CURRENT AWARENESS DOCUMENT
DIFUROXANTHONE-, DIFUROCOUMAROLACTONE- AND DIFUROANTHRAQUINONE-TYPE
ALKYLATING AGENTS
CARCINOGENICITY AND STRUCTURE ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
Prepared by:
Yin-Tak Woo, Ph.D., D.A.B.T.
David Y. Lai, Ph.D.
JRB Associates/
Science Applications
International Corporation
8400 Westpark Drive
McLean, Virginia 22102
EPA Contract No. 68-02-3948
JRB Project No. 2-813-07-409
EPA Project Officer and Scientific Editor
Joseph C. Arcos, D.Sc*.
Extradivisional Scientific Editor
Mary F. Argus, Ph.D.
February 1985
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5.3.1.1 Difuroxanthone-, Difurocoumarolactone- and Difuroanthraquinone-type
Alkylating Agents: Aflatoxins, and Related Aspergillus Toxins
5.3.1.1.1 Introduction.
The existence of aflatoxins was discovered in England in 1960 when a
Brazilian peanut meal used as a protein supplement to poultry diets caused the
acute deaths of over 100,000 young turkeys from liver damage or turkey "X"
disease (1). At the same time, an outbreak of turkey X-linked disease in
ducklings occurred in Kenya following ingestion of African peanut meals (2).
Symptoms similar to turkey X disease were also reported in outbreaks of
disease in other farm or domestic animals, fed peanut meals. Coincidentally,
an epizootic of liver cancer in hatchery-reared rainbow trout occurred in the
United States and Europe in I960 (3-6). A commercial trout feed containing
contaminated cottonseed meal was eventually found to be associated with the
trout hepatoma problem (7). The magnitude of the problem attracted intensive
research efforts by investigators throughout the world. The toxic agent in
peanut meal was narrowed down to a mixture of aflatoxins (abbreviation for
Aspergillus flavus toxins) produced by a ubiquitous mold, Aspergillus flavus
as secondary metabolites (8). The mixture could be separated into four main
components by thin layer chromatography and were designated B^, %2> Gi and G2
according to their fluorescent colors (B for blue, G for green) and relative
mobility in the chromatogram (9, 10). The molecular formulas for aflatoxins
Bj and G^ were deduced to be cu^]^206 and C17H12°7» respectively (9) and
aflatoxins 82 and G£ were shown to be dihydro derivatives of Bj and G^,
respectively (11). The correct chemical structures were finally elucidated by
Asao et al. (12) in 1963 and confirmed by further studies (13, 14).
-------
The carcinogenicity of aflatoxin mixture was first demonstrated in 1961
by Lancaster et al. (15) by feeding aflatoxin-contaminated peanut meals to
rats. Shortly afterwards, similar findings were reported by Schoental (16),
LeBreton _et__al_. (17), Salmon and Newberne (18) and other investigators (see
Section 5.3.1.1.3.2). Subsequent studies using purified aflatoxins (see
Section 5.3.1.1.3.3) indicated that the carcinogenicity associated with afla-
toxin mixture of aflatoxin-contaminated feed was mainly attributable to afla-
toxin BI (AFBj). The demonstration by Wolf and Jackson (7) that cottonseed
meal was repsonsible for the outbreak of hepatoma in hatchery-reared rainbow
trout soon led to the finding that the principal carcinogenic agent in the
contaminated cottonseed meal was AFBi (see Section 5.3.1.1.3.3). To date,
AFBj or mixture containing AFBj has been found to be carcinogenic in eleven of
twelve animal species tested including nonhuraan primates. In the most suscep-
tible species (such as rainbow trout, rats), AFBi is the most potent
"complete" hepatocarcinogen ever tested. With the realization that human food
supply is susceptible to aflatoxin contamination, the possibility has been
suggested that these mycotoxins may be involved in human liver cancer (17,
19). The first major epidemiological study was undertaken in Uganda in 1966-
1967 by Alpert _£t__al^ (20) who showed that the tribal variation of hepatoma
incidence was related to the frequency of aflatoxin contamination of their
food. Subsequently, four separate field studies in Kenya (21), Mozambique
(22), Swaziland (23), and Thailand (24, 25) demonstrated an association
between aflatoxin consumption and liver cancer incidence, and a dose-response
relationship was established (26). Short of establishing a direct causal
relationship, the data provided strong evidence for a contributory or etio-
logic role of aflatoxin in the induction of human liver cancer.
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The concern over aflatoxins as contaminants of human food supply and
animal feeds, the importance of aflatoxins as potential environmental carcino
gen for humans, and the usefulness of aflatoxins as experimental animal car-
cinogens have generated literally innumerable publications reflected by the
large number of comprehensive monographs (27-35) and reviews (36-49) on this
subject.
5.3.1.1.2 Physicochemical Properties and Biological Effects.
5.3.1.1.2.1 PHYSICAL AND CHEMICAL PROPERTIES.
The physical and chemical properties of aflatoxins and related compounds
have been extensively discussed in a variety of reviews and monographs (32,
33, 37, 38, 45, 50). Hence, only a synoptic summary is presented in this
section. The structures of a number of aflatoxins, their metabolites and
related compounds are depicted in Table I. Physical and chemical properties
of some of these compounds are summarized in Table II.
The nomenclature of aflatoxins was originally based on their fluorescent
color and chromatographic mobility. The four major aflatoxins which occur
naturally in plant products were named B,, B2, G, and Gn with B standing for
blue, G for green and subscripts designating relative positions of these
fluorescent bands on the thin-layer chromatograph. The trivial names afla-
toxins Mi, M«, GMi and GM2 were used to designate 4-hydroxy derivatives of
aflatoxins B^, ^^t Gj and 62, respectively, because AFM^ was first discovered
as an excretory product of AFBi in cow's milk (M stands for milk). The M
aflatoxins have since been detected as mammalian urinary as well as fungal
metabolites of aflatoxins. The 0-demethylated metabolite of AFBj was named
AFPi (P stands for phenolic) whereas another hydroxylated metabolite (at the
position beta to the carbonyl group in the cyclopentenone ring) was named
-------
0 0
0 0
0 0
AFB
AFP,
AflatoxicoKAFL)
AFLH
AFLM
0
OH
Sterigmatocystin Versicolorin A
0
Versicolorin B
Table I. Structural Formulas of Aflatoxins and Related Compounds.
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Table II
Physicochemical Properties of Some Aflatoxins and Related Compounds8
Compound
Aflatoxin B,
Aflatoxin B£
Aflatoxin G,
Aflatoxin G~
Aflatoxin M,
Aflatoxin B2a
Aflatoxin P,
Aflatoxin Qj
Af latoxicol
Physical appearance
of crystals
Pale yellow
White needles
Colorless needles
Colorless needles
Colorless plates
Yellow plates
—
—
Colorless
Melting
point (8C)
268-269
288-289
244-246
237-240
299
240
> 320
—
230-234
UV Absorption ( £M)C at
[o4]Du 265 nm
-558° 12,400
-430° 12,100
-556° 9,600
-473° 8,200
-280° 14,150
10,300
(256 nm)
11,200
(267 nm)
11,450
(267 nm)
10,800
(261 nm)
360-362 nm
21,800
24,000
17,700
17,100
21,250
(357 nm)
20,400
(363 nm)
15,400
(362 nm)
17,500
(366 nm)
14,100
(325 nm)
Fluorescence
emission (nm)
425 (blue)
425 (blue)
450 (green)
450 (green)
425 (blue)
425 (blue)
yellow-green
yellow-green
425 (blue)
B d
RF
0.56
0.53
0.48
0.46
0.40
0.13
—
—
0.54
Summarized from data compiled by R.W. Detroy, E.B. Lillehoj and A. Ciegler: In "Microbial Toxins," Vol. VI
(A. Ciegler, S. Kadis and S.J. Aji, eds.), Academic Press, New York, 1971; J.G. Heathcote and J.R. Hibbert,
"Aflatoxins: Chemical and Biological Aspects," Elsevier, Amsterdam, 1978; World Health Organization, ,
"Environmental Health Criteria 11: Mycotoxins," World Health Organization, Geneva, 1979.
Optical rotation; all compounds dissolved in chloroform except aflatoxin Mi (in dimethylformamide).
°Molar extinction coefficient; all compounds dissolved in methanol except aflatoxin P. (in ethanol).
Relative mobility in thin-layer chromatography. Solid phase: silica gel G; solvent: chloroform/methanol
(97:3 v/v).
-------
to distinguish it from AFP^. Aflatoxicol, a keto-reduced metabolite of AFBj,
was called aflatoxin RQ by many investigators; however, the term aflatoxicol
(abbreviated AFL) appears to be more commonly used following the discovery of
two other forms of aflatoxicol metabolites (AFLMj and AFLHj; see Section
5.3.1.1.4.1). Two systems are currently used in numbering the carbon and
oxygen atoms in the aflatoxin molecule. In the older conventional, but still
commonly used system, the atoms in the terminal furan ring are numbered 1, 2,
3, 4 starting from the oxygen. The numbering of atoms in the rest of the
molecule (shown in parentheses in the formula to the left, below) does not
Text figure -j.
appear to have been uniformly adopted. In the IUPAC system, the atoms on the
periphery of the molecule are numbered in a clockwise direction starting with
the first atom following ring fusion in the uppermost ring farthest to the
right. (For details on the nomenclature of polynuclear compounds, see Section
5.1.1.1, Vol. IIA.) Thus, the double bond in the terminal furan ring of both
AFBi and AFGi is called 2,3-double bond in the older, conventional system but
8,9-double bond for AFBj and 9,10-double bond for AFGj in the IUPAC system.
The systematic, IUPAC names (used by Chemical Abstracts) for AFB^ and AFGj are
2,3,6 ot. ,9 P(-tetrahydro-4-methoxycyclopenta[c]furo[3',2':4,5]furo[2,3-
h] [l]benzopyran-l,ll-dione and 3,4,7c/ ,10* -tetrahydro-5-methoxy-lH,12H-
furo[3*,2':4,5]furo[2,3-h]pyrano[3,4-c][l]benzopyran-l,12-dione, respec-
tively. Aflatoxin Bj and related compounds are classified as difurocou-
marocyclopentenone whereas AFG^ and related compounds belong to
difurocoumarolactone.
4
-------
1 02)
(conventional)
OCH3
(IUPAC)
Text-Figure 1^
-------
Naturally occurring aflatoxins are very slightly soluble in water (of the
order of 10-20 rag/liter), insoluble in nonpolar solvents, but freely soluble
in polar organic solvents such as chloroform, methanol and especially di-
methyIsulfoxide. As pure substances, the aflatoxins are very stable even at
relatively high temperatures. Little or no destruction of aflatoxins occurs
under ordinary cooking or pasteurization conditions. Chloroform and benzene
solutions of aflatoxins are stable for years if kept cold and in the dark.
However, aflatoxins are relatively unstable when exposed to light (especially
UV) particularly when dissolved in highly polar solvents. The lactone ring in
aflatoxins is susceptible to alkaline hydrolysis. However, if the alkaline
treatment is mild, acidification will reverse the reaction to reform afla-
toxins. Aflatoxins can be totally destroyed by autoclaving in the presence of
ammonia or by treatment with hypochlorite.
Molecular orbital calculations by Heathcote and Hibbert (32, 51) indi-
cated that the double bond in the 2,3-position in AFB, and AFGi is the most
reactive molecular site and is susceptible to epoxidation. This view is
supported by the finding that under mildly acidic condition, AFBt and AFGi are
readily hydrated to yield AFB2a and AFG2a, respectively (32, 52). Incubation
of AFBj with an appropriate chemical oxidizing agent (e.g., jn-chloroperbenzoic
acid) yields a reactive intermediate concluded to be AFB^-2,3-oxide (53,
55). The putative AFB^-2,3-oxide may react with nucleophiles (55) or is
hydrolyzed to 2,3-dihydro-2,3-dihydroxyaflatoxin B^ (AFBj-dihydrodiol). Both
AFBo- and AFBi-dihydrodiol are hemiacetals and readily tautomerize to dialde-
hyde under alkaline conditions; these can react with amino groups to form
Schiff bases (see Section 5.3.1.1.4.2). There is some evidence that the
coumarin moiety of AFBj may be oxidized to yield a variety of oxidation pro-
ducts (54). Besides chemical activation, the 2,3-double bond of AFB^ and AFGj
-------
can also be activated photochemically to yield reactive-intermediates which
can bind covalently to DNA (56). The mechanism of photoactivation has been
postulated to involve excitation of the coumaryl chromophore (similar to
photoactivation of psoralen; see Section 5.3.2.4), followed by intramolecular
energy transfer to the 2,3-double bond. The nature of the photoadduct is not
known but is believed to differ from AFB^-2,3-oxide produced by chemical
oxidation because photobinding of AFB, to DNA occurs preferentially at sites
rich in adenine and thymidine, whereas AFBj-2,3-oxide binds preferentially to
guanine moieties (56).
Sterigmatocystin, a difuroxanthone compound, and versicolorin A, a
difuroanthraquinone compound, are biosynthetic precursors of aflatoxins and
are structurally closely related (see Table 1). The physical and chemical
properties of sterigmatocystin and versicolorin A (32, 45, 50) are similar to
those of AFBj.
5.3.1.1.2.2 BIOLOGICAL EFFECTS OTHER THAN CARCINOGENICITY.
Toxicity. The toxicology of aflatoxins and related compounds has been
extensively reviewed (32-34, 37-40, 44, 57, 58). The acute toxicity of AFBj
has been tested in a wide variety of animal species. Table III summarizes the
acute, oral LD^Q data. As the data in the table indicate, considerable
species difference in response to the toxic effects of AFBj has been
observed. Ducklings, rabbits, rainbow trout, dogs, pigs, cats and newborn
rats are extremely susceptible, with LD^Q of less than 1 mg/kg, whereas
hamsters, mice and catfish are highly resistant. In virtually all animal
species, the principal toxicity target organ is the liver. Depending on the
species, the toxic effects observed may include necrosis (centrilobular or
periportal), cirrhosis, fatty liver, bile duct proliferation and fibrosis (37,
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p. 1 of 2
Table III
Acute Toxicity of Aflatoxins and Related Compounds in Various Animal Species
Compound Species
Aflatoxin B. Chick embryo
Duckling
Rabbit
Dog
Cat
Pig (weanling)
Rat (newborn)
(weanling, M)
(weanling, F)
(adult, M)
(adult, F)
Rainbow trout
Guinea pig
Sheep
Baboon
Monkey
(Macaca irus, M)
(Macaca
fascicularis , F)
Hamster
Catfish
Mouse
Aflatoxin 82 Duckling
Rat
Aflatoxin Gj Duckling
Rat
Rainbow trout
Route
injection
oral
oral or i.p.
oral
oral
oral
oral
oral
oral
oral
i.p.
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
i.p.
oral
i.p.
i.p.
LD5Q (mg/kg)
11-40 ng/egg
0.24-0.73
0.3
0.5-1.0
0.55
0.62
0.56; 1.36
1.53
5.9; 6.0
7.2
0.65; 6.0
17.9
0.81
1.4
2.0
2.0
2.2
7.8
10.2
10-15
9; 60
1.7; 1.8
>200
0.78; 1.2; 1.8
1.5-2.0
1.9
Reference
(59,
(12,
(65)
(66)
(65)
(65)
(65,
(68)
(65,
(69)
(64,
(69)
(70)
(71)
(72)
(73)
(74)
(75)
(65)
(39)
(65)
(61,
(64)
(14,
(64)
(70)
60)
61-64)
67)
68)
69)
•
64)
61, 64)
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Table III (continued)
Compound
Aflatoxin 62
Aflatoxin Mj
Aflatoxin M2
Aflatoxin Pj
Aflatoxin Q^
Tetrahydro-
deoxyaf la-
toxin BI
Sterigraato-
cystin
Species
Duckling
Rat
Duckling
Duckling
Chick embryo
Chick embryo
Rat
Monkey (vervet)
&
Rat (F)
(M)
Route
oral
i.p.
oral
oral
injection
injection
i.p.
i.p.
oral
oral
LD5Q (mg/kg)
2.8; 3.45
>200
0.32
1.22
>190 ng/egg
207 ng/egg
>200
32
120
165
Reference
(61, 64)
(64)
(63)
(63)
(76)
(60)
(64)
(77)
(78)
(78)
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39, 58, 65). Fatty infiltration and focal necrosis may also occur in the
kidney and heart. In AFBj-treated monkeys, cerebral edema may be prominent
whereas gall bladder edema and hemorrhage are distinctive features of acute
aflatoxicosis in dogs. The acute toxicity of AFBj may be modified by a
variety of endogenous and exogenous factors. Newborn animals appear to be
substantially more susceptible to the acute toxic effects of AFBj than
adults. In the rat, the adult-to-neonate LDcn ratios may be as high as 9.2
(67) or 13-32 (65). A significant sex difference has also been observed in
some strains of rats; castration removes most of the sex difference (68).
Diets high in lipid have a protective effect against aflatoxicosis in various
animal species. Deficiency in vitamin A, D or riboflavin makes animals more
sensitive to aflatoxin while thiamine deficiency has the opposite effect (79).
Acute toxicity data are relatively scant on other aflatoxins. Compara-
tive toxicity studies using ducklings, trout and rats indicate that AFB^ is
slightly more toxic than AFGj (61, 64, 70). Their 2,3-dihydro derivatives,
AFB2 and AFG2 are considerably less toxic (see Table III), suggesting that the
2,3-double bond plays an important role in the toxic effect of the afla-
toxins. Aflatoxin Mj is almost as toxic as AFBj (0.32 mg/kg vs. 0.2A mg/kg)
in ducklings; whereas its dihydro derivative AFM2 is approximately four times
less toxic (63). In chick embryo test, AFBj is considerably more toxic than
aflatoxins B2, Gj, G2 (59), Pj (76), Qx (60) and B2a (80). The estimated LD5Q
of aflatoxins Qj (60) and B2a (80) are at least 18 and 80 times higher than
that of AFBj.
Aflatoxin Bj, sterigmatocystin and a number of related compounds are
highly cytotoxic to cultured animal cells (rev. 32) as well as to a number of
microorganisms (81). Using primary kidney epithelial cells of monkey,
Engelbrecht and Altenkirk (82) carried out a structure-cytotoxicity relation-
-------
ship study using 15 compounds in the sterigmatocystin, aflatoxin_and related
structural area. The most cytotoxic compounds are, in decreasing order of
potency, sterigmatocystin, AFBj, 0-methylsterigmatocystin, AFGj and demethyl-
sterigmatocystin. Dihydrosterigmatocystin, AFB2 and open-ring furobenzofuran
compounds are only slightly cytotoxic. Engelbrecht and Altenkirk (82) sug-
gested that the two principal structural requirements are the double bond in
the terminal furan ring and the C*, ft -unsaturated carbonyl group in
o-lactone moiety of aflatoxins or in Y~Pyrone moiety of sterigmatocystin.
The cytotoxicity of aflatoxin compounds differs from their genotoxicity in
several respects. The genotoxic effects of aflatoxin compounds appear to be
expressed at a lower dose than cytotoxic effects (83). Results obtained from
studies on cultured hepatoma cells indicate that the enzymatic activities that
convert AFB^ into cytotoxic metabolites are distinct from those converting
AFBj into mutagenic metabolites (84). There is some evidence that hepatocytes
become more resistant to the cytotoxicity of AFB, during AFB,-induced carcino-
genesis (85).
Apart from poisoning of farm animals by aflatoxin-contaminated feed,
aflatoxins have been associated with a number of acute human poisonings (rev.
