Aflatoxin mutagenesis

.Mutation Research, 32 (I975) 35-53 © Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands

35

AFLATOXIN MUTAGENESIS

TONG-MAN ONG National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, N.C. 27709 (U.S.A.) (Received January 22nd, 1975) (Accepted February i4th, 1975)

(1) (2) (3) (4) (5) (6) (7) (8) (9) (±o)

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Teratogenic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of chromosomal aberrations . . . . . . . . . . . . . . . . . . . . . Induction of gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . Formation of toxic and mutagenic metabolites by mammalian liver microsomes . . . . Other genetically related activities . . . . . . . . . . . . . . . . . . . . . . . Mutagenic hazard to man . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

35 36 36 38 38 39 41 43 46 48 49

INTRODUCTION I n 196o, an u n k n o w n disease (Turkey X disease) caused the d e a t h of at least i o o o o o t u r k e y s in E n g l a n d 14. A similar type of disease was also reported in ducklings (ref. IO), pigs 66 a n d c a t t l e % The presence of Brazilian g r o u n d n u t meal in the a n i m a l rations was f o u n d to be the c o m m o n factor in the disease outbreaks. The toxic factors were extracted from the g r o u n d n u t meaP 3 a n d were identified as metabolites of the c o m m o n fungus, Aspergillus flavus 8~, which h a d grown on tile g r o u n d n u t . The collective t e r m " a f l a t o x i n " was assigned to the toxic factors to indicate their origin. Some other fungal species have also been reported to produce aflatoxin (see ref. 30 for review) a n d several different c o m p o u n d s have been isolated a n d identified. Aflatoxin B1, the most extensively studied c o m p o u n d a n d the most c o m m o n m e m b e r of the aflatoxins, is now k n o w n to be one of tile most p o t e n t hepatocarcinogens a m o n g k n o w n carcinogenic chemicals. This c o m p o u n d also possesses p o t e n t m u t a g e n i c activity. A l t h o u g h aflatoxin has been k n o w n for less t h a n 15 years, its p o t e n t i a l hazard to h u m a n health has caused great concern a n d has resulted in aflatoxin becoming one of the most studied m y c o t o x i n s in the h u m a n e n v i r o n m e n t . E x t e n s i v e studies on aflatoxin have been conducted a n d h u n d r e d s of aflatoxin-related papers have alr e a d y been published. I n a d d i t i o n to n u m e r o u s review articles, a collection of the comprehensive reviews on aflatoxin was published b y GOLDBLATTin 196947. The present paper is i n t e n d e d to review the studies on the m u t a g e n i c a n d other genetically Abbreviation: DMSO, dimethyl sulfoxide; EMS, ethyl methanesulfonate; ICR-I77, N-(2-chloroethyl) -N-ethyl-i, 3-propylenediamine •2HCt.

36

T. O N G

related activities of aflatoxin. For such purposes the chemistry, toxicity, carcinogenicity and teratogenicity of this toxin will also be briefly discussed. CHEMISTRY

In early studies on the isolation of aflatoxin, NEWBITT et al. 72 obtained two types : aflatoxin B and aflatoxin G. The former emits a blue-violet fuorescence whereas the latter has a green fluorescence. The 1964 studies of HARTLEYand his coworkers 52 showed that aflatoxin can be separated into four closely related compounds : aflatoxins B1, B2, G1 and G2. The chemical fornmlas of these four compounds have been determined as follows : B1, C17H1206; B2, C17H1406; G1, C17H1207; and G2, C17H,O~. Aflatoxins B~ and G2 are dihydro derivatives of aflatoxins B1 and G~, respectively. The amount and relative proportions of these four compounds present in culture extracts varies with the mold strains and substrates used; aflatoxin B~ is usually present in large amounts whereas aflatoxins B~ and G2 are present in small quantities. DATTON AN'I) HEATHCOTE in 196636 isolated two additional aflatoxins from cultures of A . flavors. These two compounds were found to be 2-hydroxy derivatives of aflatoxins B2 and G~; and, therefore, were named aflatoxins B2a and G2a, respectively. The milk toxin, initially isolated from milk of cows fed aflatoxin-containing rations, was designated as aflatoxin M (refs. 4, 5). This agent, which has also been isolated from Aspergillus-contaminated peanutsSL is now known to be a metabolite of aflatoxin B in several animal species, including man. HOLZAPFELet al. 54 reported that aflatoxin M can be separated into two compounds : aflatoxins M1 and Ms. Aflatoxin M1 was found to be 4-hydroxyaflatoxin Bt, and aflatoxin M2 to be 4-hydroxyaflatoxin B2. Three additional aflatoxin B~ metabolites have also been isolated, identified and added to the aflatoxin family. Aflatoxin R0, a hydroxylated compound, is reduced from aflatoxin B~ by the fungus D a c t y l i u m dendroides 26. Aflatoxin Q~ is formed by incubation of aflatoxin B~ with monkey 7° or human 16 liver microsomal preparations in a N A D P H generating system. The reduction of aflatoxin B1 to R0 occurs at the carbonyl group in the cyclopentane ring 27 while hydroxylation of B~ to Qt is found to be at the carbon atom fl to the carbonyl of the cyclopentenone ring 70. Aflatoxin P~, the Odemethylated aflatoxin B~ ,was obtained from urine of monkeys dosed with B~ (ref. 24) ; this compound was also isolated after incubation of aflatoxin B1 with a homogenate from biopsied human liver tissueTL Aflatoxin GM~, originally isolated from urine of sheep dosed with mixed aflatoxins, was identified as a 4-hydroxy derivative of aflatoxin G1 (ref. 5). At the present time, at least 12 compounds are named as aflatoxin. The chemical structures of several aflatoxins are shown in Iqg. i. TOXICITY

The toxic effect of aflatoxin in animal rations, as well as in purified form, has been demonstrated in many animal species. Ducklings, rainbow trout, guinea pigs, rabbits, dogs, turkeys et al. are highly susceptible to aflatoxin, with sheep being found the most resistant of the animal species tested. In general, aflatoxin is more toxic to young animals than to old and more toxic to males than to females. The first signs of aflatoxicosis in animals are lack of appetite, loss of weight, and reduced alertness.

37

AFLATOXIN MUTAGENESIS

90

-o- -o-

09

-ocm

koJ'o

Bt

B~,

o91"Po

9 ~ o "0

"0"

G,

v-OCH3 G2

9 I

"0" "0" v-OC, Mi

9

H3

q

(/,y

Lo'ko ocH M2

g

o

o;-o

o

I ocm °" P:

Qa

Fig. I. C h e m i c a l s t r u c t u r e s of s e v e r a l a f l a t o x i n s .

