half-life of FB1 in LLC-PK1 cells (Riley et al., 1998), which
    suggests either that the inhibition of sphinganine  N-acyltransferase
    is persistent, that sphinganine does not easily diffuse out of cells,
    or that sphinganine degradation is slow relative to its biosynthesis.
    In urine from rats fed FB1, > 95% of the free sphinganine is
    recovered in dead cells which have apparently sloughed into the urine
    (Riley et al., 1994a). Thus, in urine sphinganine is tightly
    associated with the cells.

         In rat hepatocytes, a portion of the accumulated sphinganine is
    metabolized to sphinganine 1-phosphate and then cleaved into a fatty
    aldehyde and ethanolamine phosphate (Merrill et al., 1993c), both of
    which can be redirected into other biosynthetic pathways. The enzyme
    responsible for hydrolysis of sphingosine (sphinganine) 1-phosphate is
    sphingosine-phosphate lyase (Van Veldhoven & Mannaerts, 1993). In J774
    cells exogenous sphinganine has been shown to be initially accumulated
    and then rapidly metabolized (Smith & Merrill, 1995). About one-third
    of the ethanolamine in phosphatidylethanolamine is derived from
    long-chain base catabolism when fumonisin is added (Smith & Merrill,
    1995). Similar findings have been reported using fumonisin-treated
    Chinese hamster ovary cells (Badiani et al., 1996), confirming that,
    in some cell types, accumulated free sphingoid bases are rapidly
    metabolized into ethanolamine and fatty aldehydes.  In vitro and
     in vivo, rat liver lipid composition is markedly altered by FB1
    (Wang et al., 1991; Gelderblom et al., 1997). In addition to changes
    in free sphingoid bases, more complex sphingolipids (ceramides, etc.)
    and phosphatidylethanolamine (Wang et al., 1991; Merrill et al.,
    1993c), there are many changes in the fatty acid composition of liver
    phospholipids (Gelderblom et al., 1997). The ability of cells to
    rapidly metabolize bioactive sphingoid bases into other products may
    protect cells from the toxicity associated with accumulation of free
    sphingoid bases or ceramide (Spiegel, 1999). Chronically disrupted
    sphingolipid metabolism leads to imbalances in phosphoglycerolipid and
    fatty acid metabolism. Because the accumulation of specific
    end-products and intermediates is dependent upon the balance between
    anabolic and catabolic processes, it is conceivable that changes in
    concentration of specific end-products could occur with no change in
    the steady-state concentration of free sphingoid bases.  Fumonisin disruption of sphingolipid metabolism in vivo

    a)    Equids 

         Typically free sphingosine and sphinganine are present in normal
    tissues and cells in trace amounts (Merrill et al., 1988; Riley et
    al., 1994c). This is to be expected since free sphinganine is a
    metabolic intermediate in the sphingolipid biosynthetic pathway, and

    free sphingosine is generated primarily as a consequence of
    sphingolipid turnover or degradation (Merrill, 1991; Rother et al.,
    1992; Michel et al., 1997). The first  in vivo study to test if
    dietary fumonisins could change free sphingoid base concentration used
    serum obtained from ponies fed diets containing maize screenings
    naturally contaminated with fumonisins (primarily FB1) (Wang et al.,
    1992). Upon consumption of diets containing fumonisin, all of the
    ponies exhibited large increases in sphinganine, although the
    magnitude of the changes varied among the animals. The elevation in
    serum sphinganine is reversible and the increase in free sphinganine
    and the Sa/So ratio occurred before increases in serum transaminase
    activity and clinical signs of ELEM (Wang et al., 1992). For example,
    a pony was given feed (corn screenings) containing 15 to 22 mg
    FB1/kg. The Sa/So ratio increase by day 182 was followed by an
    increase in serum biochemical indices of cellular injury by day 223
    (Wang et al., 1992). The pony died of ELEM on day 241.

         In another study, horses were fed diets containing
     F. proliferatum (M 6290 and M 6104) culture material, which
    contained primarily either FB2 (75 mg/kg) or FB3 (75 mg/kg) (Ross et
    al., 1994). Analysis of serum and tissues from these horses showed a
    qualitatively similar response to horses fed FB1 (Wang et al. 1992),
    but the magnitude of the increase in free sphinganine in serum, liver
    and kidney was much less in the horses fed FB3 diets (Riley et al.,
    1997). While two of the three ponies fed FB2 diets developed ELEM,
    the three ponies fed FB3 diets showed no clinical signs of ELEM or
    evidence of liver damage after 60 days (Ross et al., 1994).

         There was also a reduction in the amount of complex sphingolipids
    in the serum, liver and kidney of ponies fed diets containing
    fumonisins (Wang et al., 1992; Riley et al., 1997), as would be
    expected if the elevation in sphinganine was due to inhibition of
     de novo sphingolipid biosynthesis. In ponies fed diets containing
    FB2 or FB3 (75 mg/kg), the levels of complex sphingolipids in the
    liver were reduced by 88% and 72%, respectively, and the kidney was
    similarly affected (Riley et al., 1997). In contrast to ponies fed
    FB2 diets, ponies fed FB3 diets exhibited no liver or kidney
    pathology (Ross et al., 1994). Thus, prolonged exposure to fumonisins
    can result in a marked depletion of the complex sphingolipids in liver
    with no evidence of liver pathology.

    b)    Pigs 

         Similar results were obtained for pigs, confirming that there was
    a dose-response relationship between the ratio of free sphinganine to
    free sphingosine in serum and tissues and the amount of
    fumonisin-contaminated feed consumed (Riley et al., 1993). Pigs were
    fed diets formulated from naturally contaminated maize screenings at 0
    (< 1), 5, 23, 39, 101 and 175 mg/kg total fumonisins (FB1 plus
    FB2). The results showed that the Sa/So ratio was significantly
    elevated in the liver, lung and kidney from pigs consuming feeds
    containing > 23 mg/kg fumonisins. Liver injury was observed at

    fumonisin levels > 23 mg/kg. However, injury to the kidney was not
    observed at any dose even though it contained equal or greater amounts
    of free sphingoid bases. In lung tissue, free sphingoid base content
    was elevated at doses > 23 mg/kg, but lung lesions were only
    observed in pigs fed the diet containing 175 mg/kg. Smith et al.
    (1996a) showed that in pigs fed fumonisins significant effects on
    cardiovascular function were associated with significant increases in
    free sphingoid bases in heart tissue. Subsequent studies found that
    damage to pig alveolar endothelial cells,  in vivo, was preceded by
    accumulation of free sphingoid bases in lung tissue (Gumprecht et al.,

         Elevation of the Sa/So ratio in pig serum paralleled the increase
    in tissues (Riley et al., 1993). This finding supported the earlier
    hypothesis (Wang et al., 1992) that the elevated ratio in serum was
    due to the movement of free sphinganine (accumulating as a result of
    inhibition of sphinganine  N-acyltransferase) from tissues into the
    blood. Statistically significant increases in the serum ratio were
    observed at feed concentrations as low as 5 mg/kg total fumonisins
    (after 14 days) and in pigs (at higher concentrations) in which other
    serum biochemistry parameters were not changed and in which there were
    no observable gross or microscopic lesions in liver, lung or kidney.
    Thus, the increase in the Sa/So ratio was an earlier and more
    sensitive indicator of fumonisin exposure than the development of
    lesions in liver or lung in pigs detectable by light microscopy.
    Nonetheless, the increases in free sphinganine in tissues and serum
    closely paralleled the dose-dependent increases in other biochemical
    parameters measured at 14 days (Motelin et al., 1994).

         It has been proposed that the ratio of free sphinganine to free
    sphingosine and the presence of elevated levels of free sphinganine in
    serum, urine and tissue be used as indicators for consumption of
    fumonisins by farm animals (Riley et al., 1994c). However, a
    subsequent study with pigs found altered growth at doses of FB1 that
    did not cause an increase in free sphinganine (Rotter et al., 1996).
    Thus, in pigs, elevation of free sphinganine appears to occur at
    dosages that are greater than those that cause subtle changes in
    performance but lower than those that are toxic.

    c)    Poultry and other commercially important animals 

         Chickens fed diets supplemented with  F. verticillioides culture
    materials (Weibking et al., 1993a, 1995) or pure FB1 (Henry, 1993)
    exhibited elevated sphinganine levels and elevated ratios in tissues
    and serum. Similar findings have been made in the rabbit (Gumprecht,
    et al., 1995; LaBorde et al., 1997), catfish (Goel et al., 1994), mink
    (Restum et al., 1995; Morgan et al., 1997) and trout (Meredith et al.,

    d)    Laboratory animals 

         In short-term studies with rats, rabbits and mice, disruption of
    sphingolipid metabolism, as shown by statistically significant
    increases in free sphinganine concentration, occurs at or below the
    fumonisin dosages than cause liver or kidney lesions (Riley et al.,
    1994a; Martinova & Merrill, 1995; LaBorde et al., 1997; de Nijs, 1998;
    Tsunoda et al., 1998; Voss et al., 1998). In rats (Sprague-Dawley,
    RIVM:WU) and mice (BALB/c) dosed with fumonisins, the increase in free
    sphinganine concentration in the kidney and/or liver is closely
    correlated with the extent and severity of lesions (Riley et al.,
    1994a; de Nijs, 1998; Tsunoda et al., 1998; Voss et al., 1998). In two
    separate 21-day feeding studies (Fischer-344 rats), liver free
    sphinganine level was increased, although not significantly, at the
    lowest FB1 dose (50 mg/kg diet) that had liver cancer-promoting
    potential (Gelderblom et al., 1996c, 1997). In rats and rabbits, the
    concentration of fumonisin that causes nephrotoxicity and an increase
    in kidney free sphinganine concentration is lower than the fumonisin
    dose that causes hepatotoxicity (Voss et al., 1993, 1996b, 1998;
    Gumprecht et al., 1995; LaBorde et al., 1997; de Nijs, 1998). For
    example, in Sprague-Dawley rats significant elevation of free
    sphinganine levels and hepatosis were observed at > 15 mg/kg
    < 50 mg/kg dietary FB1 (Riley et al., 1994a), whereas the NOEL for
    nephrosis in male Sprague-Dawley rats is 9 mg/kg (Voss et al., 1995)
    and significant increases in kidney free sphinganine have been
    detected in rats fed AIN-76 diets containing 1 mg FB1/kg (Wang et
    al., 1999). In male RIVM:WU rats, the liver free sphinganine level was
    significantly elevated at > 0.19 < 0.75 mg FB1/kg body weight
    (equivalent to 1.9 and 7.5 mg/kg dietary FB1) in the absence of any
    evidence of hepatosis (de Nijs, 1998) and the NOEL for tubular cell
    death and significant increases in kidney free sphinganine was
    < 0.19 mg FB1/kg body weight, which was equivalent to 1.9 mg/kg in
    feed (de Nijs, 1998).

         In the US National Toxicology Program, long-term feeding study
    with Fischer-344/N Nctr BR rats, pure FB1 induced an increase in the
    Sa/So ratio in kidney tissue, which correlated with increased
    non-neoplastic and neoplastic lesions (US NTP, 1999). In B6C3F1/Nctr
    BR female mouse liver, free sphinganine and the Sa/So ratio were
    increased after 3 and 9 weeks at 50 and 80 mg FB1/kg diet, which were
    the same doses that induced liver adenoma and carcinoma (US NTP,
    1999). Livers taken from rats (BD IX) in a long-term study (2 years)
    that were fed diets containing 10 and 25 mg/kg FB1 did not show
    significant changes in liver free sphingoid bases, although the mean
    concentration of free sphinganine and free sphingosine in the liver of
    rats fed 25 mg FB1/kg diet was 8- and 3-fold, respectively, higher
    than control values (Gelderblom et al., 1997).

         In rats, rabbits and vervet monkeys, increases in free
    sphinganine concentration have been detected in the urine of animals
    fed fumonisin-containing diets (Riley et al., 1994a; Castegnaro et
    al., 1996; Shephard et al., 1996b; LaBorde et al., 1997; Merrill et
    al., 1997b; Solfrizzo et al., 1997a,b; Wang et al., 1999).

    Accumulation of free sphinganine in rat urine (associated with
    accumulation of dead cells) closely reflected the changes which
    occurred in the kidney (Riley et al., 1994a). Analysis of urine from
    rats fed commercially available chows showed a statistically
    significant correlation between the free sphinganine to free
    sphingosine ratio in urine and the fumonisin concentration in the
    chows. The FB1 concentration in the chows ranged from undetected to
    3.3 mg/kg (Merrill et al., 1997b). Feeding studies with pure FB1 in
    AIN-76 diets indicate that the no-observed-effect level (NOEL) for
    elevation of urinary free sphinganine level in Sprague-Dawley rats is
    > 1 mg/kg diet < 5 mg/kg diet (Wang et al., 1999). In other
    studies, rats fed a diet containing mixture of fumonisins (from
    culture material) for 13 days showed a NOEL of between 1 and 2 mg/kg
    diet (Solfrizzo et al., 1997b).

