UNITED NATIONS ENVIRONMENT PROGRAMME
INTERNATIONAL LABOUR ORGANISATION
WORLD HEALTH ORGANIZATION
INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
Environmental Health Criteria 219
FUMONISIN B1
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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CONTENTS
ENVIRONMENTAL HEALTH CRITERIA FOR FUMONISIN B1
PREAMBLE
ABBREVIATIONS
INTRODUCTION
1. SUMMARY, EVALUATION AND RECOMMENDATIONS
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
transformation
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. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL
METHODS
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
3. SOURCES OF HUMAN EXPOSURE
4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION
5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
6. KINETICS AND METABOLISM IN ANIMALS
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. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS
7.1. Laboratory animals and in vitro test systems
7.1.1. Single exposure
7.1.2. Repeated exposure
7.1.2.1 Body weight loss
7.1.2.2 Hepatocarcinogenicity and nephrotoxicity
7.1.2.3 Immunotoxicity
7.1.3. Skin and eye irritation
7.1.4. Reproductive toxicity, embryotoxicity and
teratogenicity
7.1.5. Mutagenicity and related end-points
7.1.6. Carcinogenicity
7.1.6.1 Carcinogenicity bioassays
7.1.6.2 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
7.3.1.1 Sphingolipids and their metabolism
7.3.1.2 Fumonisin-induced disruption of sphingolipid
metabolism in vitro
7.3.1.3 Fumonisin disruption of sphingolipid
metabolism in vivo
7.3.1.4 Tissue and species specificity
7.3.1.5 Fumonisin-induced sphingolipid alterations:
effects on growth, differentiation and cell
death
7.3.1.6 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. EFFECTS ON HUMANS
8.1. Transkei, South Africa
8.2. China
8.3. Northern Italy
9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY
9.1. Microorganisms
9.2. Plants
9.2.1. Duckweed and jimsonweed
9.2.2. Tomato
9.2.3. Maize
10. FURTHER RESEARCH
11. PREVIOUS EVALUATIONS BY INTERNATIONAL ORGANIZATIONS
REFERENCES
APPENDIX 1. NATIONAL GUIDELINES FOR FUMONISINS
APPENDIX 2. NATURAL OCCURRENCE OF FUMONISIN B1 (FB1) IN MAIZE-BASED
PRODUCTS
RESUME, EVALUATION ET RECOMMANDATIONS
RESUMEN, EVALUACION Y RECOMENDACIONES
NOTE TO READERS OF THE CRITERIA MONOGRAPHS
Every effort has been made to present information in the criteria
monographs as accurately as possible without unduly delaying their
publication. In the interest of all users of the Environmental Health
Criteria monographs, readers are requested to communicate any errors
that may have occurred to the Director of the International Programme
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
International Register of Potentially Toxic Chemicals, Case postale
356, 1219 Châtelaine, Geneva, Switzerland (telephone no. + 41 22 -
9799111, fax no. + 41 22 - 7973460, E-mail irptc@unep.ch).
* * *
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
PREAMBLE
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JMPR
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WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR FUMONISIN B1
Members
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
Africa
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
(Vice-Chairman)
Secretariat
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,
France
WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR FUMONISIN B1
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.
ABBREVIATIONS
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
INTRODUCTION
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. SUMMARY, EVALUATION AND RECOMMENDATIONS
1.1 Summary
1.1.1 Identity, physical and chemical properties, and analytical
methods
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
cerevisiae.
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
developed.
2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS
2.1 Identity
Common name: Fumonisin B1 (FB1)
Chemical formula: C34H59NO15
Chemical structure:
Relative molecular mass: 721
CAS Name: 1,2,3-Propanetricarboxylic acid,
1,1'-[1-(12-amino-4,9,11-trihydroxy-2-methyl-
tridecyl)-2-(1-methylpentyl)-1,2-ethane-diyl]
ester
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)
partition
coefficient
(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
products.
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.,
1997).
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,
1996).
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).
3. SOURCES OF HUMAN EXPOSURE
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,
1994).
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
Europe
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)
Africa
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
Zimbabwe
Miscellaneous maize foodsb Botswana, South Africa 8/17 0.03-0.35
Maize feed South Africa 16/16 0.47-8.85
Asia
Maize China, Indonesia, Nepal, Philippines, Thailand, 361/614 0.01-155
Vietnam
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
Oceania
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.,
1998).
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).
4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION
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.
5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
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
mycotoxicoses.
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
destinations.
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.,
1998a).
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)
(µg/kg)
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.
6. KINETICS AND METABOLISM IN ANIMALS
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
animals.
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
humans.
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. EFFECTS ON ANIMALS AND IN VITRO TEST SYSTEMS
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
7.1.2.1 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 7.1.6.1), 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
diets.
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).
7.1.2.2 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,
1997).
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.
7.1.2.3 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