Liver necrosis and bile duct proliferation are the most common pathological effects in animals. The toxicities of aflatoxin in different animal species have been reviewed by ALLCROFTa and B U T L E R 17. Aflatoxin B1, the most abundant member of the aflatoxin family, is also the most toxic. In one day-old ducklings, the oral 7 day LDs0 values on a 5o-g body weight basis are 18.2 #g for B1, 84.8 #g for B2, 39.2 #g for G1 and 172.5 #g for G2 (ref. 22). Aflatoxins B2~ and G2~ are relatively non-toxic 37; however, aflatoxins M~ and M2 have been reported to be as toxic as B~ and B2, respectively 78,8°. For most of the animal species tested, the LD~0 for a single dose of aflatoxin B1 is in the range of o. 5 to IO mg/kg body weight. It has been reported that aflatoxin B1 can cause significant liver injury in primates ~6. Aflatoxin has also been found to be toxic to tissue culture cells. A concentration of aflatoxin B1 as low as o.i #g/ml can kill up to 5o% of Chang liver cells and duck embryo primary cultured cells 4~. The 5o% toxic dose of aflatoxin Bt for chicken embryo primary cultured cells and H e L a cells is 5/~g/ml (ref. 41). Aflatoxin Bt at a concentration of o.o5 #g/ml totally inhibits the growth of human embryonic lung cells 60. Aflatoxins B~, B2, G1 and G2 are all toxic to human embryo liver cells93,~°8; B2 and G2, however, are considerably less toxic than B~. The relative toxicity of these four compounds to human embryonic liver cells is in the order of Bt > G~ > G~ > B~. It is

38

T. ONG

interesting to note t h a t the toxicity of aflatoxin G2 in h u m a n embryonic liver cells is 3 to 4 times higher than t h a t of B2. Aflatoxin B~ is also toxic to cultured adult h u m a n liver cells; the toxicity, however, is less than that found in embryonic liver cells 93. CARCINOGENICITY

Aflatoxin is highly carcinogenic in rats, ducklings, rainbow trout, nlice29, 99, and Rhesus monkeys 1. It has been shown t h a t all rats developed sarcomas or fibrosarcomas at the injection site when the animals were given a mixture of aflatoxins B1 and G~. The rats were injected twice weekly with doses of 50/~g and 500/~g at each injection for 60 and 8 weeks, respectively 2s. It has also been reported that after being fed for three months with rations containing 1.75 #g of a mixture of aflatoxins B~ and G1, one-third of the rats developed hepatomas one year later 1~. At a concentration as low as 15 ppb in the food rations, aflatoxin B1 was reported to induce carcinomas in all 12 male rats and all 13 female rats after 68 and 80 weeks, respeetively~05; at a dose of I ppm, all male rats developed tumors within 41 weeks. A correlation between dietary aflatoxin content and liver t u m o r incidence in rats has been demonstrated. At a concentration of 5 p p m of toxin, 93% t u m o r incidence was found in animals fed over a period of 37 ° days 73. The studies on the carcinogenic activity of aflatoxin in ducks have shown t h a t 8 of I I animals developed hepatic tumors after being fed for 14 nlonths on a diet containing 30 p p b aflatoxin from toxic groundnut meal'-". In rainbow trout, it was reported t h a t more than 50% of the fish developed liver tumors after being fed for 9 m o n t h s with diets containing 20 ppb aflatoxin B1 (ref. 91). Thus it can be seen t h a t low levels of aflatoxin B1 cause high levels of t u m o r incidences and it appears t h a t this c o m p o u n d is one of the most potent hepatocarcinogens found in the environnlent. Aflatoxin G~ has also been found to be a very potent carcinogen. It was reported t h a t feeding aflatoxin G1, at a concentration of 3/~g/ml, to rats in the drinking water resulted in 21 of 26 rats developing liver tumors after the rats had received a total dose of 6 rag. Six of the 26 animals also developed kidney tumors 19. At a total dose of I mg, aflatoxin G~ produced I liver t u m o r while B~ produced 3 liver tumors in IO animals. In this same study, no t u m o r was observed in IO rats which had been fed with aflatoxin B e to a total dose of I rag. With i.p. injection, however, aflatoxin B2 was found to be a weak hepatoearcinogen in rats 1°6. Aflatoxins B2, G~ and M1 were found to be carcinogenic in rainbow trout ~1,92. The carcinogenic activity of these agents in trout is observed to be less than t h a t of Bt. Recent studies have also shown t h a t aflatoxin B1 causes neoplastic transformations in B A L B / 3 T 3 (ref. 33) and hamster embryo ~ cells. Studies on the structure-activity relationships of the toxins have shown t h a t both the lactone portion and the dihydrofurofuran moiety of the molecules are imp o r t a n t for the carcinogenic activities of the aflatoxins~l, ~06. A review article on aflatoxin carcinogenesis has recently been published by WOOAN10~. TERATOGENIC ACTIVITY

ELlS AND DIPAOLO3s reported t h a t aflatoxin B 1 is a potent teratogen for hamsters. Injection of 4 m g / m l aflatoxin B~ on d a y 8 of pregnancy was found to be very

AFLATOXIN MUTAGENESIS

39

effective for the induction of malformation; 29.4% of the fetuses was found to be malformed and 17.6% were dead or reabsorbed if the hamsters were killed on the day following the administration of the toxin. The frequencies of malformed fetuses decreased with gestation time while the frequencies of dead or reabsorbed fetuses increased. It was also found that, if the aflatoxin was first mixed with deoxyribonucleic acid for 24 h prior to injection, the teratogenic effect was diminished. In studies on the teratogenic effect of aflatoxin in mice, DIPAOLO et al. 3~ showed no malformations or significant weight differences between fetuses from untreated and toxin-treated mothers. Mice experiments were performed using doses of aflatoxin B1 which produced rates of reabsorption and death comparable to those obtained with hamsters. In rats, LE BRETON et al. 57 observed that injection of 300 #g of aflatoxin into pregnant mothers caused fetal death, while repeated small doses ( < 200 #g) caused retardation of fetal growth. No malformations, however, were found in the fetuses. BUTLERAND WIGGLESWORTHTM reported that no toxic effects on the fetus were found when aflatoxin B , in a dose equivalent to 1/4 of the LDs0 dose for the non-pregnant rat, was given orally to rats at various stages of early pregnancy. Although the toxin caused retardation of fetal growth in rats treated on day 16 of pregnancy, such fetal growth retardation was suggested by the authors to be a secondary effect of the toxin on the mother. The literature concerning the teratogenic activity of aflatoxin is very limited. The available data indicate that the induction of malformations in animals by aflatoxin is highly species specific. Differences in metabolic pathways of aflatoxin between animals may contribute such specificity. I N D U C T I O N OF CHROMOSOMAL ABERRATIONS

