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Benomyl — A novel type of base analogue mutagen?
379
Mutation Research, 40 (1976) 379--382 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication BENOMYL -- A NOVEL TYPE OF BASE ANALOGUE MUTAGEN?
A. KAPPAS, M.H.L. GREEN, B.A. BRIDGES, A.M. ROGERS and W.J. MURIEL
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BN1 9QG (England) (Received May 4th, 1976) (Accepted June 15th, 1976)
Benomyl (methyl-l-(butylcarbamoyl)-2-benzimidazole carbamate) is a benzimidazole derivative widely used as a systemic fungicide which has been found greatly to increase somatic segregation in Aspergillus nidulans heterozygous diploid strains [7,9]. No induction of point mutations was detected in A. nidulans [7] b u t both benomyl and its breakdown product methyl-2-benzimidazole carbamate have been shown to induce low levels of base pair substitution mutations using a Salmonella typhimurium spot test [12]. The mutagenicity of benzimadazole and its derivatives has recently been reviewed by Seiler [13] who concludes that they may be incorporated into DNA as base analogues. We now report that benomyl mutates enterobacteria by means of the lex+-dependent misrepair pathway and that the premutagenic lesion is susceptible to excision-repair. It is suggested that benomyl may be a new t y p e of base analogue mutagen that, when incorporated into DNA, is detected by UV endonuclease and provokes mutations not by misincorporation during normal DNA replication, b u t by misrepair of gaps in newly-synthesized DNA. The bacterial strains used were the tryptophan-requiring Escherichia coli WP2 and its derivatives WP2 uvrA (8) and CM611 (uvrA lexA) (3,5) and the histidine requiring S. typhimurium TA1535 uvrB and TA1538 uvrB (1). The WP2 strains and TA1535 respond to base-pair mutagens and TA 1538 to frameshift mutagens. Because of the anticipated low yields of induced mutants to prototrophy, we used a simplified fluctuation test with high
~ - NHCOOCH3 /
(~ONHC/.,H9
Fig. 1. Structure of benomyL A. KaPpas was o n leave Fellowship.
by a NATO
f r o m the N u c l e a r R e s e a r c h C e n t e r " D e m o k r i t o s " , A t h e n s , G r e e c e , and s u p p o r t e d
380
sensitivity [6]. For T A 1 5 3 5 this was modified by the substitution of biotin (0.4 pg/ml) and L-histidine (0.25/~g/ml) for tryptophan. Benomyl was used in the form of Benlate (du Pont) which contains about 50% active ingredient. Concentrations given are corrected. Similar results were obtained in a limited number of experiments with pure benomyl (du Pont). The chemical was added in the form of a solution in dimethyl sulphoxide. An appropriate quantity of the latter was added to control tubes. The results are shown in Table I. Significant mutagenesis was observed in WP2 uvrA and T A 1 5 3 5 , both excision-deficient strains that respond to basepair substitution mutagens, but not in the frameshift mutagen detector TA1538, confirming previous reports [13].
TABLE 1 MUTAGENICITY OF BENOMYL TOWARDS SIMPLIFIED FLUCTUATION TEST [6]
E. C O L I
A N D S. T Y P H I M U R I U M
Benomy] concentration /zg/ml
Number of experiments
Average n u m b e r of tubes positive
0 0.125 0.25 0.5 1.0 2.5 5.0 10.0
5 3 5 3 5 3 3 3
4.0 10.0 10.2 11.0 11.2 7.3 7.0 6.0
0 0.125 0.250 1.0 5.0 10.0 15.0
5 5 5 5 5 5 2
9.0 9.0 8.8 8.8 10.0 8.2 8.5
0 0.125 0.25 0.5 1.0 5.0
5 5 5 5 5 5
6.0 7.8 4.8 4.6 6.6 3.8
Salmonella lyphimurium TA1535 0 0.125 0.25 0.5 1.0 5.0
3 3 3 3 2 2
2.3 5.7 6.0 5.7 6.5 4.0
2 2 2 2 2 2
2.5 1.0 1.5 2.0 2.0 1.0
Eschcrichia coli WP2 uvrA
WP2
CM611 uvrA e x r A
TA1538
0 0.125 0.25 0.5 1.0 5.0
STRAINS USING A
<0.01 <0.001 <0.001 <0.001
<0.05 <0.05 <0.05 <0.05
381
