11
Mutation Research, 53 (1978) 11--20 © Elsevier/North-Holland Biomedical Press
EVALUATION OF THE MUTAGENIC POTENTIAL OF MYCOTOXINS USING SALMONELLA TYPHIMURIUM AND SACCHAROMYCES CEREVISIAE
M A U R E E N H. KUCZUK, PAUL M. BENSON, H A R R Y HEATH and A. WALLACE HAYES
Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Miss. 39216, and Department of Microbiology, The University of Alabama, Ala. 35486 (U.S.A.) (Received 12 May, 1977) (Accepted 4 July, 1977)
Summary The mutagenic effects of fifteen mycotoxins on Salmonella typhimurium strains TA1535, TA1537 and TA1538 and Saccharomyces cerevisiae strain D-3 were tested. Only aflatoxin B1 and sterigmatocystin were mutagenic. Both were active against S. typhimurium strain TA1538 and S. cerevisiae strain D-3; however, both required activation by the hepatic S-9 enzyme preparation. A positive correlation between the other mycotoxins reported to be carcinogenic and the two in vitro test systems employed was not demonstrated in our hands.
Introduction Standard (toxicological) techniques routinely used to identify carcinogenic chemicals are time-consuming and expensive. It also is often difficult to perform large-scale in vivo rodent studies to evaluate the carcinogenic potential of naturally occurring chemicals and/or their animal metabolites because of compound scarcity. A promising alternative using in vitro test systems for the analysis of the carcinogenic properties of a chemical has been proposed [1]. The desired goals of cost reduction and rapid evaluation as well as improved sensitivity, increased test population numbers and diversification in endpoints are achievable with these systems. It has been suggested that a chemical reaction with DNA is the basis of drug-
All correspondence and reprint request to: Dr. A. Wallace Hayes, D e p a r t m e n t of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi 39216 (U.S.A.).
12 induced carcinogenesis [ 21]. Since mutagenesis also involved cellular DNA, the argument for use of such mutagenic systems was strengthened by the demonstration that carcinogens or their "activated" intermediates were mutagenic for microorganisms [16]. It, therefore, should be possible to apply results obtained with microorganisms as screening systems to detect mutagens for mammals. On the assumption that carcinogenesis results from a derangement or alteration of DNA structure or its metabolism, a number of procedures involving microorganisms have been devised to detect mammalian carcinogens [1,3,20,21]. Ames et al. [2] have made a strong case for the generalization that with few exceptions carcinogens are mutagens, thus giving strength to the desirability of the Salmonella test as a rapid and economical system for screening purposes. Fungi produce a variety of toxic metabolites, mycotoxins [23] which are not only cytotoxic, b u t in the case of several have been reported to be carcinogenic to laboratory animals [5,12,18,24]. Furthermore, considerable evidence is accumulating to indicate that a large proportion of human cancer may be caused by exposure to toxic chemicals in the environment, including aflatoxin B1 [24]. Aflatoxin B1 and sterigmatocystin are examples of mycotoxins activated to frame-shift mutagens by liver microsomal preparations [9,10]. This paper presents mutagenicity data on 15 mycotoxins, five (aflatoxin B1, griseofulvin, patulin, penicillic acid and sterigmatocystin) reported to be carcinogenic and t w o (aflatoxin BI and rubratoxin B) reported mutagenic in animal models [5,7,12,13,18,24], using S. typhimurium and Saccharomyces cerevisiae as the test organisms. The studies were designed to determine the correlation between mutagenicity as estimated b y the t w o in vitro microbial test systems and the reported carcinogenicity. Materials and m e t h o d s Bacterial test system The Salmonella/microsome mutagenicity test has recently been reviewed [2]. S. t y p h i m u r i u m tester strains TA1535, which detects base-pair mutagens, and T A 1 5 3 7 and TA1538, which detect frame-shift mutagens, were obtained from B.N. Ames, University of California, Berkeley. In addition to a u x o t r o p h y for histidine, each strain carries a deletion originating in the galactose operon and extending into the uvr B locus. Consequently, each strain requires biotin and shows increased sensitivity to mutagens due to the absence of excision repair. Each strain also bears a defect in a gene for lipopolysaccharide synthesis (rfa) resulting in increased permeability to large molecules and the so-called deep rough phenotype. The relevant properties of the tester strains were confirmed as described b y Ames et al. [2]. The bacteria were stored and grown as outlined by Ames et al. [ 2 ]. Each m y c o t o x i n was tested for mutagenic activity directly and in the presence of the hepatic S-9 enzyme preparation in both qualitative (data not presented) and quantitative assays as described b y Ames et al. [2]. The hepatic S-9 enzyme fraction was prepared from Sprague--Dawley male rats pretreated with a saline solution of sodium hexobarbital (60 mg/kg, i.p.) 2 days before being sacrificed. Preliminary runs had determined the a m o u n t of S-9 equivalent
13 to 20 mg of liver tissue per plate to be o p t i m u m for mutagenesis at the dose level of 0.1 pg of aflatoxin B1 per plate. All mycotoxins were dissolved in dimethylsulfoxide (Burdick and Jackman, Muskegon, Mi.); no more than 0.1 ml of dimethylsulfoxide was added per plate, an a m o u n t have no appreciable effect on the mutagenic response of the systems. Revertants were scored using a Bactronic colony counter. All experiments were performed under subdued light conditions and repeated at least once. Negative controls for the spontaneous reversion of the tester strains to histidine p r o t o t r o p h y differed from experimental tests only by the absence of a mycotoxin. Positive controls for induced reversion of strains TA1535 and TA1537 employed N-methyl-N'-nitroN-nitrosoguanidine (NG) and quinacrine dihydrochloride (~C), respectively. Aflatoxin B~ was used as a positive control for induced reversion of strain TA 1538 and as a control to confirm the ability of our S-9 enzyme preparation to activate this m y c o t o x i n which is non-mutagenic in the direct assay.