Although the carcinogenic activity of the aflatoxins was demonstrated soon after this group of compounds was isolated and identified, the mutagenic activity of aflatoxin was recognized much later. The first study on the induction of chromosomal aberrations by aflatoxin was reported by LILLY63. Roots of Vicia faba seedlings were treated at 21 ° for 3 h with 67 m M of a mixture of aflatoxins. The mixture consisted of 37.7% B1, 56.4% G1 and small amounts of B~ and G2. In this study aflatoxin was found to cause a significant increase in the number of abnormal anaphases. Most of the abnormalities consisted of chromosomal fragments with occasional bridges; at the low fixation times (3 and 6 h), the abnormalities showed stickiness and many bridges. W I T H E R S 1°°, treating cultured human leukocytes with a similar aflatoxinmixture, found a concentration of 4.7" Io-4 M, added 30 h prior to harvesting, produced the highest frequency of chromosomal aberrations. Chromatid breakage was the most common aberration while no chromosomal breakage was found. Induction of chromosomal aberrations in cultured human leukocytes was also investigated by DOLIMPIO et al24 using leukocyte cells isolated from three healthy females. The cells were treated with 40 #g/ml of aflatoxin B1 and I to 50/~g/ml of an aflatoxin mixture for either 8 or 48 h, and then incubated for a total of 7 ° h. The aflatoxin mixture contained ~5% B , 9% G~ and less than I % B~ and G2. Their results showed that the percent of cells with chromosomal aberrations increased with increasing toxin concentrations as well as with length of treatment time. The percentage of cells which showed breakage increased from 0.07% for the control to 24.4% for treated cells. Chromosomal aberrations included gaps, breaks, fragments, deletions and translocations, with each aberra-

4°

T. O N G

tion affecting usually only one chromatid. Aflatoxin was shown by these authors to affect the chromosomes in a non-random pattern; groups A and B, the largest chromosomes, have more breaks while the groups E, F and G have less than the expected frequencies of breaks. The long arms of the chromosomes were more frequently attacked by aflatoxin than either the short arms or the centromere. However, PROMCHAINANT et al.79 found that the frequencies of chromosome-type and chromatid-type aberrations did not differ greatly when male human leukocytes were first treated with 50/~g/ml of aflatoxin for 24 h and then incubated for a total of 72 h. The aflatoxin used was composed of approximately 15% B1, 7% B2 and only traces of G1 and G2. This study also showed that the larger chromosomes were more frequently affected and indicated that the relative amount of breakage of the different chromosomal groups seemed to be in proportion to the size of the chromosomes. These same authors showed an additive mutagenic effect when using a combination of 7-rays (200 R) and aflatoxin in a human leukocyte culture. Chromosomal aberrations induced by aflatoxin have also been reported in short-term phytohemagglutinin-stimulated leukocyte cultures, human diploid embryonic lung cultures, and a rat kangaroo cell line system '~5. GREEN" el al.4%5° demonstrated that in the rat kangaroo cell line system, aflatoxin and mitomycin produced chromatid-type gaps and breaks. Chromosome-type aberrations were also found with aflatoxin. In these experiments, cells were removed fro,n growth medium i or 2 days after plating and treated with either aflatoxin or mitomvcin C for 8 h. Concentrations of 12.5 and 25/~g/ml of a crude aflatoxin preparation added on day I resulted in 34°,o and 41% of cells with breaks, respectively. If toxin was added o n day 2, frequencies of cells with breaks were 23% and 42%, respectively. The frequency of breaks in control cells was only o.7%. In this system, the long arms of the autosomes are sensitive to both aflatoxin and mitomycin; the most sensitive region is the heterochromatic area of the X chromosomes; however, the degree of aflatoxin sensitivity in the heterochromatic regions of chromosomes from different species has not been investigated. Aflatoxin B1 has also been found to cause chromosome breaks and rearrangements in cultured Chinese hamster cells°t Breaks were reported to be more frequent in the first 48 h after treated with toxin for 3 h ; rearrangements, however, were found to be more frequent in the subsequent days. After treating roots of A l l i u m cepa with aflatoxin B~, REISS81 observed the presence of chromosome bridges; frequent chromosome aberrations were reported when the roots were grown for 48 h in 2oo/~g/ml of aflatoxin. Aflatoxin B, has also been shown to break DNA as determined bv both alkaline and neutral sucrose gradient techniques 97. When HeLa cells $3 are treated with IOO #g/ml of aflatoxin Bi for I h, neither single- nor double-stranded DNA is significantly affected. A DNA breakage does appear, however, if cells are treated with aflatoxin B~ at 32 #g/ml for 24 h. Recovery is observed if treated cells are incubated for an additional 24 h without toxin. It is not known why DNA breakages appear only when HeLa cells are treated with toxin for long time periods. One of the plausible interpretations is that aflatoxin must first be converted to a reactive form and that such conversion can be carried out by HeLa cells. The studies on the induction of chromosome aberrations as summarized in Table I, have been limited to aflatoxin B1 and the mixture of aflatoxins. Whether aflatoxins B2, G~, M~ and others can also cause chromosome breakages has not yet been demonstrated. The cytogenetic effects of aflatoxin in human leukocytes and in cells

41

AFLATOXIN MUTAGENESIS

d e r i v e d from r a t kangaroo k i d n e y appear to be a delay type which is somewhat similar to the effects of m i t o m y c i n C in these cellsS°, TM. This resemblance seems to suggest t h a t aflatoxin m a y act as an a l k y l a t i n g agent. INDUCTION OF GENE MUTATIONS Only a few studies have been reported on the i n d u c t i o n of gene m u t a t i o n s b y aflatoxin. Aflatoxin B~ was shown b y MAHER AND SUMMERS6s to induce m u t a t i o n s in t r a n s f o r m i n g D N A of B a c i l l u s subtilis. I n this study, wild-type t r a n s f o r m i n g D N A prepared from the donor bacteria, B . s u b t i l i s strain SBI9, was t r e a t e d with aflatoxin B~ at various t e m p e r a t u r e s for time periods of 30 to 220 min. The molar ratio of t o x i n to D N A p r e p a r a t i o n ranged from 2: i to i o : i. T r e a t e d a n d control D N A ' s were inc u b a t e d with recipient cells of B . s u b t i l i s strain T3 at 37 ° for 45 rain. I t was found t h a t TABLE I INDUCTION TEST

OF

CHROMOSOMAL

ABERRATIONS

AND

DNA

BREAKAGES

A flatoxin

Solvent

Concentrations Cells (or organism) tested

Mixture

Alcohol

670 iz2Vl

V. faba

200 #g/ml 47° #M 1-5o/zg/ml

(seedling root) A. cepa (root) Human leukocytes Human leukocytes

g 1

Mixture Mixture or B1 Mixture Mixture

DMF b DMSOb Propylene glycol or chloroform DMSOb

Mixture B1 B1

BY

AFLATOXIN

IN

VARIOUS

SYSTEMS

DMSOb

5°/zg/ml o.1-5o ppm

Human leukocytes Human embryonic lung cells 12.5 d 25 #g/ml Rat kangaroo kidney cells Chinese hamster io 75/~g/ml ceils 32/zg/ml HeLa cells

