447
Mutation Research, 44 (1977) 447--450 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY OF F U N G A L METABOLITES R E L A T E D TO A F L A T O X I N BIOSYNTHESIS
JEFFREY J. WONG, RAJENDRA SINGH and DENNIS P.H. HSIEH
Department of Environmental Toxicology, University of California, Davis, Calif. 95616 (U.S.A.) (Received December 28th, 1976) (Revision received March 3rd, 1977) (Accepted April 4th, 1977)
Summary Fungal metabolites identified as the intermediates in aflatoxin biosynthetic pathway were screened for their mutagenic activity to Salmonella typhimurium TA98. Norsolorinic acid, averufin, and versiconal acetate were found to possess questionable mutagenic activity, b u t versicolorin A, and sterigmatocystin were significant mutagens relative to aflatoxin BI. 'The mutagenic activity appears to be related to the bisfuran and n o t the anthraquinone moiety of the molecule, even though the latter is a key structure of such p o t e n t carcinogenic m y c o t o x i n as luteoskyrin.
Introduction Aflatoxins, a major group of f o o d b o r n e carcinogenic mycotoxins are produced by the fungi Aspergillus flavus and A. parasiticus. During our studies on aflatoxin biosynthesis, a number o f metabolites were isolated and identified as the intermediates in the main pathway of aflatoxin biosynthesis [5,8,12)16]. These are: norsolorinic acid, averufin, versiconal acetate, versicolorin A, and sterigmatocystin. Since these c o m p o u n d s are the biogenetic precursors of aflatoxin B,, one would expect them to occur concomitantly with aflatoxins and to be ingested along with aflatoxins by the animals feeding on contaminated feeds. Also it is possible that some genetic variants of the aflatoxigenic strains may occur in nature which would accumulate one or another of these metabolites as the major endproduct. Therefore, they should be considered as potential mycotoxins, and it is important to evaluate their toxicity, particularly their carcinogenic potential. The Salmonella typhimurium based mutagen assay developed by Dr. Bruce Ames has found a widespread use in the detection of most known animal car-
448 cinogens as bacterial mutagens [1,2,9,10,11]. A good correlation between the relative in vitro mutagenic activity of aflatoxin animal metabolites and their in vivo carcinogenicity has been demonstrated using the sensitive frame-shift tester strain S. typhimurium TA98 [15]. In this communication, we report the mutagenic activity of various aflatoxin biosynthetic intermediates assayed with the same tester strain, using the procedure developed for aflatoxins. Materials and methods
Aflatoxin and its biosynthetic intermediates Aflatoxin B~ and sterigmatocystin were purchased from Makor Chemicals, Ltd., Jerusalem, Israel. Norsolorinic acid [6], averufin [3], and versicolorin A [7] were synthesized by using the respective mutant strains of A. parasiticus. Versiconal acetate was synthesized by treating the wild type A. parasiticus cultures with dichlorvos (2,2-dichlorovinyl dimethyl phosphate) as outlined by Yao and Hsieh [16]. Norsolorinic acid from the crude fungal extract was purified as described by Hsieh et al. [5]. Other intermediates were purified by thin-layer chromatography as described previously [ 12]. Biochemicals and reagents All the chemicals used were of analytical grade and solvents were double distilled. Biochemicals: NADP, glucose-6-phosphate, histidine, and biotin, were purchased from Sigma Chemicals, St. Louis, Mo. Mu tagenicity testing S. typhimurium strain TA98, a gift from Dr. Bruce Ames of the University of California, Berkeley, was used as the bacterial indicator. The bacterial cultures were maintained and grown as outlined by McCann et al. [9]. Mutagen assay procedures, detailed by Ames et al. [1,2] and modified by Wong and Hsieh [15] for use with aflatoxins, were utilized to test the mutagenic activity of aflatoxin B1 and its biosynthetic intermediates. Results and discussion The relative mutagenicities of aflatoxin B~ and its known biosynthetic intermediates are summarized in Table I. The compounds are listed in the order of their sequence in the biosynthetic pathway, which also coincides with their increased relative mutagenic potency, with aflatoxin B1, t h e end product of this pathway, having the highest potency. Norsolorinic acid, averufin, and versiconal acetate exhibited questionable mutagenic activity. The notable feature in these three compounds is that they all contain an anthraquinone moiety. Verisoclorin A, another anthraquinone, exhibited significant mutagenic activity, indicating that the activity is due to its bisfuran ring structure. Sterigmatocystin, which consists of a xanthone moiety in place of anthraquinone, was twice as mutagenic as versicolorin A. Aflatoxin B~, in which the xanthone is transformed into a coumarin moiety, exhibited about 10 times as much activity as sterigmatocystin. It is thus evident that although the bisfuran moiety is essential for mutagenic activity, it is not the sole determinant of the activity.