Yeast test system Diploid strain D-3 of Saccharomyces cerevisiae, from J. Epler, Oak Ridge National Laboratory, Tenn., carries markers for dominant cycloheximide resistance (CYH 4), adenine a u x o t r o p h y (ade 2) and histidine a u x o t r o p h y (his 8) on one homologue of chromosome XV with the corresponding wild-type alleles on the other homologue [26]. H o m o z y g o u s ade 2 mutants accumulate a red pigment (sectors) which serves as a convenient means for detecting genetic damage resulting in homozygosity at the ade 2 locus. Mayer [14] has shown that the majority of the red sectors induced b y metabolites of dialkylnitrosamines and naphthylamines were due to mitotic recombination although a variety of mechanisms including mutagenesis, gene conversion, chromosomal deletion, aneuploidy and haploidization also could produce red sectors. The mechanism of red sector formation b y mycotoxins, however, was not investigated in the present study. Each m y c o t o x i n was tested directly and after activation by the hepatic S-9 enzyme preparation for genetic activity (induction of red colonies/sectors). For direct assay, 2.0 ml of reaction mixture containing 60 mM sodium phosphate buffer, pH 7.2, 6.25 X 106 cells/ml and 100 pg of m y c o t o x i n per ml were incubated for 3 h at 37°C. In the activated assays 1.5 ml of S-9 mix replaced the buffer and m y c o t o x i n concentrations were 50 pg/ml. Following incubation, cells were washed twice in 60 mM sodium phosphate buffer, pH 7.2, b y centrifugation and appropriately diluted for determining the frequency of red colonies/sectors and viable counts on YEPD agar (1.0% Bacto-yeast extract (Difco), 2.0% Bacto-peptone, 2.0% glucose and 1.5% Bacto-agar). After incubation for 3 days at 30°C, the plates were stored for 2 days at 5°C to enhance pigment accumulation. All experiments were repeated a minimum of t w o times. Negative controls for spontaneous frequency of red colonies and sectors differed from experimental assays only b y the absence of a m y c o t o x i n . Positive controls for detecting genetic damage to strain D-3 in the direct and S-9-activated assays employed ethyl methanesulfonate (EMS) and dimethylnitrosamine (DMN), respectively. Results were considered negative if the number of sectors per 10 s surviving cells for a test compared were less than one-third of the positive control.
TABLE
1
9 13 23
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
TA 1535 TA 1538
Diacetoxyscirpenol
A
A + B c
Griseo fulvin
Ochratoxin
Ochratoxin
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
TA 1535
Oosporein
Patulin
Penicillic acid
TA 1535 TA 1537 TA 1538
21 36 28
TA 1535 TA 1537 TA 1538
12
2 27 16
7 17 18
28 16 40
13 25
6 30 25
14
6 30 17
17 17 26
23 22 45
13 26
4 17 29
7 10 18
17 19 31
14
11 33 26
8 11 28
30 19 37
12 18
6 11 17
10 17 9
26 35 32
1
14
5 34 36
3 14 30
10 24 44
NT b NT
9 16 32
10 9 19
11 31 33
20
8 32 27
8 17 30
20 28 43
14 19
8 16 19
8 8 21
20 32 27
9
11 30 19
11 7 49
18 23 120
13 44
10 20 38
7 15 24
11 36 28
8
11 33 35
18 22 35
18 26 53
17 32
8 23 28
7 10 15
10 19 31
10
11
7 41 31
17 15 41
12 18 50
23 32
11 22 35
7 12 23
6 35 32
1
100
l0
100
DMSO
TYPHIMURIUM
Activated a Dg/plate (of test compound) 0.1
OF SALMONELLA
Non-activated pg/plate (of test compound)
Citrinin
STRAINS
per plate
IN THREE
Revertants
MYCOTOXINS
S. t y p h i murium strain
OF FIFTEEN
Mycotoxin
MUTAGENICITY
7
7 32 27
9 16 34
13 27 44
NT NT
11 22 34
11 12 21
7 31 23
0.1
9
7 34 42
7 12 33
16 19 45
20 27
7 22 33
7 12 15
9 32 27
DMSO
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
Verruculogen
Viriditoxin
Zearalenone
>1000 >1000 16
8 7 27
15 9 11
5 13 21
13 27 44
9 21 27
13 22 32
11 15 15
12 28
19 14 20
15 8 16
6 II 22
18 21 35
8 16 20
8 20 20
9 11 22
13 19
19 13 28
10 12 20
8 6 18
17 20 45
12 11 28
12 18 32
11 11 8
6 30
19 15 17
8 8 14
12 15 25
14 33 43
6 18 17
8 20 37
7 7 21
10 18
20 12 16
20 14 18
20 12 19
8 12 19
20 18 43
9 20 18
8 16 38
20 8 21
17 30
>1000 >1000 400---600
14 12 18
5 13 30
10 15 44
10 17 54
NT NT 310
6 24 29
12 9 24
4 12
i0 12 29
28 16 31
7 8 20
8 21 55
NT NT 280
12 27 18
10 7 27
16 43
a Activated with the hepatic S-9 e n z y m e preparation. b N T = not tested. c A mixture of ochratoxins A and B ( 8 5 % A and 1 5 % B). Also tested at 2 5 0 pg (98 revertants) and at 5 0 0 pg (118 revertants).
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
T-2 toxin
Controls MNNG QC Aflatoxin B 1
TA 1535 TA 1537 TA 1538
Sterigmatocystin
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
A
Rubratoxin
Penitrem
TA 1537 TA 1538
11 12 40
9 9 42
5 8 21
6 23 46
NT NT 160
6 30 26
10 9 30
23 34
10 14 23
11 19 29
10 15 13
9 20 43
NT NT 90
9 24 28
7 10 28
13 23
9 12 14
9 20 25
9 I0 28
7 I0 28
9 33 45
NT NT 14
7 20 38
9 12 15
12 33
16 o Ochratoxins A & B o Ochratoxin
A
~
o
120
Q"
80
o. c to ~c
R
D
300
50C)
40
0
100 per
plate
Fig. 1. A c o m p a r i s o n of t h e i n d u c t i o n of r e v e r t a n t s in S. t y p h i m u r i u m strain T A 1 5 3 8 b y o c h r a t o x i n A a n d a m i x t u r e o f o c h r a t o x i n s A a n d B.