Treatment Response a Refertime (h) ence

3

C

63

48 3° 8 or 48

C C C

81 IOO 34

24

C C

79 35

8

C

5°

3

C

98

24

D

97

a Response is either chromosomal aberrations (C) or DNA breakages (D). b DMF, dimethylformamide; DMSO, dimethylsulfoxide. aflatoxin B1 caused i n a c t i v a t i o n of the t r a n s f o r m a t i o n of the t r y p t o p h a n s y n t h e t a s e gene B to the e x t e n t of 87%. M u t a t i o n s from 4- to 23-fold above the s p o n t a n e o u s level were observed in genes which were linked to gene B a n d were responsible for the synthesis of i n t e r m e d i a t e s in the t r y p t o p h a n p a t h w a y before indole glycerol phosphate. Reversion studies showed t h a t 35% of the m u t a n t s i n d u c e d b y aflatoxin B1 in transforming D N A were very stable a n d were most p r o b a b l y deletion m u t a n t s eS. Aflatoxin B1 was found b y these workers to be n o n - m u t a g e n i c in i n t a c t cells of B . subtilis. Using reversion test systems, LILLY observed no m u t a g e n i c effects of aflatoxin on either i n t a c t B . s u b t i l i s or T 4 phage 65. The m u t a g e n i c i t y a n d m u t a g e n i c specificity of aflatoxin have been s t u d i e d b y OxG in the adenine-3 (ad-3) test system of N e u r o s p o r a crassa 25. Aflatoxin B1 a n d G1 were n o t m u t a g e n i c when resting conidia of N . crassa were t r e a t e d with 4 ° # g / m l of either B~ or G~ for time periods from 2 to 12 h74,75. These two compounds, however, were highly m u t a g e n i c when growing vegetative cultures were treated74,75, 77. I n the

42

T. OXG

forward m u t a t i o n experiments, conidia from 7-day-old vegetative cultures of a heterokaryotic N. crassa strain were inoculated onto the b o t t o m of a slant containing Io ml of medium and a specific a m o u n t of aflatoxin (zoo-4oo #g) in o.4 ml of 95°,;, ethanol or in o. 4 ml of z : I mixture of 95}~ ethanol and dimethyl sulfoxide (DMSO) was added either before or after the medium was autoclaved. After 7 days of incubation, conidia from the vegetative cultures were harvested and analyzed for the presence of two specific locus mutations in the ad-3 region. Aflatoxin B 1 at a concentration of 4 ° p g / m l caused a z2o- to I5o-fold increase in the n m t a t i o n frequency over the average ad-7 spontaneous frequency. At the same concentration, aflatoxin G1 caused a 2o- to 28fold increase over the spontaneous level. These results indicate that aflatoxin B1 is a potent m u t a g e n while G1 is a moderate nmtagenic agent. Aflatoxin has also been observed to have no mutagenic effect upon non-growing cells of other fungi. A t t e m p t s have been made b y LILLY64 to s t u d y the effect of aflatoxin on the reverse mutation at the ads and ad~4 loci of Aspergilhts nidulans; however, no significant increase in reversion frequencies was observed in toxin-treated conidia. W h e t h e r aflatoxin would be mutagenic in growing vegetative cultures of Aspergillus has not as yet been demonstrated. In the veast Saccharo*lgvces cerevisiae, BRUSICK (personal communication) found that aflatoxin BI is nmtagenic only in growing vegetative cultures. The mutagenic activities of aflatoxins B.~ and G~ were also tested in the ad- 2 test system of N. crassa ; however, no significant increase in the ad- 3 n m t a t i o n frequencies was observed after treating vegetative cultures of N. crassa with either of these compoundsTL It has to be noted ,however, that the concentrations of aflatoxins used for the mutagenicity tests in Neurospora were relatively low. Genetic analyses of aflatoxin-induced ad- 3 m u t a n t s reveal that the relative frequencies of genotypes, leakiness, allelic complementation and nonpolarized complementation patterns are comparable among m u t a n t s induced b y aflatoxins B, and (it (ref. 75)- This suggests t h a t both agents induce the same relative frequencies of the same types of genetic alterations. Genetic analysis and reversion studies indicate that aflatoxin-induced m u t a n t s of /Y. crassa include base-pair substitutions, fralnesbift mutations, multilocus deletions and other types of intragenic alterations 75. Aflatoxin B, has been shown by LAMB AND LILLY to induce recessive lethal mutations in Drosophila meZa~,ogaster ~6. In their study, male flies fed with aflatoxin were m a t e d to C y / B I L females and six successive broods of offspring were produced b y each male after two virgin females were provided every 3 days. A recessive lethal m u t a t i o n was scored if no wild-type flies were found in the Ira generation. This s t u d y showed t h a t the recessive lethal m u t a t i o n frequencies in broods I and II of the aflatoxin-treated were only slightly increased over those of the controls. Aflatoxin B~ however, caused significant increases in recessive lethal mutations in the later broods, especially in brood I I I . W h e t h e r this result suggests that aflatoxin B~ mainly affects spermatocytes during spermatogenesis or t h a t cell division might play a role in aflatoxin mutagenesis, is yet to be determined. Induction of streptomycin resistant (sr) m u t a n t s in Chlarns, domo~zas reinhardii b y aflatoxin B~ was recently reported by SCHIMMER AND \~rERNERS6. The cells of C. reinhardii were treated with toxin in minimal medium for I to 144 h at a concentration of IO-5O/zg/ml. After treatment, the cells were analyzed for streptomycin resistant mutations. No increase in sr m u t a t i o n frequencies over control values was observed in cells treated for one hour; the m u t a t i o n frequencies, however, increased up to IOO-

43

AFLATOXIN MUTAGENESIS

fold over the control frequencies when cells were exposed to toxin for 6 h. Aflatoxin B1 induces primarily three types of sr mutants : low resistants, s r - r and sr-2. The s r - r mutants show a Mendelian pattern of inheritance while the sr-2 show non-Mendelian patterns of inheritance. The resistant genetic marker of the latter type was suggested to be located on extranuclear DNA, possibly chloroplast DNA. This study, therefore, demonstrates that aflatoxin B1 can cause genetic changes in both nuclear and extranuclear DNA. Aflatoxin B1 has also been reported by V E L A Z Q U E Z et al. 98 to cause 8azaguanine resistant mutations in cultured Chinese hamster cells. The concentrations of aflatoxin B~ which killed 7o-9O~o of the cells caused at least a 2o-fold increase in the mutation frequency. Using a mixture of aflatoxins B~ and G , EPSTEIN AND SHAFNER39,4° showed induction of dominant lethal mutations in mice. The male mice were treated with toxin at a dose of 68 mg/kg and were then mated to untreated females. The frequency of post-implantation loss was determined by post-mortem examination of female mice at mid-term pregnancy. Aflatoxin was shown to cause early post-implantation loss and was suggested to act on the late spermatocyte. It is not known, however, whether the dominant lethal mutations induced in mice by aflatoxin resulted from gene mutations, chromosome aberrations or non-genetically related mechanisms. Among the aflatoxins, only Bt has been shown to cause gene mutations in several organisms. With the exception of mutation-induction in transforming DNA of B . s u b t i l i s , aflatoxin B~ seems to be mutagenic only when metabolically active cells are treated. The mutagenicity tests of aflatoxins G1, B~ and G2 have only been reported in N . crassa. According to the data from Neurospora, the relative mutagenicity of the aflatoxins seems to be in accord with the relative toxicity and hepatocarcinogenicity of these compounds. The differences in the mutagenicities of aflatoxins Bt and G~ are also qualitatively related to the degree of interaction of these two compounds with DNA 23. A summary of mutation-induction by aflatoxins in various organisms is given in Table II. FORMATION OF TOXIC AND MUTAGENIC