33, 34, 57). In 1967, an outbreak of poisoning of 26 persons in two villages
in Taiwan occurred as a result of consuming moldy rice (containing up to 200
ppm AFBj); three of them (children aged 4-8) died shortly afterwards. The
symptoms observed included abdominal pain, vomiting, palpable liver and edema
of the lower extremities, but no fever (86). Aflatoxin was suspected to be
associated with the death due to acute hepatitis of a 15-year-old Ugandan boy
whose diet consisted mainly of moldy cassava containing 1.7 ppm aflatoxin
(87). An epidemic outbreak of acute hepatitis occurred during the last two
months of 1974 affecting several hundred persons in 150 villages in adjacent
8
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districts of two neighboring states in northwest India (88-91). The outbreak
started after the consumption of recently harvested corn that was spoiled due
to unusual rainfalls in the areas. Subsequent analysis of samples of corn
obtained from affected families revealed the presence of the aflatoxin-
producing mold Aspergillus flavus and AFBj levels ranging from 0.25 to 15.6
ppra. Over one hundred persons died as a result of the poisoning, men being
affected approximately twice as often as women. Histopathological studies
indicated extensive liver damage. Besides liver disease, aflatoxins have been
implicated as one of the possible causes of Reye's syndrome (encephalopathy
with fatty degeneration of the viscera, EFDV), an acute, often fatal disease
affecting mainly infants and young children, that usually progresses from a
mild prodromal viral illness to severe cerebral involvement with coma.
Clustering of Reye's syndrome has been observed in northeast Thailand in
predominantly rural areas geographically and seasonally related to heavy
contamination of market food samples (92, 93). In at least two cases, the
food consumed by the patients 2-3 days prior to the onset of Reye's syndrome
was found to be heavily contaminated with aflatoxins and toxigenic molds (93,
94). Trace amounts of aflatoxins were detected in tissues, body fluids, or
gastrointestinal contents of 22/23 Thai patients who died from Reye's
syndrome; the liver specimens of two of these patients contained 47 and 93 ppb
AFBj (95). The presence of aflatoxins was also demonstrated in the liver
specimens of a number of Reye's syndrome patients in New Zealand (96),
Czechoslovakia (97) and in the United States (98, 99); however, a dietary
source of aflatoxin was not identified in any of the above cases. It should
be noted that not all Reye's syndrome patients had detectable amounts of
aflatoxins in their tissues or body fluids (100). Moreover, no unusual
clustering of Reye's syndrome has been found in other countries with high
frequencies of food contamination with aflatoxins.
9
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Mutagenicity. The mutagenicity of aflatoxins and related compounds has
been extensively investigated using a great variety of test organisms. Both
A*"8! and a mixture of AFBj and AFGj have been shown to exhibit prophage-
inducing activity in Escherichia coli p4^6, Staphylococcus aureus LM 204 and
lysogenic Bacillus megaterium (101, 102). They induce gene mutations in
transforming DNA of Bacillus subtilis (103), in a number of strains of
Salmonella typhimurium (see discussion below), in growing vegetative cultures
(but not resting conidia) of Neurospora crassa (104) and Saccharomyces
cerevisiae (Brusick, cited in 43), in Chlamydomonas reinhardii (105),
j)rosophila melanogaster (106), in cultured Chinese hamster cells (107) and
mouse lymphoma cells (108). They also produce chromosomal aberrations in
seedling roots of Vicia faba (109), in Allium cepa (110), in a kidney-cell
line derived from rat kangaroo (111), and cultured human leucocytes (112-
115). In most cases, AFBj is rautagenic only if metabolic activation system is
added to the incubation medium or when metabolically active cells are used.
In in vivo studies, Epstein and Schafner (116) reported the induction of
dominant lethal mutation in male mice treated with a high dose (68 mg/kg) of a
mixture of AFBj and AFG^. Leonard ^t__al_. (117) indicated that AFBj alone,
given at a dose of 5 mg/kg, does not produce gross structural chromosome
changes in male mouse germ cells. However, Fabry and Roberfroid (115) showed
that AFBj (5 mg/kg) is clastogenic in bone marrow cells of mice, producing
chromatid gaps and fragments and micronucleated cells. Most of the earlier
mutagenicity data were reviewed by Ong (43) in 1975 and by Hayes (34) in
1981. The following discussion focuses on mutagenicity studies using the Ames
Salmonella test, which has been widely employed as a test for the prediction
of carcinogenicity and for exploring structure-mutagenicity relationships.
10
-------
Over 20 aflatoxins and related compounds have been tested~for-mutagenic
activity using the Ames test. Among the five commonly used tester strains
(TA98, TA100, TA1535, TA1537, TA1538) of Salmonella typhimurium. strains TA98
(which detects frameshift mutagens) and TA100 (which detects base-pair substi-
tution mutagens but is also sensitive to frameshift mutagens) are considerably
more sensitive to AFBi than the other strains (118-121). Most of the muta-
genicity studies on aflatoxins and related compounds have been carried out
with these two strains. The results of these studies are summarized in Table
IV. The compounds are classified as (a) metabolites and related compounds of
AFBj and AFGj, (b) biosynthetic precursors of AFBj, and (c) derivatives and
analogs of sterigmatocystin, and are assigned potency ranking for easier
comparison.
The relationship between chemical structure and mutagenic activity of
aflatoxins and their mammalian metabolites has been extensively studied by
Wong and Hsieh (124). Using TA98 as the tester strain, these investigators
showed that AFBi is considerably more mutagenic than any other aflatoxin or
the aflatoxin metabolites. Among the metabolites of AFB^ tested, aflatoxicol
(AFL) is the most active, followed by AFMj, AFLHj and AFQj, while AFPj and
AFB2a are inactive. Aflatoxin Gj is slightly mutagenic whereas aflatoxins 82,
Go and G2a are all nonmutagenic. In terms of relative potency, AFL, AFGj,
AFMj, AFLHj and AFQj have about 22.8, 3.3, 3.2, 2.0 and 1.2% of the activity
of AFBi, respectively. All the mutagenic compounds possess a double bond in
the 2,3-position of the terminal furan ring, whereas those without the
2,3-double bond (except AFP,) are nonmutagenic. The presence of rat liver S-9
preparation is required for mutagenic activity. These findings led Wong and
Hsieh (124) to conclude that the 2,3-double bond plays a role in the mechanism
of mutagenicity of the aflatoxins, and that the 2,3-oxide generated meta-
bolically is the most probable ultimate mutagenic form of these compounds.
11
-------
p. 1 of 2
Table IV
Comparative Mutagenicity of Aflatoxins, Sterigmatocystin
and Related Compounds in the Ames Salmonella Test
Relative Mutagenic Potency
Strain TA98 Strain TA100
Compound With S-9 Without S-9 With S-9 Without S-9
Aflatoxins and Related Compounds
AFB, +••••»•+ +; - ++++ + ; -
AFL +++ - n.t. n.t.
AFGj ++ - n.t. n.t.
AFMj ++ ++
AFLHj + - n.t. n.t.
AFQj + - n.t. n.t.
AFPj - - n.t. n.t.
AFB2 - - -
AFB2a - - n.t.
AFG2 - - n.t. n.t.
AFG2a - - n.t. n.t.
2 ,3-Dichloro- n.t. +++ n.t. +++
2,3-dihydro-AFBj
2,3-Dihydro-2,3- n.t. - n.t.
dihydroxy-AFBj^
2,3-Dihydro-2- n.t. - n.t.
hydroxy-3-chloro-
AFBj
3a ,8a-Dihydrofuro- - - + -
[ 2 , 3-b ]benzof urana
Tetrahydrofuro- - - - -
References
(119-129)
(124)
(124, 128)
(119, 124)
(124)
(124)
(124)
(120, 123,
124)
(123, 124)
(124)
(124)
(123)
(123)
(123)
(126)
(126)
benzofurane
-------
p. 2 of 2
Table IV (continued)
Relative Mutagenic Potency
Strain TA98 Strain TA100
Compound With S-9 Without S-9 With S-9 Without S-9 References
Precursors of Aflatoxin B,
Sterigmatocystin +++ •«•; - +++ - (121, 122,
125, 127,
128)
Versicolorin A ++ - ++ ++ (121, 125,
127)
Versiconal - n.t. n.t. (125)
acetate3
Averufin3 - - - - (121, 125)
Averantin3 + + + + (121)
Norsolorinic - - - (121, 125)
acid3
Analogs and Derivatives of Sterigmatocystin
0-Methyl sterig- - - (127)
matocystin
0-Acetyl sterig- - + n.t. n.t. (128)
matocystin
Austocystin A3 -n-n- - ++ (127)
Austocystin Da * + +• - (127)
3For structural formulas, see below:
[Text-figure 2]
-------
P, OH 0 OH
CH3-(CH2)4-C, ' " '
HO
Norsolorinic acid
OH 0 OH
CH3-(CH2)4-CH
Averantin
OH 0
0
Averuf in
0
II
CH3CO 0 OH
v i i| §
H..C
^ A HO
0
Versiconal acetate
3a,8a-Dihydrofuro-
[2,3-b]benzofuran
Tetrahydrofuro-
benzofuran
H3CO 0 OCH3
X0 0 ^ 0'
Austocystin A
OH 0 OH
Austocystin D
Text-Figure 2_
-------
However, the 2,3-double bond is not the sole molecular moiety that deter-
mines the mutagenic activity of AFBj. Alteration of the molecular structure
elsewhere invariably leads to reduction in mutagenic activity. Thus, 0-de-
methylation of AFBj (yielding AFPj) completely abolishes the mutagenic
activity of the parent compound, while 4-hydroxylation (yielding AFMj),
replacement of the cyclopentenone ring with a terminal lactone ring (AFGj),
reduction of the keto group (yielding AFL), or hydroxylation of the cyclopen-
tenone ring (yielding AFQj), all result in significant decrease in mutagenic
activity. Formation of the highly reactive AFBj-2,3-oxide (as the ultimate
mutagenic intermediate of AFBj) is supported by the study by Swenson et al.
(123) using 2,3-dichloro-2,3-dihydro-AFBj as a model compound for the
epoxide. Like AFBj-2,3-oxide, the 2,3-dichloride derivative would be expected
to form a resonance-stabilized carbonium ion at carbon 2 and would act
directly as an electrophile. 2,3-Dichloro-2,3-dihydro-AFBj was indeed shown
to be a potent, direct-acting mutagen for both strains, TA98 and TA100. Under
similar assay conditions (without the S-9 mix), AFBj, AFB2> AFB2a and the
dihydrodiol and chlorohydrin derivatives of AFBj (hydrolysis products of
2,3-dichloro-2,3-dihydro-AFBj) all showed little or no mutagenic activity (see
Table IV).
In other comparative studies, Uwaifo and Bababumi (119) reported that
TA100 is a more sensitive tester strain for AFBj and AFMj than TA98 and that
the relative mutagenic potency of AFMj in strain TA100 (10% of that of AFBj)
correlates well with the relative carcinogenic potency of these two myco-
toxins. Ueno _et^ _al_. (128) found AFBj slightly mutagenic for strains TA98 and
TA100 without metabolic activation. Their data indicate that the mutagenic
potency of AFBj increases by at least 100 times when including the S-9 mix.
Aflatoxin Gj is also mutagenic but considerably less active than AFBj*
12
-------
Wheeler et_al_* (120) showed that AFBj displays little or no mutagenic activity
using strains TA1538 and TA1535 but is highly mutagenic in TA98 and TA100.
Aflatoxin B2 is completely inactive. Coles et^ al^. (126) found 3a ,8a-dihydro-
furo[2,3-b]benzofuran mutagenic for TA100 but not for TA98. The double bond
in the terminal furan ring appears to account for the mutagenic activity in
TA100, since tetrahydrofurobenzofuran is inactive. The lack of mutagenic
activity of 3a,8a-dihydrofuro[2,3-b]benzofuran in TA98 suggests that the rest
of molecular structure in AFBi is needed to confer frameshift mutagenic
activity.
The biosynthesis of AFB, in fungi is believed to proceed via the path-
way: norsolorinic acid ——>• averantin ^ averufin —>• versiconal
acetate ^ versicolorin A > sterigmatocystin >• AFB, (see Section
5.3.1.1.5.2). The bacterial mutagenicity of these precursors has been tested
in the Ames test (see Table IV). Using TA98 as the tester strain, Wong et al.
(125) showed that norsolorinic acid, averufin and versiconal acetate are all
practically inactive while versicolorin A and sterigmatocystin are significant
mutagens with approximately 5.8 and 10.7% of the activity of AFB}, respec-
tively. The most important difference between the mutagenic and nonmutagenic
compounds is the presence of the unsaturated bisfuran ring structure in the
former, indicating the requirement of the 2,3-double bond for the mutagenic
activity of AFBi. In accord with conclusions derived from mutagenicity
studies on mammalian metabolites of AFB,, besides the 2,3-double bond the
configuration and electronic structure of the entire molecule are important
contributory factors for mutagenic activity. Substitution of the coumarin
moiety of AFBj with xanthone (as in sterigmatocystin) or anthraquinone (as in
versicolorin A) results in a reduction in the mutagenic activity. Essentially
identical results were obtained by Dunn^t__al_. (121) in their mutagenicity
13
-------
testing of norsolorinic acid, averantin, averufin, versicolorin A and sterig-
matocystin using tester strains TA98, TA100, TA1535, TA1537 and TA1538.
However, the relative mutagenic potencies of versicolorin A and sterigmato-
cystin in tester strain TA100 were 1.8% and 53.6% of that of AFBj, respec-
tively; averantin, a relatively recently discovered precursor of AFBj,
displayed weak mutagenic activity (0.1% of activity of AFB^). It is interest-
ing that versicolorin A and averantin (both pigmented compounds with anthra-
quinone structure) display mutagenic activity even in the absence of metabolic
activation.
Several derivatives and analogs of sterigmatocystin have been tested for
mutagenicity. Wehner et al. (127) found the 0-methyl derivative of sterig-
matocystin to be devoid of mutagenic activity. The 0-acetyl derivative of
sterigmatocystin is, however, weakly mutagenic in the absence of metabolic
activation, but not significantly active in the presence of S-9 mix (128). In
contrast to the lack of mutagenic activity of 0-methylsterigmatocystin, its
close analog austocystin A (in austocystin A, the xanthone moiety is fused to
the bisfuran ring structure in a linear configuration instead of angular
configuration as in sterigmatocystin) is highly mutagenic in the frameshift
mutation tester strain TA98, with activity comparable to that of AFBj. The
closely related austocystin D, which is polyhydric and substituted with a
bulky substituent, is only weakly mutagenic.
Teratogenicity. In contrast to extensive testing of carcinogenicity and
mutagenicity of aflatoxins and related compounds, relatively few studies have
been undertaken to investigate the teratogenicity of these compounds. More-
over, most of these studies were carried out using chick embryos. Verrett et
al. (59) reported first that AFBj induced teratogenic effects (mainly leg
deformities) in White Leghorn chicks after injection into yolk-sac of the
14
-------
embryos at early stages of development. No teratogenic effects were observed
with AFBj when injected into the air sac of the embryos. The teratogenicity
of AFBj was confirmed by Bassir and Adekunle (130) using White Rock chick
embryos. The effect apppears to be nonspecific; malformations of the eyes,
skull and limbs were observed. These abnormalities were described by Bassir
and Adekunle (130) as being similar to those observed by Keraper (131) in
testing of thalidomide in chick embryos. Aflatoxin Bj was also teratogenic to
tadpoles of Rana temporaia, causing mainly limb defects (132).
Among mammalian species tested, AFB, was reported to be teratogenic in
golden hamsters, but inactive in strain C3H/He mice (133) and in Wistar rats
(134). In hamsters the greatest teratogenic response was observed when AFBj
was injected intraperitoneally on day 8 of gestation at the dose of 4 mg/kg
body weight (133, 135). Animals were sacrificed on days 9, 11, 13 and 15 of
gestation. The most severe malformations (anencephaly, disorganization of
cranial end of neural tube, ectopic cordis, head malformations) and the
O
greatest number of malformed fetuses were found on days 9 and 11. These
malformations are usually incompatible with life. On days 13 and 15 of gesta-
tion, many dead or resorbed fetuses were found; the malformations noted
(microcephaly and umbilical hernia) were less severe and probably compatible
with postuterine life. No information was provided in these reports (133,
135) to indicate whether AFB, caused a significant increase in the incidence
of malformations in the offspring at birth. In strain C3H/He mice, a single
intraperitoneal injection of 8 mg/kg body weight AFB, on day 8 or 12 of
gestation, or repeated injections of AFBj at the dose of 4 mg/kg body weight,
led to high incidences of dead or resorbed fetuses but produced no evidence of
teratogenicity (133). In Wistar rats, oral administration of AFBj (equivalent
to 1/4 of LD^g) early in pregnancy had no significant teratogenic effect.
15
-------
Retardation in fetal growth was observed when AFBj was administered late in
pregnancy (i.e., after completion of organogenesis). The effect was con-
sidered to be a secondary effect occurring as a result of maternal liver
damage (134).
In addition to AFBi, several metabolites or derivatives have been tested
using chick embryos. Aflatoxins B2a and Qj were at least 80 and 18 times less
embryotoxic than AFB,, respectively (60, 80). Aflatoxin Pj was also signifi-
cantly less embryotoxic than AFBj (76). Neither of the above three
metabolites or derivatives produced teratogenic effects in chick embryos.
Sterigmatocystin is at least 16 times less embryotoxic than AFBj in chick
embryos (136). Teratogenic effects were observed occasionally but they did
not appear to be consistent. The most common deformity was twisted feet.
There was no difference in teratogenic response between injection of Sterig-
matocystin into the air cell or into the yolk sac of the chick embryo. Afla-
toxin G, was teratogenic to tadpoles of Rana temporaia; the dose required to
produce teratogenic effects was approximately 10 times higher than that of
AFBj (132).
5.3.1.1.3 Carcinogenicity and Structure-Activity Relationships.
5.3.1.1.3.1 OVERVIEW.
Since the first report in 1961 of carcinogenicity of aflatoxin-
contaminated groundnut meal in rats, aflatoxin mixture (which mainly consists
of aflatoxin Bj and Gi and traces of Bo and Go) or purified AFBi was found to
be carcinogenic in 11 of the 12 animal species tested. Great variation in the
susceptibility of these animal species to the carcinogenic effect of AFBj was
observed. In the highly susceptible species (e.g., rainbow trout, rat, duck),
AFBi is the most potent hepatocarcinogen presently known, inducing tumors in
16
-------
animals maintained on diets containing only parts per billion (ppb) levels of
the mycotoxin.
Over 20 aflatoxins and related compounds have been tested for carcino-
genic activity. These compounds include metabolites, synthetic derivatives
and structural analogs of AFBj, AFGj and sterigmatocystin. Structurally, they
represent difurocoumarocyclopentenone, difurocoumarolactone, difuroxanthone,
difuroanthraquinone, cyclopentenonecoumarin and coumarin derivatives. The
most important finding of these studies is that the presence of a double bond
at the 2,3-position of terminal furan ring is a critical structural require-
ment for carcinogenic activity. Thus, while AFBj, AFGj, sterigmatocystin and,
to a lesser extent, versicolorin A are carcinogenic, their corresponding
dihydro derivatives, AFBo, AFGoj dihydrosterigmatocystin and versicolorin B
are practically inactive. This finding is consistent with (a) metabolism and
mutagenicity studies implicating AFBj-2,3-oxide as the genotoxic reactive
intermediate (see Section 5.3.1.1.2.2), (b) the demonstration of direct-acting
carcinogenic activity of 2,3-dichloro-2,3-dihydroaflatoxin Bj (123), which is
expected to have similar electrophilic activities as AFBj-2,3-oxide, and (c)
molecular orbital calculations (32, 51) which show that the double bond in the
2,3-position in AFB^, AFGj, sterigmatocystin and versicolorin A is the most
reactive molecular site and is susceptible to epoxidation. Besides the
2,3-double bond, the configuration and the electron distribution in the rest
of the molecule are important contributory factors in the carcinogenic activ-
ity of the aflatoxins and mold metabolites. The Bi congener appears to repre-
sent the structure most optimal for the hepatocarcinogenic activity of the
aflatoxins; ring substitution, or substitution of the coumarocyclopentenone
moiety with coumarolactone, xanthone or anthraquinone moiety, invariably leads
to a reduction in carcinogenic activity. The lower carcinogenic activity of
17
-------
hydroxylated derivatives of AFBj (e.g. , AFMj, AFQj) may be related to changes
in pharmacoki.netic properties as a result of an increase in hydrophilicity.