         In rats fed an AIN-76 diet containing 10 mg FB1/kg for 10 days
    and then put on a control diet, the urinary sphinganine concentration
    returned to control levels in 10 days. However, if the diet contained
    1 mg FB1/kg, the urinary sphinganine concentration remained markedly
    elevated for at least 10 days after changing the feed (Wang et al.,
    1999). Thus, in this study, once elevated by feeding toxic levels of
    FB1, apparently non-toxic concentrations kept the free sphinganine
    concentration significantly elevated to concentrations that were
    equivalent to those of the nephrotoxic fumonisin dosage. This result
    is, however, in contrast with the findings of Solfrizzo et al.
    (1997b), showing that the elevated levels of sphingoid bases after
    exposure to relatively high levels of fumonisins (7-15 mg/kg for 13
    days) return to their original values when rats are exposed to a
    low-fumonisin diet (1 mg/kg or less) for a period of time that is
    directly dependent on the previous level of exposure, in terms of dose
    and time (Solfrizzo et al., 1997b).  Tissue and species specificity

         The tissue specificity and the severity of the pathology in rats
    (Sprague-Dawley, Fischer-344, Wistar, RIVM:WV) and mice (BALB/c) seem
    to correlate well with the disruption of sphingolipid biosynthesis
    (Riley et al., 1994a; Tsunoda et al., 1998; Voss et al., 1998). This
    is not the case in pigs and horses, where the kidney appears to be
    equally or more sensitive than the liver with regards to the
    fumonisin-induced increase in free sphinganine (Riley et al., 1993,
    1997). There is significant liver pathology in horses and pigs (Ross
    et al., 1994; Haschek et al., 1996), with little evidence of kidney
    damage (Harvey et al., 1996).

         It has been suggested that these differences in tissue and
    species specificity may be due to differing susceptibility to the
    adverse cellular effects of disrupted sphingolipid metabolism (Voss et
    al., 1996b). For example, liver and kidney may have different
    abilities to metabolize or eliminate free sphinganine or to compensate
    for depletion of complex sphingolipids. In addition, fumonisin, free
    sphingoid bases or their metabolites, in serum may affect the function

    of the vasculature and thus indirectly affect tissues that are not
    directly affected by fumonisin inhibition of ceramide synthase
    (Ramasamy et al., 1995; Smith et al., 1996b). For example, the
    correlation between the fumonisin-induced increase in serum
    free-sphinganine (Wang et al., 1992) and the onset of ELEM could be
    explained if the vascular function in horse brain was altered due to
    elevated serum free sphinganine. In pigs, it has been hypothesized
    that cardiovascular dysfunction, subsequent to increased free
    sphingoid base concentration in the heart, is the cause of PPE (Smith
    et al., 1996b).

         Riley et al. (1996) have recommended that detection of high
    concentrations of free sphinganine in urine, serum or tissues should
    be viewed as a clinical tool to be developed and used in conjunction
    with other clinical tools in situations where animal toxicity
    resulting from exposure to fumonisins is suspected. Changes in
    sphingolipid profiles in serum and urine in vervet monkeys fed
    fumonisin-containing diets have been reported (Shephard et al.,
    1996b). Whether human exposure to fumonisins in maize and maize
    products will result in increased free sphinganine concentration in
    tissues, urine or serum is not known. However, free sphingoid bases
    can be detected in human urine (Castegnaro et al., 1996; Solfrizzo et
    al., 1997a).  Fumonisin-induced sphingolipid alterations: effects on
    growth, differentiation and cell death

         There are many ways that disruption of sphingolipid metabolism
    could account for the cell damage caused by fumonisins. In order to
    fully understand the possibilities, it is necessary to consider the
    multitude of functions of complex sphingolipids (Bell et al., 1993),
    the potent bioactivity of sphinganine and its metabolites (Merrill et
    al., 1993a), and the parallel or branch metabolic pathways that can be
    affected by disruption of sphingolipid metabolism (Riley et al., 1996;
    Merrill et al., 1997a,b). Since the steady-state concentration of many
    biologically active lipid intermediates and end-products could be
    altered, there are also many potential molecular sites that could be
    affected by fumonisin-induced disruption of sphingolipid metabolism.
    Thus, it can be expected that there will also be a diversity of
    alterations in cellular regulation.

         The earliest effect of fumonisin on sphingolipid metabolism
     in vitro is the decrease in serine incorporation into ceramide,
    followed by an increase in free sphinganine concentration (Yoo et al.,
    1992). There is also a concentration-dependent decrease in more
    complex sphingolipids (Yoo et al., 1996). Because long-chain
    (sphingoid) bases are growth inhibitory, cytotoxic and induce
    apoptosis under some conditions (Merrill, 1983; Stevens et al., 1990;
    Nakamura et al., 1996; Sweeney et al., 1996; Yoo et al., 1996), and
    are growth stimulating under certain conditions (Zhang et al., 1990,
    1991; Schroeder et al., 1994), the accumulation of sphinganine (and
    sometimes sphingosine) might account for these same effects of

    fumonisins. Yoo et al. (1992) have shown that in the renal epithelial
    cell line (LLC-PK1 cells) there is a concentration-dependent
    association between the inhibition of sphingolipid biosynthesis by
    FB1 and growth inhibition and cell death. After 24 h of exposure to
    FB1 many cells began to develop a fibroblast-like appearance, with
    loss of cell-cell contact and an elongated, spindle shape. If
    fumonisin was removed, the cells that survived resumed growth and had
    a normal epithelial morphology. Addition of exogenous sphinganine
    induces cell death at intracellular concentrations that are similar to
    those induced by FB1 (Yoo et al., 1996).

         The two most likely explanations for the increased cell death
    after inhibition of sphingolipid biosynthesis by fumonisins are: (1)
    that the free sphinganine (or a sphinganine degradation product) is
    growth inhibitory and cytotoxic for the cells, as has been seen in
    many other systems (Stevens et al., 1990; Hannun et al., 1991; Sweeney
    et al., 1996); and (2) that more complex sphingolipids are required
    for cell survival and growth, as has been proven with mutants lacking
    serine palmitoyltransferase (Hanada et al., 1990, 1992) and in studies
    with specific inhibitors of glycosphingolipid biosynthesis (Radin,
    1994; Nakamura et al., 1996). ß-Chloroalanine, a non-specific serine
    palmitoyltransferase inhibitor, in the presence of FB1 reduced the
    intracellular concentration of free sphinganine and also reduced the
    inhibition of cell growth (50 to 60%) and the extent of cell death (50
    to 60%) (Yoo et al., 1996). More recent studies with LLC-PK1 cells
    indicate that fumonisin inhibition of cell proliferation and increased
    cell death (apoptosis) are prevented by > 90% using the specific
    serine palmitoyltransferase inhibitor, myriocin (ISP-1) (Riley et al.,
    1999). Similar results have been obtained with HT29 cells, a human
    colonic cell line (Schmelz et al., 1998). However, in the LLC-PK1
    cells, the morphological changes, such as decreased cell-cell contact
    and increased fibroblast-like appearance, are not reversed. In primary
    human keratinocytes, both ß-chloroalanine and  N-acetylsphingosine
    partially protected against FB1-induced apoptosis (Tolleson et al.,
    1999). However, both exogenous sphinganine and  N-acetylsphingosine
    alone induced apoptosis in these same cells (Tolleson et al., 1999).
    Thus, in cultured cells sphingolipid-dependent mechanisms for inducing
    apoptosis include accumulation of excess ceramide or sphingoid bases,
    or depletion of ceramide, or more complex sphingolipids.

         In addition to the cell types described above, apoptosis in
    response to exposure to FB1  in vitro has been reported using turkey
    lymphocytes (Dombrink-Kurtzman et al., 1994a,b), human fibroblasts,
    oesophageal epithelial cells and hepatoma cells (Tolleson et al.,
    1996b), and CV-1 monkey kidney cells (Wang et al., 1996).

         The adverse effects of fumonisin-induced depletion of more
    complex sphingolipids have been demonstrated in numerous other
    studies. For example, in hippocampal neurons, FB1 inhibition of
    complex sphingolipid biosynthesis was correlated with decreased axonal
    growth (Harel & Futerman, 1993). The FB1 inhibition of axonal growth
    could be reversed by addition of ceramide with FB1 (Harel & Futerman,

    1993; Schwarz et al., 1995). The ability of growth factors to
    stimulate axonal cell growth is dependent on sphingolipid biosynthesis
    (Boldin & Futerman, 1997). In fibroblasts (Swiss 3T3 cells),
    fumonisin-induced morphological changes could be reversed by
    ganglioside GM1. However, GM1 did not prevent the inhibition of cell
    proliferation (Meivar-Levy et al., 1997). FB1 and/or myriocin (ISP-1)
    inhibition of glycosphingolipid biosynthesis disrupts cell substratum
    adhesion in mouse melanoma cells (Hidari et al., 1996). FB1 has also
    been shown to alter the manner in which glycosyl
    phosphatidylinositol-anchored proteins, such as the folate receptor,
    are organized and function in membranes (Hanada et al., 1993; Stevens
    & Tang, 1997). FB1 inhibition of galactosylceramide biosynthesis has
    been shown to disrupt the assembly and disassembly of cytoskeletal
    proteins responsible for lipid transport and maintenance of the
    subcellular architecture in SW13 cells (derived from a human adrenal
    carcinoma) (Gillard et al., 1996). Thus, there is no doubt that the
    loss of complex sphingolipids also plays a role in the abnormal
    behavior and altered morphology of fumonisin-treated cells.

         Currently Swiss 3T3 cells are the only type of cell that respond
    to fumonisins with increased DNA synthesis (Schroeder et al., 1994).
    It was proposed that this  in vitro model would be useful for
    understanding if, within the complex  in vivo milieu of cells in the
    liver, there might exist cells that could be inappropriately selected
    to enter the cell cycle. Defects in cell cycle control have been shown
    to promote genomic instability and progression to malignancy (Hartwell
    & Kastan, 1994). Incubation of Swiss 3T3 cells with
    DL-erythro-sphinganine caused an increase in [3H]thymidine
    incorporation into DNA. Addition of FB1 to the cells elevated
    sphinganine and induced a comparable increase in [3H]thymidine
    incorporation into DNA. These findings associated an accumulation of
    sphinganine with the induction of DNA synthesis by FB1 but did not
    prove that they were causally linked. However, this was proven using
    an inhibitor of serine palmitoyltransferase in combination with FB1.
    Reduction in cellular sphinganine when ß-fluoro-L-alanine was added to
    Swiss 3T3 cells, demonstrated that this reduction in sphinganine
    completely removed the insulin-dependent stimulation of [3H]thymidine
    incorporation into DNA by FB1. Therefore, the formation of
    sphinganine is required for stimulation of DNA synthesis by fumonisins
    in Swiss 3T3 cells (Schroeder et al., 1994).

         Fumonisin inhibition of ceramide synthesis can deregulate many
    normal cell functions including non-accidental programmed cell death.
    Some of the processes have been shown to be modulated by fumonisin
    inhibition of ceramide synthase  in vitro (Table 4).

         It is important to recognize that ceramide signaling is also
    mediated by sphingomyelin hydrolysis (Perry & Hannun, 1998) via
    enzymes that are not inhibited by fumonisin. When fumonisins are added
    to cells for the purpose of inhibiting  de novo ceramide generation,
    there is also the potential for accumulation of free sphingoid bases
    and their downstream sphingoid base 1-phosphates. Thus, there is the

    Table 4.  Examples of cell functions modulated by  de novo ceramide
              biosynthesis as shown by inhibition of the process by


    *   sphingosine-induced germinal vesicle breakdown and  Xenopus oocyte
        maturation (Strum et al., 1995)

    *   daunorubicin-activated apoptosis in P388, U937 and chicken granulosa
        cells (Bose et al., 1995; Witty et al., 1996)

    *   chemotherapeutic agent (CPT-11)-induced interleukin 1-beta
        converting enzyme (ICE) cascade-dependent apoptosis in 4B1 (L929)
        mouse fibroblasts (Suzuki et al., 1997)

    *   carnitine palmitoyltransferase inhibition-induced apoptosis in LyD9
        mouse haematopoietic precursor cells (Paumen et al., 1997)

    *   lipopolysaccharide (LPS)/platelet activating factor (PAF) induced
        arachidonic acid release in murine P388D1 macrophages (Balsinde et
        al., 1997)

    *   chemical hypoxia-induced cell death in LLC-PK1 cells (Ueda et al.,

    *   Fas-transduced-caspase-dependent T-cell proliferation (Sakata et
        al., 1998)

    *   fenretinide-induced poly-(ADP-ribose) polymerase (PARP) cleavage and
        apoptosis in HL-60 cells (DiPietrantonio et al., 1998)

    *   serum-stimulated retinoblastoma (Rb) protein dephosphorylation and
        cell cycle progression (Lee et al., 1998)

    *   multidrug resistance modulator-dependent cytotoxicity (Cabot et al.,
        1998, 1999)

    *   TNF-alpha/cycloheximide-induced endothelial cell death (Xu et al., 

    *   12- O-tetradecanoylphorbol-13-acetate (TPA)-induced apoptosis in
        prostate cancer cells (Garzotto et al., 1998)

    *   hexadecylphosphocholine-induced apoptosis in HaCaT cells (Wieder et
        al., 1998)

    *   fatty acid-induced nitric oxide synthase-dependent apoptosis in
        cultured rat prediabetic islets (Shimabukuro et al., 1998)

    *   ionizing radiation-induced DNA damaged and cell death in various
        cell types (Liao et al., 1999)

    potential for misinterpreting the results of experiments using
    fumonisins as an inhibitor of ceramide biosynthesis as was recently
    pointed out by Lemmer et al. (1998).