METABOLITES

BY MAMMALIAN LIVER MICRO-

SOMES

It is not yet clear whether aflatoxin is mutagenic and carcinogenic p e r se or whether it is one of the identified, or unidentified, metabolites which is the active form for carcinogenesis and mutagenesis. The studies on tumor induction at the site of injection and on mutations in transforming DNA seem to suggest that aflatoxin is active p e r se. Other studies, however, indicate that metabolic activation of aflatoxin is necessary for the expression of the biological activities : mutation-induction is observed to occur only in metabolically active cells ; the degree of toxicity, carcinogenicity, and teratogenicity is species-related; tumorogenesis shows a definite organ specificity. A metabolic conversion of aflatoxin to substances that are responsible for the carcinogenicity of aflatoxin has been suggested 48. A recent study by MALLING69 has shown that mutagenicity of a chemical agent in which metabolic activation is necessary for the expression of mutagenic activity can be detected by application of liver mixed function oxidase in the mutagenic assay system. The studies on the metabolic activation of aflatoxin to toxic and/or mutagenic metabolites have been performed extensively by several workers. SCAIFEs5 reported that aflatoxin B1 can be converted to

44

T. ONG

T A B L E II INDUCTION

OF G E N E M U T A T I O N S B Y A F L A T O X I N IN V A R I O U S O R G A N I S M S

d flatoxin

Solvent

Concentra- Organism tion tested (l~g/ml)

~'Vlutation a Response b Reference assayed

B1

95 °'o ethanol + DMSO or DMSO 950,/o e t h a n o l + DMSO or DMSO DMSO

2 : I to lO: i e 5°

B. subtilis

FM

B. subtilis

FM

68

• 200

B. subtilis

RM

65

B1 Mixture

+

68

transforming DNA

or T 4 phage BI or G~ B 2 o r G~

B~ or G~

95 o~/.oethanol + DMSO - o/ ethanol 9~/o + DMSO 95 % e t h a n o l + DMSO

Mixture

io-4o

N. crassa

FM

75

vegetative cultures 4°

N. crassa

FM

-

vegetative cultures 4°

N. crassa

FM

-

conidia 200

A . nidulans

RM

77 75 64

conidia B1

S. cerevisiae

FM

+

JC~RUSICK,

+

personal communication 86 56 98 39, 4 °

growing cells

B1

Chloroform DMSO

B1 BI+G1

DMSO

B 1

lO-5O 75 68 a

FM RL Chinese hamster cells FM Mouse DL

C. reinhardii D. melanogaster

+ 4-

a Abbreviations for the m u t a t i o n assayed are as follows: FM, forward mutation; RM, reverse mutation; RL, recessive lethal; DL, dominant lethal. b + , mutations are induced by aflatoxin; --, no significant increase in the m u t a t i o n frequency over the control. e Molar ratio of aflatoxin B 1 added to DNA preparation. d mg/kg. a m o r e p o t e n t c y t o t o x i n b y r a t l i v e r cells. GARNER et al. a6 d e m o n s t r a t e d t h a t a f l a t o x i n B I a l o n e is n o t t o x i c t o s t r a i n s G46, C2o7, T A I 5 3 O a n d T A I 5 3 I of S a l m o n e l l a t y p h i m u r i u m . G 4 6 is a h i s t i d i n e - r e q u i r i n g b a s e - p a i r s u b s t i t u t i o n m u t a n t w h e r e a s C2o 7 is a h i s t i d i n e - r e q u i r i n g f r a m e s h i f t m u t a n t . T A I 5 3 O is G 4 6 w i t h a s i n g l e d e l e t i o n t h r o u g h the galactose operon, biotin operon, excision-repair system for DNA and chlorater e s i s t a n c e g e n e s , as T A I 5 3 I is C2o76. T h e s u r v i v a l of T A I 5 3 O a n d T A I 5 3 I w a s d r a s t i c a l l y r e d u c e d a f t e r cells w e r e i n c u b a t e d w i t h B1, a r a t l i v e r h o m o g e n a t e , a n d N A D P H - g e n e r a t i n g s y s t e m ; h o w e v e r , n o r e v e r s e m u t a t i o n s w e r e o b s e r v e d in e i t h e r s t r a i n . GARNER et al. 45 f u r t h e r d e m o n s t r a t e d t h a t l i v e r h o m o g e n a t e s f r o m r a t , g u i n e a pig, m o u s e , h a m s t e r a n d h u m a n c o u l d all c o n v e r t a f l a t o x i n BI t o a t o x i c m e t a b o l i t e for TAI53O. The liver homogenates from hamster, mouse and a single sample from h u m a n l i v e r a p p e a r t o h a v e h i g h e r a c t i v i t y for t h e m e t a b o l i c a c t i v a t i o n of a f l a t o x i n B1. T h e t o x i c i t y of t h e a f l a t o x i n m e t a b o l i t e t o b a c t e r i a d e c r e a s e d w h e n D N A or R N A w a s a d d e d t o t h e a s s a y s y s t e m , p r o b a b l y a r e s u l t of b i n d i n g of t h e a f l a t o x i n m e t a b o l i t e t o n u c l e i c acid*a, 4~. S e v e r a l studiesa~,45,51,oa, 95 h a v e s h o w n t h a t t h e r e a c t i v e m e t a b o l i t e of a f l a t o x i n B1 b i n d s c o v a l e n t l y t o R N A . T h e b i n d i n g w a s f o u n d t o b e m o r e e f f e c t i v e w h e n D N A w a s u s e d i n s t e a d of R N A or w h e n p o l y G w a s u s e d i n s t e a d of o t h e r p o l y r i b o n u c l e o t i d e s a a , 45. T e s t i n g t h e t o x i c i t y of d i f f e r e n t a f l a t o x i n s i n T A I 5 3 O of S. t y p h i m u r i u m u s i n g r a t l i v e r m i c r o s o m e s , GARNER et al. 45 s h o w e d t h a t a f l a t o x i n s BI a n d GI w e r e h i g h l y t o x i c , a n d t h a t a f l a t o x i n M1 h a d low a c t i v i t y , w h e r e a s B2, G2 a n d