Mycotoxins The following mycotoxins * were investigated: aflatoxin B1, citrinin, diacetoxyscirpenol, griseofulvin, ochratoxin A, an ochratoxin A and B mixture (85 and 15%, respectively), oosporein, patulin, penicillic acid, penitrem A, rubratoxin B, sterigmatocystin, T-2 toxin, verruculogen, viriditoxin and zearalenone. Except in the case of the mixture of ochratoxin A and B, the purity of each m y c o t o x i n was established b y melting point, thin-layer chromatography and infrared spectroscopy. All biochemicals, griseofulvin, NADP ÷, glucose 6-phosphate, histidine and biotin, were purchased from Sigma Chemicals, St. Louis, Mo. Results
All of the m y c o t o x i n s were negative in the qualitative Salmonella test (activated and non-activated) although a faint ring was observed with strain TA1538 in the presence of ochratoxins A + B mixture and the S-9 mix. A summary of the data obtained over the concentration range of 0.1 to 100 pg * M y c o t o x i n s w e r e o b t a i n e d f r o m B.J. Wilson, V a n d e r b i l t U n i v e r s i t y , Nashville, T e n n . , p e n i t r e m A ; N.D. Davis, A u b u r n U n i v e r s i t y , A u b u r n , Ala., citrinin a n d o c h r a t o x i n ; J.L. R i c h a r d , N A D L , A m e s , I o w a , T-2 t o x i n ; R.J. Cole, U S D A , D a w s o n , G e o r g i a , o o s p o r e i n a n d v e r r u c u l o g e n ; J.V. R o d r i c k s , F D A , W a s h i n g t o n , D.C., z e a x a l e n o n e a n d s t e r i g m a t o c y s t i n ; D.H. Wilson, p a t u l i n a n d penicillic acid.
17 of each m y c o t o x i n per plate with the three strains of S. t y p h i m u r i u m are presented in Table 1. In the non-activated system, the number of revertants per test plate did not differ from the number of revertants per control plate regardless of the m y c o t o x i n tested. In the activated system, the number of revertants per test plate did not differ from the controls except for aflatoxin B~, sterigmatocystin and the ochratoxins A + B mixture using strain TA1538. A comparison of the results obtained with strain TA1538 and different concentrations of the ochratoxins A and B mixture and an ochratoxin A sample purified by high pressure liquid chromatography are presented in Fig. 1. A dose response was apparent with the mixture whereas the number of revertants per plate in the presence of 500 pg of the purified preparation of ochratoxin A was only slightly elevated over control plates. A summary of data obtained after screening the m y c o t o x i n s in the presence of S. cerevisiae D-3 for potential genetic damage is presented in Table 2. The results in the non-activated system were negative indicating that the mycotoxins were not capable of causing mitotic crossing-over under these conditions. When the system was activated with the S-9 enzyme preparation, negative data were obtained except in the case of aflatoxin B1 and sterigmatocystin indicating that these two mycotoxins apparently were capable of causing mitotic recombination in the D-3 strain of S. cerevisiae when activated by the hepatic S-9 enzyme preparation. TABLE
2
MUTAGENICITY Mycotoxin
OF FIFTEEN
b
MYCOTOXINS
IN THE SACCHAROMYCES
Non-activated Freq. c
CEREVISIAE
D-3 TEST
Activated a Survival (%)
Freq.
Survival (%)
Aflatoxin B 1 Citrinin Diacetoxyscirpenol Griseo fulvin Ochratoxin A Oosporein Patulin Penicillic acid Penitrem A Rubratoxin B
3.1 3.4 2.0 12.0 3.8 1.7 17.6 7.1 5.8 5.7
82 85 69 71 76 84 24 81 84 75
610.4 6.6 5.4 3.8 10.1 5.2 2.6 1.3 7.3 1.3
99 93 92 100 99 96 99 96 103 96
Sterigmatocystin T-2 toxin Vertuculogen Virid ito xin Zearalenone
4.5 2.0 8.7 7.1 11.5
63 72 66 81 74
470.2 7.8 4.0 2.5 3.8
95 113 95 99 99
6.5 92.3 NT
100 93 NT
2.2 NT 76.9
100 NT 88
Controls Negative control EMS DMN
(minus toxin)
a A c t i v a t e d w i t h t h e h e p a t i c S-9 e n z y m e p r e p a r a t i o n . b 100 and 50 ~g/0.1 ml DMSO per plate for non-activated and activated, respectively. c F r e q u e n c y o f r e d c o l o n i e s a n d r e d s e c t o r s a p p e a r i n g i f a p o p u l a t i o n o f 105 y e a s t c o l o n i e s w e r e s c r e e n e d , V a l u e s are m e a n s o f t w o e x p e r i m e n t s .