         The ability of fumonisin inhibition of ceramide biosynthesis to
    protect cells is of interest since primary rat hepatocyte necrotic
    cell death has been shown to be mediated by ceramide-induced (but not
    dihydroceramide) mitochondrial dysfunction (Arora et al., 1997). The
    activity of the enzyme responsible for desaturation of the inactive
    dihydroceramide (dihydroceramide desaturase) is regulated by the
    intracellular redox state of the cell (Michel et al., 1997). Taken
    together, these findings suggest that ceramide metabolism is sensitive
    to oxidative stress and that fumonisin-inhibition of ceramide will
    modify apoptosis induced by mitochondrial damage or oxidative stress.

         In primary rat hepatocytes and rat liver slices, large increases
    in free sphinganine occur at FB1 concentrations ranging from 0.1 to 1
    µM (Wang et al., 1992; Gelderblom et al., 1996b; Norred et al., 1996)
    that are 300- to 3000-fold less than those that are cytotoxic and 10-
    to 100-fold less than those that cause inhibition of epidermal growth
    factor-induced [3H]thymidine incorporation into DNA (Gelderblom et
    al., 1996b). There appears to be no relationship between
    fumonisin-induced increases in free sphinganine and fumonisin-induced
    inhibition of [3H]thymidine incorporation and cell death in primary
    rat hepatocytes (Gelderblom et al., 1996a,b) or the cytotoxicity in
    rat liver slices (Norred et al., 1997). At the moment there is no
    adequate explanation for the resistance of primary rat hepatocytes and
    rat liver slices to fumonisin-induced cytotoxicity or inhibition of
    cell proliferation. This is puzzling in light of the fact that other
    primary cell cultures, such as rabbit renal epithelial cells, are very
    sensitive to fumonisin-induced inhibition of cell growth (Counts et
    al., 1996) and that in liver,  in vivo, the intracellular
    concentrations of FB1 that cause hepatotoxicity are relatively low
    based on pharmacokinetic considerations (see previous sections).  Sphingolipid-mediated cellular deregulation and fumonisin

         Sphingolipids have been associated with many facets of cellular
    regulation (Merrill et al., 1993a; Bell et al., 1993; Ballou et al.,
    1996; Merrill et al., 1997b; Kolesnick & Krönke, 1998; Perry & Hannun,
    1998) that could contribute to or modify the expression of
    fumonisin-associated diseases (Table 5).

         Chronic fumonisin disruption of sphingolipid metabolism has been
    hypothesized to be a contributing factor leading to cellular
    deregulation and organ toxicity (Merrill et al., 1993c; Riley et al.,
    1994b; Schroeder et al., 1994; Tolleson et al., 1996a,b, 1999). The
    consequences of disrupted sphingolipid metabolism  in vitro are
    specific to cell type.

    Table 5.  Examples of cellular regulatory processes that have been
              shown to be modulated by sphingolipids and are known to be
              important in the control of normal cell growth,
              differentiation, apoptosis and immune responsea


    Sphingoid bases and their metabolites

    *   inhibition of protein kinase C

    *   activation of phospholipase D/inhibition of phosphatidic acid

    *   activation of the epidermal growth factor (EGF) receptor kinase
        (probably via mitogen-activated protein kinase)

    *   control of intracellular calcium (seemingly via sphingosine 1-

    *   control of plasma membrane potassium permeability in myocytes

    *   inhibition of DNA primase and increases in transcription factor
        AP-1, an early step in the growth of some cell types


    *   second messenger in cytokine signal transduction

    *   activates protein kinases, phosphatases and MAP kinases

    *   inhibits phospholipase D

    More complex sphingolipids

    *   binding of cytoskeletal proteins

    *   participation in cell-cell communication and cell-substratum

    *   protein transport, sorting and targeting

    a  For additional processes regulated by ceramide generated  de novo, 
       see Table 4 and the reviews cited in the text.

         The evidence for fumonisin-induced disruption of sphingolipid
    metabolism in target tissues has been demonstrated repeatedly in many
    independent studies. Nonetheless, the precise mechanism by which
    disrupted sphingolipid metabolism contributes to the increased organ
    toxicity in rodents is unclear. The current understanding of the
    sphingolipid signalling pathways (Merrill et al., 1997a,b; Kolesnick &
    Krönke, 1998; Perry & Hannun, 1998; Spiegel, 1999) indicates that the
    balance between the intracellular concentration of sphingolipid
    effectors that protect cells from apoptosis (decreased ceramide,
    increased sphingosine 1-phosphate) and the effectors that induce
    apoptosis (increased ceramide, increased free sphingoid bases,
    increased fatty acids) will determine the observed cellular response
    (the critical set-points will be cell-type specific). Since the
    balance between the rates of apoptosis and proliferation are critical
    determinants in the process of tumorigenesis, in cells exposed to
    fumonisins, those cells sensitive to the proliferative effect of
    decreased ceramide and increased sphingosine 1-phosphate should be
    selected to survive and proliferate when the conditions under which
    the cells are exposed to fumonisins are such that increased
    intracellular free sphingoid base concentration is not growth
    inhibitory. Conversely, when the rate of increase in free sphingoid
    bases exceeds a cell's ability to convert sphinganine/sphingosine to
    dihydroceramide/ceramide or their sphingoid base 1-phosphate, then
    free sphingoid bases will accumulate. In this case cells that are
    sensitive to sphingoid base-induced growth arrest will cease growing
    and insensitive cells will survive. Another condition that would
    promote increased apoptosis would be if the block on ceramide synthase
    was either reduced or ceramide synthesis was increased while free
    sphinganine levels were still high.

    7.3.2  Altered fatty acid metabolism in liver

         Gelderblom et al. (1995) reported that there was no relationship
    between the fumonisin-induced increase in free sphinganine and the
    mitoinhibitory effect or the cytotoxicity of FB1 in primary rat
    hepatocytes. In addition it was found that free sphinganine
    concentration increased markedly even in primary rat hepatocytes that
    had not been exposed to FB1. However, in cultured cells the simple
    act of changing the culture medium can result in a transient increase
    in free sphingoid bases (Smith & Merrill, 1995; Smith et al., 1997).
    In fumonisin-treated primary rat hepatocytes, the Sa/So ratio was
    maximally elevated at 1 µM FB1, whereas cytotoxicity was observed at
    > 250 µM FB1 (Gelderblom et al., 1995, 1996b). Polyunsaturated
    fatty acids (PUFAs) were shown to accumulate at the cytotoxic doses
    (Gelderblom et al., 1996b).

         In other studies, fumonisins were shown to create a multitude of
    changes in liver cholesterol, phospholipids, sphingoid bases and free
    fatty acid composition (Gelderblom et al., 1996a, 1997). In both the
    short-term and the long-term feeding studies, changes in fatty acid
    profiles indicated that FB1 treatment altered the n-6 fatty acid
    metabolic pathway. In the long-term study (2 years), significant

    changes were observed in livers from rats fed 10 and 25 mg FB1/kg
    diet (Gelderblom et al., 1997). These data suggested that the increase
    in the n-3 fatty acid content of liver could, through altered
    eicosanoid biosynthesis, modulate hepatocyte proliferation (Gelderblom
    et al., 1997). Recently, fumonisin treatment has been shown to
    increase the extent of lipid peroxidation in rat (Fischer-344) primary
    hepatocytes and liver  in vivo in a concentration- and dose-dependent
    manner (Abel & Gelderblom, 1998). The increased susceptibility to
    lipid peroxidation may be a consequence of the other lipid changes
    described above.

    7.3.3  Other biochemical changes

         Numerous studies using fumonisins have found changes in cellular
    regulation and cell function (Table 6). Many of these effects could be
    relevant to the organ toxicity of fumonisins.

         In conclusion, fumonisin-induced disruption of sphingolipid
    metabolism is observed both  in vitro and  in vivo. With the
    exception of primary rat hepatocytes, disruption of sphingolipid
    metabolism is closely correlated in both a time- and
    concentration-dependent manner with alterations in cell proliferation
    and increased cell death.  In vivo, evidence  for  disruption  of 
    sphingolipid  metabolism is closely correlated with the onset and
    progression of  F. verticillioides-associated diseases in pigs,
    horses, rabbits, mice and rats. However, disrupted sphingolipid
    metabolism is also observed in tissues that are not considered target
    organs (i.e., pig and horse kidney, pig heart, endothelial cells).
    Thus, fumonisin-induced disruption of sphingolipid metabolism could
    contribute both directly and indirectly to the diseases known to be
    caused by consumption of fumonisins. Fumonisins also affect other
    sites of cellular regulation that are apparently independent of the
    disruption of sphingolipid metabolism. However, disruption of various
    aspects of lipid metabolism and signal transduction pathways mediated
    by lipid second messengers appears to be an important aspect of all
    the various proposed mechanisms of action.

    7.4  Factors modifying toxicity; toxicity of metabolites

         Voss et al. (1996c) found that nixtamalization of
     F. verticillioides (MRC 826) culture material effectively eliminated
    FB1, but the resulting material (containing hydrolysed FB1) was less
    hepatotoxic but equally nephrotoxic when fed to rats. In a recent
    abstract, it was reported that pure FB1 at 50.5 and 101 mg/kg diet
    (70 and 140 µmol/kg diet) was toxic to female B6C3F1/Nctr mice when
    fed for 28 days, but FB2, FB3 and AP1 were not hepatotoxic (Howard
    et al., 1999). These considerations are important in the evaluation of
    the potential of calcium hydroxide treatment for the detoxification of
    fumonisin-contaminated maize (Sydenham et al., 1995).

    Table 6.  Summary of studies using fumonisins that have found
              changes in cellular regulation and cell function


    *   repression of expression of protein kinase C (PKC), AP-1-dependent
        transcription, stimulation of a cyclic AMP response element in CV-I
        African green monkey kidney cells (1-10 µM FB1, 3 to 16 h) (Huang et
        al., 1995)

    *   decreased phorbol dibutyrate binding, increased cytosolic PKC
        activity, with both exogenous sphinganine and FB1 to J774A.1 cells
        (Smith et al.,1997)

    *   inhibited phorbol dibutyrate binding in short-term incubations using
        crude cerebrocortical membrane preparation and both FB1 and exogenous
        sphingosine (Yeung et al., 1996)

    *   activation of the mitogen-activated protein kinase (MAPK) in Swiss
        3T3 cells with FB1 (Wattenberg et al., 1996)

    *   inhibition of serine/threonine phosphatases (PP5, IC50 of 80 µM) in
        isolated enzyme preparations (Fukuda et al., 1996)

    *   over-expression of nuclear cyclin D1 and increased cyclin-dependent
        kinase 4 (CDK4) activity in rat livers obtained from a long-term
        feeding study and a 21-day feeding study with FB1 (an abstract
        Ramljak et al., 1996)

    *   dephosphorylation of the retinoblastoma protein, repression of CDK2,
        and induction of two CDK inhibitors in CV-1 cells with FB1 
        (Ciacci-Zanella et al., 1998)

    *   apoptosis inhibitor and protease inhibitor protection of CV-1 cells
        and primary human cells from FB1-induced apoptosis (Ciacci-Zanella
        & Jones, 1999)

    *   increased TNF secretion in LPS-activated intraperitoneal macrophages
        from FB1-treated mice (Dugyala et al., 1998)

    *   altered calcium homeostasis in frog  (Rana esculenta) atrial muscle
         in vitro (Sauviat et al., 1991)

    *   glutathione depletion and lipid peroxidation in cultured cells
        (Azuka et al., 1993; Kang & Alexander, 1996; Sahu et al., 1998;
        Abado-Becognee et al., 1998; Yin et al., 1998) and  in vivo (Lim et
        al., 1996; Abel & Gelderblom, 1998)

    *   stimulation of nitric oxide production (Rotter & Oh, 1996)

         In naturally contaminated maize, the simultaneous occurrence of
    multiple fumonisins is likely. Therefore, the relative toxicity of the
    various fumonisins is of concern for hazard assessment. Gelderblom et
    al. (1993) found that the aminopentols (AP1 and AP2) of FB1 and
    FB2 did not act as cancer initiators in orally dosed male Fischer
    rats, although they were more toxic than the parent compounds in
    primary cultures of rat hepatocytes. AP1 is less toxic than FB1
    (Flynn et al., 1994, 1997). The aminopentols of FB1, FB2 and FB3
    are also less effective inhibitors of sphinganine  N-acyltransferase
    in rat primary hepatocytes and liver slices (Merrill et al., 1993c;
    Norred et al., 1997). In gestation day 9.5 rat embryos exposed
     in vitro to 0, 3, 10, 30, 100 or 300 µM AP1 throughout the entire
    45-h cultured period, significant increase in the incidence of
    abnormal embryos including neural tube defects (NTD) were observed at
    concentrations of 100 µM and above (Flynn et al., 1997). A recent
    study by Norred et al. (1997) found that the following mycotoxins had
    no effect on sphinganine levels in rat liver slices: aflatoxin B1,
    cyclopiazonic acid, beauvericin, T-2 toxin, sterigmatocystin,
    luteoskyrin, verrucarin A, scirpentriol and zearalenone. Fumonisins
    FB1, FB2, FB3, FB4, FC4, and hydrolysed FB1, FB2, FB3 and
    Aal-toxin all caused significant elevation in free sphinganine (Norred
    et al., 1997). Fumonisin B4, C4 and AAl-toxin are the most effective
    inhibitors of sphinganine  N-acyltransferase based on sphinganine
    accumulation in rat liver slices (Norred et al., 1997). However, their
    toxicity  in vivo is unknown. Pure FB3 was less effective than FB1
    or FB2 in causing reduced weight gain in rats (Gelderblom et al.,
    1993) but FB2 and FB3 are equally effective inhibitors of
    sphinganine  N-acyltranferase (Norred et al., 1997). A diet
    containing FB3 was less effective than a diet containing FB2 in
    inducing ELEM in ponies (Ross et al., 1994). The ability of diets
    containing FB3 to disrupt sphingolipid metabolism  in vivo was less
    than that of diets containing FB2 (Riley et al., 1997). However, in
    primary rat hepatocytes, FB3 was found to be more cytotoxic
    (Gelderblom et al., 1993). Acetylated FB1 had no effect (relative to
    controls) on weight gain in rats nor did it have any cancer-initiating
    activity (Gelderblom et al., 1993). It does not cause sphinganine
    accumulation in liver slices (Norred et al., 1997) but did cause
    sphinganine accumulation in a study with primary rat hepatocytes (van
    der Westhuizen et al., 1998).