AFLATOXINMUTAGENESIS

45

B2~ were essentially inactive. From these results GARNER et al. suggest that the 2-3 double bond of the aflatoxins is the crucial site for metabolic activation. According to molecular orbital calculations, as reported by HEATHCOTE AND HIBBERT53, the 2- 3 pi-bond has the highest bond order value and, therefore, is the most reactive bond within the aflatoxin molecule. In Neurospora, the spectra of genetic alterations among aflatoxin-induced ad- 3 mutants appear to be similar to those induced in N. crassa by the alkylating agents EMS and ICR-I77 (ref. 75). This seems to suggest that the active mutagenic metabolite is an alkylating agent. It has been suggested that the 2,3 expoxide of aflatoxin 131is the active metabolite of aflatoxin B1 (refs. 45, 87, 94, 95). Recent works of GARNER42 and SWENSON et a/.94,95 have provided chemical evidence that aflatoxin 131 is probably metabolically transformed to aflatoxin 131-2,3-oxide by rat liver in vivo and by human and hamster liver microsomes in vitro. Mild acid hydrolysis of the covalently-bound 131 metabolite and nucleic acid show the presence of a 2,3-dihydrodiol of aflatoxin 131 indistinguishable compound strongly suggests that aflatoxin 13i-2,3-oxide is formed during aflatoxin metabolic transformation 9~. Whether all 2,3 double-bonded derivatives of aflatoxin 131 can be converted to their respective 2,3-oxide forms is still not known. The mechanism for the formation of 2,3-dihydrodiol aflatoxin B 1 as shown in Fig. 2 has been proposed by GARNER42and SWENSONet a/.94,95. GARNER AND WRIGHT44 showed that the aflatoxin B1 metabolite is toxic in Escherichia coli KI2 and induces reverse mutations in TAI53O and T A I 5 3 I of S. typhimuriurn. These authors also showed that the production of a reactive aflatoxin metabolite can be increased by pretreating rats with phenobarbital. The sensitivity of E. coli to the aflatoxin metabolite is directly related to the genetic markers carried by the tester strains. A recombination and repair deficient strain is extremely sensitive while the wild-type is relatively resistant. The induction of reverse mutations in TAI53O and T A I 5 3 I seems to suggest that the mutagenic metabolite can cause basepair substitutions as well as frameshift mutations. This study is in agreement with 0t! .

0it

0 I.

metabolic activation

DNA or RNA

Aflatoxin BI

Aflatoxin B ,2,3- oxide

0

o b or RNA DNA-or RNA-bound forms

hydrolysis

0

I

HO~. HO

0

0

OCH 3

2,3- dihydro- 2 , 3 dihydroxy-aflatoxin Br

Fig. 2. Suggested mechanism for the activation of aflatoxin B1 by liver microsomes and for the formation of 2,3-dihydro-2,3-dihydroxy-aflatoxin B1 from the hydrolysis of nucleic acid-bound aflatoxin derivative.

46

T. ONG

that from Neurospora which indicates that mutations induced by aflatoxin include base-pair substitutions, frameshift mutations as well as other types of genetic alterations 7a. The conversion of aflatoxin B~ to a mutagenic metabolite by liver microsomes from either rat or human has also been reported by AMES et al. 7. Their study, however, showed that only TAI537 and TAI538, but not TAr535 or TAI536, of S. t y p h i m u r i u m can be reverted by the aflatoxin metabolite. TAr535 and TAI536 are essentially similar to TAI53O and T A I 5 3 I respectively, except that TAI535 and TAI536 carry the additional genetic marker, deep rough (rfa). Incorporation of rfa into TAI53O and T A I 5 3 I increase the sensitivity of these strains to mutagenic agents 8. TAI537 is a + I frameshift mutant whereas TAI538 is a - - I frameshift mutant. The induction of reverse mutations in TAI537 and TAI538 indicate that the mutagenic metabolite of aflatoxin can revert both base-pair addition as well as deletion mutants. The reason for the differences in the mutagenic responses of S. t y p h i m u r i u m tester strains to aflatoxin metabolites, as reported by various workersT,44,46, is not known. This could be due to differences in samples of liver microsomes or to differences in test conditions. In our laboratory, we have found that aflatoxin B1 can also be converted, by hamster liver microsomes, to a metabolite which is mutagenic in N . c r a s s a (MATZINGERAND ONG, unpublished results). Aflatoxin Bt is not mutagenic if conidia of N . crassa are treated without liver microsolnes. The ad-3 mutation frequency increases more than IOO times over the spontaneous frequency if conidia are treated with a concentration of 0.67 mM aflatoxin B1 and a liver mixed function oxidase for 2 h. There seems to be little doubt that aflatoxin can be converted to toxic and/or mutagenic metabolites by mammalian liver mierosomes. It is not clear, however, whether the toxic and mutagenic activities of aflatoxin are caused by aflatoxin Bx-2,3oxide. If epoxyaflatoxin is the active principle of aflatoxin, the low toxicity and carcinogenicity of the 2,3-dihydro-aflatoxins could be due to the fact that these compounds cannot be directly converted to the active form. The studies on the metabolic activation of aflatoxins to toxic and/or mutagenic metabolites are summarized in Table I I I . OTHER GENETICALLY RELATED ACTIVITIES

Aflatoxin has been shown to induce phage production in lysogenic bacteria and to cause morphological changes and inhibition of mitosis in the cells of prokaryotes and/or eukaryotes. LEGATOR~s showed that at a concentration as low as o.o6/~g/ml either a crude aflatoxin preparation or crystallized aflatoxin ]31 could induce phage production in a lysogenic E. coli, P46, and in a lysogenic Staphylococcus aureus, LM2o 4. The induction of lysogenic bacteria by aflatoxin Bt was also demonstrated by LILLEHOJ AND CIEGLER61. An approximately I5O-fold increase in plaque-forming units over the control was found after a lysogenic strain of Bacillus megaterium NNRLB-3695 was treated with 25/~g aflatoxin B~/ml for 2 h. No plaque-forming units were induced after a non-lysogenic strain was treated. WRASS et al. 1°7 reported that, when E. coli were incubated with either I or 5/ag of crude aflatoxin or crystalline aflatoxin B1 per ml of culture medium at 37 ° or at 42o , elongated filamentous cells were observed. Filamentous cells were also found by LILLEHOJet al. 6~ after the cells ofFlavobacterium aurantiacure were grown at 3 °° in culture medium containing I o - I o o /~g aflatoxin B~/ml. BEUCHAT AND LECHOWICHla found that, at a concentration of 3.8 #g/ml, aflatoxin B~