No mutagenesis was observed in the excision-proficient strain WP2 indicating that a DNA lesion is involved in the mutagenic action of b e n o m y l that is detectable and excisable by UV endonuclease. Similarly, the negative result in the misrepair and excision-deficient CM611 shows that the lexA÷-dependent misrepair pathway is necessary for benomyl mutagenesis. These properties have not previously been found for mutation induction by base analogues in bacteria. Suggestions of similar properties of 5-bromodeoxyuridine based on work with bacteriophage ~ [10] have not been confirmed in E.coli [16]. These properties are, however, n o t inconsistent with the chemical structure of benomyl. Although the butylcarbamoyl group is removed the carbamate group at the C-8 position would be expected to remain on the molecule when incorporated. It might then appear similar to a purine bearing a large alkylation group. UV endonuclease is known to act in such cases [14]. The benzene ring differs from the purine ring in being unable to pair with other bases by hydrogen bonding [13]. In this respect it is similar to a dimerized pyrimidine and distinct from 5bromouracil or 2-aminopurine. Pyrimidine dimers are known to cause gaps in newly-synthesized DNA, [11] and a specific proportion of these [4] lead to mutations via the lexA÷-dependent error-prone repair pathway [2,15]. An important feature of the mutagenic action of b e n o m y l is the lack of a dose response (see Table I). From 0.125 to 1 pg/ml benomyl shows an essentially constant number of induced mutants; at higher doses there is a decline which may be partly attributable to toxic effects (unpublished data). Although such a response is completely uncharacteristic of alkylating agents, it is perhaps explicable for benomyl in the light of its requirement for incorporation into DNA. One needs only to postulate a saturation at low concentrations of an enzyme involved in incorporation, and an inhibition at high concentrations. If our model for the action of benomyl is correct, however, an entire replication cycle would normally intervene between incorporation and replication of the incorporated analogue. This could, at least in principle, permit complete excision to occur and result in total non-mutagenicity in Uvr ÷ strains. The reported non-mutagenicity in A. nidulans (7) could be explained in this way. In conclusion, we believe that the action of benomyl may be understood if it is seen as a novel type of mutagen needing to be incorporated into DNA b u t once incorporated, seen by the cell as a non-pairing purine with a large alkyl or aryl group attached. References 1 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 w i t h the 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 . 2 B r i d g e s , B . A . , R . E . D e n n i s a n d R . J . M u n s o n , D i f f e r e n t i a l i n d u c t i o n and repair o f u l t r a v i o l e t d a m a g e l e a d i n g t o t r u e reversions and e x t e r n a l suppressor m u t a t i o n s o f an o c h r e c o d o n i n Escherichia coli B / r W P 2 , G e n e t i c s , 57 ( 1 9 6 7 ) 8 9 7 - - 9 0 8 . 3 B r i d g e s , B . A . , R . P . M o t t e r s h e a d , M . A . R o t h w e l l a n d M . H . L . G r e e n , R e p a i r - d e f i c i e n t bacterial strains suitable for m u t a g e n i c i t y screening: t e s t s w i t h t h e f u n g i c i d e c a p t a n , C h e m . Biol. I n t e r a c t i o n s 5 (1972) 77--84. 4 D o u b l e d a y , O . P . , B . A . Bridges and M . H . L . G r e e n , M u t a g e n i c D N A r e p a i r in Escherichia coll. II. F a c t o r s a f f e c t i n g loss o f p h o t o r e v e r s i b i l i t y o f U V - i n d u c e d m u t a t i o n s , M o l e c . G e n . G e n e t . , 1 4 0 ( 1 9 7 5 ) 221--230. 5 G r e e n , M . H . L . a n d W . J . M u r i e l , M u t a t i o n t e s t i n g using Trp ÷ reversion in Escherichia coli, M u t a t i o n Res., 38 (1976) 3--32.