         Because fumonisins occur naturally in combination with other
    fungal toxins (Chu & Li, 1994; Bottalico et al., 1995; Logrieco et
    al., 1995; Yamashita et al., 1995; Ueno et al., 1997), the possibility
    of toxic synergisms exists. There are numerous reports of additive
    effects but the dosages have been much greater than those known to
    occur naturally (Kubena et al., 1995a,b). Toxic synergisms have been
    reported in growing pigs fed diets containing culture material with
    FB1 (50 mg/kg diet) and deoxynivalenol-contaminated wheat (4 mg/kg
    diet) (Harvey et al., 1996) and in a similar study with aflatoxin (2.5
    mg/kg diet) and FB1 (100 mg/kg diet) culture material (Harvey et al.,

         Several reports have been published indicating that no toxic
    synergism is observed in poultry. In turkeys fed a ration containing
    200 mg FB1 and 100 mg moniliformin/kg diet from 1 to 21 days of age,
    no additive or synergistic effects were observed (Bermudez et al.,
    1997). Female turkey poults (Nicholas Large Whites) from day of hatch
    to 3 week of age fed diets containing 300 mg FB1, as well as 4 mg
    diacetoxyscirpenol or 3 mg ochratoxin A, exhibited additive or less
    than additive toxicity, but not toxic synergy (Kubena et al., 1997a).
    In male broiler chicks from day of hatch to 19 or 21 days of age fed
    diets containing 300 mg FB1, as well as 5 mg T-2 toxin/kg diet or 15
    mg deoxynivalenol/kg diet from naturally contaminated wheat, toxic
    synergy was not observed for either of these toxin combinations
    (Kubena et al., 1997b).

         Fumonisins inhibit the  in vitro biosynthesis of
    glycosphingolipid receptors for cholera toxin and shiga-like toxins
    (Sandvig et al., 1996) and inhibit the accumulation of
    glycosphingolipids believed to be responsible for multidrug-resistance
    in certain cancer cells (Lavie et al. 1996). Glycosphingolipids are
    known to be receptors and adhesion sites for viruses, bacteria and


         There has been one report of a disease outbreak characterized by
    abdominal pain, borborygmi and diarrhoea in India suspected to be
    associated with foodborne FB1 (Bhat et al., 1997).

    8.1  Transkei, South Africa

         The only studies available were correlation studies, most of
    which indicated some relationship between oesophageal cancer rates and
    the occurrence of  F. verticillioides (IARC, 1993).

         A very high incidence of oesophageal cancer among the black
    population of the Transkei, South Africa, has been reported in several
    surveys (Jaskiewicz et al., 1987c; Makaula et al., 1996), some of
    which have been reviewed by IARC (1993). The incidence was higher in
    both sexes in the south (Butterworth and Kentani Districts) compared
    to the northern parts of the region (Bizana and Lusikisiki Districts).

         Based on the performance of hybrids in small experimental plots,
    maize grows well in both areas (Rheeder et al., 1994). The sites are
    about 200 km apart and the northern area is about 500 m higher.
    Although some soil fertility factors were different between the high
    and low oesophageal cancer areas, there is no evidence that any
    nutrient was limiting at least for hybrid maize production (Rheeder et
    al., 1994). Farmers grow open-pollinated maize of varying genotypes
    passed on from farm to farm and season to season. Kernel types include
    large flour-maize kernels as well as dent and flint-type white, yellow
    and blue kernels. Both areas depend on home-grown maize for around
    50-100% of the year's supply, the remaining being purchased from
    commercial sources or imports.

         Maize porridge is the staple diet (up to 100% of calories).
    Adults also consume beer deliberately made from mouldy maize selected
    by the housewife from the harvest. This maize has been found to
    contain up to 118 mg/kg fumonisins (Rheeder et al., 1992). Based on
    experiments conducted on beer made from wort containing added FB1
    (Scott et al., 1995), such beers could contain fumonisin
    concentrations of 30 mg/litre beer.

         Contamination of home-grown maize in Transkei by a number of
    toxigenic  Fusarium species, particularly  F. verticillioides, has
    been observed since the early 1970s (Marasas et al., 1979a, 1981,
    1988b, 1993; Marasas, 1993, 1994, 1995, 1996, 1997). Another
     Fusarium species associated with maize in Transkei is
     F. graminearum, and the mycotoxins deoxynivalenol and zearalenone
    produced by this fungus occur in home-grown maize intended for human
    consumption (Marasas et al., 1979b). However, the occurrence of
     F. graminearum in maize kernels was found to be greater in low-risk
    than in high-risk areas for oesophageal cancer in later studies
    (Marasas et al., 1981, 1988b), and Sydenham et al. (1990b) confirmed
    that deoxynivalenol and zearalenone levels were significantly higher

    in home-grown maize from areas with low rates of oesophageal cancer
    than from those with high rates.

         In contrast, the occurrence of  F. verticillioides in maize
    kernels was significantly correlated to oesophageal cancer rates.
    However, the rules for selection of families was different between the
    two populations. The prevalence of  F. verticillioides was greater in
    home-grown maize collected in 12 households in the high-incidence area
    compared to a similar collection in a low-incidence area. Households
    in the high-incidence area were selected on the basis of cytological
    examination of cells collected from the oesophagus (Marasas et al.,
    1988b). Subsequent studies conducted after the chemical
    characterization of the fumonisins in 1988 also found significantly
    higher levels of  F. verticillioides and fumonisins (20 times higher)
    in areas with high rates of oesophageal cancer in Transkei than in
    areas with low rates (Sydenham et al., 1990a,b; Rheeder et al., 1992).

          F. verticillioides and  F. graminearum cause maize ear disease
    under quite different ecological conditions. The environmental
    conditions that prevail in the areas in the Transkei with high rates
    of oesophageal cancer clearly favour colonization of maize ears by
     F. verticillioides. Significant fumonisin accumulation in maize
    occurs periodically in all such environments examined so far,
    primarily in relation to drought and other environmental stressors.
    Taking this into account, the South African studies have shown that
    the level of fumonisin in home-grown maize has been consistently high
    in the areas in the Transkei with high rates of oesophageal cancer.
    Cancer registry data have shown these areas to have consistently high
    rates of oesophageal cancer since 1955 (Jaskiewicz et al., 1987c;
    Makaula et al., 1996).

    8.2  China

         The only studies available were correlation studies where there
    was no clear picture on the association of either fumonisin or
     F. verticillioides contamination with oesophageal cancer.

         Maize is consumed as a staple in a number of areas in China,
    including Linxian and Cixian counties in Henan province (Zhen, 1984;
    Chu & Li, 1994; Yoshizawa et al., 1994). Mortality rates for males in
    the high-risk areas ranged from 26 to 36 per 100 000 in the low-risk
    counties and from 76 to 161 per 100 000 in the high-risk counties. The
    incidence of  F. verticillioides has been reported to be higher in
    maize in high- than low-risk areas, but the mycological data are
    fragmentary (Zhen, 1984) and difficult to evaluate.

         Maize samples from Linxian and Cixian Counties, both
    high-incidence areas of oesophageal cancer in China, were analysed for
    FB1 by Chu & Li (1994). All 31 samples contained FB1 at levels
    ranging from 18 to 155 mg/kg. These results established that
    home-grown maize in high incidence areas of oesophageal cancer in
    China may be contaminated with very high levels of FB1. Another

    investigation carried out on 246 maize samples showed that people
    residing in an area with high incidence of human oesophageal cancer
    are more exposed to fumonisins, although the exposure varied greatly
    (Zhang et al., 1997). However, no relationship between fumonisin and
    human oesophageal cancer incidence was evident from this study. In a
    comparative study of FB1 levels in maize from high (Linxian) and low
    (Shangqiu) oesophageal cancer areas, Yoshizawa et al. (1994) found no
    significant differences between the areas, which was further confirmed
    in a subsequent study (Gao & Yoshizawa, 1997). Levels of FB1 in 13/27
    samples from the high incidence area were 0.18-2.9 (mean 0.87) mg/kg,
    and in 5/20 samples from the low incidence areas, FB1 levels were
    0.19-1.7 (mean 0.89) mg/kg (Yoshizawa et al., 1994).

         In a comparative study of maize samples from high-risk (Haimen)
    and low-risk (Penlai) areas for human primary liver cancer in China,
    Ueno et al. (1997) reported significantly higher levels ( P < 0.01)
    of total fumonisins in the high- than the low-risk area. Fumonisin
    levels in 80/120 samples from the high-risk area for liver cancer were
    0.14-34.9 mg/kg and from the low-risk area were 0.08-15.1 mg/kg in
    54/120 samples for 2 of the 3 years under investigation (Ueno et al.,

    8.3  Northern Italy

         One analytical study was reported from Northern Italy.

         Pordenone Province in the northeast of Italy has the highest
    mortality rate for oral and pharyngeal cancers and oesophageal cancer
    in Italy and amongst the highest in Europe (Franceschi et al., 1990).
    Risk factors identified included alcohol and tobacco use, and
    significant associations with maize consumption were found for oral
    cancer (179 cases; odds ratios 3.3; confidence intervals 2.0-5.3),
    pharyngeal cancer (170; 3.2; 2.0-5.3) and oesophageal cancer (68; 2.8;
    1.5-5.1). There were 505 hospital controls. The elevated risk of upper
    digestive tract cancer was, however, limited to persons consuming more
    than 42 weekly drinks of alcohol (Franceschi et al., 1990). The
    possibility of reporting bias can not be excluded and no measures of
    fumonisin or  F. verticillioides contamination were available. The
    analysis was restricted to men.

         In this region, most maize is locally produced and eaten as
    cooked maize meal (polenta). Fumonisin-producing  Fusarium species
    were found on maize produced in Northern Italy (Logrieco et al.,
    1995). One study showed that 20 samples of polenta produced in Italy
    in 1993 and 1994 contained 0.15-3.76 mg FB1/kg (Pascale et al.,


    9.1  Microorganisms

         In the only available study on the effects of fumonisins on
    bacteria, Becker et al. (1997) reported that FB1 at concentrations
    from 50 to 1000 µM (36-721 mg/litre) did not inhibit the growth of
    various Gram-positive and Gram-negative bacteria. There was also no
    indication that FB1 was metabolized by any of the bacteria tested.

         Fumonisin was reported not to affect ethanol production
    (presumably by  Saccharomyces) in distillers wash made from maize
    contaminated with 15 and 36 mg FB1/kg (Bothast et al., 1992).
    Fumonisin concentrations of 25-100 mg/litre resulted in altered
    sphingolipid precursors in  Pichia ciferri (Kaneshiro et al., 1992).
    This species accumulated some trihydro fatty acids in the presence of
    50 mg FB1/litre. However, in  Rhodotorula species, FB1 depressed
    production of the same compounds (Kaneshiro et al., 1993). Pure FB1
    inhibits cell growth of  Saccharomyces cerevisiae and causes
    accumulation of free sphingoid bases and disruption of lipid
    metabolism (Wu et al., 1995).

    9.2  Plants

    9.2.1  Duckweed and jimsonweed

         Because of their structural similarity to AAL toxins from
     Alternaria alternata f.sp.  lycopersici (also called TA toxins;
    Bottini et al., 1981; Mirocha et al., 1992), fumonisins were suspected
    of being phytotoxic and virulence factors by several investigators.
    Fumonisin reduced chlorophyll synthesis by 59% in duckweed  (Lemma 
     minor) fronds at 10-6 M (Vesonder et al., 1992). Photobleaching
    occurred in excised jimsonweed  (Datura stramonium) leaves, also in
    the µM range, and at approximately 10-4 M damage to mesophyll cells
    occurred after 6 h (Abbas et al., 1992). Fumonisin apparently causes
    membrane damage, as shown by electrolyte loss in jimsonweed (Abbas et
    al., 1991, 1993). Additionally, fumonisin disrupts the synthesis of
    sphingolipids in these plants (Abbas et al., 1994).