AFLATOXIN

TA13LE

47

MUTAGENESIS

III

SUMMARY

OF

THE

FORMATION

OF

TOXIC

AND

MUTAGENIC

METABOLITES

BY

MAMMALIAN

LIVER

MICROSOMES

A flatoxin

Concentration

Source of liver Type of homogenates a metabolite produced

131

o.I-Io#M

Rat

Toxic

Organism tested

Reference

S. typhimurium

45, 46

TAI53 o, TAI53I G1

i - i o/~M

Rat

Toxic

S. typhimurium

45

TA 153o M1

io 6o#g/ml

Rat

Toxic

S. typhimurium

45, 46

TAI53 o 13~,G~, B2a or P1 B1

IO 60/~g/ml

Rat

ob

S. typhimurium

45

TAI53° 0.2/~3I

131 131

60 #M 60 #M

Guinea pig, Toxic mouse, hamster or human Rat Toxic Rat Mutagenic

B1

I/~g/nll

Rat

S. typhimurium

45

TA153° E. coli S. typhimurium

44 44

TAI53O, TAI53I 131

I #g/ml

Human

Toxic and mutagenic Mutagenic

S. typhimurium

7

TAI537, TAI538 S. typhimurium

7

TAI538 131

0.67 mM

Hamster

Toxic and mutagenic

N.crassa

MATZlNGERAND ONG, unpublished results

a Supernatant of liver homogenates after 9ooo g centrifugation was used in all experiments listed. b No significant lethal effect was found. caused the p r o d u c t i o n of a b e r r a n t filamentous cells in B . m e g a t e r i u m . Although it has been suggested t h a t in E . coli a single gene controls the formation of cross platesL it is d o u b t f u l t h a t the i n d u c t i o n of the filamentous morphology b y aflatoxin is a m u t a t i o n a l e v e n t . I t has been shown b y BEUCHATAND LECHOWICHt h a t aflatoxin-induced a b e r r a n t cells r e t u r n to n o r m a l morphology if these cells are transferred to non-aflatoxin cont a i n i n g m e d i u m . Morphologically a b n o r m a l cells i n d u c e d b y aflatoxin in c u l t u r e d heteroploid h u m a n e m b r y o n i c lung cells have been reported b y LEGATOR a n d his associates 6°. A 92% increase in g i a n t cells over the control (0.3%) was observed after L-I32 cells were exposed to I p p m crystallized aflatoxin BI for 8 - I 2 h. The n u m b e r of g i a n t cells, however, decreased, if cells which h a d been exposed to I p p m of aflatoxin for 24 h, were i n c u b a t e d in n o n - t o x i n c o n t a i n i n g m e d i u m for more t h a n 12 h. The studies on the b i n d i n g of aflatoxin to D N A a n d the i n h i b i t i o n of DNA, R N A a n d protein synthesis b y aflatoxin have been extensively carried out. These studies a n d the studies related to other biochemical aspects of this t o x i n group have been extensively reviewed b y WOGANI°I,1°4. Aflatoxin has been reported to i n h i b i t mitosis in several cell culture studies. LEGATOR AND WITHROW59 showed that, in heteroploid a n d diploid h u m a n e m b r y o n i c lung cells, mitotic frequency was reduced a p p r o x i m a t e l y 50% from t h a t of the control when the cells were t r e a t e d with 0.5 p p m of either a crude aflatoxin m i x t u r e or aflatoxin B1. R e d u c t i o n in mitosis occurred 4 h after the cells were exposed to the toxin a n d reached a m a x i m u m in 8 to 12 11. GCAIFE85 d e m o n s t r a t e d t h a t aflatoxin B1 at the c o n c e n t r a t i o n of IO # g / m l inhibits mitosis in h u m a n k i d n e y T cells a n d t h a t mitosis was m a r k e d l y reduced if the cells were t r e a t e d with toxin which

48

T. ONG

had been p r e i n c u b a t e d with Chang or rat liver cells. Mitosis i n h i b i t i o n b y aflatoxin was also found in roots of V . f a b a seedlings 6a, in roots of A . cepa sI, a n d in cultured h u m a n leukocytes a4 The m i t o t i c index of h u m a n leukocytes dropped from 4.2 for control cells to 1. 4 for cells t r e a t e d for 48 h with 5o # g / m l of toxin. A s t u d y b y GREEN et al. 50 showed t h a t c o n c e n t r a t i o n of aflatoxin as low as 25 # g / n i l significantly inhibited mitosis in a r a t kangaroo k i d n e y cell line. I n vivo studies on the i n h i b i t i o n of mitosis in rat liver b y aflatoxin have been carried out b y ROGERS AND NEWBERNE s2 in which rats received aflatoxin B~ in a dose of 3 m g / k g of b o d y weight. The reduction of mitosis in the hepatic p a r e n c h y m a l cells began within I to 3 h after a d m i n i s t r a t i o n of the compound. This rapid decrease in nfitosis, as indicated b y the authors, suggests t h a t aflatoxin not only inhibits mitosis i n d i r e c t l y t h r o u g h i n h i b i t i n g D N A synthesis, b u t m u s t also directly inhibit cellular mitosis. Table IV summarizes some of the genetically related activities of aflatoxin. MUTAGENIC

HAZARD

TO

MAN

A . f l a v u s a n d other aflatoxin-producing fungi grow in a wide t e m p e r a t u r e range a n d are c o m m o n l y f o u n d in both t e m p e r a t e a n d tropical geological areas. These fungi produce air-borne spores a n d can grow on almost a n y type of agricultural product. Consequently, aflatoxin has been found as n a t u r a l c o n t a m i n a t e in various cattle, as well as h u m a n , food stuffs, e.g., peanuts, p e a n u t meal, rice, peas, cassava, corn, cottonseed meal, soybeans, cocoa, coconut, wheat, a n d sweet potatoes. M a n y of these commodities c o n t a i n biologically significant levels of the c o m p o u n d s m, although, as indicated b y some workers, it is not k n o w n to what e x t e n t agricultural commodities in a given area are c o n t a m i n a t e d with aflatoxin or to what e x t e n t aflatoxin contamin a t e d commodities are used as food. Nevertheless, aflatoxin could enter the h u m a n

TABLE IV INDUCTION

OF S O M E O T H E R G E N E T I C A L L Y

RELATED

BIOLOGICAL CHANGES BY AFLATOXIN

A flatoxin

Concentration

Mixture or B 1 Bt

0.06/,g/ml

Lysogenic strain of

25/*g/ml

E. coli or S. aureus B. megaterium

Treatment Organism or time ( h) cells treated

2

lysogenic strain Mixture or B1 B1

I 5 #g/nil

E. coli

IO-IOO/~g/ml

F. aurantiacum

B1

3.8#g/ml

3.5 or 5.5

B. megaterium

B1

o. i - i ppm

8-12

Mixture or B 1 t31 Mixture Mixture 131 Mixture

o. 5 ppm

4-12

IO/~g/ml 670 # M 5o #g/ml 2o0/zg/ml 25/zg/ml

24 48 48

Human embryonic lung cells Human embryonic lung cells Human kidney cells V. faba seedling root Human leukocytes