382 6 G r e e n , M . H . L . , W . J . M u r i e l a n d B . A . B r i d g e s , Use o f a s i m p l i f i e d f l u c t u a t i o n t e s t t o d e t e c t l o w levels of mutagens, Mutation Res. 38 (1976) 33--42. 7 Hastie, A.C., B e n l a t e - i n d u c e d i n s t a b i l i t y o f A s p e r g i l l u s d i p l o i d s , N a t u r e 2 2 6 ( 1 9 7 0 ) 7 7 1 . 8 Hill, R . F . , U l t r a v i o l e t - i n d u c e d l e t h a l i t y a n d r e v e r s i o n t o p r o t o t r o p h y in E s c h e r i c h i a coli s t r a i n s w i t h normal and reduced dark repair ability, Photochem. Photobiol. 4 (1965) 563--568. 9 K a p p a s , A., S . G . G e o r g o p o u l o s a n d A . C . H a s t i e , O n t h e g e n e t i c a c t i v i t y o f b e n z i m i d a z o l e a n d t h i o p h a n a t e f u n g i c i d e s o n d i p l o i d A s p e r g i l l u s n i d u l a n s , M u t a t i o n Res., 2 6 ( 1 9 7 4 ) 1 7 - - 2 7 . 1 0 P i e t r z y k o w s k a , I., O n t h e m e c h a n i s m o f b r o m o u r a c i l i n d u c e d m u t a g e n e s i s ~ M u t a t i o n R e s . , 19 ( 1 9 7 3 ) 1--9. 11 R u p p , W . D . a n d P. H o w a r d - F l a n d e r s , D i s c o n t i n u i t i e s in t h e D N A s y n t h e s i z e d in a n e x c i s i o n - d e f e c t i v e s t r a i n o f E s c h e r i c h i a coli f o l l o w i n g u l t r a v i o l e t i r r a d i a t i o n , J. M o l . Biol., 31 ( 1 9 6 8 ) 2 9 1 - - 3 0 4 . 1 2 Seiler, J . P . , T h e m u t a g e n i c i t y o f b e n z i m i d a z o l e a n d b e n z i m i d a z o l e d e r i v a t i v e s . I. F o r w a r d a n d reverse m u t a t i o n s in S a l m o n e l l a t y p h i m u r i u m c a u s e d b y b e n z i m i d a z o l e a n d s o m e o f its d e r i v a t i v e s , M u t a t i o n Res. 15 (1972) 273--276. 1 3 Seiler, J . P . , T o x i c o l o g y a n d g e n e t i c e f f e c t s o f b e n z i m i d a z o l e c o m p o u n d s , M u t a t i o n R e s . 3 2 ( 1 9 7 5 ) 161--168. 1 4 T a r m y , E . M . , S. V e n i t t a n d P. B r o o k e s , M u t a g e n i c i t y o f t h e c a r c i n o g e n 7 - b r o r n o m e t h y l b e n z (a) a n t h r a c e n e ; a q u a n t i t a t i v e s t u d y in r e p a i r - d e f i c i e n t s t r a i n s o f E s c h e r i c h i a coli, M u t a t i o n R e s . , 19 (1973) 153--166. 1 5 W i t k i n , E . M . , M u t a t i o n - p r o o f a n d m u t a t i o n - p r o n e m o d e s o f survival in d e r i v a t i v e s o f E s c h e r i c h i a coli B d i f f e r i n g in s e n s i t i v i t y t o u l t r a v i o l e t l i g h t , B r o o k h a v e n S y m p . Biol., 2 0 ( 1 9 6 7 ) 1 7 - - 5 5 . 1 6 W i t k i n , E . M . a n d E . C . Parisi, B r o m o u r a c i l m u t a g e n e s i s : m i s p a i r i n g o r m i s r e p a i r ? M u t a t i o n R e s . , 2 5 (1974) 407--409.
Mutation Research, 40 (1976) 379--382 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication BENOMYL -- A NOVEL TYPE OF BASE ANALOGUE MUTAGEN?
A. KAPPAS, M.H.L. GREEN, B.A. BRIDGES, A.M. ROGERS and W.J. MURIEL
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BN1 9QG (England) (Received May 4th, 1976) (Accepted June 15th, 1976)
Benomyl (methyl-l-(butylcarbamoyl)-2-benzimidazole carbamate) is a benzimidazole derivative widely used as a systemic fungicide which has been found greatly to increase somatic segregation in Aspergillus nidulans heterozygous diploid strains [7,9]. No induction of point mutations was detected in A. nidulans [7] b u t both benomyl and its breakdown product methyl-2-benzimidazole carbamate have been shown to induce low levels of base pair substitution mutations using a Salmonella typhimurium spot test [12]. The mutagenicity of benzimadazole and its derivatives has recently been reviewed by Seiler [13] who concludes that they may be incorporated into DNA as base analogues. We now report that benomyl mutates enterobacteria by means of the lex+-dependent misrepair pathway and that the premutagenic lesion is susceptible to excision-repair. It is suggested that benomyl may be a new t y p e of base analogue mutagen that, when incorporated into DNA, is detected by UV endonuclease and provokes mutations not by misincorporation during normal DNA replication, b u t by misrepair of gaps in newly-synthesized DNA. The bacterial strains used were the tryptophan-requiring Escherichia coli WP2 and its derivatives WP2 uvrA (8) and CM611 (uvrA lexA) (3,5) and the histidine requiring S. typhimurium TA1535 uvrB and TA1538 uvrB (1). The WP2 strains and TA1535 respond to base-pair mutagens and TA 1538 to frameshift mutagens. Because of the anticipated low yields of induced mutants to prototrophy, we used a simplified fluctuation test with high
~ - NHCOOCH3 /
(~ONHC/.,H9
Fig. 1. Structure of benomyL A. KaPpas was o n leave Fellowship.