18 Discussion None of the mycotoxins tested were mutagenic in the unactivated systems employing S. typhimurium strains TA1535, 1537 or 1539 or S. cerevisiae D-3. We have interpreted this to mean that the parent compounds were not mutagenic. However, when these compounds were activated with the hepatic S-9 enzyme preparation obtained from rats treated with hexobarbital, aflatoxin B1, sterigmatocystin and the mixture of ochratoxins A and B were mutagenic in the Ames' system and aflatoxin B, and sterigmatocystin were active in the yeast system. It is possible that the result obtained from the combination of ochratoxins A and B was due to ochratoxin B alone. Since the number of reverrant colonies per plate in the presence of ochratoxin A did not differ from control values, ochratoxin A may require metabolic activation to ochratoxin B for mutagenic activity along the lines proposed by McCann and Ames [17] for dehalogenation of certain chlorinated hydrocarbons. However, this does not seem likely since ochratoxin B is considerably less toxic than ochratoxin A. It also is possible that the increase in the number of revertant colonies per plate was caused by a synergistic effect of the two toxins or that a contaminant in the mixture was responsible for the observed effect. The results obtained using the D-3 yeast system confirmed the lack of mutagenicity for ochratoxin A. Our data support the concern about false negatives in light of the reported carcinogenicity of penicillic acid and patulin [5]. Furthermore, the negative results obtained in this study and by Ueno et al. [22] with the carcinogenic antifungal agent, griseofulvin, offers additional concern about the adequacy of such screening systems. Our failure to detect the mutagenicity of rubratoxin B in either microbial system contradicts the report of the mutagenic effect of rubratoxin B in mice [7,8]. As pointed out by De Serres [4], when an assay is developed for forward mutations which would detect any type of genetic alteration rather than reverse mutation which only detects particular types of genetic alterations, some of the problems with false negatives should be eliminated. Improvements in the sensitivity of tester strains also will help as was the case with two aflatoxin B, metabolites. Aflatoxicol H~ and aflatoxin Q~ were determined to be non-mutagenic using S. typhimurium TA1538 [11,19] but weakly mutagenic with strain TA098 [25]. Engel and Von Milczewski [6], however, were unable to detect an increase in the mutation rate of S. typhimurium TA98 or TA100 by either patulin or penicillic acid. Our studies, therefore, should be repeated with a more sensitive strain before the case on griseofulvin, patulin and penicillic acid is closed. Even though most of the mycotoxins were not mutagens, negative results cannot be considered conclusive at this time. According to our data, using the S. typhimurium tester strains TA1535, TA1537 and TA1538 and the S. cerevisiae D-3 strain, only aflatoxin B1 and sterigmatocystin were mutagenic; it is conceivable that griseofulvin, patulin and penicillic acid, reported mammalian carcinogens, would have been eliminated from additional toxicological testing based on these results. However, Ueno et al. [22] recently have reported that penicillic acid and patulin but not griseofulvin were positive in their recombination-deficient m u t a n t cells of Bacillus subtilis. Mayer and Legator [15] have reported the production of petite mutants in a haploid strain of S. cerevisiae
19 by patulin. Thus, a series of in vitro microbial tests should be employed for screening purposes. Although some carcinogens may escape detections, these systems remain a useful battery of screens for chemicals that may have the potential for interacting with cellular genomes. Acknowledgments Support by U.S. Public Health Services grants ES-01351 and ES-01352 from the National Institute of Environmental Health Sciences. References 1 A m e s , B.N., T h e d e t e c t i o n o f c h e m i c a l m u t a g e n s w i t h e n t e r i c b a c t e r i a , in: C h e m i c a l m u t a g e n s ; principles a n d m e t h o d s f o r t h e i r d e t e c t i o n , vol. 1, A. H o l l a e n d e r ( E d . ) , c h a p t e r 9 ( 1 9 7 1 ) , P l e n u m Press, New York. 2 A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s f o r d e t e c t i o n of c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l m o n e l l a / m a m m a l i a n - - m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 Brusick, D.J. a n d V.W. M a y e r , N e w d e v e l o p m e n t s in m u t a g e n i c i t y s c r e e n i n g t e c h n i q u e s w i t h y e a s t , Environ. Health Perspectives, 6 (1973) 83--96. 4 De Serres, F.J., T h e u t i l i t y o f s h o r t - t e r m t e s t s for m u t a g e n i c i t y in t h e t o x i c o l o g i c a l e v a l u a t i o n o f e n v i r o n m e n t a l a g e n t s , M u t a t i o n Res., 33 ( 1 9 7 5 ) 1 1 - - 1 5 . 5 D i c k e n s , F. and H . E . H . J o n e s , C a r c i n o g e n i c a c t i v i t y of a series of r e a c t i v e l a c t o n e s a n d r e l a t e d subs t a n c e s , Brit. J. C a n c e r , 15 ( 1 9 6 1 ) 8 5 - - 1 0 0 . 6 Engel, G. a n d K . E . 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G a r n e r a n d R.S, H a n s o n , F o r m a t i o n o f a f a c t o r l e t h a l for S. t y p h i m u r i u m T A 1 5 3 0 and T A 1 5 3 1 o n i n c u b a t i o n of a f l a t o x i n B I w i t h r a t liver m i c r o s o m e s , B i o c h e m . B i o p h y s . Res. C o m m u n . , 4 5 ( 1 9 7 1 ) 7 7 4 - - 7 8 0 . 10 G a r d n e r , R.C., E.C. Miller a n d J . A . Miller, L i v e r m i c r o s o m a l m e t a b o l i s m o f a f l a t o x i n B I t o a reactive d e r i v a t i v e t o x i c to S a l m o n e l l a t y p h i m u r i u r n T A 1 5 3 0 , C a n c e r R e s e a r c h , 32 ( 1 9 7 2 ) 2 0 5 8 - - 2 0 6 6 . 