Heathcote and Hibbert (32, 51) attributed the weaker carcinogenic activity of
versicolorin A to the presence of a reactive L-region.* Moreover, as among
other polynuclear carcinogens (see Section 5.1.1.2, Vol. IIA), the absence of
angular annelation of the rings may be an additional feature responsible for
the lower carcinogenicity of versicolorin A. From the structural standpoint
the aflatoxins and sterigmatocystin bridge the distance between the lactone
and polynuclear carcinogens, 'and bear a relationship to the heteroaromatic
polynuclear lactone carcinogens investigated in some detail by Buu-Hoi and
coworkers (see "Notes" following Section 5.1.1.4, Vol. IIA).
5.3.1.1.3.2 CARCINOGENICITY OF AFLATOXIN MIXTURES OR AFLATOXIN-
CONTAMINATED FEEDS.
Most of the early carcinogenicity studies on aflatoxins were conducted
using aflatoxin-contaminated feed or semipurified aflatoxin mixtures consist-
ing of approximately equal amounts of AFB, and AFG, and traces of AFB^ and
AFG2- Lancaster £t^^l_- (15) were the first to report in 1961 the hepatocar-
cinogenic effect of aflatoxin-contaminated groundnut meal in rats. Continuous
feeding of a purified diet containing 20% highly toxic groundnut meal for 6
months led to the induction of liver tumors in 9 of 11 rats. Shortly after-
ward, similar findings were reported by Schoental (16), LeBreton et al. (17),
*According to the "electronic K-L region theory" actively promoted between
about 1945 and 1970 by the French school of theoretical chemists led by the
Pullmans to account for the carcinogenic activity of polycyclic hydrocarbons
and related polynuclears (see Section 5.1.1.6.1, Vol. IIA), these compounds
must have — in order to be carcinogenic — a reactive double bond (termed the
"K-region" and equated in the angular polynuclears with the meso-phenanthrenic
double bond) and if a compound also has a meso-anthracenic region (termed
"L-region") it must be rather unreactive.
18
-------
Salmon and Newberne (18) and other investigators (see Table V). The final
concentrations of aflatoxin (mainly Bi and Gi) in the contaminated diets that
elicited a carcinogenic response ranged from 0.8-4 ppm (144). Using a par-
tially purified aflatoxin Bj and Gj mixture, Barnes and Butler (147) showed
that feeding a diet containing 1.75 ppm aflatoxin for 89 days (corresponding
to a total dose of only about 2.5 mg/animal) was sufficient to induce liver
tumors in rats. In addition to the induction of hepatocarcinomas, carcinomas
of the glandular stomach, kidney, lung, lachrymal duct and salivary gland were
occasionally seen (144-146). Carnaghan (139) showed that a single oral
administration of a sublethal dose of a mixture of AFBj and AFGj (equivalent
to 5.1 mg AFBj/kg body weight) leads to the induction of hepatic tumors in
7/16 rats with an average latency period of 26 months. In a more recent
study, Fong and Chan (148) found that feeding for 22 months a purified diet,
in which aflatoxin-contaminated peanut oil (obtained from local markets) was
used as the fat source, induced sarcomas in 3/76 rats. None of the 90 control
rats developed malignant tumors. The estimated AFBj content in the diet was
5-7 ppb. Norred and Morrissey (143) found a liver tumor incidence of 100% in
rats fet a diet containing aflatoxin-contaminated corn for 91 weeks; the esti-
mated dietary levels were 150 ppb AFBj, 8 ppb AFGj, and 18 ppb AFB2- The
hepatocarcinogenicity of the feed was completely abolished if the corn was
subjected to decontamination with ammonia.
Besides oral administration, aflatoxin-contaminated feed or partially
purified aflatoxin mixture was shown to be carcinogenic in rats by subcuta-
neous injection, intratracheal administration, or by the transplacental/lacta-
tional route. Dickens et al. (137, 138, 149) reported that twice-weekly
subcutaneous injections of 2, 10, 50 or 500 ug of a mixture of aflatoxins B^
and Gj induce local sarcomas with a virtually 100% incidence. No distant
19
-------
Table V
Carcinogenicity of Aflatoxin Mixtures or Aflatoxin-Contaminated Feed
in Various Animal Species3
Animal Species
Mouse
Rat
Syrian hamster
Rainbow trout
Duck
Ferret
Sheep
Monkey
Route
B.C.
oral
>
s .c
intratracheal
transplacental
and/or lactational
oral or i.p.
oral
oral
oral
oral
i.ra. and oral
Principal Organs
Affected
Local sarcoma
Liver
Liver, stomach,
multiple sites
Liver, colon,
s.c. tissue
Local sarcoma
Trachea, liver
Liver
Harderian gland
Liver
Liver
Liver
Liverb (?)
Liverb
References
(137)
(15-18, 138-143)
(144-147)
(148)
(137, 138,
(138)
(150)
(151)
(152-155)
(156)
(141)
(157)
(158, 159)
149)
aSee Table VI for carcinogenicity study on purified aflatoxin Bi,
Only a small number of animals were used in the study.
-------
tumors were noted. By intratracheal administration (300 ug in 30 ul oil,
twice weekly for 30 weeks), the aflatoxin mixture induced a variety of local
and distant tumors including tracheal squamous carcinomas in 3/6 and hepatomas
in 4/6 rats. One rat developed a renal adenoma, a carcinoma at the pyloric
portion of the intestine, as well as a hepatoma. Grice _et_ _al/ (150) fed 6
groups of 10 pregnant Wistar rats a diet containing either 25% or 50% toxic
groundnut meals (contaminated with 10 ppm AFBi and 2 ppm AFB£) from (a) day 10
of gestation to parturition, (b) day 1 to day 10 post-partum, or (c) day 10 of
gestation to day 10 post-partum. The females and the progeny were then main-
tained on uncontaminated diet and observed for up to 36 months. Liver tumors
were found in 1/36 male offspring in group (a), 1/28 female offspring in group
(b), and 2/38 female offspring in group (c); in addition, a number of off-
spring displayed hyperplastic changes in the liver. None of the 60 control
females and 65 control progeny developed liver tumors. The data provide some
suggestive evidence that low levels of AFB, or its metabolites (such as AFM,),
possibly reach embryos or nursing neonates through the placenta or milk.
In addition to the rat, aflatoxin-contaminated feed or partially purified
aflatoxin mixture has been shown to induce liver tumors in the rainbow trout,
duck, ferret, sheep and monkey (see Table V). The rainbow trout is the
species most susceptible to the hepatocarcinogenic effect of the aflatoxins;
studies in different laboratories (152-155) concur that aflatoxins produce
hepatoma in rainbow trout when fed as low as at parts per billion (ppb) level
(see also Section 5.3.1.1.3.3). In a study by Jackson et_ al_. (155) the esti-
mated carcinogenic dietary level of aflatoxin in rainbow trout was 0.4 ppb.
Ducks and ferrets are also quite sensitive to aflatoxin carcinogenesis.
Carnaghan (156) reported that 8 of 11 ducks fed a diet containing 0.5% toxic
groundnut meal (which contained 7 ppm aflatoxin, assayed as AFBi, giving a
20
-------
final dietary concentration of approx. 30 ppb) developed liver tumors
(parenchymal tumor nodules, bile duct adenomas) after 14 months; none occurred
in 10 controls. Lancaster (141) noted that all ferrets (of an unspecified
number) maintained on a diet containing 3% toxic groundnut meal (which con-
tained about 10 ppm aflatoxins) had liver tumors after 31 months. The
carcinogenicity studies reported on sheep and monkeys involved a limited
number of animals. Lewis et al. (157) found a parenchymal cell carcinoma in a
sheep which died after 3.5 years on a diet containing toxic groundnut meal.
Go pa Ian ^t__al^. (158) and Tilak (159) each reported the induction of a liver
tumor in a rhesus monkey exposed to a mixture of aflatoxins (44% AFBj, 44%
AFGj, 2% AFB2 and AFG2) initially through intramuscular injections (50-100
ug/day, 5 days/week for 1 year) and subsequently by gavage (100-200 ug/day for
4.5 years).
In contrast to the above studies, Herrold (151) found Syrian hamsters to
be highly resistant to the hepatocarcinogenic effect of aflatoxins. She
observed no evidence of regenerative nodules, cirrhosis, or tumors of the
liver in 20 female hamsters which received (by intragastric administration)
0.1 mg aflatoxin mixture (mostly AFBj^ and AFGj with traces of AFB2 and AFG2) ,
2 times a week for 10-11 months or by intraperitoneal injection 0.2 mg afla-
toxin mixture weekly for 6-8.5 months; the hamsters were then observed for the
rest of their lifespan. Four of the 20 animals developed tumors of the
Harderian gland, histologically identified as solid and papillary cyst-
adenomas. In mice, twice weekly subcutaneous injections of 10 ug aflatoxins
(mixture of Bj and Gi) led to the induction of local sarcomas in 15/17 animals
(137).
21
-------
5.3.1.1.3.3 CARCINOGENICITY OF PURIFIED AFLATOXIN Bj.
The carcinogenicity of purified AFBj has been tested in nine animal
species (see Table VI). The results indicate that the carcinogenicity of
aflatoxin mixtures or of aflatoxin-contaminated feed is mainly attributable to
AFBj. Significant species and age differences in susceptibility to carcino-
genesis by AFBi have been observed.
Among the animal species tested, the mouse is the most refractory. In
this species significant carcinogenic effects were observed only in studies in
which infant mice or very high AFB, doses were used; however, there are
notable strain differences in susceptibility. Wogan (160) found no signifi-
cant carcinogenic effects of AFBj in adult random-bred and inbred strains of
mice maintained on a diet containing 1,000 ppm AFBj for up to 70 weeks. On
the other hand, using 2-month-old female A/He mice, Wieder et al. (161) noted
lung adenomas in all the 14 mice that survived 20 weeks after 12 thrice-weekly
intraperitoneal injections of a very high AFBi dose (20 rag/kg body weight).
In contradistinction to the above strain, in newborn Charles River CD-I mice,
Swenson et al. (123) detected only a small increase in the incidence of lung
adenomas (10/107 experimental vs. 2/95 control) 14 months after 3 intraperi-
toneal injections of 0.32 iimole/kg AFBj. In the same study, topical applica-
tions (0.34 uraole/application, twice weekly for 20 weeks) of AFBj failed to
elicit any significant carcinogenic effect. Four- and 7-day-old male (C57 x
C3H)Fj mice were, on the other hand, highly responsive to the hepatocarcino-
genic effect of AFB^ (162). A total dose of 4 umole/kg, given intraperi-
toneally from the 4th to 16th day of age in 5 administrations, was sufficient
to induce hepatomas in 89% of the mice after 82 weeks. A liver tumor inci-
dence of 23% was noted among male mice that received a single dose of 6.4
umole/kg AFBj the first day after birth. Parallel studies indicate that
22
-------
Table VI
Carcinogenicity of Purified Aflatoxin Bj in Various Animal Species3
Animal Species
Mouse
Rat
Syrian golden
hamster
Rainbow trout
Coho salmon
Guppy
Tree shrew
Marmoset
Monkey
Route
oral
i.p.
topical
oral
i.p.
s .c.
transplacental
and oral
oral
oral
i.p.
embryonic
exposure
oral
oral
oral
oral
oral and i.p.
Principal Organs
Affected
None
Lung
Liver
Nonec
Liver
Liver, kidney
Liver, colon
Liver
Local sarcoma
Liver, local
sarcoma
Liver, colon
Liver
Liver
Liver
Liver
None
Liver
Liver
Liver
Liver, pancreas,
multiple sites
References
(160)
(123, 161)
(162)
(123)
(64, 139, 140,
163-169)
(170, 171)
(140, 172, 173)
(64)
(64, 137)
(123)
(173)
(169)
(155, 174-181)
(182)
(179, 183-185)
(174, 175)
(186)
(187)
(188)
(189, 190)
aSee also Table V for carcinogenicity studies using aflatoxin-contaminated
feed or aflatoxin mixtures.
Mice were treated during infancy.
cActive when used as a tumor-initiator followed by promotion with croton oil.
-------
female mice are much less susceptible; liver tumors, with an incidence of 7%,
were detected only in the group that received AFBi at day 7 of age.
Studies in various laboratories (summarized in Table VI) show that puri-
fied AFBj is a highly potent hepatocarcinogen in the rat and clear dose-
response relationships have been established (140, 165, 170). In male Fischer
rats, a dietary level as low as 1 ppb AFBj is demonstrably carcinogenic. The
incidence of liver carcinomas in rats fed diets containing 0, 1, 5, 15, 50 and
100 ppb AFB1 was 0 (0/18), §.1 (2/22), 4.5 (1/22), 19 (4/21), 80 (20/25) and
100% (28/28), respectively. The time of appearance of the earliest tumor in
the AFBj-treated groups was 104, 93, 96, 82 and 54 weeks, respectively
(165). There is some evidence for strain difference in the susceptibility of
rats to AFBj carcinogenesis. For example, feeding a diet containing 100 ppb
AFBj leads to the induction of liver tumors in 100% (28/28) male Fischer rats
(165) but in only 48% (24/50) male Sprague-Dawley rats (172). For male Wistar
rats, the liver tumor incidence was 61% (8/13) in a group fed 250 ppb AFB^
(170). Wistar strain rats are also unusual in being especially prone to
develop kidney tumors when exposed to AFB,; the renal tumor incidence ranged
from 25 to 50% in rats fed 0.25 to 3.0 ppm AFBj (170, 171). A low incidence
of colon carcinomas was observed in Fischer and Sprague-Dawley rats (140,
172); the incidence was enhanced by vitamin A-deficient diets (see Section
5.3.1.1.3.6). However, a more recent study (173) with a thorough examination
of the colon at autopsy, indicated that the incidence of colon carcinoma may
be as high as 9 to 40% in Fischer rats exposed to AFB, (2 ppm in diet) for
lifetime either from conception (i.e., prenatal plus lifetime exposure) or
from 6-7 weeks of age. Also, AFBj appears to have significant local carcino-
genic activity, indicated by the high incidence of local sarcomas at or near
the site of multiple subcutaneous injections of AFBj (64, 123, 137). In one
23
-------
of these studies, subcutaneous injections of AFB, led to induction of liver
tumors as well as local sarcomas (123). The carcinogenicity of AFBj may be
modified by different factors, principally sex hormones and the composition of
the diet; these topics will be discussed in some detail in Section
5.3.1.1.3.6.
An early study by Herrold (151) using a crude aflatoxin mixture indicated
that Syrian golden hamsters are refractory to the hepatocarcinogenic effect of
the aflatoxins (see Section 5.3.1.1.3.2). The refractoriness of hamsters has
been re-examined by Moore et al. (169) using high doses of purified AFBj in
view of the finding that microsomes from hamsters actively metabolize AFB, to
its reactive intermediate, AFBj-2,3-oxide. Male hamsters were given, by
gavage, 2 mg/kg AFBj, 5 days/week for 6 consecutive weeks and were then
observed for up to 78 weeks. Parallel studies were conducted using male
Fischer rats. The hepatic parenchymal cells of hamsters were indeed much more
resistant to the carcinogenic effect of AFBj than were those of rats. Only 1
of 25 hamsters developed hepatocellular carcinoma as compared to all of 25
similarly treated rats. In contrast, the bile duct cells of hamsters were
susceptible; of the 25 hamsters, 8 had cholangiocarcinoma and 17 had micro-
scopic cholangiomas. As in the study of Herrold (151), adenomas of the
Harderian gland were observed; however, they were not attributed to AFB,
treatment because of their occurrence in control hamsters.
Similarly to the rat, the rainbow trout (Salmo gairdneri) is another
species which is extremely susceptible to the hepatocarcinogenic effect of
AFBj. In accord with studies using aflatoxin-contaminated feed, studies using
purified AFBj indicate that lifetime feeding of only ppb levels of AFBi induce
hepatocellular carcinomas in the fish. For rainbow trout of the Mt. Shasta
strain, the reported incidences of hepatomas after 12 months of feeding of
24
-------
diets containing various levels of AFBj were: 0.5 ppb, 20% (181); 2 ppb, 61%
(181); 4 ppb, 25-48% (176, 177, 179); 8 ppb, 70% (176); and 20 ppb, 78-83%
(176, 179, 180). A very short exposure duration of only 10 days to a diet
containing 20 ppb AFBi is sufficient to induce liver tumors in 45% of the fish
(181). The embryos of rainbow trout are even more sensitive to AFBi than are
juvenile trout exposed to an AFBj-containing diet. A single 1-hour exposure
of 14-day-old trout embryos (i.e., fertile eggs) to an aqueous solution con-
taining 0.5 ppm AFBi produced liver tumors in 40-53% of the hatched fish one
year later (179, 183-185). Using 21-day-old embryos, Wales et^ _al_. (184)
determined the amount of AFB^ that could be absorbed by an egg during the
1-hour exposure and found it to be as much as 67 times less than the amount
ingested by a fish during 12-month feeding of a diet containing 4 ppb AFBj.
Besides feeding and embryonic exposure, liver tumors were also induced in
rainbow trout by intraperitoneal injections (182); twice weekly injection of
50 tig/kg AFB, for 25 weeks produced liver tumors in 16 of 24 fish that sur-
vived 50 weeks. There is some evidence of possible strain differences in the
susceptibility of rainbow trout to AFB,. Rainbow trout from the Netherlands
appear to be not as susceptible as are those from Mt. Shasta; after 16-month
the liver tumor incidence in Dutch trout fed a diet containing 5.8 ppb AFBj
was less than 13% (178). Also, steelhead trout is less susceptible to AFB^
carcinogenesis than the Mt. Shasta strain rainbow trout (184) when the eggs of
the trout were exposed to the aflatoxin.
In addition to the rainbow trout, two other species of fish have been
tested for susceptibility to AFB^ carcinogenesis. Halver ^t__al_. (175) found
that, in sharp contrast to the rainbow trout, the coho salmon (Oncorhynchus
kisutch) is refractory to hepatoma induction by AFBj. Chronic feeding of a
diet containing 20 ppb AFBj for 20 months did not induce any liver tumors in
25
-------
coho salmon. Even embryonic exposure to AFBj failed to induce tumors in coho
salmon (J.D. Hendricks and R.O. Sinnhuber, unpublished data, cited in 191).
Sato et al. (186) fed 1-month-old guppies (Lebistes reticulatus) a diet con-
taining 6 ppm AFBi for several months. Hepatic tumors were found in 2 of 5
fish after 9 months and 7 of 11 fish after 11 months. Thus, the guppy is also
responsive to AFBi carcinogenesis although its susceptibility appears to be
much lower than that of the rainbow trout.
Besides rodents and fish, AFBi has been shown to be hepatocarcinogenic in
several species of nonhuman primates. Reddy et al. (187) fed 18 tree shrews
(Tupaia glis) a diet containing 2 ppm AFBj intermittently for up to 172
weeks. Among the 12 animals that survived for more than 74 weeks, 6 of 6
females and 3 of 6 males developed hepatocellular carcinomas. The estimated
total dose ranged from 24 to 66 mg. None of the eight controls had liver
tumors. The tree shrews are small squirrel-like mammals found throughout
Southeast Asia and are regarded as primitive primates. Lin et al. (188)
induced hepatocellular carcinomas in 1 of 9 marmosets fed AFBj at a dietary
level of 2 ppm, and in 2 of 7 marmosets injected with hepatitis virus along
with AFBj feeding. Adamson et al. (189) administered various doses of AFBj to
a group of 20 rhesus (Macaca mulatta), 20 cynomologus (Macaca fascicularis),
and 2 African green (Cercopithecus aethiops) monkeys for periods of up to 9
years. At the time of the report (189), 27 of the 42 monkeys were still
alive. Of the 15 monkeys that were necropsied, 3 bore malignant primary liver
cancers. The estimated total AFBj doses administered to these three tumor-
bearing monkeys were 99, 119 and 842 mg. A more recent update (190) of the
study (including 5 additional monkeys) reported that 13 of 35 monkeys necrop-
sied at the end of 13 years developed one or more malignant tumors, yielding
an overall tumor incidence of 28%. There were 5 primary liver cancers, 2
26
-------
osteogenic sarcomas, 6 carcinomas of the gall bladder of the bile duct, 3
tumors of the pancreas and its duct, and one carcinoma of the urinary
bladder. Fifteen of the 22 necropsied monkeys without tumors showed
histological evidence of liver damage. These results indicate the
susceptibility of primates to AFBj hepatocarcinogenesis and lend support to
the likelihood that humans exposed to AFBj are at risk of developing liver
cancer.