449 TABLE 1 M U T A G E N I C I T Y OF A F L A T O X I N B 1 AND ITS B I O S Y N T H E T I C I N T E R M E D I A T E S Compound
D o s e range ( # g / p l a t e )
Revertants a/#g
Relative mutagenic p o t e n c y (%)
ON0 OH H 3 C ~ O H O Norsolorfnic Acid OH
0.0--8.0
31 -+
a
0.18
33 -+
3
0.19
37 +
5
0.21
0.0---0.8
9 9 5 -+ 3 1 0
5.83
0.0--0.1
1 , 8 2 0 -+ 4 9 0
10.66
0.0--0.1
17,066 + 1337
100.00
O OH
H 3 C ~ ~ o
OH
0.0--4.0
Averufin o
H3C-C-O 0 OH H O C ~ ~ o H OH O Versiconal Acetate
0.0--4.0
OH O OH o
Versicolorin A
oiH 3
Ster igmatocystin 0 O
Aflatoxin {31 a M e a n r e v e r t a n t value o f at least three separate e x p e r i m e n t s . Each value d e t e r m i n e d f r o m a d o s e - r e s p o n s e curve c o n s i s t i n g o f five data p o i n t s , w i t h triplicate p l a t e s per p o i n t . T h e b a c k g r o u n d r e v e r t a n t value ranged f r o m 2 7 t o 4 0 revertants.
The configuration and electronic structure of the entire molecule are also important factors. This observation is consistent with that of the mutagenicity of animal metabolites of aflatoxin BI, where changes in the molecular configuration of either the bisfuran or the coumarin moiety invariably resulted in reduction of mutagenic activity of aflatoxin B] [15]. It has been proposed that the mutagenically active form of aflatoxin B~ is generated by the epoxidation of the 2,3-vinyl ether double bound in the terminal furan ring [4,13,14]. Since both versicolorin A and sterigmatocystin possess this ring system, a common mechanism concerning their mutagenicity and implied carcinogenicity can be reasonably presumed. Both compounds have exhibited DNA targeted bactericidal activity upon microsomal activation [4], an observation serving to confirm our data. Sterigmatocystin, a known carcinogen, has been previously reported to be less mutagenic in S. typhimurium mutagen assays relative to aflatoxin B1 [1,10]. The simultaneous absence of the
450 unsaturated bisfuran structure and significant mutagenic activity, in norsolorinic acid, averufin, and versiconal acetate, further supports the requirement of this structure in mutagenicity, and presumably carcinogenicity of the aflatoxins and related compounds.
Acknowledgements The authors thank Mark Fukayama and Donald Fitzell for their technical assistance. This investigation was supported in part by USPHS Grants ES 00612 and ES 00814 and Western Regional Research Project W-122.