11 Hsieh, D . P . H . , A.S. S a l h a b , J.J. W o n g a n d S.L. Y a n g , T o x i c i t y of a f l a t o x i n Q1 as e v a l u a t e d w i t h the chicken e m b r y o and bacterial auxotrophs, Toxicol. Appl. Pharmacol., 30 (1974) 237--242. 12 H u r s t , E.W. a n d G.E. P a g e t , P r o t o p o r p h y r i n , cirrhosis a n d h e p a t o m a s in t h e livers of m i c e g i v e n griseofulvin, Brit. J. D e r m a t o l . , 75 ( 1 9 6 3 ) 1 0 5 - - 1 1 8 . 13 L e g a t o r , M.S., M u t a g e n i c e f f e c t s o f a f l a t o x i n , J. A m e r . V e t . Med. Assoc., 1 5 5 ( 1 9 6 9 ) 2 0 8 0 - - 2 0 8 3 . 14 M a y e r , V.W., I n d u c t i o n o f m i t o t i c c r o s s i n g - o v e r in S a c c h a r o m y c e s cerevisiae b y b r e a k d o w n p r o d u c t s of dimethyinitrosamine, diethylnitrosamine, 1-naphthylamine and 2-naphthylamine formed by an in v i t r o h y d r o x y l a t i o n s y s t e m , G e n e t i c s , 74 ( 1 9 7 3 ) 4 3 3 - - 4 4 2 . 15 M a y e r , V.W. a n d M.S. L e g a t o r , P r o d u c t i o n of p e t i t e m u t a n t s of S a c c h a r o m y c e s cerevisiae b y p a t u l i n , Agr. F o o d C h e m . , 17 ( 1 9 6 9 ) 4 5 4 - - 4 5 6 . 16 M c C a n n , J., E. Choi, E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s in t h e S a l m o n e l l a / m i c r o s o m e t e s t ; A s s a y o f 3 0 0 c h e m i c a l s , Proc. Natl. A c a d . Sci. (U.S.), 72 ( 1 9 7 5 ) 5 1 3 5 - 5139. 17 M c C a n n , J., a n d B.N. A m e s , D e t e c t i o n o f c a r c i n o g e n s at m u t a g e n s in t h e S a l m o n e U a / m i c r o s o m e t e s t ; Assay o f 3 0 0 c h e m i c a l s , P a r t II, Proc. Natl. A c a d . Sci. (U.S.), 73 ( 1 9 7 6 ) 9 5 0 - - 9 5 4 . 18 P u r c h a s e , I . F . H . a n d J.J. V a n d e r W a t t , C a r c i n o g e n i c i t y o f s t e r i g m a t o c y s t i n , Fd. C o s m e t . T o x i c o l . , 8 ( 1 9 7 0 ) 289. 19 Salhab, A.S. a n d D . P . H . H s i e h , A f l a t o x i n H l ; A m a j o r m e t a b o l i t e of a f l a t o x i n B 1 p r o d u c e d b y h u m a n a n d r h e s u s m o n k e y liver in v i t r o , Res. C o m m u n . C h e m . P a t h o l . P h a r m a c o l . , 10 ( 1 9 7 5 ) 4 1 9 - - 4 3 1 . 20 S c h o e n h a r d , G . L . , D.J. Lee, S.E. H o w e l l , N.E. P a w l o w s k i , L.M. L i b b e y a n d R.O. S i n n h u b e r , A f l a t o x i n B 1 m e t a b o l i s m to a f l a t o x i c o l a n d d e r i v a t i v e s l e t h a l t o Bacillus s u b t i l i s G S Y 1 0 5 7 b y r a i n b o w t r o u t ( S a l m o gairdneri) liver, C a n c e r R e s e a r c h , 36 ( 1 9 7 6 ) 2 0 4 0 - - 2 0 4 5 . 21 Slater, E . A . , M.D. A n d e r s o n a n d H.S. R o s e n k r a n z , R a p i d d e t e c t i o n of m u t a g e n s a n d c a r c i n o g e n s , C a n c e r R e s e a r c h , 31 ( 1 9 7 1 ) 9 7 0 - - 9 7 3 .
20 2 2 U e n o , Y o s h i o a n d K . K u b o t a , D N A - a t t a c h i n g a b i l i t y o f c a r c i n o g e n i c m y c o t o x i n s in r e c o m b i n a t i o n d e f i c i e n t m u t a n t cells o f Bacillus subtilis, C a n c e r R e s e a r c h , 3 6 ( 1 9 7 6 ) 4 4 5 - - 4 5 1 . 2 3 W i l s o n , B.J. a n d A.W. H a y e s , M i c r o b i a l t o x i n s , in: T o x i c a n t s o c c u r r i n g n a t u r a l l y in f o o d , F.M. S t r o n g (Ed.), Natl. Acad. of Sciences (1973), Washington, D.C. 2 4 W o g a n , G . N . , D i e t a r y f a c t o r s a n d special e p i d e m i o l o g i c a l s i t u a t i o n s o f liver c a n c e r in T h a i l a n d a n d Africa, Cancer Research, 35 (1975) 3499--3502. 25 Wong, J.J. and D.P.H. Hsieh, Mutagenicity of afiatoxins related to their metabolism and carcinogenic p o t e n t i a l , P r o c . N a t l . A c a d . Sci. (U.S.), 7 3 ( 1 9 7 6 ) 2 2 4 1 - - 2 2 4 4 . 26 Z i m r n e r m a n , F . K . , I n d u c t i o n o f m i t o t i c g e n e c o n v e r s i o n b y m u t a g e n s , M u t a t i o n Res., 11 ( 1 9 7 1 ) 327--337.
Mutation Research, 53 (1978) 11--20 © Elsevier/North-Holland Biomedical Press
EVALUATION OF THE MUTAGENIC POTENTIAL OF MYCOTOXINS USING SALMONELLA TYPHIMURIUM AND SACCHAROMYCES CEREVISIAE
M A U R E E N H. KUCZUK, PAUL M. BENSON, H A R R Y HEATH and A. WALLACE HAYES
Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Miss. 39216, and Department of Microbiology, The University of Alabama, Ala. 35486 (U.S.A.) (Received 12 May, 1977) (Accepted 4 July, 1977)
Summary The mutagenic effects of fifteen mycotoxins on Salmonella typhimurium strains TA1535, TA1537 and TA1538 and Saccharomyces cerevisiae strain D-3 were tested. Only aflatoxin B1 and sterigmatocystin were mutagenic. Both were active against S. typhimurium strain TA1538 and S. cerevisiae strain D-3; however, both required activation by the hepatic S-9 enzyme preparation. A positive correlation between the other mycotoxins reported to be carcinogenic and the two in vitro test systems employed was not demonstrated in our hands.
Introduction Standard (toxicological) techniques routinely used to identify carcinogenic chemicals are time-consuming and expensive. It also is often difficult to perform large-scale in vivo rodent studies to evaluate the carcinogenic potential of naturally occurring chemicals and/or their animal metabolites because of compound scarcity. A promising alternative using in vitro test systems for the analysis of the carcinogenic properties of a chemical has been proposed [1]. The desired goals of cost reduction and rapid evaluation as well as improved sensitivity, increased test population numbers and diversification in endpoints are achievable with these systems. It has been suggested that a chemical reaction with DNA is the basis of drug-
All correspondence and reprint request to: Dr. A. Wallace Hayes, D e p a r t m e n t of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi 39216 (U.S.A.).