    9.2.2  Tomato

         FB1 has similar toxicity to AAL-toxin-susceptible tomato
    cultivars and is not active against AAL-resistant lines. Leaf necrosis
    was reported at 0.4 µM by Mirocha et al. (1992) and at > 0.1 µM by
    Lamprecht et al. (1994). Fumonisins were reported to cause a
    dose-dependent reduction in shoot and root length and dry mass in
    tomato seedlings (Lamprecht et al., 1994). As with duckweed, fumonisin
    has been shown to disrupt sphingolipid metabolism in tomato (Abbas et
    al., 1994). In contrast, FB1 added directly to excised shoots has
    been reported to induce callus and roots at what appear to be high
    doses (Bacon et al., 1994).

    9.2.3  Maize

         Despite reports to the contrary (Abbas & Boyette, 1992), FB1 is
    toxic to maize cells. FB1 exposure did not reduce maize seed
    germination but reduced radicle elongation when the solution
    concentration was above 10-4 M, and seed amylase production was
    inhibited (Doehlert et al., 1994). Fumonisin at concentrations in the
    10 µM range decreased shoot length, shoot dry mass and root length
    (Lamprecht et al., 1994). FB1 incorporated into plant tissue culture
    media reduced the growth of maize callus at 10-6 M (Van Asch et al.,
    1992). FB1 has been shown to disrupt sphingolipid metabolism in maize
    seedlings (Riley et al., 1996). In crosses of high- and
    low-fumonisin-producing strains of  F. verticillioides, only progeny
    that produced high concentrations of fumonisin  in vitro caused
    significant stem rot (Nelson et al., 1993). These data provide
    indirect evidence that fumonisins play a role in the pathogenicity of
     F. verticillioides to maize (Miller, 1995).


    *    There is urgent need for an internationally available standard of
         pure fumonisin B1.

    *    An understanding of the fate of fumonisins in maize food
         processing and cooking, particularly in developing countries, is
         urgently required.

    *    There is urgent need to develop a validated biomarker for human
         exposure to fumonisin.

    *    Epidemiological studies on the effects of fumonisins on human
         health need to be conducted, based on sound intake estimates and

    *    Valid methods for sampling for fumonisins in maize and for
         sampling, extracting and quantifying fumonisins in foods need to
         be developed.

    *    The influence of fumonisin on the carcinogenicity of other
         agriculturally important mycotoxins (e.g., aflatoxin) and
         carcinogenic infectious agents requires further study.

    *    The importance of other routes of exposure to fumonisins,
         including occupational exposure through inhalation, needs to be

    *    There is urgent need for increased research on non-carcinogenic
         end-points including hepatotoxicity, nephrotoxicity,
         neurotoxicity, immunotoxicity, gastrointestinal toxicity,
         cardiovascular toxicity, and the mechanistic basis for the
         organ-selective toxicities.

    *    Research is needed to assess further the genotoxicity of
         fumonisin in both germ and somatic cells  in vitro and
          in vivo.

    *    The basis for the sex differences in animals in the response to
         fumonisin requires further investigation.

    *    The environmental fate of fumonisin in the ecosystem needs to be


         The International Agency for Research on Cancer evaluated FB1 in
    1992 (IARC, 1993) and reached the following conclusions.

         There is  inadequate evidence in humans for the carcinogenicity
    of toxins derived from  F. verticillioides. 

         There is  sufficient evidence in experimental animals for the
    carcinogenicity of cultures of  F. verticillioides that contain
    significant amounts of fumonisins.

         There is  limited evidence in experimental animals for the
    carcinogenicity of FB1.

         Overall Evaluation: Toxins derived from  Fusarium 
     verticillioides are  possibly carcinogenic to humans (Group 2B).


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         The US Food and Drug Administration, Center for Veterinary
    Medicine unofficial guidelines (Miller et al., 1996) recommend that
    the non-roughage portion of feeds for equine species should be less
    than 5 ppm FB1 (< 5 mg/kg), for porcine species the total diet
    should contain less than 10 ppm FB1 (< 10 mg/kg), for beef cattle
    the non-roughage portion should be less than 50 ppm FB1
    (< 50 mg/kg), and for poultry the complete feed should contain less
    than 50 ppm FB1 (< 50 mg/kg).

         An official tolerance value for dry maize products (1 mg/kg FB1
    plus FB2) has been issued in Switzerland (Swiss Federal Office of
    Public Health, 1997).


    Product           Country        Positive/     FB1           Reference
                                     total         (mg/kg)

    North America
    maize             Canada         1/1           0.08          Stack & Eppley
    maize             Canada         9/98          <1-2.5        Miller et al. (1995)
    maize flour       Canada         1/2           0.05          Sydenham et al.
    maize feed        USA            3/3           37-122        Wilson et al. (1990)
    maize feed        USA            2/2           12-130        Plattner et al. (1990)
    maize feed        USA            81/93         <1-126        Ross et al. (1991a)
    maize feed        USA            158/213       <1-330        Ross et al. (1991b)
    maize feed        USA            15/15         1.3-5.2       Thiel et al. (1991b)
    maize feed        USA            2/2           105-155       Colvin & Harrison
    maize feed        USA            29/29         3-330         Osweiler et al. (1992)
    maize feed        USA            20/21         <1-73 a       Bane et al. (1992)
    maize             USA            6/6           1.7-196.5     Stack & Eppley
     screenings                                                  (1992)
    maize feed        USA            1/1           86.0          Park et al. (1992)
    maize feed        USA            14/14         1.3-27.0      Sydenham et al.
    maize feed        USA            160/160       0.1-239       Murphy et al. (1993)
    maize feed        USA            85/85         2.6-32 a      Price et al. (1993)
    maize feed        USA            5/5           0.22-1.41     Hopmans & Murphy
    maize feed        USA            0/29          -             Chamberlain et al.
    maize feed        USA            0/1           -             Holcomb et al. (1993)
    maize feed        USA            5/5           0.77-6.2      Rumbeiha & Oehme
    maize             USA            6/6           0.14-16.31    Stack & Eppley (1992)
    maize             USA            13/99         1.2-3.2       Price et al. (1993)
    maize             USA            155/175       < 1-37.9      Murphy et al. (1993)
    maize             USA            24/28         av. 0.87      Chamberlain et al.
    maize             USA            116/322       1-> 10        Shelby et al. (1994a)
    maize flour       USA            13/25         0.05-0.35     Rumbeiha & Oehme
    maize flour       USA            7/7           0.40-6.32     Pestka et al. (1994)
    maize flour       USA            15/16         0.05-2.79     Sydenham et al.
    maize flour       USA            16/16         0.28-2.05     Stack & Eppley

    APPENDIX 2.  (contiued)

    Product           Country        Positive/     FB1           Reference
                                     total         (mg/kg)

    maize flour       USA            6/6           0.21-0.84     Hopmans & Murphy
    maize grits       USA            10/10         0.11-2.55     Sydenham et al.
    maize grits       USA            5/5           0.14-0.27     Stack & Eppley
    maize flakes      USA            0/2           -             Sydenham et al.
    maize flakes      USA            2/5           0.01          Stack & Eppley
    maize cereals     USA            7/12          0.06-0.33     Stack & Eppley
     (bran, fibre,                                               (1992)
    popcorn           USA            1/18          0.07          Rumbeiha & Oehme
    sweet maize       USA            1/1           0.026         Hopmans & Murphy
    sweet maize       USA            37/97         0.004-0.35    Trucksess et al.
    tortillas         USA            1/3           0.05-0.06     Sydenham et al.
    miscellaneous     USA            4/4           0.09-0.70     Sydenham et al.
     maize foods b                                               (1991)
    miscellaneous     USA            6/11          0.01-0.12     Stack & Eppley
     maize foods b                                               (1992)
    miscellaneous     USA            4/4           0.02-0.32     Hopmans & Murphy
     maize foods b                                               (1993)
    miscellaneous     USA            3/5           0.05-1.21     Pestka et al. (1994)
     maize foods b

    Latin America
    maize             Argentina      17/17         1.11-6.70     Sydenham et al.
    maize             Argentina      51/51         0.18-27.05    Visconti et al.
                                                                 (1995); Ramirez et
                                                                 al. (1996)
    maize feed        Brazil         20/21         0.2-38.5      Sydenham et al.
    maize             Brazil         47/48         0.6-18.5      Hirooka et al. (1996)
    tortillas         Texas-Mexico   50/52         av. 0.19      Stack (1998)
    masas             Texas-Mexico   8/8           av. 0.26      Stack (1998)

    APPENDIX 2.  (contiued)

    Product           Country        Positive/     FB1           Reference
                                     total         (mg/kg)

    maize flour       Peru           1/2           0.66          Sydenham et al.
    alkali-treated    Peru           0/2           -             Sydenham et al.
     kernels                                                     (1991)
    maize feed        Uruguay        13/13         0.26-6.3      Piñeiro et al. (1997)
    maize             Uruguay        11/22         0.17-3.7      Piñeiro et al. (1997)
    maize snacks      Uruguay        4/10          0.15-0.31     Piñeiro et al. (1997)
    frozen maize      Uruguay        1/7           0.16          Piñeiro et al. (1997)
    polenta           Uruguay        3/12          0.1-0.43      Piñeiro et al. (1997)
    maize flour       Venezuela      1/1           0.07          Stack & Eppley

    maize             Austria        6/9           1-15          Lew et al. (1991)
    maize flour       Austria        -/3 f         0.05-1.15     Sydenham et al.
    maize flour       Bulgaria       -/15 f        0.05-0.21     Sydenham et al.
    maize             Croatia        11/19         0.01-0.06     Doko et al. (1995)
    maize flour       Czech          22/22         0.01-0.49 a   Ostry & Ruprich
    maize pastes      Czech          6/11          0.01-0.51 a   Ostry & Ruprich
    maize-extruded    Czech          30/35         0.01-1.8 a    Ostry & Ruprich
     bread                                                       (1998)
    polenta           Czech          6/7           0.01-1.2 a    Ostry & Ruprich
    porridge          Czech          18/19         0.01-0.79 a   Ostry & Ruprich
    maize feed        France         43/58         0.02-8.82     Doko et al. (1994)
    maize feed        France         35/35         0.02-2.17     Dragoni et al. (1996)
    maize flour       France         1/1           1.24          Sydenham et al.
    miscellaneous     France         10/22         0.02-1.50     Visconti et al. (1995)
     maize foods b
    maize             Germany        49/458        0.007-4.83    Meister et al. (1996)
    imported maize    Germany        21/21         0.014-1.11 a  Meister et al. (1996)
    maize grits       Germany        1/2           0.01          Usleber et al. (1994a)
    grits flour,      Germany        60/71         0.01-16.00    Usleber et al.
     semolina                                                    (1994b) c
    semolina          Germany        10/11         0.01-1.23     Usleber et al. (1994a)
    popcorn           Germany        13/29         0.01-0.16     Usleber et al.
                                                                 (1994b) c

    APPENDIX 2.  (contiued)

    Product           Country        Positive/     FB1           Reference
                                     total         (mg/kg)

    popcorn           Germany        4/6           0.01-0.11     Usleber et al. (1994a)
    infant foods      Germany        0/91          -             Usleber et al.
                                                                 (1994b) c
    maize             Hungary        56/92         0.05-75.10    Fazekas et al. (1998)
    maize feed        Italy          23/25         0.02-8.40     Minervini et al. (1992)
    maize screen      Italy          3/3           55.2-70.0     Caramelli et al. (1993)
    maize feed        Italy          1/1           60            Doko & Visconti
    maize             Italy          7/7           0.1-5.3       Doko & Visconti
    maize             Italy          6/6           125-250       Bottalico et al. (1995)
    maize             Italy          26/26         0.01-2.33     Doko & Visconti
     genotypes                                                   (1994)
    commercial        Italy          7/7           0.10-5.31     Doko & Visconti
     maize kernels                                               (1994)
    maize flour       Italy          7/7           0.42-3.73     Doko & Visconti
    maize grits       Italy          1/1           3.76          Doko & Visconti
    polenta           Italy          20/20         0.15-3.76     Pascale et al. (1995)
    popcorn,          Italy          6/10          0.01-0.06     Doko & Visconti
     maize flakes,                                               (1994)
     tortilla chips
    extruded maize    Italy          6/6           0.79-6.10     Doko & Visconti
    sweet maize       Italy          5/5           0.06-0.79     Doko & Visconti
    maize flour       Netherlands    5/7           0.008-0.09    de Nijs et al. (1998c)
    maize for bread   Netherlands    8/19          0.008-0.38    de Nijs et al. (1998c)
    maize for         Netherlands    2/10          0.008-0.11    de Nijs et al. (1998c)
    maize foods       Netherlands    12/42         0.008-1.43    de Nijs et al. (1998c)
    imported maize    Netherlands    61/62         0.03-3.35     de Nijs et al. (1998b)
    maize             Poland         2/7           0.01-0.02     Doko et al. (1995)
    maize             Portugal       9/9           0.09-2.30     Doko et al. (1995)
    maize             Romania        3/6           0.01-0.02     Doko et al. (1995)
    maize feed        Spain          136/171       av. 3.3       Castella et al. (1997)
    maize kernels     Spain          1/1           0.72          Visconti et al. (1995)
    maize flour       Spain          1/3           0.05-0.07     Sanchis et al. (1994)
    maize flour       Spain          16/17         < 0.50 a      Burdaspal & Legarda
    maize grits       Spain          3/15          0.05-0.09     Sanchis et al. (1994)