References 1 A m e s , B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d F.D. Lee, C a r c i n o g e n s are m u t a g e n s : a s i m p l e test s y s t e m c o m b i n i n g liver h o m o g e n a t e s for a c t i v a t i o n a n d b a c t e r i a f o r d e t e c t i o n , Proc. Natl. A c a d . Sci., 70 (1973) 2281--2285. 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 for d e t e c t i n g 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 Salmonella/Mammalian-Microsome M u t a g e n i c i t y T e s t , Mut. Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 D o n k e r s l o o t , J . A . , R.I. M a t e l e s a n d S.S. Y a n g , I s o l a t i o n of a v e r u f i n f r o m a m u t a n t o f Aspergillus parasiticus i m p a i r e d in a f l a t o x i n b i o s y n t h e s i s , B i o c h e m . Biophys. Res. C o m m . , 47 ( 1 9 7 2 ) 1 0 5 1 - 1055. 4 G a r n e r , R.C., T h e role of c p o x i d e s in b i o a c t i v a t i o n a n d c a r c i n o g e n e s i s , in J.W. Bridges a n d L.F. Chass e a u d (eds.), Progress in D r u g M e t a b o l i s m , J o h n Wiley a n d Sons, L o n d o n , N e w Y o r k , S y d n e y a n d T o r o n t o , 1 9 7 6 , pp. 7 7 - - 1 2 8 . 5 Hsieh, D.P.H., M.T. Lin, R.C. Y a o a n d R. Singh, B i o s y n t h e s i s o f a f l a t o x i n : c o n v e r s i o n of n o r s o l o r i n i c acid and o t h e r h y p o t h e t i c a l i n t e r m e d i a t e s into a f l a t o x i n B1, J. Agr. F o o d C h e m . , 24 ( 1 9 7 6 ) 1 1 7 0 - 1174. 6 Lee, L.S., J.W. B e n n e t t , L.A. G o l d b l a t t a n d R.E. L u n d i n , N o r s o l o r i n i e acid f r o m a m u t a n t strain of AspergiUus parasiticus, J. A m e r . Oil C h e m . Soc., 48 ( 1 9 7 1 ) 9 3 - - 9 4 . 7 Lee, L.S., J.W. B e n n e t t , A.F. C u c u l l u a n d J.B. S t a n l e y , S y n t h e s i s of v e r s i c o l o r i n A b y a m u t a n t strain of Aspergillus parasiticus d e f i c i e n t in a f l a t o x i n p r o d u c t i o n , J. Agr. F o o d C h e m . , 23 ( 1 9 7 5 ) 1 1 3 2 - 1134. 8 Lin, M.T., D.P.H. Hsieh, R.C. Y a o a n d J . A . D o n k e r s l o o t , C o n v e r s i o n of a v e r u f i n into a f l a t o x i n s b y Apergillus parasiticus, B i o c h e m i s t r y , 12 ( 1 9 7 3 ) 5 1 6 7 - - 5 1 7 1 . 9 M c C a n n , J., N.E. S p i n g a r n , J. K o b o r 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 : b a c t e r i a l t e s t e r strains w i t h R f a c t o r p l a s m i d s , Proc. Natl. A c a d . Sci., 72 ( 1 9 7 5 ) 9 7 9 - - 9 8 3 . 10 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 o f c a r c i n o g e n s as m u t a g e n s in t h e Salmonella/Microsome Test: assay of 3 0 0 c h e m i c a l s , Proc. Natl. A c a d . Sci., 72 ( 1 9 7 6 ) 5 1 3 5 - - 5 1 3 9 . 11 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 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 c Test: assay of 3 0 0 c h e m i c a l s (Discussion), Proc. Natl. A c a d . Sci., 73 ( 1 9 7 6 ) 9 5 0 - - 9 5 4 . 12 Singh, R. a n d D.P.H. Hsieh, A f l a t o x i n b i o s y n t h e t i c p a t h w a y : e l u c i d a t i o n b y using b l o c k e d m u t a n t s of Aspergillus parasiticus, A r c h . B i o c h e m . B i o p h y s . , 1 7 8 ( 1 9 7 7 ) 2 8 5 - - 2 9 2 . 13 S w e n s o n , D.H., J . A . Miller a n d E.C. Miller, T h e r e a c t i v i t y a n d c a r c i n o g e n i c i t y o f a f l a t o x i n B1-2',3d i c h l o r i d e , a m o d e l for t h e p u t a t i v e 2 , 3 - o x i d e m e t a b o l i t e o f a l f a t o x i n B 1, C a n c e r Res., 35 ( 1 9 7 5 ) 3811--3823. 14 S w e n s o n , D.H., J.K. Lin, E.C. Miller a n d J . A . Miller, A f l a t o x i n B i - 2 ' , 3 - o x i d e as a p r o b a b l e interm e d i a t e in t h e c o v a l e n t b i n d i n g of a f l a t o x i n B I a n d B 2 to rat liver D N A a n d r i b o s o m a l R N A in vitro, C a n c e r Res., 37 ( 1 9 7 7 ) 1 7 2 - - 1 8 1 . 15 W o n g , J.J. a n d D.P.H. Hsieh, M u t a g e n i c i t y o f a f l a t o x i n s r e l a t e d to t h e i r m e t a b o l i s m a n d c a r c i n o g e n i c p o t e n t i a l , Proc. Natl. A c a d . Sci., 73 ( 1 9 7 6 ) 2 2 4 1 - - 2 2 4 4 . 16 Y a o , R.C. a n d D.P.H. Hsieh, Step of d i e h l o r v o s i n h i b i t i o n in t h e p a t h w a y of a f l a t o x i n b i o s y n t h e s i s , A p p l . Microbiol., 28 ( 1 9 7 4 ) 5 2 - - 5 7 .