12 induced carcinogenesis [ 21]. Since mutagenesis also involved cellular DNA, the argument for use of such mutagenic systems was strengthened by the demonstration that carcinogens or their "activated" intermediates were mutagenic for microorganisms [16]. It, therefore, should be possible to apply results obtained with microorganisms as screening systems to detect mutagens for mammals. On the assumption that carcinogenesis results from a derangement or alteration of DNA structure or its metabolism, a number of procedures involving microorganisms have been devised to detect mammalian carcinogens [1,3,20,21]. Ames et al. [2] have made a strong case for the generalization that with few exceptions carcinogens are mutagens, thus giving strength to the desirability of the Salmonella test as a rapid and economical system for screening purposes. Fungi produce a variety of toxic metabolites, mycotoxins [23] which are not only cytotoxic, b u t in the case of several have been reported to be carcinogenic to laboratory animals [5,12,18,24]. Furthermore, considerable evidence is accumulating to indicate that a large proportion of human cancer may be caused by exposure to toxic chemicals in the environment, including aflatoxin B1 [24]. Aflatoxin B1 and sterigmatocystin are examples of mycotoxins activated to frame-shift mutagens by liver microsomal preparations [9,10]. This paper presents mutagenicity data on 15 mycotoxins, five (aflatoxin B1, griseofulvin, patulin, penicillic acid and sterigmatocystin) reported to be carcinogenic and t w o (aflatoxin BI and rubratoxin B) reported mutagenic in animal models [5,7,12,13,18,24], using S. typhimurium and Saccharomyces cerevisiae as the test organisms. The studies were designed to determine the correlation between mutagenicity as estimated b y the t w o in vitro microbial test systems and the reported carcinogenicity. Materials and m e t h o d s Bacterial test system The Salmonella/microsome mutagenicity test has recently been reviewed [2]. S. t y p h i m u r i u m tester strains TA1535, which detects base-pair mutagens, and T A 1 5 3 7 and TA1538, which detect frame-shift mutagens, were obtained from B.N. Ames, University of California, Berkeley. In addition to a u x o t r o p h y for histidine, each strain carries a deletion originating in the galactose operon and extending into the uvr B locus. Consequently, each strain requires biotin and shows increased sensitivity to mutagens due to the absence of excision repair. Each strain also bears a defect in a gene for lipopolysaccharide synthesis (rfa) resulting in increased permeability to large molecules and the so-called deep rough phenotype. The relevant properties of the tester strains were confirmed as described b y Ames et al. [2]. The bacteria were stored and grown as outlined by Ames et al. [ 2 ]. Each m y c o t o x i n was tested for mutagenic activity directly and in the presence of the hepatic S-9 enzyme preparation in both qualitative (data not presented) and quantitative assays as described b y Ames et al. [2]. The hepatic S-9 enzyme fraction was prepared from Sprague--Dawley male rats pretreated with a saline solution of sodium hexobarbital (60 mg/kg, i.p.) 2 days before being sacrificed. Preliminary runs had determined the a m o u n t of S-9 equivalent
13 to 20 mg of liver tissue per plate to be o p t i m u m for mutagenesis at the dose level of 0.1 pg of aflatoxin B1 per plate. All mycotoxins were dissolved in dimethylsulfoxide (Burdick and Jackman, Muskegon, Mi.); no more than 0.1 ml of dimethylsulfoxide was added per plate, an a m o u n t have no appreciable effect on the mutagenic response of the systems. Revertants were scored using a Bactronic colony counter. All experiments were performed under subdued light conditions and repeated at least once. Negative controls for the spontaneous reversion of the tester strains to histidine p r o t o t r o p h y differed from experimental tests only by the absence of a mycotoxin. Positive controls for induced reversion of strains TA1535 and TA1537 employed N-methyl-N'-nitroN-nitrosoguanidine (NG) and quinacrine dihydrochloride (~C), respectively. Aflatoxin B~ was used as a positive control for induced reversion of strain TA 1538 and as a control to confirm the ability of our S-9 enzyme preparation to activate this m y c o t o x i n which is non-mutagenic in the direct assay.
Yeast test system Diploid strain D-3 of Saccharomyces cerevisiae, from J. Epler, Oak Ridge National Laboratory, Tenn., carries markers for dominant cycloheximide resistance (CYH 4), adenine a u x o t r o p h y (ade 2) and histidine a u x o t r o p h y (his 8) on one homologue of chromosome XV with the corresponding wild-type alleles on the other homologue [26]. H o m o z y g o u s ade 2 mutants accumulate a red pigment (sectors) which serves as a convenient means for detecting genetic damage resulting in homozygosity at the ade 2 locus. Mayer [14] has shown that the majority of the red sectors induced b y metabolites of dialkylnitrosamines and naphthylamines were due to mitotic recombination although a variety of mechanisms including mutagenesis, gene conversion, chromosomal deletion, aneuploidy and haploidization also could produce red sectors. The mechanism of red sector formation b y mycotoxins, however, was not investigated in the present study. Each m y c o t o x i n was tested directly and after activation by the hepatic S-9 enzyme preparation for genetic activity (induction of red colonies/sectors). For direct assay, 2.0 ml of reaction mixture containing 60 mM sodium phosphate buffer, pH 7.2, 6.25 X 106 cells/ml and 100 pg of m y c o t o x i n per ml were incubated for 3 h at 37°C. In the activated assays 1.5 ml of S-9 mix replaced the buffer and m y c o t o x i n concentrations were 50 pg/ml. Following incubation, cells were washed twice in 60 mM sodium phosphate buffer, pH 7.2, b y centrifugation and appropriately diluted for determining the frequency of red colonies/sectors and viable counts on YEPD agar (1.0% Bacto-yeast extract (Difco), 2.0% Bacto-peptone, 2.0% glucose and 1.5% Bacto-agar). After incubation for 3 days at 30°C, the plates were stored for 2 days at 5°C to enhance pigment accumulation. All experiments were repeated a minimum of t w o times. Negative controls for spontaneous frequency of red colonies and sectors differed from experimental assays only b y the absence of a m y c o t o x i n . Positive controls for detecting genetic damage to strain D-3 in the direct and S-9-activated assays employed ethyl methanesulfonate (EMS) and dimethylnitrosamine (DMN), respectively. Results were considered negative if the number of sectors per 10 s surviving cells for a test compared were less than one-third of the positive control.