    APPENDIX 2.  (contiued)

    Product           Country        Positive/     FB1           Reference
                                     total         (mg/kg)

    maize flakes      Spain          2/12          0.05-0.10     Sanchis et al. (1994)
    maize starch      Spain          1/13          0.03          Burdaspal & Legarda
    sweet maize       Spain          3/3           0.72          Visconti et al. (1995)
    miscellaneous     Spain          2/20          0.05-0.20     Sanchis et al. (1994)
     maize foods b
    maize flour       Sweden         1/1           0.13          Visconti et al. (1995)
    popcorn           Sweden         1/1           0.13          Visconti et al. (1995)
    maize feed        Switzerland    6/22          av. 0.24      Pittet et al. (1992)
    maize flour       Switzerland    2/7           av. 0.09      Pittet et al. (1992)
    maize grits d     Switzerland    34/55         av. 0.26      Pittet et al. (1992)
    maize grits,      Switzerland    27/27         0.01-2.20     Zoller et al. (1994)
     flour d
    maize flakes      Switzerland    1/12          0.06          Pittet et al. (1992)
    popcorn           Switzerland    8/13          0.005-0.25    Zoller et al. (1994)
    sweet maize       Switzerland    1/7           0.07          Pittet et al. (1992)
    miscellaneous     Switzerland    0/17          -             Pittet et al. (1992)
     maize foods b
    maize feed        UK             24/29         0.05-4.55     Scudamore & Chan
    maize             UK             65/67         0.03-24       Scudamore et al.
    polenta           UK             16/20         0.02-2.12 a   Patel et al. (1997)
    maize snacks      UK             31/40         0.01-0.22 a   Patel et al. (1997)
    popcorn           UK             6/22          0.01-0.78 a   Patel et al. (1997)

    maize             Benin          9/11          0.02-2.63     Doko et al. (1995)
    maize flour       Botswana       5/5           0.18-0.45     Sydenham et al.
    miscellaneous     Botswana       6/6           0.03-0.35     Doko et al. (1996)
     maize foods b
    maize flour       Egypt          2/2           1.78-2.98     Sydenham et al.
    maize kernels     Kenya          1/1           0.78          Doko et al. (1996)
    maize flour       Kenya          -/3 f         0.05-0.11     Sydenham et al.
    maize kernels     Malawi         7/8           0.02-0.11     Doko et al. (1996)
    maize kernels     Mozambique     3/3           0.24-0.29     Doko et al. (1996)
    maize feed        South Africa   15/15         0.47-4.34     Viljoen et al. (1994)
    mixed feed        South Africa   1/1           8.85          Shephard et al. (1990)
    maize             South Africa   3/3           10-83         Sydenham et al.

    APPENDIX 2.  (contiued)

    Product           Country        Positive/     FB1           Reference
                                     total         (mg/kg)

    maize             South Africa   60/60         0.2-46.9      Sydenham et al.
    maize             South Africae  62/74         0.05-117.5    Rheeder et al. (1992)
    maize             South Africa   24/68         0.05-0.87     Sydenham (1994) c
    maize flour       South Africa   46/52         0.05-0.48     Sydenham et al.
    maize flour       South Africa   2/2           0.06-0.07     Doko et al. (1996)
    maize grits       South Africa   10/18         0.05-0.19     Sydenham et al.
    maize flakes      South Africa   0/3           -             Sydenham et al.
    miscellaneous     South Africa   2/8           0.05-0.09     Sydenham et al.
     maize foods b                                               (1991)
    maize kernels     Tanzania       8/9           0.02-0.16     Doko et al. (1996)
    maize kernels     Uganda         1/1           0.60          Doko et al. (1996)
    maize             Zambia         20/20         0.02-1.42     Doko et al. (1995)
    maize flour       Zambia         1/1           0.74          Doko et al. (1996)
    maize kernels     Zimbabwe       1/2           0.12          Doko et al. (1996)
    maize flour       Zimbabwe       3/3           1.06-3.63     Sydenham et al.
    maize flour       Zimbabwe       4/4           0.05-1.91     Doko et al. (1996)

    maize             China          2/5           5.3-8.4       Ueno et al. (1993)
    maize             China          18/47         0.18-2.9      Yoshizawa et al.
    maize             China          27/68         0.01-1.4      Kang et al. (1994)
    maize             China e        34/34         18-155        Chu & Li (1994)
    maize             China e        134/240       0.08-34.87    Ueno et al. (1997)
    maize             China e        37/54         0.08-21       Gao & Yoshizawa
    maize flour       China          0/3           -             Sydenham et al.
    maize flour       China          3/4           0.06-0.2      Ueno et al. (1993)
    gluten            India          1/1           0.7           Scudamore & Chan
    maize             Indonesia      7/12          0.05-1.8      Yamashita et al.
    maize             Indonesia      16/16         0.05-2.44     Ali et al. (1998)
    maize grits       Japan          14/17         0.20-2.60     Ueno et al. (1993)
    sweet maize       Japan          0/8           -             Ueno et al. (1993)
    maize snack       Japan          0/31          -             Ueno et al. (1993)
    maize soup        Japan          0/7           -             Ueno et al. (1993)

    APPENDIX 2.  (contiued)

    Product           Country        Positive/     FB1           Reference
                                     total         (mg/kg)

    maize feed        Korea          5/12          0.05-1.33     Lee et al. (1994)
    maize             Nepal          12/24         0.05-4.6      Ueno et al. (1993)
    maize             Philippines    26/50         0.05-1.8      Yamashita et al.
    maize             Philippines    9/10          0.3-10.0      Bryden et al. (1996)
    miscellaneous     Taiwan         52/153        0.07-2.39     Tseng & Liu (1997)
     maize foods b
    maize             Thailand       19/27         0.06-18.8     Yamashita et al.
    maize feed        Thailand       5/22          0.05-1.59     Wang et al. (1993)
    maize flour       Thailand       6/6           0.48-0.88     Wang et al. (1993)
    maize grits       Thailand       5/5           0.25-1.82     Wang et al. (1993)
    maize             Vietnam        12/12         0.3-9.1       Bryden et al. (1996)
    maize             Vietnam        8/15          0.27-3.45     Wang et al. (1995)
    maize powder      Vietnam        15/17         0.27-1.52     Wang et al. (1995)

    maize             Australia      67/70         0.3-40.6      Bryden et al. (1996)
    maize flour       New Zealand    0/12          -             Sydenham et al.

    a   Fumonisins B1 + B2 + B3
    b   Includes maize snacks, canned maize, frozen maize, extruded maize, bread, 
        maize-extruded bread, biscuit, cereals, chips, flakes, pastes, starch, sweet 
        maize, infant foods, gruel, purée, noodles popcorn, porridge, tortillas, 
        tortilla chips, masas, popped maize, soup, taco, tostada
    c   From: Shephard et al. (1996a)
    d   Maize grits and flour samples analysed were imported cereals (mainly from 
    e   Fumonisin levels in low- and high-risk area for human oesophageal cancer
    f   The number of positive samples was not indicated in the original report

    1.  Résumé

    1.1  Identité, propriétés physique et chimiques et méthodes

         La fumonisine B1 (FB1), de formule brute C34H59NO15, est le
    diester de l'acide propane-1,2,3-tricarboxylique et du
    2-amino-12,16-diméthyl-3,5,10,14,15-pentahydroxyeicosane (masse
    moléculaire relative: 721). C'est la plus abondante des fumonisines,
    qui constituent une famille de toxines dont on a identifié au moins 15
    membres. A l'état pur, ce composé se présente sous la forme d'une
    poudre hygroscopique de couleur blanche, soluble dans l'eau, le
    mélange eau-acétonitrile et le méthanol. Elle est stable dans le
    mélange eau-acétonitrile à 1:1 et instable dans le méthanol. Elle est
    stable aux températures utilisées pour la transformation des denrées
    alimentaires ainsi qu'à la lumière.

         Plusieurs méthodes d'analyse ont été proposées, notamment la
    chromatographie sur couche mince ou en phase liquide, la spectrométrie
    de masse, la chromatographie en phase gazeuse après hydrolyse et un
    certain nombre de méthodes immunochimiques. En fait, la plupart des
    dosages se font par chromatographie en phase liquide d'un dérivé

    1.2  Sources d'exposition humaine

         La FB1 est produite par plusieurs espèces de  Fusarium, mais
    essentiellement par  Fusarium verticillioides (Sacc.) Nirenberg
    (=  Fusarium moniloforme Sheldon) qui est l'un des parasites
    cryptogamiques les plus fréquents du maïs. La FB1 peut s'accumuler en
    quantités importantes dans le maïs lorsque les conditions
    météorologiques sont favorables à l'apparition de la fusariose.

    1.3  Transport, distribution et transformation dans l'environnement

         On est fondé à penser que les fumonisines peuvent être
    métabolisées par certains microorganismes terricoles. On sait
    toutefois peu de choses de leur devenir dans l'environnement une fois
    qu'elles ont été excrétées ou que les produits qui en contiennent ont
    été transformés. 

    1.4  Concentrations dans l'environnement et exposition humaine

         On a mis en évidence de la FB1 dans le maïs et les produits qui
    en dérivent partout dans le monde à des concentrations de l'ordre du
    mg/kg, parfois en association avec d'autres mycotoxines. Des
    concentrations de cet ordre ont également été observées dans des
    denrées alimentaires destinées à la consommation humaine. Lors de la
    mouture à sec du maïs, la fumonisine se répartit dans le son, le germe
    et la farine. Lors d'essais de mouture par voie humide, on a mis en
    évidence la toxine dans l'eau de macération, dans le gluten, dans les

    fibres et les germes, à l'exclusion de l'amidon. La FB1 reste stable
    dans le maïs et la polenta, mais elle s'hydrolyse dans les aliments à
    base de maïs traités par des solutions alcalines à chaud.

         La FB1 est absente du lait, de la viande et des oeufs provenant
    d'animaux nourris avec du maïs dont la teneur en toxine ne représente
    aucun danger pour eux. On estime que l'exposition humaine journalière
    aux Etats-Unis, au Canada, en Suisse, aux Pays-Bas et au Transkei
    (Afrique du Sud) varie entre 0,017 et 440 µg/kg de poids corporel. On
    ne possède aucune donnée sur l'exposition professionnelle par

    1.5  Cinétique et métabolisme chez l'animal

         On ne dispose d'aucune donnée sur la cinétique ou le métabolisme
    de la FB1 chez l'Homme. Chez les animaux de laboratoire, le composé
    est peu résorbé après ingestion; il disparaît rapidement du courant
    sanguin et se retrouve inchangé dans les matières fécales. Il est
    excrété en quantité importante par la voie biliaire et en faible
    proportion dans les urines. Chez les primates non humains et certains
    ruminants, la fumonisine peut subir une dégradation hydrolytique
    partielle dans l'intestin. Elle subsiste en petite quantité dans le
    foie et les reins.

    1.6  Effets sur l'animal et les systèmes d'épreuve in vitro

         La FB1 s'est révélée hépatotoxique pour les espèces animales sur
    lesquelles elle a été testée, notamment le rat, la souris, les
    équidés, le lapin, le porc et les primates non humains. Sauf dans le
    cas du hamster doré, on n'observe d'effets embryotoxiques ou
    tératogènes que concurremment ou postérieurement aux manifestations
    toxiques qui se produisent chez la mère. Les fumonisines sont
    néphrotoxiques chez le porc, le rat, le mouton, la souris et le lapin.
    Dans le cas du rat et du lapin, la néphrotoxicité se manifeste à des
    doses plus faibles que l'hépatotoxicité. On sait que la
    leucoencéphalomalacie équine et l'oedème pulmonaire porcin observés
    après consommation de provendes à base de maïs sont dus à la présence
    de fumonisines. Les données dont on dispose sur les propriétés
    immunologiques de la FB1 sont limitées. A la dose de 50 mg/kg de
    nourriture, le composé a provoqué des cancers du foie chez une souche
    de rats et des cancers du rein chez une autre souche; dans les mêmes
    conditions de dosage et d'administration, il a également provoqué des
    cancers du foie chez des souris femelles. Il semble qu'il existe une
    corrélation entre les effets toxiques sur les organes et l'apparition
    de cancers. La FB1 a été le premier inhibiteur du métabolisme des
    sphingolipides qui ait été découvert et on l'utilise beaucoup depuis
    lors pour étudier le rôle des sphingolipides dans la régulation
    cellulaire. La FB1 inhibe la croissance cellulaire; elle entraîne
    l'accumulation de bases sphingoïdes libres et modifie le métabolisme
    des lipides chez les animaux, les plantes et certaines levures. Elle
    ne provoque pas de mutation génique chez les bactéries et mise en
    présence d'une culture primaire d'hépatocytes de rat, elle n'entraîne

    pas une synthèse non programmée de l'ADN. On a constaté par contre
    qu'elle pouvait provoquer des aberrations chromosomiques à faible dose
    dans ces mêmes cultures cellulaires.