Mutation Research, 44 (1977) 447--450 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY OF F U N G A L METABOLITES R E L A T E D TO A F L A T O X I N BIOSYNTHESIS
JEFFREY J. WONG, RAJENDRA SINGH and DENNIS P.H. HSIEH
Department of Environmental Toxicology, University of California, Davis, Calif. 95616 (U.S.A.) (Received December 28th, 1976) (Revision received March 3rd, 1977) (Accepted April 4th, 1977)
Summary Fungal metabolites identified as the intermediates in aflatoxin biosynthetic pathway were screened for their mutagenic activity to Salmonella typhimurium TA98. Norsolorinic acid, averufin, and versiconal acetate were found to possess questionable mutagenic activity, b u t versicolorin A, and sterigmatocystin were significant mutagens relative to aflatoxin BI. 'The mutagenic activity appears to be related to the bisfuran and n o t the anthraquinone moiety of the molecule, even though the latter is a key structure of such p o t e n t carcinogenic m y c o t o x i n as luteoskyrin.
Introduction Aflatoxins, a major group of f o o d b o r n e carcinogenic mycotoxins are produced by the fungi Aspergillus flavus and A. parasiticus. During our studies on aflatoxin biosynthesis, a number o f metabolites were isolated and identified as the intermediates in the main pathway of aflatoxin biosynthesis [5,8,12)16]. These are: norsolorinic acid, averufin, versiconal acetate, versicolorin A, and sterigmatocystin. Since these c o m p o u n d s are the biogenetic precursors of aflatoxin B,, one would expect them to occur concomitantly with aflatoxins and to be ingested along with aflatoxins by the animals feeding on contaminated feeds. Also it is possible that some genetic variants of the aflatoxigenic strains may occur in nature which would accumulate one or another of these metabolites as the major endproduct. Therefore, they should be considered as potential mycotoxins, and it is important to evaluate their toxicity, particularly their carcinogenic potential. The Salmonella typhimurium based mutagen assay developed by Dr. Bruce Ames has found a widespread use in the detection of most known animal car-
448 cinogens as bacterial mutagens [1,2,9,10,11]. A good correlation between the relative in vitro mutagenic activity of aflatoxin animal metabolites and their in vivo carcinogenicity has been demonstrated using the sensitive frame-shift tester strain S. typhimurium TA98 [15]. In this communication, we report the mutagenic activity of various aflatoxin biosynthetic intermediates assayed with the same tester strain, using the procedure developed for aflatoxins. Materials and methods
Aflatoxin and its biosynthetic intermediates Aflatoxin B~ and sterigmatocystin were purchased from Makor Chemicals, Ltd., Jerusalem, Israel. Norsolorinic acid [6], averufin [3], and versicolorin A [7] were synthesized by using the respective mutant strains of A. parasiticus. Versiconal acetate was synthesized by treating the wild type A. parasiticus cultures with dichlorvos (2,2-dichlorovinyl dimethyl phosphate) as outlined by Yao and Hsieh [16]. Norsolorinic acid from the crude fungal extract was purified as described by Hsieh et al. [5]. Other intermediates were purified by thin-layer chromatography as described previously [ 12]. Biochemicals and reagents All the chemicals used were of analytical grade and solvents were double distilled. Biochemicals: NADP, glucose-6-phosphate, histidine, and biotin, were purchased from Sigma Chemicals, St. Louis, Mo. Mu tagenicity testing S. typhimurium strain TA98, a gift from Dr. Bruce Ames of the University of California, Berkeley, was used as the bacterial indicator. The bacterial cultures were maintained and grown as outlined by McCann et al. [9]. Mutagen assay procedures, detailed by Ames et