TABLE
1
9 13 23
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
TA 1535 TA 1538
Diacetoxyscirpenol
A
A + B c
Griseo fulvin
Ochratoxin
Ochratoxin
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
TA 1535
Oosporein
Patulin
Penicillic acid
TA 1535 TA 1537 TA 1538
21 36 28
TA 1535 TA 1537 TA 1538
12
2 27 16
7 17 18
28 16 40
13 25
6 30 25
14
6 30 17
17 17 26
23 22 45
13 26
4 17 29
7 10 18
17 19 31
14
11 33 26
8 11 28
30 19 37
12 18
6 11 17
10 17 9
26 35 32
1
14
5 34 36
3 14 30
10 24 44
NT b NT
9 16 32
10 9 19
11 31 33
20
8 32 27
8 17 30
20 28 43
14 19
8 16 19
8 8 21
20 32 27
9
11 30 19
11 7 49
18 23 120
13 44
10 20 38
7 15 24
11 36 28
8
11 33 35
18 22 35
18 26 53
17 32
8 23 28
7 10 15
10 19 31
10
11
7 41 31
17 15 41
12 18 50
23 32
11 22 35
7 12 23
6 35 32
1
100
l0
100
DMSO
TYPHIMURIUM
Activated a Dg/plate (of test compound) 0.1
OF SALMONELLA
Non-activated pg/plate (of test compound)
Citrinin
STRAINS
per plate
IN THREE
Revertants
MYCOTOXINS
S. t y p h i murium strain
OF FIFTEEN
Mycotoxin
MUTAGENICITY
7
7 32 27
9 16 34
13 27 44
NT NT
11 22 34
11 12 21
7 31 23
0.1
9
7 34 42
7 12 33
16 19 45
20 27
7 22 33
7 12 15
9 32 27
DMSO
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
Verruculogen
Viriditoxin
Zearalenone
>1000 >1000 16
8 7 27
15 9 11
5 13 21
13 27 44
9 21 27
13 22 32
11 15 15
12 28
19 14 20
15 8 16
6 II 22
18 21 35
8 16 20
8 20 20
9 11 22
13 19
19 13 28
10 12 20
8 6 18
17 20 45
12 11 28
12 18 32
11 11 8
6 30
19 15 17
8 8 14
12 15 25
14 33 43
6 18 17
8 20 37
7 7 21
10 18
20 12 16
20 14 18
20 12 19
8 12 19
20 18 43
9 20 18
8 16 38
20 8 21
17 30
>1000 >1000 400---600
14 12 18
5 13 30
10 15 44
10 17 54
NT NT 310
6 24 29
12 9 24
4 12
i0 12 29
28 16 31
7 8 20
8 21 55
NT NT 280
12 27 18
10 7 27
16 43
a Activated with the hepatic S-9 e n z y m e preparation. b N T = not tested. c A mixture of ochratoxins A and B ( 8 5 % A and 1 5 % B). Also tested at 2 5 0 pg (98 revertants) and at 5 0 0 pg (118 revertants).
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
T-2 toxin
Controls MNNG QC Aflatoxin B 1
TA 1535 TA 1537 TA 1538
Sterigmatocystin
TA 1535 TA 1537 TA 1538
TA 1535 TA 1537 TA 1538
A
Rubratoxin
Penitrem
TA 1537 TA 1538
11 12 40
9 9 42
5 8 21
6 23 46
NT NT 160
6 30 26
10 9 30
23 34
10 14 23
11 19 29
10 15 13
9 20 43
NT NT 90
9 24 28
7 10 28
13 23
9 12 14
9 20 25
9 I0 28
7 I0 28
9 33 45
NT NT 14
7 20 38
9 12 15
12 33
16 o Ochratoxins A & B o Ochratoxin
A
~
o
120
Q"
80
o. c to ~c
R
D
300
50C)
40
0
100 per
plate
Fig. 1. A c o m p a r i s o n of t h e i n d u c t i o n of r e v e r t a n t s in S. t y p h i m u r i u m strain T A 1 5 3 8 b y o c h r a t o x i n A a n d a m i x t u r e o f o c h r a t o x i n s A a n d B.
Mycotoxins The following mycotoxins * were investigated: aflatoxin B1, citrinin, diacetoxyscirpenol, griseofulvin, ochratoxin A, an ochratoxin A and B mixture (85 and 15%, respectively), oosporein, patulin, penicillic acid, penitrem A, rubratoxin B, sterigmatocystin, T-2 toxin, verruculogen, viriditoxin and zearalenone. Except in the case of the mixture of ochratoxin A and B, the purity of each m y c o t o x i n was established b y melting point, thin-layer chromatography and infrared spectroscopy. All biochemicals, griseofulvin, NADP ÷, glucose 6-phosphate, histidine and biotin, were purchased from Sigma Chemicals, St. Louis, Mo. Results
All of the m y c o t o x i n s were negative in the qualitative Salmonella test (activated and non-activated) although a faint ring was observed with strain TA1538 in the presence of ochratoxins A + B mixture and the S-9 mix. A summary of the data obtained over the concentration range of 0.1 to 100 pg * M y c o t o x i n s w e r e o b t a i n e d f r o m B.J. Wilson, V a n d e r b i l t U n i v e r s i t y , Nashville, T e n n . , p e n i t r e m A ; N.D. Davis, A u b u r n U n i v e r s i t y , A u b u r n , Ala., citrinin a n d o c h r a t o x i n ; J.L. R i c h a r d , N A D L , A m e s , I o w a , T-2 t o x i n ; R.J. Cole, U S D A , D a w s o n , G e o r g i a , o o s p o r e i n a n d v e r r u c u l o g e n ; J.V. R o d r i c k s , F D A , W a s h i n g t o n , D.C., z e a x a l e n o n e a n d s t e r i g m a t o c y s t i n ; D.H. Wilson, p a t u l i n a n d penicillic acid.