    1.7  Effets sur l'Homme

         On ne dispose d'aucune donnée confirmée relative à la toxicité
    aiguë des fumosinines pour l'Homme. Des études effectuées au Transkei
    (Afrique du Sud) sur la corrélation entre divers effets toxiques et la
    présence de fumonisines dans la ration alimentaire suggèrent
    l'existence d'un lien entre ces composés et le cancer de l'oesophage.
    Cette corrélation a été observée dans des circonstances où il y avait
    une forte exposition aux fumonisines et où les conditions
    environnementales étaient favorables à une importante accumulation de
    toxines dans le maïs, qui constitue une denrée alimentaire de base
    dans la région. Des études du même genre ont également été effectuées
    en Chine. Ces dernières n'ont toutefois pas permis de dégager une
    relation claire entre la contamination par les fumonisines ou
     F. verticillioides et le cancer de l'oesophage. Fautes de données
    concernant l'exposition aux fumonisines, il n'est pas possible non
    plus de tirer de conclusions d'une étude cas-témoins effectuée en
    Italie sur des sujets de sexe masculin et qui révèle l'existence d'une
    association entre la consommation de maïs et les cancers des voies
    digestives supérieures chez les gros buveurs.

         Il n'existe pas de marqueurs biologiques valables de l'exposition
    à la FB1.

    1.8  Effets sur les autres organismes en laboratoire

         La FB1 inhibe la croissance cellulaire et provoque
    l'accumulation de bases sphingoïdes libres ainsi que la modification
    du métabolisme des lipides chez  Saccharomyces cerevisiae. 

         La FB1 est phytotoxique, elle provoque des lésions de la
    membrane cellulaire et réduit la synthèse chlorophyllienne. Elle
    perturbe également la biosynthèse des sphingolipides chez les végétaux
    et pourrait jouer un rôle dans les maladies du maïs dues aux espèces
    de  Fusarium qui produisent des fumonisines

    2.  Evaluation des risques pour la santé humaine

    2.1  Exposition

         L'Homme est exposé partout dans le monde puisque la FB1 est
    présente dans le maïs destiné à la consommation humaine. Toutefois,
    des différences notables existent entre les régions du culture de
    cette céréale. Cette constatation s'impose lorsque l'on compare pays
    développés et pays en développement. Par exemple, même si aux
    Etats-Unis, au Canada et en Europe occidentale la FB1 est présente
    dans les produits tirés du maïs, la consommation de ces produits y
    reste à un niveau modeste. Dans certaines régions d'Afrique,

    d'Amérique centrale et d'Asie, il y a des populations dont l'apport
    calorique alimentaire est constitué pour une large part de farine de
    maïs et la contamination par la FB1 peut y être importante (voir
    Appendice 2). Le maïs naturellement contaminé par la FB1 peut
    également l'être par d'autres toxines de  F. verticillioides ou
     F. proliferatum ou encore par des toxines d'importance agricole
    telles que le désoxynivalénol, la zéaralénone, l'aflatoxine ou

         Les procédés de transformation des denrées alimentaires utilisés
    en Amérique du Nord ou en Europe occidentale n'ont aucun effet sur la
    stabilité de la FB1. Le traitement du maïs par une base ou son lavage
    à l'eau réduisent sensiblement la teneur en toxine. Cependant on
    constate toujours des effets hépatotoxiques et néphrotoxiques chez les
    animaux de laboratoire. On sait peu de choses sur la manière dont les
    techniques de préparation des aliments utilisées dans le monde en
    développement peuvent agir sur la FB1 présente dans les produits
    tirés du maïs.

    2.2  Nature des dangers

         Le rôle étiologique de la FB1 dans la leucoencéphalomalacie
    équine est établi. D'importantes flambées de cette zoonose mortelle se
    sont produites aux Etats-Unis au cours du 19ème siècle et plus
    récemment en 1989-1990. De même, on a également montré que cette
    toxine était à l'origine d'une zoonose tout aussi mortelle, l'oedème
    pulmonaire porcin. Une faible dose de FB1 peut également être
    mortelle pour le lapin, comme on a pu l'observer sur des lapines
    gravides. Chez toutes les espèces animales étudiées, y compris les
    primates non humains, on a constaté que cette toxine provoquait des
    lésions rénales et hépatiques. La FB1 provoque une
    hypercholestérolémie chez plusieurs espèces animales, dont les
    primates non humains. On de bonnes raisons de penser que dans les
    maladies animales dues à une exposition à la FB1, il y a modification
    du métabolisme des lipides. Les effets toxiques observés  in vivo ou
     in vitro sont précédés ou accompagnés d'une perturbation du
    métabolisme des sphingolipides. L'utilisation des fumonisines dans
    l'étude de la fonction des sphingolipides a montré que ces composés
    sont nécessaires à la croissance cellulaire et qu'il peuvent affecter
    les molécules signal de différentes manières, avec pour conséquence la
    mort cellulaire par apoptose ou nécrose, la différenciation cellulaire
    ou encore la modification de la réponse immunitaire. Après exposition
    à la FB1, on observe communément une modification du métabolisme des
    lipides et de l'expression ou de l'activité des enzymes qui jouent un
    rôle clé dans la progression du cycle cellulaire. La FB1 n'exerce pas
    d'effets toxiques sur le développement chez le rat, la souris ou le
    lapin. En revanche, elle est foetotoxique chez le hamster doré à forte
    dose même quand elle n'a pas d'effet sur la mère.

         Chez les rongeurs, la cancérogénicité de la FB1 varie selon
    l'espèce, la souche et le sexe. La seule étude qui ait été effectuée
    sur des souris B6C3F1 a montré que la toxine provoquait des cancers

    du foie chez les femelles à la dose de 50 mg/kg de nourriture. Des
    cancers primitifs du foie et des cholangiomes ont été observés chez
    des rats mâles BD IX qui avaient reçu, pendant une durée allant
    jusqu'à 26 mois, une alimentation contenant 50 mg/kg de FB1. Chez des
    rats mâles F344/N alimentés dans les mêmes conditions de dosage, on a
    mis en évidence des adénomes et des carcinomes au niveau des tubules
    rénaux. Il semble qu'il y ait corrélation entre la toxicité pour tel
    ou tel organe et l'apparition de cancers à ce niveau.

         Les études de génotoxicité sont en nombre limité. Celles qui
    portent sur des bactéries n'ont pas révélé d'effets mutagènes. Dans
    des cultures de cellules mammaliennes on n'a pas non plus décelé de
    synthèse non programmée de l'ADN mais selon une étude, la FB1 a
    provoqué des cassures chromosomiques dans des hépatocytes de rat.
    Selon d'autres travaux, la FB1 accroît la peroxydation des lipides
     in vivo et  in vitro. Il est possible qu'il existe une relation de
    cause à effet entre la peroxydation des lipides et les cassures

         Les teneurs en FB1 supérieures à 100 mg/kg constatées dans le
    maïs consommé par l'Homme en Afrique et en Chine pourraient
    probablement provoquer selon le cas, des leucoencéphalomalacies, des
    oedèmes pulmonaires et des cancers si on donnait à manger ce maïs à
    des chevaux, des porcs, des rats ou des souris. On connaît des cas où
    l'exposition humaine à cette toxine est très importante, mais aucun
    cas d'intoxication aiguë par une fumonisine n'a été décrit. Les études
    de corrélation effectuées au Transkei (Afrique du Sud) incitent à
    penser qu'il pourrait y avoir un lien entre une exposition à la
    fumonisine par voie alimentaire et le cancer de l'oesophage. On a
    effectivement constaté un taux élevé de cancers de l'oesophage là où
    l'exposition à cette toxine était importante et où les conditions
    environnementales étaient favorables à l'accumulation de fumonisines
    dans le maïs, qui constitue un aliment de base dans ces régions.

         Une étude cas-témoins réalisée en Italie a mis en évidence une
    association entre la consommation de maïs et les cancers des voies
    digestives supérieures, et notamment le cancer de l'oesophage chez les
    gros buveurs. Aucune donnée sur l'exposition à la toxine n'a été

    2.3  Relation dose-réponse

         La dose la plus faible capable de provoquer l'apparition de
    cancers du foie chez l'animal de laboratoire est égale à 50 mg par kg
    de nourriture dans le cas de rats mâles BD IX et de souris femelles
    B6C3F1/Nctr; aucun cancer n'a été observé aux doses respectives de 25
    ou 15 mg/kg de nourriture. Dans chaque cas et avec les mêmes souches
    de rats et de souris, on a observé des signes d'hépatotoxicité ou de
    modification du métabolisme des lipides à ces mêmes doses ou à des
    doses inférieures. La dose la plus faible ayant provoqué des cancers
    du rein chez des rats mâles de souche F344/Nctr était égale à 50 mg/kg
    de nourriture; aucun effet cancérogène n'a été observé à la dose de 15

    mg/kg de nourriture. Ces études ont également montré qu'à des doses
    inférieures à celles qui provoquaient l'apparition de cancers, il y
    avait apoptose et prolifération des cellules des tubules rénaux, avec
    des modifications au niveau des sphingolipides tissulaires et

         On ne possède aucune donnée qui puisse permettre d'établir une
    relation quantitative entre l'exposition à la FB1 et d'éventuels
    effets sur l'organisme humain.

    2.4  Caractérisation du risque

         La FB1 est cancérogène pour la souris et le rat et alle provoque
    des maladies mortelles chez le porc et le cheval à des concentrations
    auxquelles l'Homme pourrait être exposé. Le Groupe de travail n'a pas
    été en mesure de formuler une estimation quantitative du risque pour
    la santé humaine mais il a été d'avis qu'il y avait urgence à cet

    3.  Recommandations pour la protection de la santé humaine

    a)   Il faudrait établir les limites d'exposition par voie
         alimentaire. On devra s'attacher en particulier aux populations
         dont l'apport calorique provient pour une grande part de la
         farine de maïs.

    b)   Des mesures devraient être prises pour limiter l'exposition aux
         fumonisines et la contamination du maïs par ces toxines; elles
         pourraient consister

         *    à changer de culture là où le maïs n'est pas vraiment adapté

         *    à mettre au point des variétés de maïs qui résistent à la

         *    à mieux gérer les cultures

         *    à éliminer les grains parasités

    c)   On veillera à faire prendre conscience suffisamment tôt du risque
         de contamination alimentaire en faisant en sorte qu'il y ait une
         meilleure communication entre les vétérinaires et les
         responsables de la santé publique en cas de flambées de
         mycotoxicoses parmi les animaux domestiques.

    d)   Il faudrait mettre au point une méthode de recherche de la
         contamination du maïs par les fumonisines qui soit bon marché,
         simple et peu sensible aux conditions d'application.


    1.  Resumen

    1.1  Identidad, propiedades físicas y químicas y métodos analíticos

         La fumonisina B1 (FB1) tiene la fórmula empírica C34H59NO15 y
    es el diéster del ácido propano-1,2,3-tricarboxílico y el 2-amino-12,
    16-dimetil-3, 5, 10, 14, 15-pentahidroxieicosano (masa molecular
    relativa: 721). Es la más frecuente de las fumonisinas, familia de
    toxinas con 15 miembros identificados por lo menos. La sustancia pura
    es un polvo higroscópico blanco, soluble en agua, acetonitrilo-agua o
    metanol, estable en acetonitrilo-agua (1:1), inestable en metanol y
    estable a la temperatura de elaboración de los alimentos y la luz.

         Se han notificado varios métodos analíticos, en particular la
    cromatografía en capa fina y la cromatografía líquida, la
    espectrometría de masas, la cromatografía de gases poshidrólisis y
    métodos inmunoquímicos, aunque la mayoría de los estudios se han
    realizado utilizando análisis por cromatografía líquida de un derivado

    1.2  Fuentes de exposición humana

         La FB1 se produce en varias especies de  Fusarium, 
    principalmente  Fusarium verticillioides (Sacc.) Niremberg
    (=  Fusarium moniliforme Sheldon), que es uno de los hongos más
    comunes asociados con el maíz en todo el mundo. En el maíz hay una
    acumulación importante de FB1 cuando las condiciones climatológicas
    favorecen la podredumbre del grano debida a  Fusarium. 

    1.3  Transporte, distribución y transformación en el medio 

         Hay pruebas de que algunos microorganismos del suelo pueden
    metabolizar las fumonisinas. Sin embargo, se sabe poco acerca del
    destino de las fumonisinas en el medio ambiente tras la excreción o la

    1.4  Niveles en el medio ambiente y exposición humana

         Se ha detectado FB1 en el maíz y sus productos en todo el mundo
    en concentraciones de varios mg/kg, a veces combinada con otras
    micotoxinas. Se han notificado también concentraciones de mg/kg en
    alimentos para consumo humano. Como consecuencia de la elaboración en
    seco del maíz, la fumonisina se distribuye en el salvado, los gérmenes
    y la harina. En la elaboración en húmedo experimental, se detectó
    fumonisina en el agua de remojo, el glúten, la fibra y los gérmenes,
    pero no en el almidón. La FB1 es estable en el maíz y la polenta,
    mientras que se hidroliza en los alimentos nixtamalizados a base de
    maíz, es decir, alimentos elaborados con soluciones alcalinas

         La FB1 no está presente en la leche, la carne o los huevos de
    animales alimentados con grano que contiene FB1 en concentraciones
    que no afectarían a la salud de los animales. Las estimaciones de la
    exposición humana para los Estados Unidos, el Canadá, Suiza, los
    Países Bajos y el Transkei (Sudáfrica) oscilaron entre 0,017 y
    440 µg/kg de peso corporal al día. No se dispone de datos sobre la
    exposición por inhalación en el puesto de trabajo.