5.3.1.1.3.4 CARCINOGENICITY OF COMPOUNDS RELATED TO AFLATOXIN Bj —
STRUCTURE ACTIVITY RELATIONSHIPS.
The carcinogenicity of close to 20 metabolites, derivatives and struc-
tural analogs of AFBj has been tested in the two most susceptible test species
— the rat and the rainbow trout — and in the mouse. The results of these
studies are summarized in Tables VII and VIII.
Comparative carcinogenicity studies of aflatoxins B,, Hy, G, and 62 and
several metabolites of AFBj have been conducted by Sinnhuber et al (176, 177,
179, 180, 192), Wogan_e£^l_. (64, 193), Butler et_ al_. (164) and Canton et al.
(178) using the rat and the rainbow trout. The results obtained from studies
on these two so different animal species are remarkably concordant (see Table
VII). Except for the induction of kidney tumors in rats by high doses of
AFBj, the liver is virtually the only carcinogenicity target organ of the
various aflatoxins. Among the four naturally occurring aflatoxins, the rela-
tive carcinogenic potency follows the order: AFBj > AFGj » AFB2 in the rat
and AFBj > AFG^ _>. AFB~ > AFG2 in the rainbow trout. Using male Fischer rats,
Wogan £t^ jl_. (64) found that all the 51 rats given small doses of AFB,, total-
ing 0.5, 1.0 or 1.5 mg by stomach tube or 1.3 mg by i.p. injections over a
period of 4-8 weeks, developed hepatocellular carcinomas within 74 weeks. Two
27
-------
Table VII
Relative Potency of Aflatoxins and Related Compounds in the Induction of Liver Tumors in
Rats or Rainbow Trout After Oral or Intraperitoneal Administration
p. 1 of 3
Compound3
Aflatoxin Bj (AFBj)
Aflatoxin BZ (AFB2>
Aflatoxin Gj (AFGj)
Aflatoxin G0 (AFG0)
Ratc
Dietary
level
Total dose (mg) % Incidence References (ppb)
0.277 40 (192) 4
0.5; 1.0; 1.3; 1.5 100 (64, 193)
1.0; 2.0 (MRC) 30; 63d (164) 20
5.8
1.0; 150 0; 33 (64) 20
1.0 (MRC) 0 (164)
0.7; 1.4; 2.0 0; 60; 100e (164) 20
1.0; 2.0; 6.0 (MRC) 10; 10f; 81£ (164)
20
Rainbow trout^
% Incidence
25; 48; 48
(12-month)
* 60 (16-month)
56 (8-month)
78; 83; 83
(12-month)
13 (16-month)h
5 (12-month)
0 (16-month)
5 (12-month)
17 (16-month)
0 (16-month)
References
(176, 177,
179)
(177)
(180)
(176, 179,
180)
(178)
(176)
(176)
(176)
(176)
(176)
-------
Table VII (continued)
p. 2 of 3
t
Compound3 Total dose (rag)
Aflatoxin Mj (AFM^
AFMi (racemic mixture) 1.0
Aflatoxin 0^ (AFQj)
Aflatoxicol (AFL)
Aflatoxicol diastereomer
(AFL')
AFLrAFL1 (1:1 mixture) 0.279; 1.12
Tetrahydrodeoxy-AFBj
5 ,7-Dimethoxycyclopente- 156
Ratc
Dietary
level
% Incidence References (ppb)
4; 16;
32; 64
5.9; 27.3
3 (193)
20; 100
29
61
20; 70 (192)
20
0 (64) 10
Rainbow trout*
% Incidence
13; 70; 61; 60
(12-month)
40; 65; 95; 95
( 16-month)
0; 2 (16-month)h
0; 11 (12-month)
26 (8-month)
81 (12-month)
0 (8-raonth)
24 (12-month)
1 (12-month)
0 (12-month)
References
(177)
(177)
(178)
(179)
(180)
(180)
(180)
(180)
(176)
(176)
none[2,3-c]coumarin
-------
p. 3 of 3
Table VII (continued)
Compound3 Total dose (rag)
5, 7-Diraethoxycyc lopent e- 156
nonef 3,2-c Jcoumarin
5 , 7-Dimethoxycyc lopente- 156
none [c ] coumar in
Isobergaptene
7-Ethoxy-4-methylcoumarin
Ratc
Dietary
level
% Incidence References (ppb)
0 (64)
0 (64)
20
20
Rainbow trout*5
% Incidence References
0 (12-month) (176)
0 (12-month) (176)
aSee Table I for structural formulas.
For structural formulas see below:
cExcept where indicated, male Fischer rats were used in these studies.
Two of 15 male rats also developed kidney tumors.
eFour of 26 male rats also developed kidney tumors.
Five of 15 male rats in the medium dose group and 6 of 11 male rats in the high dose group also developed kidney
tumors.
^Except where noted, rainbow trout of the Mt. Shasta strain were used.
Rainbow trout of Dutch origin.
[Text-Figure 3]
-------
0 0
OCH3
Tetrahydrodeoxy-AFB]
H,CO
5,7-Dimethoxycyclopentene-
[c] coumarin
5,7-Dimethoxycyclopenten-
one-[2,3-c] coumarin
Isobergaptene
H,CO
5,7-Dimethoxycyclopenten-
one-[3,2-c| coumarin
7-Ethoxy-4-methylcoumarin
Text-Figure 3_
-------
Table VIII
Comparative Carcinogenicities of Derivatives of Aflatoxin Bi in Rodents
by Subcutaneous or Topical Administration3
Compound
Aflatoxin Bj (AFB^)
2 , 3-Dichloro-2 , 3-d ihydro-AFBj
( AFBj-2 , 3-d ichlor ide)
Aflatoxin B?
Aflatoxin ftja
Species
and Route
Mouse, topical
Rat, s.c.
Mouse, topical
Rat, s.c.
Rat, s.c.
Mouse, topical
2,3-Dihydro-2,3-dihydroxy-AFBj Mouse, topical
2,3-Dihydro-2-hydroxy-3-
chloro-AFBj
Aflatoxin Gi
aExcept where indicated, the
Res. 15_, 3811 (1975)].
Expressed as umole compound
Mouse, topical
Rat, s.c.
Total Doseb
0.34 or 0.39
0.39 + COC
0.13
0.64
1.28d
2.3-4.7e
0.034
0.1; 0.34
0.39 + CO
0.13; 0.64
38. 2d
0.39 + CO
0.39 + CO
0.39 + CO
3.6-7.9e
data were summarized from D.H.
per animal.
cTest for tumor-initiating activity only; CO =
Summarized from G.N. Wogan, G.S. Edwards and
croton oil .
P.M. Newberne
Duration
of Study
Neoplasm
70 or 33 weeks None
29 weeks Skin carcinoma
20 months
20 months
58 weeks
18-37 weeks
70 weeks
70 weeks
29 weeks
20 months
78-86 weeks
29 weeks
29 weeks
29 weeks
30-65 weeks
Swenson, J.A.
[Cancer Res.
None
Liver carcinoma
Local sarcoma
Local sarcoma
Local sarcoma
Skin papilloma
Skin carcinoma
Skin carcinoma
Local sarcoma
None
Skin carcionma
None
None
Local sarcoma
Incidence
2/29
3/15
5/15
9/9
6/6
2/22
9/24; 14/17
14/24
6/15; 12/15
—
1/30
—
—
4/6
Miller and E.G. Miller [Cancer
31, 1936 (1971)].
Summarized from F. Dickens and H.E.H. Jones (Br. J. Cancer 19, 392 (1965)].
-------
rats in the highest dose group (40 x 37.5 ug over 8 weeks) developed hepato-
cellular carcinomas within 6 weeks after the termination of dosing. For AFGj,
the incidences of hepatocellular carcinomas were 0/3, 3/5 and 18/18 for rats
receiving, by stomach tube, a total dose of 0.7, 1.4 and 2.0 mg, respec-
tively. In the high dose group (40 x 50 ug over 8 weeks), 4 of 26 rats also
developed renal adenocarcinomas. No tumors were found in rats given orally a
total dose of 1.0 mg AFB2 (10 x 100 ug over 2 weeks) after 78 weeks. Only 3
of 9 rats which received a considerably higher total dose of AFB2 (150 mg; 40
x 3.75 mg over 8 weeks) by i.,p. injection developed hepatocellular carcinomas
after 57-59 weeks; for comparison, 9 of 9 rats given a total i.p. dose of 1.3
mg AFBi had liver tumors within 46 weeks. The same relative order of carcino-
genic potency of aflatoxins Bi, Gi and Bo in the induction of liver tumors was
found by Butler et_ _aJU (164) using MRC rats of both sexes. The respective
incidences of liver tumors were 30 and 63% for rats receiving a total dose of
1 and 2 mg AFBj via drinking water, 10, 10 and 81% for rats receiving 1,2 and
6 mg AFGj, and 0% for rats receiving 1 mg AFB2. However, AFG^ appears to be a
more potent renal carcinogen in male rats, inducing kidney tumors in 5/15 and
6/11 rats in the medium (2 mg) and high (6 mg) dose groups compared to 2/15 in
rats receiving 2 mg AFBj. There was no sex difference in the incidence of
liver tumors in the MRC rats. Ayres et al. (176) reported that in rainbow
trout of the Mt. Shasta strain, fed a diet containing 20 ppb AFBj, AFGj, AFB2
or AFGo> the incidences of hepatomas at 12 months were 78, 5, 5 and 0%,
respectively. In another experiment by the same investigators, the 16-month
hepatoma incidences in fish fed 20 ppb AFG^, AFB2 and AFG2 were 17, 0 and 0%,
respectively.
Several metabolites of AFB^ have been tested for carcinogenic activity in
rats and rainbow trout. The relative carcinogenic potency follows the
28
-------
order: AFBj _>. AFL » AFMj in the rat and AFBj > AFL > AFMj > AFQj in the
rainbow trout. Studies by Sinnhuber and associates using rainbow trout of the
Mt. Shasta strain indicated that aflatoxicol (AFL) was close to one half as
potent as AFBj. At 8 months, the incidence of hepatomas at 8 months in fish
fed 29 ppb AFL was 26% compared to 56% in fish fed 20 ppb AFBj. At 12 months,
the difference (81% for AFL versus 83% for AFBj) was less evident because the
carcinogenic response to AFB, was near maximum expression. An unnatural
diastereomer (AFL1) of AFL, obtained by chemical reduction of AFBi, was con-
* 1
siderably less carcinogenic than (about 15% as active as) its natural isomer;
the incidences of hepatomas in fish fed 61 ppb AFL1 were 0 and 24% at 8 and 12
months, respectively (180). Comparison of the hepatoma incidences at 12
months (13% versus 48%) in rainbow trout of the Mt. Shasta strain, fed 4 ppb
AFMj or AFBj, suggested that AFMi was about one third as potent as AFB^
(177). In a comparative study (178) using the less susceptible strain of
rainbow trout obtained from the Netherlands AFM, appeared to be even much less
potent, at least 20-30 times less than AFBj. Aflatoxin Qi has a potency about
l/100th that of AFBj in Mt. Shasta strain rainbow trout; only 11% of the fish
fed a diet containing 100 ppb of the compound developed hepatomas after 12
months compared to 48% of the fish fed 4 ppb AFB^ (179). In studies using
male Fischer rats, Wogan and Paglialunga (193) found that a synthetic, racemic
mixture of AFM^ was a very weak carcinogen. Only one of 29 rats given the
AFMi mixture by stomach tube in 40 doses totalling 1 mg developed a hepatocel-
lular carcinoma at 96 weeks. For comparison, all the 9 rats given a total
dose of 1 mg AFBj developed liver tumors within 53 weeks. Nixon £t__al_. (192)
reported the unexpected finding that a synthetic, racemic mixture of AFL was
highly carcinogenic in male Fischer 344 rats. The incidences of liver tumors
were 20 and 70% in rats fed diets containing 50 and 200 ppb AFL:AFL' mixture
29
-------
(equivalent to a total dose of 0.279 or 1.12 ing), respectively, compared to
40% of rats fed 50 ppm AFBj. Considering the fact that the racemic mixture
contains 50% of the considerably less active, unnatural diastereomer (AFL1),
the data suggest that in male Fischer 344 rats the natural diastereomer (AFL)
may be nearly as carcinogenic as AFBj .
Six derivatives and structural analogs of AFBi , namely, tetrahydrodeoxy-
AFBj , 5 , 7-dimethoxycyclopentenone [ 2 , 3-c ] coumarin , 5 , 7-d imethoxycyclopente-
none[3,2-c]coumarin, 5,7-dimethoxycyclopentenone[c]coumarin, isobergaptene and
7-ethoxy-4-raethylcoumarin (see Table VII for structural formulas) have been
tested for carcinogenic activity in either the rat or the rainbow trout by
oral administration. With the exception of a questionable carcinogenic activ-
ity (a tumor incidence of only 1%) of tetrahydrodeoxy-AFBj in the rainbow
trout, none of these compounds exhibited any carcinogenic activity. These
results suggest that in AFBi the coumarocyclopentenone moiety is not directly
responsible for the carcinogenic activity of AFBj, but is necessary for pro-
viding a favorable molecular size, shape and/or electronic structure.
Comparative carcinogenicity studies on aflatoxins Bj, Gj and 62*
several metabolites and derivatives of AFBi have been carried out in rodents
also by subcutaneous injection and topical application (see Table VIII). Like
the studies involving oral or intraperitoneal administration, bioassays by
subcutaneous administration indicate that the carcinogenic potency of the
three naturally occurring aflatoxins follows the order: AFBj > AFGj » AFB£
(inactive). A study by Dickens and Jones (137) showed that AFBj (20 ug doses,
twice weekly for up to 65 weeks) induced tumors in more rats and more rapidly
than equal doses of AFGj. Local sarcomas were found within 18-37 weeks at the
site of subcutaneous injection in 6 of 6 rats given AFB,. Four of 6 rats
given AFGj developed local sarcomas, with the first appearing at 30 weeks and
30
-------
the last at 50 weeks. Wogan _et^ _al_. (64) induced local sarcomas in 9 of 9 rats
by twice weekly s.c. injections of 10 ug AFBj for 20 weeks. Aflatoxin B2> at
30 times higher doses, induced no tumors in rats after 78 weeks.
Structure-activity correlation and metabolism studies have strongly
implicated the highly unstable metabolite AFBj-2,3-oxide as the probable
ultimate carcinogen of AFBi. To test this hypothesis, Swenson et al. (123)
synthesized a 2,3-dichloro derivative of AFB, and showed that the derivative
had chemical properties (e.g. , electrophilic carbon-2, high reactivity, rapid
hydrolysis, interaction with nucleic acid or protein) expected for AFB^-2,3-
oxide. Comparative carcinogenicity studies indicated that 2,3-dichloro-2,3-
dihydro-AFBi was indeed a more potent direct-acting carcinogen than AFBj.
Subcutaneous injection of a total dose of 0.13 or 0.64 iimole 2,3-dichloro-2,3-
dihydro-AFB, led to the induction of local sarcomas after 20 months in 6/15
and 12/15 rats, respectively; the corresponding incidences for AFBj were 0/15
and 5/15. In mouse skin carcinogenesis studies, 2,3-dichloro-2,3-dihydro-AFB^
was active as a "complete" carcinogen (while AFBj was not) and was a more
potent skin tumor-initiator than AFBj. Among the hydrolysis products of
2,3-dichloro-2,3-dihydro-AFB1, aflatoxin B2a had a marginal skin tumor-
initiating activity while the chlorohydrin (2,3-dihydro-2-hydroxy-3-chloro-
AFBj) and the dihydrodiol (2,3-dihydro-2,3-dihydroxy-AFBj) were devoid of
tumor-initiating activity.
5.3.1.1.3.5 STERIGMATOCYSTIN, VERSICOLORIN AND RELATED COMPOUNDS.
The carcinogenicity of sterigmatocystin has been tested in three species
of rodents and two species of fish. The compound is carcinogenic in mice,
rats, rainbow trout and medaka; its potency is generally lower than that of
AFBj. The results of the studies on sterigmatocystin and several of its
derivatives are summarized in Table IX.
31
-------
Table IX
Carcinogenicity of Sterigmatocystin, Versicolorin and Related Compounds
Compound
Sterigmatocystin
Dihydrosterigmato-
cystin
0-Methyl Sterigmato-
cystin
0-Acetyl sterigraato-
cystin
Versicolorin A
Versicolorin B
Species and Route
Mouse, oral
Mouse, s.c.
Mouse, oral
Rat, oral
Rat , i . p .
Rat , s.c.
Rat, topical
Guinea pig, oral
Rainbow trout ,
embryo exposure
Medaka, oral
Rat, i.p.
Medaka, oral
Medaka, oral
Rat, i.p.
Rainbow trout,
embryo exposure
Medaka, oral
Medaka, oral
Principal
Organs Affected
Lung
Liver, brown fat
tissue, multiple
sites
Lung, liver
Liver, miltiple
sites
Liver
Peritoneum,
liver
Local sarcoma,
liver
Skin, liver
None
Liver
Liver
None
None
Liver
Liver
Liver
Liver
None
References
(194)
(195)
(196)
(197)
(198-200)
(201)
(138)
(197)
(202)
(185)
(203)
(201)
(203)
(203)
(201)
(185)
(203)
(203)
-------
Zwicker et al. (194) fed groups of three-week-old ICR Swiss mice of both
sexes diets containing 5 ppm sterigmatocystin (in pure form or in a rice
culture of Aspergillus versicolor) for periods of two weeks alternating with
two-week periods on control diet, for a total of 54-58 weeks. Pulmonary
adenomas developed in 21/25 (84%) and 33/55 (60%) of the treated mice, respec-
tively, compared to 4/37 (11%) in control mice. There was no significant sex
difference in the incidence of pulmonary adenomas. Pulmonary adenocarcinoraas
were also observed in 9/25 (36%) and 3/55 (6%) of the treated mice, but not in
control mice. In the group fed pure sterigmatocystin, most of the pulmonary
adenocarcinomas developed in female mice (8/10 females vs. 1/15 males). The
incidence of other tumors was not affected by feeding the mycotoxin. Enomoto
et ^1. (195) confirmed the carcinogenicity of sterigmatocystin using female
(C57BL/6NCr x DBA/2NCrj)Fj mice. However, the principal carcinogenicity
target tissues of the compound in this strain of mice are the liver and the
dorsal brown-fat tissue (which is known to be rich in vascular tissue), with
angiosarcoma the predominant tumor type. Of the 53 mice that survived more
than 43 weeks of feeding 30 ppm sterigmatocystin (equivalent to a daily intake
of about 2.5 mg/kg body weight), 34 (64.2%) developed hepatic angiosarcomas,
14 (26.4%) had hepatic hemangioendotheliomas and 6 (11.3%) had angiosarcomas
in the subcutaneous brown-fat tissue located between the bilateral scapulas of
the back. When exposed to a higher dose, 120 ppm, 27/51 (52.9%) of the mice
developed angiosarcomas in the dorsal brown-fat tissue. Interestingly, no
angiosarcomas of the liver were found in the high-dose group. A few angio-
sarcomas of the ovary and the lung, and increases in the incidences of lung
and hepatocellular adenomas were also observed in both test groups. Newborn
mice are much more susceptible to the carcinogenic effects of sterigmatocystin
in agreement with the results of carcinogenicity studies with AFB^ (see
32
-------
Section 5.3.1.1.3.3 above). Fujii _et_ al_. (196) reported that a single s.c.
injection of 5 or 1 iig/kg body weight sterigmatocystin (suspended in 1%
gelatin solution) to (BALB/c x DBA/2)Fj mice within 24 hours after birth gave
rise to a significant increase in the incidence of lung and liver adenomas in
the animals sacrificed at the end of one year. There was a sex difference in
the incidences of these tumors, with males being substantially more suscept-
ible than females. The results show that even a very small dose of sterig-
matocystin may be tumorigenic in newborn animals.