17 of each m y c o t o x i n per plate with the three strains of S. t y p h i m u r i u m are presented in Table 1. In the non-activated system, the number of revertants per test plate did not differ from the number of revertants per control plate regardless of the m y c o t o x i n tested. In the activated system, the number of revertants per test plate did not differ from the controls except for aflatoxin B~, sterigmatocystin and the ochratoxins A + B mixture using strain TA1538. A comparison of the results obtained with strain TA1538 and different concentrations of the ochratoxins A and B mixture and an ochratoxin A sample purified by high pressure liquid chromatography are presented in Fig. 1. A dose response was apparent with the mixture whereas the number of revertants per plate in the presence of 500 pg of the purified preparation of ochratoxin A was only slightly elevated over control plates. A summary of data obtained after screening the m y c o t o x i n s in the presence of S. cerevisiae D-3 for potential genetic damage is presented in Table 2. The results in the non-activated system were negative indicating that the mycotoxins were not capable of causing mitotic crossing-over under these conditions. When the system was activated with the S-9 enzyme preparation, negative data were obtained except in the case of aflatoxin B1 and sterigmatocystin indicating that these two mycotoxins apparently were capable of causing mitotic recombination in the D-3 strain of S. cerevisiae when activated by the hepatic S-9 enzyme preparation. TABLE
2
MUTAGENICITY Mycotoxin
OF FIFTEEN
b
MYCOTOXINS
IN THE SACCHAROMYCES
Non-activated Freq. c
CEREVISIAE
D-3 TEST
Activated a Survival (%)
Freq.
Survival (%)
Aflatoxin B 1 Citrinin Diacetoxyscirpenol Griseo fulvin Ochratoxin A Oosporein Patulin Penicillic acid Penitrem A Rubratoxin B
3.1 3.4 2.0 12.0 3.8 1.7 17.6 7.1 5.8 5.7
82 85 69 71 76 84 24 81 84 75
610.4 6.6 5.4 3.8 10.1 5.2 2.6 1.3 7.3 1.3
99 93 92 100 99 96 99 96 103 96
Sterigmatocystin T-2 toxin Vertuculogen Virid ito xin Zearalenone
4.5 2.0 8.7 7.1 11.5
63 72 66 81 74
470.2 7.8 4.0 2.5 3.8
95 113 95 99 99
6.5 92.3 NT
100 93 NT
2.2 NT 76.9
100 NT 88
Controls Negative control EMS DMN
(minus toxin)
a A c t i v a t e d w i t h t h e h e p a t i c S-9 e n z y m e p r e p a r a t i o n . b 100 and 50 ~g/0.1 ml DMSO per plate for non-activated and activated, respectively. c F r e q u e n c y o f r e d c o l o n i e s a n d r e d s e c t o r s a p p e a r i n g i f a p o p u l a t i o n o f 105 y e a s t c o l o n i e s w e r e s c r e e n e d , V a l u e s are m e a n s o f t w o e x p e r i m e n t s .
18 Discussion None of the mycotoxins tested were mutagenic in the unactivated systems employing S. typhimurium strains TA1535, 1537 or 1539 or S. cerevisiae D-3. We have interpreted this to mean that the parent compounds were not mutagenic. However, when these compounds were activated with the hepatic S-9 enzyme preparation obtained from rats treated with hexobarbital, aflatoxin B1, sterigmatocystin and the mixture of ochratoxins A and B were mutagenic in the Ames' system and aflatoxin B, and sterigmatocystin were active in the yeast system. It is possible that the result obtained from the combination of ochratoxins A and B was due to ochratoxin B alone. Since the number of reverrant colonies per plate in the presence of ochratoxin A did not differ from control values, ochratoxin A may require metabolic activation to ochratoxin B for mutagenic activity along the lines proposed by McCann and Ames [17] for dehalogenation of certain chlorinated hydrocarbons. However, this does not seem likely since ochratoxin B is considerably less toxic than ochratoxin A. It also is possible that the increase in the number of revertant colonies per plate was caused by a synergistic effect of the two toxins or that a contaminant in the mixture was responsible for the observed effect. The results obtained using the D-3 yeast system confirmed the lack of mutagenicity for ochratoxin A. Our data support the concern about false negatives in light of the reported carcinogenicity of penicillic acid and patulin [5]. Furthermore, the negative results obtained in this study and by Ueno et al. [22] with the carcinogenic antifungal agent, griseofulvin, offers additional concern about the adequacy of such screening systems. Our failure to detect the mutagenicity of rubratoxin B in either microbial system contradicts the report of the mutagenic effect of rubratoxin B in mice [7,8]. As pointed out by De Serres [4], when an assay is developed for forward mutations which would detect any type of genetic alteration rather than reverse mutation which only detects particular types of genetic alterations, some of the problems with false negatives should be eliminated. Improvements in the sensitivity of tester strains also will help as was the case with two aflatoxin B, metabolites. Aflatoxicol H~ and aflatoxin Q~ were determined to be non-mutagenic using S. typhimurium TA1538 [11,19] but weakly mutagenic with strain TA098 [25]. Engel and Von Milczewski [6], however, were unable to detect an increase in the mutation rate of S. typhimurium TA98 or TA100 by either patulin or penicillic acid. Our studies, therefore, should be repeated with a more sensitive strain before the case on griseofulvin, patulin and penicillic acid is closed. Even though most of the mycotoxins were not mutagens, negative results cannot be considered conclusive at this time. According to our data, using the S. typhimurium tester strains TA1535, TA1537 and TA1538 and the S. cerevisiae D-3 strain, only aflatoxin B1 and sterigmatocystin were mutagenic; it is conceivable that griseofulvin, patulin and penicillic acid, reported mammalian carcinogens, would have been eliminated from additional toxicological testing based on these results. However, Ueno et al. [22] recently have reported that penicillic acid and patulin but not griseofulvin were positive in their recombination-deficient m u t a n t cells of Bacillus subtilis. Mayer and Legator [15] have reported the production of petite mutants in a haploid strain of S. cerevisiae
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