    1.5  Cinética y metabolismo en los animales

         No hay informes sobre la cinética o el metabolismo de la FB1 en
    el ser humano. En animales experimentales se absorbe muy poco cuando
    se administra por vía oral, se elimina rápidamente de la circulación y
    se recupera sin metabolizar en las heces. La excreción biliar es
    importante y se excretan pequeñas cantidades en la orina. Se puede
    degradar a FB1 parcialmente hidrolizada en el intestino de primates
    no humanos y en algunos rumiantes. Se retiene una pequeña cantidad en
    el hígado y el riñón.

    1.6  Efectos en los animales y en los sistemas de prueba in vitro

         La FB1 es hepatotóxica en todas las especies animales sometidas
    a prueba, en particular ratones, ratas, équidos, conejos, cerdos y
    primates no humanos. Con la excepción de los hámsteres sirios, sólo se
    observa embriotoxicidad o teratogenicidad cuando se produce toxicidad
    materna o después de ella. Las fumonisinas son nefrotóxicas en cerdos,
    ratas, ovejas, ratones y conejos. En ratas y conejos se produce
    toxicidad renal a dosis inferiores a las de la hepatotoxicidad. Se
    sabe que las fumonisinas producen leucoencefalomalacia equina y
    síndrome de edema pulmonar porcino, asociados ambos con el consumo de
    piensos a base de maíz. Es limitada la información sobre las
    propiedades inmunológicas de la FB1. Fue hepatocarcinogénica para las
    ratas machos de una raza y nefrocarcinogénica en otra raza, utilizando
    la misma dosificación (50 mg/kg de alimentos) y fue
    hepatocarcinogénica con 50 mg/kg de alimentos en ratones hembra.
    Parece haber una correlación entre la toxicidad en los órganos y la
    aparición de cáncer. La FB1 fue el primer inhibidor específico del
    metabolismo de los esfingolípidos  de novo que se descubrió y se está
    utilizando ampliamente en la actualidad para estudiar su función en la
    regulación celular. La FB1 inhibe el crecimiento celular y produce la
    acumulación de bases esfingoideas libres y la alteración del
    metabolismo lipídico en animales, plantas y algunas levaduras. No
    indujo mutaciones génicas en bacterias o síntesis no programada de ADN
    en hepatocitos primarios de rata, pero sí un aumento de las
    aberraciones cromosómicas dependiente de la dosis con concentraciones
    bajas en un estudio sobre los hepatocitos primarios de rata.

    1.7  Efectos en el ser humano

         No hay registros confirmados de toxicidad aguda de la fumonisina
    en el ser humano. Los estudios de correlación disponibles procedentes
    de Transkei (Sudáfrica) parecen indicar una vinculación entre la
    exposición a la fumonisina en los alimentos y el cáncer de esófago.

    Esto se ha observado en lugares donde se ha demostrado una exposición
    relativamente alta a la fumonisina y donde las condiciones ambientales
    favorecen la acumulación de fumonisina en el maíz, que es el alimento
    básico. También hay estudios de correlación de China. Sin embargo, no
    se obtuvo una imagen clara de la relación entre la contaminación bien
    por fumonisina o bien por  F. verticillioides y el cáncer de esófago.
    Debido a la ausencia de datos sobre la exposición a la fumonisina, no
    se puede llegar a ninguna conclusión a partir de un estudio de casos y
    testigos de varones en Italia que mostraba una asociación entre el
    consumo de maíz y el cáncer en la parte superior del aparato
    gastrointestinal en personas con un elevado consumo de alcohol.

         No hay biomarcadores validados para la exposición humana a la

    1.8  Efectos en otros organismos en el laboratorio

         La FB1 inhibe el crecimiento celular y produce acumulación de
    bases esfingoideas libres y la alteración del metabolismo lipídico en
     Saccharomyces cerevisiae. 

         La FB1 es fitotóxica, provoca lesiones en las membranas
    celulares y reduce la síntesis de clorofila. También altera la
    biosíntesis de esfingolípidos en las plantas y puede desempeñar una
    función en la patogenicidad del maíz por las especies de  Fusarium 
    que producen fumonisina.

    2.  Evaluación de los riesgos para la salud humana

    2.1  Exposición

         La exposición humana, demostrada por la presencia de FB1 en el
    maíz para consumo humano, es común en todo el mundo. Hay diferencias
    considerables en el grado de exposición humana entre las diferentes
    regiones de cultivo de maíz. Esto se pone de manifiesto sobre todo
    cuando se establece una comparación entre países plenamente
    desarrollados y en desarrollo. Por ejemplo, aunque puede haber FB1 en
    productos de maíz en los Estados Unidos, el Canadá y Europa
    occidental, el consumo humano de estos productos es pequeño. En
    algunas partes de Africa, América del Sur y Central y Asia, algunas
    poblaciones consumen un elevado porcentaje de sus calorías como harina
    de maíz, cuya contaminación por FB1 puede ser alta (véase el apéndice
    2). El maíz contaminado de forma natural por FB1 puede estar
    contaminado simultáneamente por otras toxinas de  F. verticillioides 
    o  F. proliferatum o por otras toxinas importantes desde el punto de
    vista agrícola, en particular el deoxinivalenol, la zearalenona, la
    aflatoxina y la ocratoxina.

         La FB1 es estable en los métodos de elaboración de alimentos que
    se utilizan en América del Norte y Europa occidental. El tratamiento
    del maíz con bases y/o el lavado con agua reduce de manera eficaz las
    concentraciones de FB1. Sin embargo, en animales experimentales
    siguen siendo evidentes su hepatotoxicidad y/o nefrotoxicidad. Se sabe

    poco acerca de la influencia de las técnicas de elaboración de
    alimentos utilizadas en el mundo en desarrollo en la FB1 en los
    productos de maíz.

    2.2  Identificación de peligros

         Se ha demostrado la función causal de la exposición a la FB1 en
    la leucoencefalomalacia equina. Durante el siglo XIX se produjeron en
    los Estados Unidos brotes en gran escala de esta enfermedad letal, y
    también en épocas tan recientes como 1989-1990. Se ha establecido
    asimismo la función causal de la exposición a la FB1 en la enfermedad
    mortal del edema pulmonar porcino. Tal como se observó en hembras
    preñadas, una exposición baja a la FB1 es letal para los conejos. Se
    ha demostrado que la exposición provoca toxicidad renal y
    hepatotoxicidad en todas las especies animales estudiadas, incluidos
    los primates no humanos. La exposición a la FB1 produce
    hipercolesterolemia en varias especies animales, en particular en
    primates no humanos. Hay pruebas convincentes de que en las
    enfermedades animales asociadas con la exposición a la FB1 se altera
    el metabolismo lipídico. Es manifiesta la perturbación del metabolismo
    de los esfingolípidos antes de la toxicidad  in vitro e  in vivo o
    coincidiendo con ella. El uso de fumonisinas como instrumento para
    estudiar la función de los esfingolípidos ha puesto de manifiesto que
    éstos se requieren para el crecimiento celular y afectan de varias
    formas a la señalización de las moléculas, provocando muerte celular
    apoptótica y necrótica, diferenciación celular y respuestas
    inmunitarias alteradas. Parece que son factores comunes tras la
    exposición a la FB1 la alteración del metabolismo lipídico y los
    cambios en la actividad y/o la expresión de enzimas fundamentales
    encargados del funcionamiento normal del ciclo celular. La FB1 no es
    tóxica para el desarrollo en ratas, ratones o conejos. A dosis
    elevadas sin toxicidad materna induce fetotoxicidad en el hámster

         La carcinogenicidad de la FB1 en roedores varía en función de
    las especies, las razas y el sexo. El único estudio con ratones
    B6C3F1 puso de manifiesto que la FB1 era hepatocarcinogénica para
    las hembras a 50 mg/kg en los alimentos. En ratas BD IX macho que
    recibieron alimentos con 50 mg de FB1/kg durante un período de hasta
    26 meses se observó la inducción de carcinomas hepatocelulares
    primarios y carcinomas colangiales. Se detectaron adenomas y
    carcinomas de los túbulos renales en ratas F344/N Nctr macho a las que
    se suministraron 50 mg de FB1/kg. Parece existir una correlación
    entre la toxicidad en los órganos y la aparición de cáncer.

         El número de estudios de genotoxicidad disponible es limitado. La
    FB1 no fue mutagénica en valoraciones bacterianas. En un estudio
    realizado con células de mamífero  in vitro no se detectó síntesis de
    ADN no programado, pero la FB1 provocó roturas cromosómicas en
    hepatocitos de rata. En otros estudios se ha puesto de manifiesto que
    la FB1 provoca un aumento de la peroxidación de los lípidos  in 

     vivo e  in vitro. Es posible que los efectos de la rotura de los
    cromosomas y la peroxidación de los lípidos tengan una relación

         Las concentraciones de FB1 superiores a 100 mg/kg, notificadas
    en el maíz de consumo humano en Africa y China, probablemente
    provoquen leucoencefalomalacia, síndrome de edema pulmonar o cáncer si
    se administran a caballos, cerdos y ratas o ratones, respectivamente.
    A pesar de estos casos de exposición humana muy alta, no hay datos
    confirmados de intoxicación aguda por fumonisina en personas. Los
    estudios de correlación disponibles del Transkei, Sudáfrica, parecen
    indicar una relación entre la exposición a la fumonisina a través de
    los alimentos y el cáncer de esófago. Se han observado índices
    elevados de cáncer de esófago donde se ha demostrado una exposición
    relativamente elevada a la fumonisina y donde las condiciones
    ambientales favorecen la acumulación de fumonisina en el maíz, que es
    el alimento básico.

         En un estudio de casos y testigos realizado con varones en Italia
    se observó una asociación entre el consumo de maíz y la aparición de
    cáncer en la parte superior del aparato digestivo, incluido el cáncer
    de esófago, entre personas habituadas a un consumo de alcohol alto. No
    había datos sobre la exposición a la fumonisina.

    2.3  Evaluación de la respuesta en función de la dosis

         La dosis más baja de FB1 que indujo hepatocarcinomas en animales
    experimentales fue de 50 mg/kg de alimentos en ratas BD IX macho y en
    ratones B6C3F1/Nctr hembra; no se observó inducción de cáncer con 25
    ó 15 mg/kg de alimentos, respectivamente. En cada caso, se detectaron
    indicios de hepatotoxicidad o alteraciones lipídicas con dosis iguales
    o inferiores en estudios con esas mismas razas de ratas y ratones. La
    dosis más baja de FB1 que indujo carcinomas renales en ratas F344/N
    Nctr macho fue de 50 mg/kg de alimentos; no se observó inducción de
    cáncer con 15 mg/kg de alimentos. Se produjo apoptosis tubular renal y
    proliferación celular, así como cambios en los esfingolípidos
    tisulares y urinarios, con dosis inferiores a las normalmente
    necesarias para la inducción de cáncer en esos estudios.

         No se dispone de datos para evaluar cuantitativamente la relación
    entre la exposición a la FB1 y los posibles efectos en el ser humano.

    2.4  Caracterización del riesgo

         La FB1 es carcinogénica en ratones y ratas e induce enfermedades
    letales en cerdos y caballos en concentraciones de exposición a las
    que están sometidas los seres humanos. El Grupo Especial no estuvo en
    condiciones de realizar una estimación cuantitativa de los riesgos
    para la salud humana, pero consideró que se necesitaba con urgencia
    dicha estimación.

    3.  Recomendaciones para la protección de la salud humana

    a)   Se deben establecer límites para la exposición de las personas a
         través de los alimentos. Se debe prestar particular atención a
         las poblaciones que consumen un porcentaje elevado de sus
         calorías como harina de maíz.

    b)   Se deben adoptar medidas para limitar la exposición a la
         fumonisina y la contaminación del maíz mediante:

         *    plantación de cultivos alternativos en zonas donde el maíz
              no esté bien adaptado;

         *    obtención de maíz resistente a la podredumbre del grano por

         *    aplicación de mejores prácticas de cultivo;

         *    separación de los granos mohosos.

    c)   Se debe aumentar la sensibilización temprana acerca de la
         posibilidad de contaminación de los alimentos, mejorando la
         comunicación entre los veterinarios y los funcionarios de salud
         pública sobre los brotes de micotoxicosis en animales domésticos.

    d)   Se debe elaborar un método sólido, de bajo costo y sencillo para
         la detección de la contaminación por fumonisina en el maíz.


    See Also:
       Toxicological Abbreviations
       Fumonisin B1 (IARC Summary & Evaluation, Volume 82, 2002)