Rats are relatively more susceptible to the carcinogenic effects of
sterigmatocystin than are mice. Purchase and Van der Watt (197) administered
to weanling Wistar-derived rats daily doses of 0.15-2.25 mg sterigmatocystin
(about 88% pure) in the diet (10-150 ppm) or by gavage, 5 days/week for 52
weeks. Among the rats that survived more than 42 weeks of treatment, 39/50
(78%) eventually developed hepatocellular carcinomas. Eight other types of
tumors were found in the uterus, ovary, omenturn and liver; moreover,
acanthotic changes occurred in the stomachs of 30 out of 39 rats examined.
The acanthomas were not considered malignant, although "pearls" and marked
folding of the basal cell layers were observed. No tumors occurred in 19
control rats. Based on a comparison of tumor yield, Purchase and Van der Watt
(197) concluded that sterigmatocystin is at least one tenth as potent as AFBj
in this strain of rats by oral administration. The potent hepatocarcinogenic-
ity of sterigmatocystin in rats has also been demonstrated by using rice
contaminated with Aspergillus versicolor. Enomoto et_ _al_. (198) reported that
hepatic tumors developed in 2/6 Fischer rats fed moldy rice containing only 1
ppm sterigmatocystin for 40 weeks. Ohtsubo et al. (199) found hepatic tumors
(mostly hepatocellular carcinomas) in 23/36 male Donryu rats given a diet to
which moldy rice culture was added to provide 5 or 10 ppm sterigmatocystin
33
-------
(equivalent to daily intake of 75 or 150 ug/rat) for 709 days beginning at
about 6 weeks of age. The average latent period was 470 days. These authors
(199) considered sterigmatocystin a carcinogen nearly equipotent to AFBj.
Maekawa^jiK (200) fed 11-week-old male ACI/N rats a diet containing 0.1, 1
or 10 ppm pure sterigmatocystin for their lifespan. Liver tumors were
observed in the 1 ppm and 10 ppm groups; however, the incidences (1/29 and
5/26, respectively) were substantially lower than those reported in the
studies described above. The authors (200) suggested that ACI/N rats may be
more resistant to the hepatocarcinogenic effect of sterigmatocystin than the
other strains. Moreover, the age of the animals at the start of the experi-
ment also plays a role in susceptibility to carcinogenesis.
In addition to its systemic hepatocarcinogenic activity, sterigmatocystin
displays direct-acting carcinogenic activity in rats. Dickens ^t^ _al^ (138)
injected subcutaneously 0.5 mg sterigmatocystin to a group of 6 rats twice
weekly for 24 weeks. Local sarcomas were observed in 3/6 animals at the end
of the 65-week study; no such tumors occurred in control rats. However,
sterigmatocystin is a substantially weaker local carcinogen than AFBi in the
rat by subcutaneous route. Dickens et al. (138) considered a 0.5 mg dose of
sterigmatocystin to be comparable to a 2 ug dose of AFB,. Purchase and Van
der Watt (204) applied 1 mg sterigmatocystin (dissolved in dimethyl sulfoxide
or acetone) onto the shaved dorsal skin of male Wistar-derived rats twice
weekly for 70 weeks. By 40 weeks, skin papillomas developed and by 70 weeks
all skin-painted rats had either papillomas (7/20 rats) or squamous cell
carcinomas (13/20 rats). None of the control animals had any skin tumors.
The authors (204) emphasized the potential carcinogenic risk of human exposure
through skin contact. Terao (201) injected approximately 1 mg sterigmato-
cystin (dissolved in a 50% aqueous solution of dimethyl formamide) into the
34
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peritoneal cavity of 40 male Wistar rats once a week for 23 weeks and observed
the animals for an additional 57 weeks. Twenty-five rats were alive at 20
weeks, 21 at 40 weeks and 5 at 80 weeks. Twenty of the rats developed meso-
theliomas in the peritoneal cavity with the first tumor appearing at 40
weeks.* None of the 30 control rats had any mesothelioma. In each of the
above three studies, liver tumors were also observed in the rats, but the
incidences (2/6 in the study by Dickens etal., 12/20 in the study by Purchase
and Van der Watt, and 1/40 in the study by Terao) were lower than those of
local tumors.
In addition to mice and rats, sterigmatocystin has been tested for car-
cinogenic activity in guinea pigs, rainbow trout, and medaka (Orizias latipes,
a small aquarium fish). Mabuchi (202) reported that guinea pigs were very
sensitive to the toxicity of the mycotoxin but no tumors developed. Hendricks
et al. (185) exposed 14-day rainbow trout (Salmo gairdneri) embryos to an
aqueous suspension of 5 ppm sterigmatocystin for one hour and observed 1 year
later a 13% incidence of hepatocellular carcinomas (compared to 0% for
controls) among survivors. The relative carcinogenic potency of sterigmato-
cystin was estimated to be about one fourth of that of AFB,. Terao et al.
(203) gave medaka a diet containing 5 ppm sterigmatocystin for 12 weeks and a
control diet for an additional 12 weeks. At 24 weeks, when the medaka were
killed, 10/18 (56%) of the fish were found to have hepatomas.
*The induction of mesotheliomas by sterigmatocystin is highly unusual in view
of the fact that virtually all mesothelioma-inducing substances are in fibrous
or in crystalline form (see Section 5.5). Although sterigmatocystin is known
to form fine needle-like crystals in a 50% aqueous dimethylformamide solution,
two closely related analogs (dihydrosterigmatocystin and 0-acetylsterigmato-
cystin) fail to induce mesotheliomas under similar condition.
35
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Only three derivatives of sterigmatocystin have been testecMror carcino-
genic activity. Dihydrosterigmatocystin was found inactive in both rats and
raedaka (see Table IX) indicating that, like aflatoxins, the presence of a
double bond in the terminal furan ring of sterigmatocystin is an essential
structural requirement for carcinogenicity. 0-Acetylation of sterigmatocystin
appears to modify the carcinogenicity target tissue of the mycotoxin admini-
stered intraperitoneally to rats. Terao (201) reported that 0-acetylation
eliminated the local carcinogenic activity of sterigmatocystin in the peri-
toneal cavity, but enhanced its hepatocarcinogenic activity. He suggested
that 0-acetylsterigmatocystin may be readily absorbed from the peritoneum,
thus eliminating the possibility to display local carcinogenic effects.
Another 0-substituted derivative, 0-methylsterigamtocystin, has been tested by
feeding to medaka (203). Under comparable conditions (5 ppm in diet for 12
weeks followed by 12 weeks control diet), the 0—methyl derivative induced
slightly more hepatomas (18/27 medaka, or 66%) than the parent compound (10/18
medaka, or 56%). Versicolorin A, a biosynthetic precursor of sterigmato-
cystin, has also been tested in the same study; the compound was only weakly
carcinogenic inducing hepatomas in 3/44 (7%) medaka. Versicolorin B, the
dihydro derivative of Versicolorin A, was completely inactive indicating the
requirement of double bond in the terminal furan ring for carcinogenic activ-
ity. Versicolorin A has also been shown to be hepatocarcinogenic in rainbow
trout. Hendricks et al. (185) exposed 21-day trout embryos to 3% dimethyl
sulfoxide solutions containing 5 or 25 ppm Versicolorin A and observed hepato-
cellular carcinoma incidences of 42 and 68%, respectively, among survivors
after one year. The authors (185) estimated that the carcinogenic potency of
versicolorin A is between 1/50 to 1/16 of that of AFBj. In the same study,
*
the carcinogenic potency of sterigmatocystin is about one-fourth of that of
36
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AFBj. Thus, the study of both lerao et_£l_. (203) and Hendricks et_ al_. (185)
indicate that the linearly annelated (acene-type) versicolorin A is less
carcinogenic than the angularly annelated (phene-type) sterigmatocystin.
5.3.1.1.3.6 MODIFICATION OF AFLATOXIN CARCINOGENESIS.
The carcinogenicity of aflatoxins may be modified by a variety of host
and environmental factors. These include hormonal status, nutrients, simul-
taneous exposure to environmental chemicals, viral infection as well as (arti-
ficial) sunlight. The study of these factors is of great importance in
assessing the carcinogenic risk of human exposure to aflatoxin in the environ-
ment and in developing strategies for cancer prevention. A comprehensive
review of the factors that may modify aflatoxin carcinogenesis has been pub-
lished by the World Health Organization (33) in 1979. In this subsection,
only the major findings of modification studies are discussed, with emphasis
on recent studies. A more detailed discussion will be presented in Vol. IV of
this series of monographs.
Effect of diet. Dietary modification of aflatoxin carcinogenesis has
attracted much attention in view of the occurrence of nutritional deficiencies
in certain parts of the world where aflatoxin exposure can be considerable.
The dietary factors studied include protein, lipid, lipotropic agents,
vitamins, and natural and synthetic dietary components.
The effect of dietary protein on AFBj-induced hepatocarcinogenesis has
been studied by a number of investigators. Early studies in 1966 and 1968 by
Newberne _e£_al_. (163, 205) indicated that rats maintained on a low (9%)
protein diet developed more tumors in a shorter time in response to AFB, than
those maintained on a high (20%) protein diet. However, subsequent studies
tend to support the opposite view. Madhavan and GopaIan (206) reported that
37
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AFBj-treated rats fed a 5% casein diet developed fewer liver tumors than those
fed a 20% casein diet. Wells et al. (207) maintained rats on diets with three
different levels of casein and found that the incidence of AFBj-induced liver
tumors increased as the level of dietary protein increased. Lee et al. (208)
found that the incidence of AFBi-induced hepatoma was significantly higher in
rainbow trout fed a diet containing 49% fish protein than those fed a 32%
protein diet. Bailey _et_^U (181) showed that the enhancing effect of protein
diet on AFBi hepatocarcinogenesis was even more dramatic with higher protein
concentration; the 9-month hepatoma incidences in rainbow trout fed 20 ppb
AFBj and a 40, 50, 60 or 70% protein diet were 33, 48, 68 and 90%, respec-
tively. The enhancing effect was evident even in fish exposed to AFBj as
embryos and then reared on high protein diets suggesting that high protein
enhanced transformation following DNA damage by AFB,. There is some evidence
that low dietary protein inhibits the initiation (adduct formation) stage
(209) as well as the promotion stage (210) of AFBi hepatocarcinogenesis in the
rat. In the rainbow trout, high dietary protein has no significant effect on
covalent binding of AFB, to liver DNA suggesting that it enhances hepatocar-
cinogenesis through a promotional mechanism (181, 191).
There is growing evidence that dietary lipids may play a significant
contributory role in the induction of cancer (this topic will be discussed in
detail in Vol. IV of this series of monographs). Specially interesting
constituents of lipids, principally of plant origin, are the cyclopropenoid
fatty acids (CPFA). Studies by Sinnhuber and associates have shown that CPFA
act as potent synergists or promoters in hepatocarcinogenesis by AFBj (180,
181, 211), AFMj (177), AFQj (179), and AFL (180) in rainbow trout. For
example, in a 9-month feeding study, the incidence of hepatocellular carci-
nomas was 0% for fish fed 0.5 ppb AFB^ 2% for fish fed 20 ppm CPFA but
38
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increased to 63% for fish fed 0.5 ppb AFBj plus 20 ppm CPFA (181)._ The
enhancing effect of CPFA on AFBj in rats, however, appears to be much less
prominent (212) or negligible (213, 214). The major constituents of CPFA are
malvalic acid and sterculic acid. They occur in triglycerides of plants in
the order Malvales (215) and may be present in human foods derived from
cottonseed or kapok oil (181).
Newberne and Rogers have studied the modifying effect of lipotropic
agents on AFBj carcinogenesis in rats. Diets high in fat and marginally
deficient in the lipotropic agents methionine, choline and vitamin B,^ pro-
tected rats against the acute toxic effects of AFBj, but enhanced the inci-
dence of liver tumors and shortened the latent period of tumor induction (216-
218) . The enhancing effect was attributed to marginal deficiency in lipo-
tropic agents because high dietary fat content alone appeared to inhibit
rather than contribute to enhancement of AFBj carcinogenesis (Rogers et al.,
cited in 33). However, in other experiments severe dietary deficiency in
lipotrops caused inhibition rather than enhancement of AFB^ carcinogenesis
(218, 219).
Several vitamins and micronutrients have been tested as potential modi-
fiers of aflatoxin carcinogenesis. Temcharoen et al. (220) reported that
dietary supplementation with vitamin B,~ (a weakly lipotropic agent) enhanced
liver tumor incidence in rats fed a mixture of AFB^ and AFGj and maintained on
a 20% protein diet; however, in rats maintained on a 5% protein diet, vitamin
Bio appeared to reduce rather than enhance tumor incidence. Newberne and
Rogers (172) showed that marginal deficiency in vitamin A had no significant
effect on AFBj hepatocarcinogenesis in rats; however, an apparent increase in
the incidence of colon carcinomas was observed. This finding was confirmed in
a further study by Newberne and Suphakarn (221); moreover, supplementation
39
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with excessive vitamin A had no protective effect against AFB, carcinogenesis
in either the liver or the colon. Dietary selenium (222) and ascorbic acid
(223) also appeared to have no effect on AFB^ carcinogenesis in rats. There
is some evidence that photosensitized riboflavin may protect rat against AFBj
hepatocarcinogenesis (see discussion on "Effect of other environmental
factors").
Sinnhuber and associates have examined the ability of a number of natural
and synthetic dietary components to modify AFBj hepatocarcinogenesis in rain-
bow trout. Several flavonoid and indole compounds such as ^-naphthoflavone,
indole-3-carbinol, and possibly quercitin and a tangeritin-nobilitin mixture
have been shown to inhibit AFB, hepatocarcinogenesis when fed prior to and
during AFBi exposure (181). On the other hand, dietary cruciferous vegetables
(such as cauliflower, broccoli, brussel sprouts) and associated isothiocyanate
compounds does not exhibit any modifying activity (224). The possible
mechanism by which A-naphthof lavone inhibits AFB, hepatocarcinogenesis in
trout has been explored (181). The mechanism of this inhibition by
ft-naphthoflavone (a well known inducer of microsomal mixed-function oxidases)
appears to be to redirect AFBi metabolism from the production of the highly
carcinogenic metabolite, aflatoxicol to the less carcinogenic metabolite,
AFMj. In the rat, there is some evidence that dietary cauliflower and cabbage
act as inhibitors of AFB^ hepatocarcinogenesis, whereas ingestion of beets as
part of the diet has the opposite effect (168, 225); the mechanisms of these
modifying effects remain to be elucidated.
Effect of hormones. Investigations with aflatoxin-contaminated feed or
purified AFBj suggest that, in some strains of rats (e .g. , Fischer), females
are less susceptible to AFBi hepatocarcinogenesis than males (e.g., 140,
163). Consistent with this finding, Newberne and Williams (226) noted that
40
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the incidence of AFB^-induced liver carcinomas in male rats was significantly
reduced from 71% to 20% by simultaneous oral administration of the synthetic
estrogen, diethylstilbestrol. Cardeilhac and Nair (227) reported that castra-
tion of male rats soon after weaning abolished the hepatocarcinogenic response
of the rats to a combined treatment of AFB^ and carbon tetrachloride (which
act synergistically). In vitro DNA binding studies by Gurtoo et al. (228)
indicate that the sex difference in susceptibility is associated with dif-
ference in the binding of AFBi to DNA. Besides castration, hypophysectomy has
a dramatic modifying effect on the carcinogenic response of rats to AFBi.
Goodall and Butler (229) showed that 14 of 14 rats fed 4 ppm AFBj developed
liver carcinomas within 49 weeks, whereas none of the 14 hypophysectomized
rats developed tumors at the same time period despite receiving slightly
higher amounts of AFBj. Hypophysectomy of rats has also been shown to inhibit
in vivo binding of AFBj to DNA (223).
Effect of chemical agents. A variety of pharmacologically active com-
pounds have been tested for their ability to modify or act synergistically
with aflatoxin carcinogenesis. Carcinogens which are known at present to act
synergistically with AFBj are ethionine (in rats) (205), AFB2 (i-n trout)
(176), carbon tetrachloride (in mice) (230) and cyclopropenoid fatty acids (in
rainbow trout) (181). There is no evidence for syncarcinogenesis between
urethan and aflatoxin in rats (231). Lasiocarpine, a pyrrolizidine alkaloid,
alters the histopathology but does not affect the incidence of AFBj-induced
liver tumors in rats (232). In contrast, o^-hexachlorocyclohexane (c<-BHC) is
a potent inhibitor of AFBj hepatocarcinogenesis (167) possibly by inducing
enzymes detoxifying AFBj. in mouse skin carcinogenesis studies, AFB^ showed
tumor-initiatory activity which can be promoted by croton oil (233). There is
some evidence that 3-methylcoumarin (a noncarcinogenic compound which is
41
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structurally related to AFBj) may function as cocarcinogen when fed simul-
taneously with AFBi to trout (176).
Phenobarbital, an inducer of microsomal mixed-function oxidases, has been
shown in two studies (142, 223) to inhibit AFBj hepatocarcinogenesis in
rats. In another study in which rats and Syrian golden hamsters were fed
large doses of AFBi, phenobarbital apparently had no modifying effect (169).
Novi (234) reported the interesting finding that reduced glutathione admini-
stered JJ__t£_2^jn££ths_afte_r_ AFB^ treatment to rats, bearing AFBj-induced
liver tumors, caused regression of tumor growth. Novi recommended further
investigation on the use of glutathione as a potential antitumor drug.
0
In addition to the studies reviewed above, a number of other mycotoxins
have been tested as potential modifiers of AFBj carcinogenesis. Rubratoxin B
displayed no effect on AFB, carcinogenesis when fed simultaneously with AFBj
to rats for 60 weeks (64). Two Fusarium toxins (T-2 toxin and diacetoxy-
scirpenol) were ineffective as tumorigenesis promoters in mouse skin initiated
by painting with AFBj (233).
Effect of other environmental factors. The effect of exposure to light
on the carcinogenicity of AFBi was investigated by Joseph-Bravo et al.
(166). Two groups of rats were given intragastrically a very large dose of
riboflavin, followed by 25 jug AFBi 5 days/week for 3 weeks. Thirty minutes
after dosing with AFB,, one group of rats were irradiated for 2 hours with
artificial sunlight and the other group served as unirradiated control. At
the end of the 53-week study, all (11/11) unirradiated rats developed liver
tumors whereas only 5 of 12 irradiated rats had such lesions. The investi-
gators postulated that photosensitized ribloflavin may complex with AFB^ and
prevents its activation to a carcinogenic metabolite. Lin et al. (188)
42
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studied the long-term effects of a combined exposure to AFB, and viral
hepatitis on marmoset liver. Liver cirrhosis was much more severe after
exposure to both AFB^ and hepatitis virus than after exposure to either agent
alone. However, the effect of combined exposure on carcinogenesis appears to
be nil: liver tumors were observed in 2/7 marmosets given AFBj and hepatitis
virus, 3/9 marmosets given AFBi alone and 0/7 marmosets given the virus alone.
5.3.1.1.4 Metabolism and Mechanism of Action
5.3.1.1.4.1 METABOLISM
It is now generally accepted that most of the toxic actions, particularly
carcinogenicity and mutagenicity, of naturally occurring aflatoxins require
metabolic activation. Owing to their potent carcinogenicity and ubiquitous
i
environmental occurrence, the metabolism of aflatoxins has been extensively
studied. The role of metabolism in the activation of aflatoxins has been the
subject of many reviews (e.g., 32-34, 44, 46, 49).