al. [1,2] and modified by Wong and Hsieh [15] for use with aflatoxins, were utilized to test the mutagenic activity of aflatoxin B1 and its biosynthetic intermediates. Results and discussion The relative mutagenicities of aflatoxin B~ and its known biosynthetic intermediates are summarized in Table I. The compounds are listed in the order of their sequence in the biosynthetic pathway, which also coincides with their increased relative mutagenic potency, with aflatoxin B1, t h e end product of this pathway, having the highest potency. Norsolorinic acid, averufin, and versiconal acetate exhibited questionable mutagenic activity. The notable feature in these three compounds is that they all contain an anthraquinone moiety. Verisoclorin A, another anthraquinone, exhibited significant mutagenic activity, indicating that the activity is due to its bisfuran ring structure. Sterigmatocystin, which consists of a xanthone moiety in place of anthraquinone, was twice as mutagenic as versicolorin A. Aflatoxin B~, in which the xanthone is transformed into a coumarin moiety, exhibited about 10 times as much activity as sterigmatocystin. It is thus evident that although the bisfuran moiety is essential for mutagenic activity, it is not the sole determinant of the activity.
449 TABLE 1 M U T A G E N I C I T Y OF A F L A T O X I N B 1 AND ITS B I O S Y N T H E T I C I N T E R M E D I A T E S Compound
D o s e range ( # g / p l a t e )
Revertants a/#g
Relative mutagenic p o t e n c y (%)
ON0 OH H 3 C ~ O H O Norsolorfnic Acid OH
0.0--8.0
31 -+
a
0.18
33 -+
3
0.19
37 +
5
0.21
0.0---0.8
9 9 5 -+ 3 1 0
5.83
0.0--0.1
1 , 8 2 0 -+ 4 9 0
10.66
0.0--0.1
17,066 + 1337
100.00
O OH
H 3 C ~ ~ o
OH
0.0--4.0
Averufin o
H3C-C-O 0 OH H O C ~ ~ o H OH O Versiconal Acetate
0.0--4.0
OH O OH o
Versicolorin A
oiH 3
Ster igmatocystin 0 O
Aflatoxin {31 a M e a n r e v e r t a n t value o f at least three separate e x p e r i m e n t s . Each value d e t e r m i n e d f r o m a d o s e - r e s p o n s e curve c o n s i s t i n g o f five data p o i n t s , w i t h triplicate p l a t e s per p o i n t . T h e b a c k g r o u n d r e v e r t a n t value ranged f r o m 2 7 t o 4 0 revertants.
The configuration and electronic structure of the entire molecule are also important factors. This observation is consistent with that of the mutagenicity of animal metabolites of aflatoxin BI, where changes in the molecular configuration of either the bisfuran or the coumarin moiety invariably resulted in reduction of mutagenic activity of aflatoxin B] [15]. It has been proposed that the mutagenically active form of aflatoxin B~ is generated by the epoxidation of the 2,3-vinyl ether double bound in the terminal furan ring [4,13,14]. Since both versicolorin A and sterigmatocystin possess this ring system, a common mechanism concerning their mutagenicity and implied carcinogenicity can be reasonably presumed. Both compounds have exhibited DNA targeted bactericidal activity upon microsomal activation [4], an observation serving to confirm our data. Sterigmatocystin, a known carcinogen, has been previously reported to be less mutagenic in S. typhimurium mutagen assays relative to aflatoxin B1 [1,10]. The simultaneous absence of the
450 unsaturated bisfuran structure and significant mutagenic activity, in norsolorinic acid, averufin, and versiconal acetate, further supports the requirement of this structure in mutagenicity, and presumably carcinogenicity of the aflatoxins and related compounds.
Acknowledgements The authors thank Mark Fukayama and Donald Fitzell for their technical assistance. This investigation was supported in part by USPHS Grants ES 00612 and ES 00814 and Western Regional Research Project W-122.
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