]31

3 mg/kg

1- 3

3

A. cepa

Rat kangaroo kidney cells Rat

Response

Rej)rence

Phage production Phage production Filamentous cells formation Filamentous cells formation Filamentous cells formation Giant cells formation Mitotic inhibition Mitotic inhibition Mitotic inhibition Mitotic inhibition Mitotic inhibition Mitotic inhibition Mitotic inhibition

58 61 lO7 62 13 60 59 85 63 34 8I 5° 82

AFLATOXIN MUTAGENESIS

49

body either directly, through agricultural products or indirectly, through contaminated farm animals. Indeed, aflatoxin has been found to be present in human urine 2°, milk 9 and several organs 89. Little is known in respect to the metabolic fate of aflatoxin in man. Several studies indicated that aflatoxin B1 could be converted to other forms of aflatoxins. Aflatoxin M1 was recovered by CAMPBELLet al. 2° from urine of humans who had consumed aflatoxin-contaminated food. BOCHI e t a l . TM reported that the 9000 g supernatant of fresh human liver homogenates could convert 3O~o of aflatoxin B1 to Q1 in 30 miD. Small amounts of aflatoxins M1 and P1 were recovered by MERRILL A~D CAMPBELL71 after aflatoxin B~ was incubated for 6.5 h with human liver homogenates from biopsy tissue. No aflatoxin P~ was found when autopsy liver tissue was used. Aflatoxin B 1 probably could also be converted to aflatoxin B1-2,3-oxide by human liver microsomes 95. Although it is assumed that the conversion of aflatoxin B1 to the 2,3 oxide of aflatoxin B~ is an activation process, it is not clear whether demethylation and hydroxylation of aflatoxin B1 are also activation processes. The conversion of aflatoxins B~ to P1, M~ or Q~ may facilitate the elimination of the toxin from the human body. Direct evidence that aflatoxin causes cancer in humans is still lacking; several lines of study, however, suggest that aflatoxin is probably responsible for the high incidence of cancer in certain geographic areas~5,88,9°,1°2. Aflatoxin has also been implicated in the death of Thai children with acute encephalopathy and fatty degeneration of the visceral~, 89. Aflatoxin causes chromosomal aberrations in animal as well as in plant cells and induces gene mutations in several organisms. In N . crassa, the inutagenic activity of aflatoxins B1 and G1 is retained even after autoclaving at 15 lb/in. 2 of pressure (12o °) for 30 miD 7.. Thus it appears that the mutagenic activity of aflatoxin cannot be affected by ordinary cooking. It is not known whether aflatoxin and/or its metabolites are mutagenic in humans. However, studies such as the induction of chromosomal aberrations in cultured hmnan cells and the conversion of aflatoxin to its mutagenic metabolites in bacteria by human liver microsomes strongly suggest that aflatoxin possesses a potential mutagenic hazard to man. F~xtensive studies on the mutagenic activity of B1 as well as other aflatoxins and their metabolites need to be carried out in a variety of test systems. Only when additional data are available can the potential mutagenic hazard of aflatoxin to man be meaningfully evaluated. As aflatoxins are known to be potent chemical carcinogens and mutagens, these agents have to be handled with care. The contaminated materials and glassware have to be detoxified; soaking all contaminated materials in commerical chlorox is probably the most commonly used and a very effective detoxification process. REFERENCES I ADAMSON, R. H., P. CORREA AND D. W. DALGARD, Brief c o m m u n i c a t i o n : Occurrence of a p r i m a r y liver carcinoma in a R h e s u s m o n k e y fed aflatoxin B1, J. Natl. Cancer Inst., 5 ° (1973) 549 553. 2 ADLER, H. I., AND A. A. HARDIGREE, Analysis of gene controlling cell division and sensitivity to radiation in Escherichia coli, J. Bacteriol., 87 (1964) 720-726. 3 ALLC•OFT, R., Aflatoxicosis in f a r m animals, in L. A. GOLDBLATT (Ed.), Aflatoxin : Scientific Background, Control and Implications, Academic Press, N e w York, 1969, pp. 237 264. 4 ALLCROFT, R., AND R. B. D. CARNAGHAN, G r o u n d n u t toxicity: An e x a m i n a t i o n for t o x i n in h u m a n food p r o d u c t s f r o m animals fed toxic g r o u n d n u t meal, Vet. Rec., 75 (1963) 259-263.

50

T. 0NG

5 ALLCROFT, t~., H. ROGERS, G. LEWIS, J. NABNEY AND P. E. BEST, Metabolism of aflatoxin in sheep: E x c r e t i o n of the " m i l k t o x i n " , Nature (London), 209 (1966) 154 155. 6 AMES, B. N., The detection of chemical m u t a g e n s w i t h enteric bacteria, in A. HOLLAENDER (Ed.), Chemical Mutagens: Principles and Methods for their Detection, Vol. I, Plenum, New York, 1971 , pp. 267-287 . 7 AMES, B. N., W. E. DURSTON, E. YAMASAKI AND F. D. LEE, Carcinogens are m u t a g e n s : a simple test s y s t e m combining liver h o m o g e n a t e s for activation and bacteria for detection, Proc. Natl. Aead. Sci. (U.S.A.), 7 ° (1973) 2281-2285 . 8 AMES, B. N., F. D. LEE AND ~V. E. DURSTON, An i m p r o v e d bacteria test s y s t e m for the detection and classification of m u t a g e n s and carcinogens, Proc. Natl. Aead. Sci. (U.S.A.), 7 ° (1973) 782-786. 9 ARRHENIUS, E., M y c o t o x i e o s i s - - a n old h e a l t h h a z a r d with new dimensions, Ambio, 2 (1973) 49-56 . IO ASPLIN, F. D., AND R. B. A. CARNAGHAN, The toxicity of certain g r o u n d n u t meals for p o u l t r y w i t h special reference to their effect on ducklings and chickens, Vet. Rec., 73 (19611 1215-1219. i I AYRES, J. L., D. L. LEE, J. H. WALES AND R. O. SINNHUBER, Aflatoxin s t r u c t u r e and hepatocarcinogenicity in r a i n b o w t r o u t (Salmo gairdneri), J. Natl. Cancer Inst., 46 (1971) 561-564. 12 BARNES, J. M., AND W. H. BUTLER, Carcinogenic activity of aflatoxin to rats, Nature (London) 202 (1964) lO16. 13 BEUCHAT, L. R., AND R. V. LECHO~,VICH, Morphological alterations in Bacillus megaterium as p r o d u c e d b y aflatoxin B1, Appl. Microbiol., 21 (1971) 124-131. 14 BLOUNT, \~7. p., T u r k e y " X " disease, J. Br. Turkey Fed., 9 (19611 52, 55-58, 61, 77. 15 BOURGEOIS, C. H., R. C. SHANK, R. A. GROSSMANN, D. O. JOHNSON, ~V. L. WOODING AND P. CHANDAVIMOL, Acute aflatoxin B 1 toxicity in the m a c a q u e and its similarities to Reye's s y n d r o m e , Lab. 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