Metabolism of Aflatoxin B, (AFBj). Aflatoxin Bj is actively metabolized
in a variety of animal species. Figure 1 depicts the map of known metabolic
pathways of AFB^. The relative importance of each individual pathway varies
substantially depending on the animal species and the experimental condi-
tions. Essentially, initial metabolism of AFB, involves three principal types
of reactions: (a) hydroxylation, (b) epoxidation, and (c) ketoreduction. The
former two reactions are believed to be carried out principally by a micro-
somal mixed-function oxidase system while the latter by a cytosolic NADPH-
dependent reductase. In most animal species, the hydroxylated AFB, metabo-
lites may undergo phase II metabolism by conjugating with glucuronic acid or
sulfate. The role of each individual metabolic pathway in the overall toxic
effects of AFBj is discussed below.
43
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0 OH
II
0
OH
OCH3
Aflatoxicol(AFL)
0 OH
GSH-conjugate'
AFBr2,3-oxide
Covalent binding to DMA,
RNA and protein
SrXX'^^OH
AFPi
0 0
II
0'
HO,
icrV^
AFBr2,3-dihydrodiol
Fig. 1. Metabolic pathways of aflatoxin B
-------
Legend to Figure 1 -
Fig. 1. Metabolic pathways of aflatoxin Bj. (Aflatoxin Bj metabolites
containing 2,3-double bond can be further metabolized to corresponding
2,3-oxide. The conversion of AFBi to AFBoa predominantly occurs nonenzyraatic-
ally under acidic condition. Both AFB2a and AFB1~2,3-dihydrodiol can bind
covalently to protein via Schiff base formation.)
-------
Epoxidation of the 2,3-double bond (or 8,9-double bond by _the__IUPAC
nomenclature) of AFBi is now generally accepted to be the key metabolic reac-
tion eliciting the carcinogenic or mutagenic effect of the mycotoxin. That
the 2,3-double bond is a critical structural requirement for carcinogenicity
and mutagenicity has been pinpointed by structure-activity relationships
studies on a variety of structural analogs of AFBj (see Section 5.3.1.1.2.2
and 5.3.1.1.3.4). Moreover, quantum mechanical calculations have shown that
the 2,3-double bond is the most reactive site in the molecule (32, 51).
Although attempts to isolate the putative reactive intermediate, AFBi-2,3-
oxide, have been unsuccessful because of its instability, its formation can be
deduced from its reaction products with cellular constituents and its hydrol-
ysis products. Aflatoxin Bi binds covalently to nucleic acids after in vitro
metabolic activation by liver microsomes (53, 55, 235-239) as well as in in
vivo studies (49, 191, 223, 238, 240-243). The major acid hydrolysis products
of AFBj-DNA adducts were identified as 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-
aflatoxin Bi and 2,3-dihydro-2-(2,6-diaraino-4-oxo-3,4-dihydropyrimid-5-yl-
formamido)-3-hydroxyaflatoxin B^ (see Section 5.3.1.1.4.2) indicating the
involvement of AFB^-2,3-oxide. In the absence of exogenous nucleophiles,
2,3-dihydro-2,3-dihydroxyaflatoxin B^, an expected hydrolysis product of
AFB^-2,3-oxide, is a major metabolite in the incubation of AFBj with rat,
hamster and trout liver microsomes (244-247). An AFB^-GSH conjugate, tenta-
tively identified as 2,3-dihydro-2-(_S_-glutathionyl)-3-hydroxyaflatoxin Bj was
also detected as an in vitro as well as in vivo metabolite of AFBj in the
rat. The AFBj-GSH conjugate accounted for about 10% of the administered AFBj
dose in this study (248).
The hepatic microsomal enzyme system that catalyzes 2,3-epoxidation of
exhibits the typical characteristics of a mixed-function oxidase (239,
44
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249, 250). Pretreatment of animals with phenobarbital (but not with 3-methyl-
cholanthrene) greatly enhances the in vitro formation of adducts of AFBj with
nucleic acids (235, 239, 249, 251, 252) suggesting the involvement of a cyto-
chrome P-450-dependent system. However, somewhat inconsistent results have
been obtained from reconstitution experiments using purified cytochromes. In
two of these studies (253, 254) purified hepatic cytochrome P-448 species
obtained from polychlorinated biphenyl- or 3-methylcholanthrene-treated rats
were more effective than purified phenobarbital-induced cytochrome P-450, in
catalyzing the formation of a reactive intermediate that binds to DNA or
exerts a mutagenic effect. In a third study (247), purified phenobarbital-
induced rat liver cytochrorae P-450 was more active than /3-naphthof lavone-
induced cytochrome P-448 in catalyzing the formation of AFBj-DNA adduct. A
purified form of trout liver cytochrome P-450 was found to be at least 10
times more active than rat liver P-450. There are also discrepancies between
in vitro and in vivo studies; the in vivo formation of AFBi-DNA adducts was
reduced rather than enhanced by phenobarbital pretreatment (223, 240). The
difference was attributed to a possible change in the pharmacokinetics of AFB,
metabolism with more metabolism proceeding via detoxification pathways (such
as conversion to AFQ^ which is also enhanced by phenobarbital) in the animal,
than in an in vitro system in the presence of excess substrate and cofactors
(240). In this respect, it is interesting to note that the apparent K for
conversion of AFBj to AFQj (l^ = 0.07 mM) by rat liver microsomes (255)
appeared to be substantially lower than that of metabolic activation of AFB^
to DNA-binding metabolites (l^ = 1-97 mM) (256). Besides liver microsomes,
rat liver nuclei (257, 258) and mitochondria (259, 260) have also been shown
to contain mixed-function oxidase systems that are capable of catalyzing
covalent binding of AFBj to DNA. In view of the high reactivity and insta-
45
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bility of AFBj-2,3-oxide, nuclear activation is potentially more important in
the covalent binding of AFBi to DNA than microsomal activation, because of
spatial proximity. Mitochondrial activation may also be of great importance
because of the presence of mitochondria! DNA. In fact, a recent study by
Niranjan _et^ jil^ (261) showed that the in vivo covalent binding of AFB, to
mitochondrial DNA was three to four times higher than that of nuclear DNA (see
Section 5.3.1.1.4.2).
The formation of 2,3-dihydro-2,3-dihydroxyaflatoxin B^ (AFBj-dihydrodiol)
as an in vitro metabolite of AFBi was not demonstrated until relatively
recently. The difficulty stems from the fact that the dihydrodiol is highly
unstable at alkaline or neutral pH, and may bind to microsomal protein, gluta-
thione or amines (including the widely used tris-hydroxymethyl aminomethane
"Tris" buffer). Lin ^t_ ^1_. (244) found that less than 3% of AFBj was con-
verted to AFB,-dihydrodiol when incubated with rat and hamster liver micro-
somes and NADPH (at pH 6.5) in the absence of an exogenous nucleophile. Much
smaller amounts of the dihydrodiol were detected at physiological pH or in the
presence of exogenous DNA. Addition of inhibitors of epoxide hydrase to the
incubation medium did not lower the yield of the dihydrodiol, suggesting that
the hydrolysis of AFBj-2,3-oxide to the dihydrodiol is mainly nonenzymatic.
Neal and Colley (246) presented evidence that AFBj-dihydrodiol is indeed a
major metabolite of AFBj produced by rat liver microsomes, but it is removed
by binding to microsomal proteins. When Tris is used as the buffer, most of
the dihydrodiol binds to Tris, forming Tris-diol. Williams and Buhler (247)
reported that using a purified form of cytochrome P-450 from trout liver in a
reconstituted system, the formation of AFBj-dihydrodiol (including those
trapped by Tris buffer) accounted for as much as 72% of total AFBi metabo-
lized. Besides hydrolysis, reduced glutathione (GSH) may compete for inter-
46
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action with AFB1~2,3-oxide. Lotlikar ^ jil^ (262) observed that the addition
of cytosolic fraction inhibits the hamster hepatic microsome-mediated covalent
binding of AFBj to DNA. Depletion of GSH or heat treatment of the cytosolic
fraction abolishes the inhibitory activity indicating the involvement of
glutathione S-transferase. The inhibition of AFBj-binding to DNA suggested
the formation of an AFBj-GSH conjugate. Indeed, an AFBj-GSH conjugate was
detected as a major in vivo metabolite of AFB, in the rat (248).
Besides epoxidation, interconversion between AFBj and aflatoxicol (APL;
also called aflatoxin RQ) may also play a role in AFBj carcinogenesis. Afla-
toxicol has been shown to be the most mutagenic (see Section 5.3.1.1.2.2) and
carcinogenic (see Section 5.3.1.1.3.3) metabolite of AFB^. Its carcinogenic
potency is about one-half that of AFBj in the rainbow trout (180) and is only
slightly less than or may be even comparable to that of AFB, in the rat
(192). In vitro studies by various investigators (255, 263-266) have indi-
cated that the conversion of AFB, to AFL proceeds through a NADPH-dependent
reductase present in the cytosolic (100,000 x g) fraction of liver homogenate
from a number of animal species. The reaction is reversible (264) possibly
through the action of a NADPH-dependent AFL dehydrogenase (266), which is
inhibited by steroid hormones (267). Species comparison studies have shown a
reasonably good correlation between susceptibility to AFBi carcinogenesis and
API-forming activity. This metabolic pathway is highly active in two very
susceptible species, the rainbow trout (265, 266) and the duck (255, 263, 264)
as well as in rabbits (266) (susceptibility not known), but is low or inactive
.-•
in less susceptible or resistant species, such as the guinea pig, mouse,
hamster and monkey (255, 266, 268). In vitro studies, using as enzyme source
the liver of a highly susceptible species, the rat, showed little (266) or no
(255) AFL-forming activity; however, an in vivo study by Wong and Hsieh (268)
47
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found that AFL is in fact the major metabolite of AFBj in^rat plasma. In the
same in vivo study, AFL was not detected in the plasma of AFBj-treated rhesus
monkey and mouse. In vitro studies using human liver samples indicated low
(266) or negligible (255) AFL-forming activity. The finding by Bailey et al.
(181) that /3-naphthoflavone, which protects rainbow trout against AFB^ car-
cinogenesis, alters the metabolic profile by lowering AFL but increasing AFM^
production is consistent with the view that AFL may play a contributory role
in AFBi carcinogenesis. The possible role by which the AFL pathway may con-
tribute to AFBj carcinogenesis is not clearly understood. The reversible
nature of the pathway suggests that the formation of AFL may serve as a meta-
bolic storage reservoir of AFB^ (264, 269). Alternatively, the possibility
that AFL may act as a proximate carcinogen of AFBj cannot be ruled out.
The metabolism of AFB, to AFM,, AFPi and AFQ, is generally regarded as
detoxification pathways. Both AFM^ and AFQ^ are substantially less carcino-
genic and mutagenic than AFB, while AFP, is not mutagenic in the Ames test
(see Sections 5.3.1.1.2.2 and 5.3.1.1.3). Moreover, the hydroxylated metabo-
lites are more polar than the parent compound and are expected to be more
readily excreted. Conjugation of the hydroxylated metabolites with glucuronic
acid or sulfate further facilitates renal excretion. Aflatoxin M, has been
detected in the milk or urine of the cow (270; rev. 271), sheep (272, 273),
rat (274), mouse (274), guinea pig (275), monkey (276, 277) and of humans
(278) exposed to AFBj. The excretion of AFMj (which still retains some car-
cinogenic activity) in the milk of farm animals fed contaminated feed has been
an agricultural problem of concern (see Section 5.3.1.1.5.2). The conversion
of AFBj to AFM^ has also been demonstrated using liver microsomal or post-
mitochondrial fractions from rats (251, 256, 279-282), trout (181), mice
(252), monkey (281, 283), humans (284), dogs (266, 282), chickens (281),
48
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hamsters, cows, lambs and pigs (282). Considerable species differences in
AFMj-forming activity have been observed (266, 281, 282). The in vitro AFMj-
fortning activity of liver microsomes from rat, mouse and rainbow trout is
markedly enhanced by pretreatment of the animals with 3-methylcholanthrene,
2,3,7,8-tetrachiorodibenzo-p-dioxin, polychlorinated biphenyls, or ft -naphtho-
t
flavone (181, 252, 253, 256, 282, 285). These results, along with studies
using inbred strains of mice with genetically different responsiveness to
enzyme induction (252, 286), suggest that a cytochrome P-448-dependent enzyme
system is responsible for 4-hydroxylation of AFBj to AFMj. This has been con-
firmed using purified cytochrome P-448 in a reconstitution experiment (253).
Aflatoxin P^ is a major metabolite (mostly as glucuronide and sulfate) of
AFBj in the urine of monkey following intraperitoneal administration of AFBj
(276). However, AFP^ is only a minor urinary metabolite when AFBj is given
orally to monkeys (277). The 0-demethylation of AFBj appears to be a minor
p."t:hway in the in vitro metabolism of AFB, by most animal species. Aflatoxin
p, was not detected in an in vitro study by Masri et al. (281) using monkey,
rat and chicken liver. Roebuck and Wogan (255) detected the generation of
AFPi by human, monkey and mouse liver, but not by duck and rat liver, incu-
bated with AFBj. Dahms and Gurtoo (251), however, reported that mouse liver
microsomes actively 0-demethylate AFBi; the amount of AFPj produced by mouse
liver microsomes was 10 times greater than that by rat liver microsomes.
Pretreatment of mouse or rat with phenobarbital slightly increased the in
vitro AFP|-forming activity (251).
Aflatoxin Qj is the major in vitro metabolite produced by human (255,
287), monkey (255, 281, 283), and rat (251, 255, 256) liver. The in vitro
AFQj-forming activity is relatively low using mouse (251, 2 52 )_or...chicken
(281) liver, and negligible with duck liver (255). The conversion of AFE^ to
49
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AFQj by liver microsomes can be substantially enhanced by pretreatment of
animals with phenobarbital but not with 3-methylcholanthrene, suggesting the
involvement of a cytochrome P-450-dependent enzyme system (245, 251, 252, 256,
281, 285, 288). However, in reconstitution experiments using purified cyto-
chromes, both cytochrome P-448 and P-450 can convert AFBj to AFQ^ (253).
Aflatoxin B2a (2,3-dihydro-2-hydroxyaflatoxin Bj) is a potential degrada-
tion product of AFBj. Being a hemiacetal, AFB2a is unstable at physiological
pH and may readily bind covalently to microsomal protein (see Section
5.3.1.1.4.2). Aflatoxin &2a ^s relatively nontoxic and nonmutagenic (see
Section 5.3.1.1.2.2); however, it is believed to contribute to the toxic (but
not the carcinogenic) action of AFB, if formed metabolically in target organs
(44). Aflatoxin B2a can be formed nonenzymatically by acid hydration of AFB,
(52); it has been detected in acidic gastric juices (289) and in urine (290)
of animals fed AFBj. Several investigators (250, 280, 291, 292) reported in
•eduction of "AFB2a" from AFBi by rat liver microsomes; the reaction
re^. • tfADPH and is inhibited by SKF-525A, a typical inhibitor of microsomal
mixed-function oxidases (250). However, a more recent study by Lin et al.
(244) indicates that the metabolite of AFB^ generated by a NADPH-dependent
system reported in the above study was most likely 2,3-dihydro-2,3-dihydroxy-
aflatoxin Bj rather than AFB2a.
In addition to the various metabolites discussed in the preceding para-
graphs, two other in vitro metabolites of AFB^ have been identified. Salhab
and Hsieh (293) detected the formation of aflatoxicol Hj (AFLHj; see Fig. 1
for structural formula) as a major metabolite of AFBi produced by human and
rhesus monkey liver in vitro. Both the microsomal hydroxylase and the cyto-
solic reductase system are required for the formation of AFLHj- It is not
known whether AFLH^ is formed by reduction of AFQi or hydroxylation of AFL or
50
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both. The compound is nonmutagenic in Ames test. Another relatively new
metabolite is aflatoxicol Mj (AFLMj) (294). Aflatoxicol Mj may be produced by
hydroxylation of AFL using dog liver microsomes or by ketoreduction of AFMj by
rabbit liver cytosol. It may be oxidized to AFMj by a carbon monoxide-
insensitive dehydrogenase present in human liver microsomes. Aflatoxicol M,
has also been detected as an in vitro metabolite of AFBj using trout liver
microsomes (181). No information is available on the genotoxic potential of
this metabolite.
In addition to microsomal activation, AFB, may also be photochemically
activated. Shieh and Song (56) irradiated a solution of AFBj^ and calf thyraus
DNA with near-UV light under anaerobic condition and detected a significant
amount of covalent binding of AFBj to DNA (1 AFBj per 1,300 nucleotides). The
AFBj-DNA adduct showed a substantial inhibition of its DNA template activity
for DNA synthesis and for RNA transcription in vitro. The photobinding of
AFBj to DNA was greater than that of AFGj. The photobinding of their dihydro
derivatives, AFBo and AFGo. was low or negligible consistent with the require-
ment for the 2,3-double bond in the terminal furan ring. The nature of photo-
adduct remains to be explored. The mechanism for photobinding of AFBj is
believed to differ from that occurring by microsomal activation, because
photobinding to DNA occurs preferentially at the A-T sequence rather than at
the G-C sequence. Shieh and Song (56) proposed a multi-step activation
mechanism involving the photoexcitation of the coumaryl chromophore (similar
to photoactivation of psoralen) and an intramolecular energy transfer from the
coumaryl chromophore to activate the 2,3-double bond of AFBj. The biological
significance of photoactivation of aflatoxins is unclear. It would be of
interest to investigate in this context whether near-UV light may contribute
to skin carcinogenesis by AFBi.
51
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Metabolism of other aflatoxins and related compounds. Compared to AFB,,
relatively little information is available on the metabolism of other afla-
toxins and related compounds. In general, it is probable that the map of
metabolic pathways of AFBj is valid to most of the other aflatoxins. The
metabolism of aflatoxin Bo (AFB2) includes 2-hydroxylation to form AFB2a
(290), 4-hydroxylation to form AFM2 (252, 290, 291), ketoreduction to
2,3-dihydroaflatoxicol (292). It was suggested that a small amount of AFB«
may be converted to AFBj which accounts for the weak carcinogenicity of AFB2
(about 1% of potency of AFBj) (64). An in vivo study by Swenson et_ al_. (223)
indicates that AFB2 may indeed be converted to AFBj in the rat liver in vivo
to an extent (about 1%), which supports the postulate that AFB2 exerts its
carcinogenic action via AFBi. Aflatoxin Mi is metabolized at a much slower
rate by rodent liver microsomal (245) or postmitochondrial (282) fraction than
AFBj. Aflatoxicol Mi has been identified as a metabolite of AFM> after incu-
bation with rabbit liver cytosolic preparation; the reaction appears to be
reversible and may be analogous to. interconversicn between AFBj and AFL
(294). Aflatoxin M^ may bind to nucleic acid through the formation of reac-
tive 2,3-oxide intermediate as indicated by the detection of 2,3-dihydro-2-
(N -guanyl)-3-hydroxyaflatoxin M, (49, 243). Aflatoxin P, was reported to be
not metabolized by rat liver microsomes (245); however, 2,3-dihydro-2-(N'-
guanyl)-3-hydroxyaflatoxin P^ has been detected in the liver of rat given AFB^
(49). Aflatoxin Qi may be metabolized by rat liver microsomes in the absence
of NADPH to an unidentified metabolite; there is some evidence that in the
presence of NADPH a small amount of AFQj may be metabolized back to AFBj
(245). One possible metabolite of AFQj is AFLH]^ through ketoreduction
(293). Aflatoxin GX can be 4-hydroxylated to form aflatoxin GMj (290, 291),
2-hydroxylated to form aflatoxin G2a (290) and 0-demethylated (as evidenced by
52
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the detection of formaldehyde in the incubation media) to some as yet uniden-
tified metabolite(s) (295). Covalent binding of AFGj to nucleic acid
presumably involves 2,3-epoxidation because its 2,3-dihydro derivative (AFG2)
does not bind to any significant extent (293, 296). The metabolic activation
of sterigmatocystin has also been shown to involve epoxidation of the double
bond in the terminal furan ring as deduced from the chemical structure of the
sterigmatocystin-DNA adduct (243, 297).
5.3.1.1.4.2 Mechanism of Action.
There is now a considerable body of evidence to indicate that covalent
binding of metabolically activated aflatoxins to cellular macromolecules
(particularly DNA) is principally responsible for their mutagenic and carcino-
genic actions. Mechanistically, the presence of adducts in DNA should lead to
infidelity of replication or transcription, alteration of gene function, gene
rearrangement, or induction of an error-prone repair process indirectly caus-
ing fixation of molecular lesions. These initiating events ultimately lead to
transformation of normal cells into cancer cells by processes that are still
not clearly understood. The high potency of AFB, as a carcinogen has been
attributed to its preferential binding to DNA, nonrandom distribution of this
binding and persistency of the AFBi-DNA adduct.
The covalent binding of AFBi to cellular macromolecules has been exten-
sively studied. Both in vitro (235, 239) and in vivo (223, 240, 241) studies
showed that metabolically activated AFBi binds to nucleic acids much more
effectively than to protein. With nucleic acids from various sources
different degrees of binding efficiency were observed, with the relative order
being micrococcus DNA > calf thymus DNA = rat liver DNA > rat liver RNA > rat
liver tRNA (239). The binding of AFBj to DNA does not appear to proceed in a
53
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random fashion. Activated AFB^ preferentially binds to guanine residues in
DNA (see discussion on the nature of AFBi-DNA adduct). Studies by Misra,
Muench and Humayun (298, 299) revealed that guanine residues in DNA strands
show a "sequence-specific" proneness to serve as targets for AFBj-2,3-oxide
binding. In general, guanine residues flanked by AT sequences are poor
targets while certain, but not all, guanine residues in GC base-paired
clusters are very susceptible targets. For example, guanine residues in
sequence such as 5'-CCG-3" and its complement 5'-GGC-3' are excellent targets
for AFBj alkylation. It appears that a sequence-specific "precovalent" asso-
ciation between double-stranded DNA and AFBj occurs before covalent binding
actually takes place. The nonrandora binding of AFB, is believed to have a
greater chance of causing DNA damage on both strands at closely set sites than
random distribution of binding throughout both the strands (298). Aflatoxin
Bj binding to DNA also displays a "domain-specificity." Bailey et al. (300)
showed that AFB, preferentially binds to internucleosomal DNA in rainbow
trout. Yu (301) found that AFBi, after either ir. vivo or in vitro metabolic
activation, binds preferentially to the active regions of rat liver nucleolar
chromatin. This binding specificty is apparently conferred by the nucleolar
chromosomal proteins. The binding of AFBj to an active region of chromatin
may be expected to have a more highly deleterious effect than binding to a
dormant region because of the greater chance of propagating the DNA lesion.
Mitochondrial DNA is also a preferential target for AFE^ binding. In an in
vivo study using rats, Niranjan et al. (261) found that the level of covalent
binding of AFBi to mitochondrial DNA was three to four times higher than that
of nuclear DNA. They suggested that the mitochondrial genetic system may play
a role in AFBj hepatocarcinogenesis.
54
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The nature of AFB^-DNA adducts and the efficiency of excision repair of
these adducts have been the focus of attention of many investigators. The
most abundant AFB^-DNA adduct present in the acid hydrolysis products of DNA
from livers of AFBi-treated animal or from in vitro incubation in presence of
activated AFBj is 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin Bj (AFBj-N7-
Gua) (55, 236, 242, 302-305). The absolute configuration of AFBj-N^Gua has
been unambiguously established (49; see Fig. 2). Using H-labeled AFBi , it
has been established that AFBj-N -Gua accounts for more than 80% of all the
AFBj-DNA adducts present in rat liver (49, 236, 302, 304). The second most
abundant AFBi-DNA adduct has been characterized as 2,3-dihydro-2-(2,6-diamino-
4-oxo-3,4-dihydropyrimid-5-yl-formaroido)-3-hydroxyaflatoxin B^ (AFB^-FAPy)
which results from the hydroxyl ion-catalyzed opening of the imidazole ring of
AFBj-^-Gua (49, 302, 304, 306, 307) (see Fig. 2). Other DNA adducts present
in the livers of AFBj-treated rats include AFPj-N7-Gua and AFMi~N7-Gua (49),
suggesting that the two hydroxylated metabolites of AFB, can undergo metabolic
activation to 2,3-oxides and then bind to DNA. Small amounts of AFBj-N-*-
adenine adduct have also been detected (308).
The AFBi-N -Gua adducts in DNA are chemically unstable, because the
imidazole ring of the modified guanine residue carries a positive charge (see
Fig. 2). They may undergo three major spontaneous reactions: (a) release of
2,3-dihydro-2,3-dihydroxy-AFB, with reconstitution of the guanine residues
intact, (b) release of AFBj-N7-Gua due to hydrolysis of the N-glycosidic bond
with the concomitant formation of apurinic sites in DNA, and (c) conversion to
AFBj-FAPy adducts as described above. The half-life of AFB^-N7-Gua adducts in
DNA repair-deficient cultured human fibroblasts from a xerodenna pigmentosum
patient is of the order of 20 hours, while that in normal fibroblasts is only
5-6 hours (304). In the rat, the apparent half-life of AFB^-N7-Gua adduct in
55
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N
DMA
AFB,-
2,3-oxide
0 R
If
DNA
H®
0 R
H2N
AFB,-N-Gua
0 R
H
N OH
DNA
e
Q R
H2
DNA
0
ziyrV^NHg"
AFB,-FAPy
OH
0 R
DNA
H®
s ?
H-N-~VNH H
Adduct A
Fig. 2. Mechanism of formation of the major AFB..-DNA adducts.
-------
Legend to Figure 2
Fig. 2. Mechanism of formation of the major AFBj-DNA adducts, AFB^-N -
Gua, and its conversion to formamidopyrimidine derivatives. The chemical
names of the adducts are: AFB^-N -Gua, 2,3-dihydro-2-(N'-guanyl)-3-hydroxy
aflatoxin B^; AFB^-FAPy, 2,3-dihydro-2-(2,6-diamino-4-oxo-3,4-dihydropyrimid-
5-yl formamido)-3-hydroxy aflatoxin Bj; adduct A, 2,3-dihydro-2-(2-amino-6-
formamido-4-oxo-3,4-dihydropyrimid-5-yl araino)-3-hydroxy aflatoxin Bj.
(Adapted from J.M. Essigmann, R.G. Croy, R.A. Bennett and G.N. Wogan: Drug
Metab. Rev. _13_, 581 [1982], P.J. Hertzog, J.R. Lindsay Smith and R.C.
Garner: Carcinogenesis 3, 723 [1982]; the heavy arrow indicates the
predominant pathway.)
-------
liver DNA is approx. 7.5 hours (49, 309). In contrast, the AFB,rFAPy adduct
in DNA is highly stable and persistent. Interestingly, in cultured human
fibroblasts, the level of AFB|-FAPy adducts increased during the posttreatment
incubation period up to a maximum of 20 hours (after removal of AFBj from the
incubation medium). There was no evidence of spontaneous removal or excision
repair during this period (304). A similar pattern was observed in the rat in
an in vivo study (309). The level of AFBi-FAPy adducts accumulating during
the posttreatment period reached a maximum at about 24 hours, at which time
AFBj-FAPy became the most abundant adduct. Approximately 20% of the AFBj-N7-
Gua adducts initially formed were converted to AFBj-FAPy. The AFBj-FAPy
adducts in DNA were slowly removed, if at all, during a 72-hour study
period. Thus, the conversion of AFBj-N^-Gua to AFBj-FAPy adduct represents a
transformation of a repairable lesion into a nonrepairable one. It appears
reasonable to speculate that the persistence of AFBi-FAPy adducts in rat liver
DNA may provide a longer expression time for AFB, to register its carcinogenic
or mutagenic effect. As previously mentioned, the mitochondrial DNA in rat
liver is a preferential target for AFBi binding. It is interesting to note
that although the mitochondrial DNA-AFBi adducts are also very persistent,
there was no evidence of excision repair of the lesions after 24 hours (261).
In addition to AFBi, the covalent binding of a number of other aflatoxins
and of sterigmatocystin to cellular macromolecules has been demonstrated.
Essigmann et al. (49, 243) reported that administration of a racemic mixture
of AFMj to rat liver by perfusion results in covalent binding of AFMj to
DNA. Like AFBj, AFM, is metabolically activated to AFMj-2,3-oxide and
preferentially binds to guanine residues. The conversion of AFMj-N^-Gua to
its imidazole-ring-opened derivative has also been observed. Aflatoxin ^2a
and 2,3-dihydro-2,3-dihydroxyaflatoxin Bj (AFBj-dihydrodiol) bind covalently
56
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to microsomal protein after their in vitro formation at physiological pH (123,
244, 246, 250). The mechanism involves alkaline hydrolysis to dialdehyde
intermediates (see Fig. 3) and subsequent condensation of the aldehyde groups
with terminal amino groups in protein to form Schiff bases (Aflatoxin-CH=N-
Protein). It was suggested (44, 310) that AFB2a could play a role in the
acute toxicity of AFBi by binding to and inhibiting key enzymes of cellular
metabolism. It is conceivable that the same could apply to AFB,-dihydro-
diol. In fact, it has been suggested (244) that the "AFB2a" identified in
earlier metabolic studies should actually be AFB^-dihydrodiol. The covalent
binding of sterigmatocystin to rat liver DNA has been studied by Essigmann et
aj. (243, 297). The results indicate that sterigmatocystin is metabolically
activated via epoxidation of the double bond in the terminal bisfuran ring in
the same manner as is AFB^. The epoxide of sterigmatocystin preferentially
binds to the N -position of guanine residues in DNA; the major adduct in the
acid hydrolysate of sterigmatocystin-treated DNA is the sterigmatocystin-N -
guanine.
The relationship between covalent binding to DNA and carcinogenic action
of aflatoxins has been extensively studied (revs. 32, 44, 49). Organotropism,
species comparison and modification studies all indicate a positive correla-
tion between in vivo covalent binding to DNA and carcinogenicity of AFB^.
Garner and Wright (241) compared the in vivo covalent binding of AFBj to
cellular macromolecules in the rat (a susceptible species) and the hamster (a
resistant species). Rat liver DNA bound four times as much AFBi (approxi-
mately 20 ng AFBj/mg DNA) and rRNA ten times as much AFBj (approximately 45 ng
AFB^/mg rRNA) as hamster liver DNA and rRNA 6 hours after carcinogen admini-
stration, at which time the binding reached its maximum. No significant
difference in covalent binding to liver protein and to kidney (a nontarget
57
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Oe 0
OHCX/R QX^N
OHC
a
s?^S>
OCH,
Fig. 3. Mechanism of alkaline hydrolysis of AFB (R = H) and 2,3-dihydro-
2a
2,3-dihydroxyaflatoxin B^ (R = OH) to dialdehyde intermediates.
-------
tissue) macromolecules was observed. Consistent with the critical importance
of DNA binding for carcinogenesis, Croy and Wogan (309) found that the level
of covalent binding of AFBj to rat liver DNA was ten times higher than kidney
DNA (1,250 vs.' 124 AFBj modification/107 nucleotides) 2 hours after AFBj
administration. Similarly, the level of covalent binding to liver DNA and
kidney DNA were 23 and 76, respectively, in the mouse (a resistant species).
The data are consistent with a comparative study of Lutz et al. (311) who
reported that the "covalent binding index" of AFBj, defined as (umol afla-
toxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal) was 10,400 for rat liver
DNA and 240 for mouse liver DNA 6-8 hours after AFBj administration. The
covalent binding index of AFMi (which is about 1/3 as potent as AFBj in Mt.
Shasta strain rainbow trout) in rat liver DNA was 2,100. Pig liver DNA showed
very high covalent binding indexes, 19,100 and 13,300 at 24 and 48 hours after
AFBi administration. It would be interesting to investigate whether the pig
is acutally highly susceptible to AFBi carcinogenesis as predicted by the
covalent binding index. The finding of Witham et al. (191) is significant in
this regard, showing that the covalent binding of AFB, to liver DNA in coho
salmon, which is refractory to AFBi carcinogenesis, is more than 20-fold lower
than that in the rainbow trout (10.6 vs. 243 pmol AFB^/mg DNA) at 24 hours
after the administration of the mycotoxin. A number of inhibitory agents and
factors of AFBj carcinogenesis, such as phenobarbital (223, 240); ^3-naphtha-
•
flavone (181, 191), and hypophysectomy (223) have been shown to inhibit JLn
vivo binding of AFBj to DNA. In vitro DNA binding data may explain the
greater susceptibility of male Sprague-Dawley rats than females to AFBj
hepatocarcinogenesis (228). However, liver microsomes from phenobarbital-
pretreated rats enhanced rather than inhibited the in vitro covalent binding
of AFBj to DNA (240, 247); the difference between in vivo and in vitro binding
data was attributed to the complex pharmacokinetics in AFBj metabolism (240).
58
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The molecular mechanism of carcinogenesis which proceeds following cova-
lent binding of AFBi to DNA is poorly understood. As a bulky adduct, AFB^ may
be expected to affect the template function of DNA (312); yet there is no
direct evidence for a cause-effect relationship between N -guanine adduct
formation by AFBj and mutagenesis and carcinogenesis. It appears that the
secondary lesions such as apurinic sites and AFBj-FAPy adducts may be more
important for AFB^-induced mutagenesis/carcinogenesis than the primary
AFBj-N'-Gua adducts. Although depurination has been considered to be nonmuta-
genic under normal conditions (313), there is evidence that depurination
causes mutations in "SOS-induced" cells (314). Although the "SOS response,"
an inducible, error-prone DNA repair pathway, has generally been observed in
Escherichia coli (315), a similar response may exist in mammalian cells (316,
317). Since AFB, binding to guanine appears to display sequence-specific
clustering (298), the resulting depurination could lead to double strand DNA
breaks which can lead to micromutation (small deletion) and gross chromosomal
rearrangement. Although alkylation at the N -position of guanine is generally
not expected to affect template function to a significant extent, an imidazole
ring-opened adduct (2,6-diamino-4-hydroxy-5-N-inethyl-formamidopyriinidine) does
affect template activity of DNA if present in sufficient amount (318). This
finding suggests that the corresponding AFBj-FAPy adducts may be potentially
more deleterious to the cells than AFB,-N^-Gua adducts. Amstad j;t_ jil_. (305)
have recently conducted an experiment which suggests that secondary lesions
such as AFBj-FAPy adducts and possibly apurinic sites may indeed be more
important for AFBj-induced in vitro cell transformation than the primary
AFBj-N'-Gua adducts. These investigators treated confluent cultures of mouse
embryo fibroblasts (C3H/10T1/2 cells) with AFBj and then allowed the resulting
AFBj-N -Gua adducts to undergo spontaneous or enzymatic conversion to
59
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secondary lesions by incubating in fresh media for 0, 8, 16 or 40 hours. At
the end of the various incubation periods, the cells were harvested for deter-
mination of the concentrations of AFBj-N'-Gua and AFB,-FAPy adducts and then
replated at low density (i.e., released from confluency holding) in order to
determine the potential to develop into transformed foci. It was found that
the level of AFBj-N -Gua declined rapidly with time whereas the level of AFB^-
FAPy increased with time reaching a maximum at 16 hours and remained unchanged
up to 40 hours. The potential to form transformed foci increased with time
reaching a maximum at 16 hours but declined afterwards. Thus, while there was
no simple relationship between the concentrations of adducts and cell trans-
formation, the results did indicate the greater importance of secondary
lesions.
The covalent binding of AFB, to mitochondrial DNA leads to inhibition of
both mitochondrial transcription and translation (261, 319). These results,
coupled with the finding that mitochondrial DNA is a preferential target for
AFBj binding, led these investigators to suggest that the mitochondrial
genetic system may be one of the direct targets of AFB^ hepatocarcinogenesis.
The modification of mitochondrial DNA may directly or indirectly contribute to
carcinogenic process through mitochondrial mutational events or through long-
term inhibition of mitochondrial biosynthetic processes, respectively.
Apart from direct interaction with DNA, AFB, induces a variety of bio-
chemical effects which may contribute to possible epigenetic mechanisms of
carcinogenesis. It is not known, however, whether any of these effects is
causally related to carcinogenesis. Several studies have demonstrated that
AFBi inhibits protein synthesis through a number of different mechanisms;
random inhibition of selective protein synthesis could lead to disturbance of
gene expression and loss of control of nuclear function. Bhattacharya and
60
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Aboobaker (320) showed that AFBj inhibits the acceptor activity of rat liver
tRNA. The most significantly inhibited tRNA species were those for proline,
aspartic acid and leucine. These functional changes of tRNA were attributed
to covalent interaction of activated metabolite of AFBj with tRNA. Wagner and
Unterreiner (321) reported that chronic administration of AFBj lead to reduc-
tion of protein synthesis mainly through inhibition of rat liver aminoacyl-
tRNA synthetases. Sidransky et al. (322) observed disaggregation of both
hepatic free and membrane-bound polyribosomes by AFBj. An in vivo study by
Sarasin and Moule (323) suggests that AFB, inhibits protein synthesis in rat
liver initially (up to 5 hours) at the polyribosome level by blocking polypep-
tide chain elongation and termination, and later (beyond 7 hours) at the
initiation step (as a consequence of transcription impairment). Kensler et
al. (324) found that AFB, significantly inhibits the nuclear binding capacity
of the glucocorticoid-cytosol receptor complex. The effect was near maximal
after 2 hours and persisted for at least 36 hours. Although the relevance of
this finding to AFBi carcinogenesis is unclear, hormones are known to modify
AFBj carcinogenesis (see Section 5.3.1.1.3.6).
5.3.1.1.5 Environmental Significance.
5.3.1.1.5.1 EPIDEMIOLOGICAL EVIDENCE.
There is a considerable body of evidence to implicate aflatoxins as an
important etiologic factor in human liver cancer (revs. 26, 33, 47, 57).
Epidemiologic studies established a positive association between the geo-
graphic distribution of areas of high liver cancer incidence and that of
prevalence of aflatoxin contamination of foodstuffs. A dose-response rela-
tionship between aflatoxin consumption and liver cancer incidence has been
demonstrated using data from four separate field studies in Kenya, Mozambique,
61
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Swaziland and Thailand. Aflatoxins have been detected with a relatively high
frequency in the tissues and body fluids of liver cancer patients. Although
the evidence does not provide information on latent period, and does not
constitute a definitive scientific proof of the principal etiologic role of
aflatoxins in human liver cancer, the weight of the evidence appears to be
quite strong. Future follow-up studies on survivors of the recent outbreak of
aflatoxin-induced acute toxic hepatitis in India (see Section 5.3.1.1.2.2) or
other heavily exposed population groups may shed more light on the importance
of aflatoxin as a human hepatocarcinogen.
The hypothesis that mycotoxins may play an etiologic role in the induc-
tion of human liver cancer was first proposed by Oettle (19) in 1965. The
first major epidemiologic study was undertaken in Uganda in 1966-1967 by
Alpert et al. (20). They found that the variation of hepatoma incidence in
various tribes in different parts of the country was related to the frequency
of aflatoxin contamination of their food. Keen and Martin (325) attributed
the difference in hepatoma incidences between two tribes in Swaziland to their
eating and cooking habits, particularly to the consumption of groundnuts
stored under conditions that favored aflatoxin contamination. A subsequent
study by Peers _et_ jil^ (23) showed a positive correlation between tumor inci-
dence and aflatoxin consumption. An extensive series of studies by Shank et
al. (24, 25, 326-328) showed that the high incidence of liver cancer in tropi-
cal southeast Asia, particularly Thailand, is associated with dietary exposure
to aflatoxin-contaminated food. Peers and Linsell (21) selected the popula-
.;
tions of three areas of different altitude in the Murang's district of Kenya
for studies on the hepatoma incidences as related to the extent of food con-
tamination by aflatoxins. As predicted, the difference in the climates of
these three areas yielded differences in the extent of food contamination with
62
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aflatoxin, which correlated positively the hepatoma incidences of the three
population groups. Perhaps the highest incidence of primary liver cancer in
the world (110 cases/100,000 persons/year for males; 29 cases/100,000
persons/year for females) was reported in Lourenco Marques, Mozambique
(329). Van Rensburg _et^ al_. (22) found that the liver cancer incidence in the
Inhambane district of Mozambique was also unusually high, with values of 35.5
and 24.5/100,000/year for the 1964-1968 and 1969-1971 periods. The food
consumed by the population in the Inhambane district was found to be heavily
*
contaminated with aflatoxins. The estimated daily intake was 15 jug per adult
(or 222.4 ng/kg body weight), probably the highest value ever found in a
population group. Besides the studies mentioned above, food contamination
with aflatoxin or Aspergillus flavus has been suspected to contribute to the
induction of liver cancer in individuals or population groups in Senegal
(330), Taiwan (331), the Philippines (332), in Poland (333), Transkei (334)
and Hong Kong (335).
Convincing evidence for a dose-response relttionship between aflatoxin
consumption and liver cancer incidence has been provided by Peers and Linsell
(26). Combining the data from four separate field studies in Kenya (21),
Thailand (24, 25), Mozambique (22) and Swaziland (23), Peers and Linsell (26)
demonstrated a high degree of positive correlation between the estimated daily
intake of aflatoxin [expressed as ng/kg body weight/day (X)], on one hand, and
the adult incidence rate of liver cancer [expressed as cases/10* adults/year
(Y)], on the other hand (see Table X). The correlation equation was given
as: Y = 7.6 log X - 3.6 (significant at p < 0.001).
A number of factors may affect or confound the relationship between
aflatoxin consumption and liver cancer incidence. Using data from Kenya and
Swaziland, Peers and Linsell (26) showed that men were substantially more
63
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Table X
Summary of Data Supporting a Dose-Response Relationship
Between Aflatoxin Consumption and Liver Cancer Incidence3
Country
Kenya
Thailand
Swaziland
Kenya
Swaziland
Kenya
Swaziland
Thailand
Swaziland
Mozambique
Area
High altitude
Songkhla
High veld
Middle altitude
Mid veld
Low altitude
Lebombo
Ratburi
Low veld
Inhambane
aAdapted from F.G. Peers and
Aflatoxin
intake
(ng/kg/day)b
3.5
5.0
5.2
5.9
8.9
10.0
15.4
45.0
43.1
222.1
C.A. Linsell
Number
of cases
4
2
11
33
29
49
4
6
42
>101
[Ann. Nutr
Liver cancer
Incidence
(no./100,000 persons/yr)c
1.23
2.00
2.18
2.51
3.83
4.01
4.27
6.00
9.18
16. 1-25. 4d
. Alira. 31, 1005 (1977)]
and World Health Organization: "Environmental Health Criteria 11: Myco-
toxin," WHO, Geneva, 1979. Periods covered were 1 year in Thailand, 3 years
in Mozambique, and 4 years each in Kenya and Swaziland.
Estimated average daily intake (excluding native beers) of aflatoxin by
adults expressed as ng aflatoxin/kg body weight/day.
clncidence expressed as number of new cases per 100,000 population per year.
A recent revision by Van Rensburg (cited in WHO, 1979) gave an incidence of
13.0.
-------
susceptible to the apparent carcinogenic effect of aflatoxin than women. The
correlation equation was Y = 21.96 log X - 11.17 (p < 0.001) for males and
Y = 4.14 log X - 0.80 (p < 0.05) for females. It is interesting to note that
in areas of high liver cancer incidence in the world, the male to female ratio
for hepatocellular carcinoma is considerably higher than that in areas of low
liver cancer incidence. For example, the male to female ratio was 3:1 in
Kenya (21), 3.5:1 in Singapore (336), 4:1 in Swaziland (23, 325), and between
5:1 and 6:1 in Thailand (25) compared to between 1:1 and 1.5:1 in areas of low
liver cancer incidence (47). Hepatitis B viral infection has been considered
to be a possible confounding factor (26, 47). Hepatitis B infection is common
in countries with a high incidence of primary liver cancer. It has been sug-
gested (337, 338) that tumors may arise as a late manifestation of liver
cirrhosis resulting from viral hepatitis. It is also possible that aflatoxin
and viral hepatitis act synergistically in the induction of liver cancer.
Moreover, the nutritional status is an important modifying factor in aflatoxin
carcinogenesis (see Section 5.3.1.1.3.6) in animals; whether the same holds
for humans remains to be investigated.
In addition to reports on the induction of human cancer due to ingestion
of aflatoxin-contaminated foodstuffs, there are two reports of cancer induc-
tion due to occupational exposure. Van Nieuwenhuize et al. (339) followed up
on a group of 55 workers known to be exposed for 2-9 years to dust containing
aflatoxin in a mill crushing groundnut and other oil seeds. The estimated
O
airborne aflatoxin level in the workplace ranged from 0.87 to 72 ng/m . The
total amount of aflatoxin, to which the mill workers were exposed throughout
the exposure period, was calculated to range from 160 to 395 tig. Eleven of
these 55 exposed workers developed cancers (including one liver cholangiocar-
cinoma) within an observational period of up to 11 years; only 4 cases of
64
-------
cancer (no liver cancer) were found in an age-matched group of 55 workers from
a different factory in the same area. Deger (340) reported two cases of
induction of carcinoma of the colon in two research workers (in the same
laboratory) who had been involved in purifying substantial amounts of afla-
toxins for research purposes. These reports emphasize the extreme precaution
needed in handling aflatoxin in the workplace.
There are a number of reports of detection of aflatoxin in the liver or
body fluids of liver cancer patients in various countries. Pang, Husaini and
Karyadi (cited in 33) reported that in a 2-year study in Indonesia, AFBi was
chromatographically detected in extracts of liver tissue biopsy samples from
41 of 71 (57.7%) patients with primary liver cancer; extracts of liver tissues
from 15 patients without liver cancer did not contain aflatoxin. Phillips et
al. (341) detected AFB, (estimated to be 520 ng/gm wet weight) in a sample of
liver biopsy from a 56-year-old rural resident of Missouri, U.S.A., suffering
from cancer of the liver and rectum. Stora et al. (342) found evidence (by
direct fluorescence microscopic examination) of the presence of AFBj in liver
extracts from 13 of 15 liver cancer patients in Czechoslovakia; in at least 5
cases, the presence of AFBi was confirmed by chemical identification on thin-
layer chromatography. Onyemelukwe'et al. (343) detected aflatoxins Bj, 62, G^
and Go i-n sera of 20 of 20 Nigerian patients with primary liver cell
carcinoma; in 3 of these patients, the concentration of AFBi exceeded 150
ng/ml. Aflatoxin Bi was also detected in the serum of a U.S. resident
suffering from primary hepatoma (344). These reports provide additional
supporting evidence for an association between aflatoxin intake and induction
of liver cancer in humans.
65
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5.3.1.1.5.2 ENVIRONMENTAL FORMATION, OCCURRENCE AND EXPOSURE.
Environmental formation. The environmental formation and the biosyn-
thesis of aflatoxins, sterigmatocystin and related mycotoxins, and the
environmental conditions that are conducive to mycotoxin production, have been
extensively reviewed by Detroy et_ al_. (38), Stoloff (345, 346), Heathcote and
Hibbert (32), and the World Health Organization (33). Aflatoxins are produced
by certain strains of two species of fungi, Aspergillus flavus Link and_A.
parasiticus Speare, in a geographic area where environmental conditions are
suitable for the development of the mold. Both species are members of the _A.
flavus group; they are practically indistinguishable from each other to inves-
tigators untrained in the fine details of mold taxonomy. There is some
evidence that j\. parasiticus is more likely to be encountered in warmer
environments and is the species more likely to produce a greater variety of
aflatoxins. Not all strains of A. flavus produce aflatoxins; however, depend-
ing on the substrate, a high proportion (20-98%) of the strains isolated are
aflatoxin-producers. The presence of extensive mold growth in foodstuffs is
not, by itself, indicative of presence of aflatoxin because some strains are
non-producers and producing strains can be affected by the presence of compet-
ing microorganisms. There are a number of reports of aflatoxin production by
a variety of other fungi such as A. oryzae (used for the production of miso
and soy sauce), Penicillium sp. (present in moldy peanuts), Streptomyces sp.
and Rhizopus sp. (rev. 38); however, none of these findings could be confirmed
by other investigators (38, 346, 347). The precursors of aflatoxins, sterig-
matocystin and versicolorin A are produced by certain strains of A^. versicolor
as well as of _A. flavus and _A. parasiticus (rev. 32).
The biosynthesis of aflatoxins and related mycotoxins has J>een exten-
sively studied (rev. 32). Under laboratory conditions, Mg, Mo, Zn and Fe are
66
-------
essential minerals required for aflatoxin production. The molds, can use most
carbohydrates as sole carbon source, although optimum production occurs in
media supplemented with glucose or sucrose. The initial step of biosynthesis
is believed to involve head-to-tail assembly or condensation of acetate to
form polyhydroxyanthraquinones (e.g., norsolorinic acid), but the exact molec-
ular mechanism has not clearly established. The resulting polyhydroxyanthra-
quinones are then further modified to yield various aflatoxins. For AFB^,
recent studies (32, 348-351) suggest the following biosynthetic pathway:
norsolorinic acid • > averantin —^ averufin —V- versiconal acetate —>•
versicolorin A—y sterigmatocystin—^- aflatoxin Bj (see Tables I and IV for
chemical structures).
The environmental conditions known to affect aflatoxin production in
agricultural commodities and their products are: (a) humidity and moisture,
(b) temperature, (c) aeration, and (d) the type and condition of the substrate
(revs. 32, 33, 38, 352). The relative humidity is a crucial factor for the
growth of A. flavus which appears to be more fastidious in moisture require-
ment than many other molds. Depending on the moisture content of the
substrate, a relative humidity of 80-85% is considered to be a minimal
requirement; beyond 95%, the yield of aflatoxin production generally increases
with an increase of relative humidity. The lower limit of moisture content
that supports growth of ^. flavus is 18.3-18.5% on a wet weight basis for
starchy, cereal grains such as maize, rice, oat, wheat, barley and sorghum,
and 9-10% for commodities with a higher oil content such as groundnuts, tree
nuts, copra and sunflower seeds (33). Aspergillus flavus has been classified
as a mesophilic fungus. Depending on the humidity and the type of substrate,
the minimal, optimal and maximal temperature ranges for aflatoxin production
are 11-12°C, 25-32°C, and 40-42°C, respectively. The biosynthesis of afla-
67
-------
toxin is an aerobic process. Adequate aeration is an important factor for
optimal aflatoxin production in fermentors. Atmospheric gases can have a
significant effect on aflatoxin production by A. flavus growing on a solid
substrate such as groundnut. Decreasing oxygen levels below 5% or increasing
carbon dioxide levels above 20% significantly reduce the yield of aflatoxin
(38). The yield of aflatoxin is also dependent on the type of substrate
used. Freshly grated coconut, wheat, rice and cottonseed are among the list
of favorable solid substrates for aflatoxin production (38). Damaged agricul-
tural commodities, either mechanically or by insects, often provide favorable
opportunities for mold growth (33, 346). A study by Lillehoj et al. (353)
showed that contamination with A. flavus (and aflatoxin) in Iowa (U.S.A.) corn
before harvest occurred exclusively in insect-damaged ears of corn.
Environmental occurrence and exposure. Human exposure to aflatoxins
occurs mainly as a result of consumption of contaminated foodstuffs. The
environmental occurrence of aflatoxins has been reviewed by Campbell and
Stoloff (354), Stoloff (346), Rodricks and Stoloff (355), and the World Health
Organization (3_3). As may be expected from the favorable environmental
conditions for mold growth, aflatoxin contamination of agricultural commodi-
ties and their products is mainly confined to the tropical and subtropical
regions of the world. Seasonal variations and unusual weather conditions may
play a significant role in encouraging mold growth, by providing a humid
environment or by causing crop damage. It should be noted that reports of
aflatoxin occurrence in the scientific literature are not always reliable; a
scrutiny of the methodology used is often needed to assess the reliability of
the data [for a recent review of the recommended analytical methods, see
Ciegler et_a±. (356)].
68
-------
The agricultural commodities that are most susceptible to aflatoxin
contamination include corn, peanuts, cottonseed and copra. Small grains
(wheat, oats, rice, rye, grain sorghum, millet) are rarely contaminated unless
improperly stored or prepared. In most contaminated samples, AFBj is the
principal aflatoxin; AFGj and, to a lesser extent, AFM^, AFB2 and AFG2 have
also been detected. Table XI summarizes some of the representative data of
occurrence of AFBj in selected agricultural commodities and their products in
four countries. In addition to the commodities tabulated in Table XI, cotton-
seed and related products are often contaminated with aflatoxin. Stoloff
(345) reported that AFBj was detected in 6.5-8.8% of more than 3,000 samples
of unprocessed cottonseed collected in three successive crop years (1964-1967)
in U.S.A. an in 12.8-21.5% of more than 3,000 samples of cottonseed meal.
Relatively high levels (maximum 2.58 mg/kg) of aflatoxin were found in an area
in southern California. A survey by Marsh et al. (357) of cottonseeds in
11-13 locations in corn belt of U.S.A. showed aflatoxin contamination in three
regions. Considerable levels, as high as 200-300 mg AFBj per kg cottonseed,
were found in seeds from some individual lots. Vedanayagam et al. (358) found
frequent aflatoxin contamination of cottonseeds in India; samples from humid
areas were more than two times (78%j/s_. 31%) more likely to be contaminated
than those from dry areas. Coconut copra is another frequently contaminated
commodity; Stoloff (345) found aflatoxin in 88% of 72 samples of copra with
concentration ranging from trace to 30 ug/kg. Edible oil extracted from poor-
grade peanuts or cottonseed may be contaminated with aflatoxins. Fong et al.
(335) detected AFBi in 3 of 11 samples of peanut oil purchased from local
markets in Hong Kong; the concentration of AFB, ranged from 98-150 ug/kg. The
contamination was presumed to originate from the use of poor-grade peanuts for
the extraction of oil. Analysis of 10 samples of such peanuts indicated the
69
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Table XI
Occurrence of Aflatoxin B, in Selected Foodstuffs
Country
Philippines
(1967-1969)
Thailand
(1967-1969)
Uganda
(1966-1967)
United States
(1964-1975)
Number of
contaminated
samples /number
Foodstuff examined
Peanut butter
Peanuts, whole
Peanut candies
Corn, whole
Corn products
Beans
Root and tuber products
Rice products
Peanuts
Corn
Chili peppers
Wheat and barley
Dried fish/shrimp
Beans
Cassava starch
Rice
Corn
Beans
Peanuts
Sorghum
Millet
"Cassava
Rice
Corn and corn meals
Peanut products
Grain sorghum
Oats
Wheat
Rye
Rice
Barley
145/149
80/100
47/60
95/98
22/32
26/29
48/62
1/72
106/216
22/62
12/106
5/44
7/139
10/322
2/65
7/364
19/48
15/64
29/150
16/69
6/55
2/34
0/11
72/175*
44/1, 763b
269/1, 416d
10/786
3/416
3/1,828
2/35
1/170
0/254
Average
AFBj in
contaminated
samples
(ug/kg) Reference
213
98
38
110
32
35
44
< 1
870
270
80
38
104
106
60
10
133
500
363
152
26
879
0
44
< lc
lc
12
6
8
trace
5
0
(332)
(327)
(20)
(346)
aSamples collected from Southeast regions in 1969-1970 and 1974.
bSamples collected from Corn Belt in 1964-1965, 1967 and 1974.
cAverage of all samples.
Including some samples collected from Canada.
-------
presence of AFBj in all the samples with concentrations of 95 ug/kg to 1.05
mg/kg. Abalaka and Elegbede (359) found AFBj at levels of 450-860 yug/kg in
peanuts and cottonseeds used in a Nigerian oil extraction plant. The crude
and refined peanut oil contained 98 and 9 iig/kg AFBj while the crude and
refined cottonseed oil contained 65 and 23 ug/kg AFB^, respectively.
Interestingly, fresh vegetables are not known to be susceptible to ^. flavus
and aflatoxin contamination. However, a recent study by Mertz et al. (360)
indicated that lettuce seedlings grown in soil adulterated with AFBj can
absorb and retain measurable amounts of the mycotoxin. In view of the common
practice of disposal of large amounts of aflatoxin-contaminated plant material
in the soil, agricultural commodities grown on such soil should be closely
monitored for possible aflatoxin contamination.
Apart from plant foods and their products, a lesser source of human
exposure to aflatoxins is through consumption of milk or edible tissues of
animals that may have received aflatoxin in their feed. The occurrence of
aflatoxin in animal feeds has been frequently encountered (revs. 33, 355). In
rural areas, moldy food rejected by humans are often fed to farm animals.
Surveys in Belgium (361), Germany (362-364), India (365), the Netherlands
(366), South Africa (367) and in the U.S.A. (368) have indicated the presence
of AFMj in 0-67% of liquid or dried cow's milk or milk products; the highest
concentration reported was 13.3 ug/kg in one milk sample collected in India.
A number of studies (369-371; jrev. 355) have reported the detection of AFBj
and AFMi residues in eggs and meat animals intentionally dosed with AFBj-
contaminated feed. The liver contained the highest amount of aflatoxin resi-
due. The estimated ratios of AFBi concentration in the feed to the expected
AFBj concentration in the liver of common meat animals were: beef, 14,000:1;
swine, 800:1; broiler chicken, 1,200:1. The corresponding ratio for eggs was
70
-------
2,200:1 (346, 355). This led to the recommendations that the use of contami-
nated feed for dairy cows should be more strictly controlled and that uncon-
taminated or low contamination feed should be fed to meat animals in the
period shortly before slaughtering.
In addition to ingestion, human exposure to aflatoxin may also occur via
inhalation under occupational settings. Van Nieuwenhuize _et__al_. (339) esti-
mated that the level of airborne aflatoxin in the work place of a Dutch mill
for crushing peanuts and oil seeds ranged from 0.87 to 72 ng/m . Thus, the
exposed workers appeared to have an increased liver cancer risk (see Section
5.3.1.1.5.1). Workers and farmers handling contaminated commodities are also
expected to be at risk from inhaling contaminated grain dust. An approach to
analyze the potential risk associated with such exposure has been discussed by
Baxter _£t_ jO_« (372).
The importance of aflatoxin as a potential environmental carcinogen for
humans has prompted worldwide efforts to control aflatoxin contamination
through prevention of molding as well as decontamination', of aflatoxin-
containing foodstuffs. This topic has been the subject of several extensive
reviews (32, 373, 374). Education of farmers to improve crop quality and
storage, close monitoring of foodstuffs and animal feeds for aflatoxin con-
tamination, and application and development of effective food-processing tech-
nology to remove contaminated crop or to destroy aflatoxins in situ are
several components of effective programs to control aflatoxin contamination
discussed in the above reviews. Approaches to regulatory control of aflatoxin
in foodstuffs and feeds have varied from one country to another; the tolerance
71
-------
limits for aflatoxin in foodstuffs ranged from zero (or the lowest limit of
detection) to 50 iig/kg (346). These limits are merely intended as guidelines
to implement aflatoxin control programs and should not be mistaken as provid-
ing an absolute protection against carcinogenic risk associated with aflatoxin
exposure. The problems associated with assessment of carcinogenic risk of
human exposure to low levels of dietary aflatoxin have been discussed by Shank
(375). In the absence of reliable human carcinogenicity data for low level
exposure and the limitations of extrapolation from trout and rat carcinogenic-
ity data to assessment of human risk, it would be prudent to adopt a conserva-
tive approach to the control of aflatoxin.
72
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