by a NATO
f r o m the N u c l e a r R e s e a r c h C e n t e r " D e m o k r i t o s " , A t h e n s , G r e e c e , and s u p p o r t e d
380
sensitivity [6]. For T A 1 5 3 5 this was modified by the substitution of biotin (0.4 pg/ml) and L-histidine (0.25/~g/ml) for tryptophan. Benomyl was used in the form of Benlate (du Pont) which contains about 50% active ingredient. Concentrations given are corrected. Similar results were obtained in a limited number of experiments with pure benomyl (du Pont). The chemical was added in the form of a solution in dimethyl sulphoxide. An appropriate quantity of the latter was added to control tubes. The results are shown in Table I. Significant mutagenesis was observed in WP2 uvrA and T A 1 5 3 5 , both excision-deficient strains that respond to basepair substitution mutagens, but not in the frameshift mutagen detector TA1538, confirming previous reports [13].
TABLE 1 MUTAGENICITY OF BENOMYL TOWARDS SIMPLIFIED FLUCTUATION TEST [6]
E. C O L I
A N D S. T Y P H I M U R I U M
Benomy] concentration /zg/ml
Number of experiments
Average n u m b e r of tubes positive
0 0.125 0.25 0.5 1.0 2.5 5.0 10.0
5 3 5 3 5 3 3 3
4.0 10.0 10.2 11.0 11.2 7.3 7.0 6.0
0 0.125 0.250 1.0 5.0 10.0 15.0
5 5 5 5 5 5 2
9.0 9.0 8.8 8.8 10.0 8.2 8.5
0 0.125 0.25 0.5 1.0 5.0
5 5 5 5 5 5
6.0 7.8 4.8 4.6 6.6 3.8
Salmonella lyphimurium TA1535 0 0.125 0.25 0.5 1.0 5.0
3 3 3 3 2 2
2.3 5.7 6.0 5.7 6.5 4.0
2 2 2 2 2 2
2.5 1.0 1.5 2.0 2.0 1.0
Eschcrichia coli WP2 uvrA
WP2
CM611 uvrA e x r A
TA1538
0 0.125 0.25 0.5 1.0 5.0
STRAINS USING A
<0.01 <0.001 <0.001 <0.001
<0.05 <0.05 <0.05 <0.05
381
No mutagenesis was observed in the excision-proficient strain WP2 indicating that a DNA lesion is involved in the mutagenic action of b e n o m y l that is detectable and excisable by UV endonuclease. Similarly, the negative result in the misrepair and excision-deficient CM611 shows that the lexA÷-dependent misrepair pathway is necessary for benomyl mutagenesis. These properties have not previously been found for mutation induction by base analogues in bacteria. Suggestions of similar properties of 5-bromodeoxyuridine based on work with bacteriophage ~ [10] have not been confirmed in E.coli [16]. These properties are, however, n o t inconsistent with the chemical structure of benomyl. Although the butylcarbamoyl group is removed the carbamate group at the C-8 position would be expected to remain on the molecule when incorporated. It might then appear similar to a purine bearing a large alkylation group. UV endonuclease is known to act in such cases [14]. The benzene ring differs from the purine ring in being unable to pair with other bases by hydrogen bonding [13]. In this respect it is similar to a dimerized pyrimidine and distinct from 5bromouracil or 2-aminopurine. Pyrimidine dimers are known to cause gaps in newly-synthesized DNA, [11] and a specific proportion of these [4] lead to mutations via the lexA÷-dependent error-prone repair pathway [2,15]. An important feature of the mutagenic action of b e n o m y l is the lack of a dose response (see Table I). From 0.125 to 1 pg/ml benomyl shows an essentially constant number of induced mutants; at higher doses there is a decline which may be partly attributable to toxic effects (unpublished data). Although such a response is completely uncharacteristic of alkylating agents, it is perhaps explicable for benomyl in the light of its requirement for incorporation into DNA. One needs only to postulate a saturation at low concentrations of an enzyme involved in incorporation, and an inhibition at high concentrations. If our model for the action of benomyl is correct, however, an entire replication cycle would normally intervene between incorporation and replication of the incorporated analogue. This could, at least in principle, permit complete excision to occur and result in total non-mutagenicity in Uvr ÷ strains. The reported non-mutagenicity in A. nidulans (7) could be explained in this way. In conclusion, we believe that the action of benomyl may be understood if it is seen as a novel type of mutagen needing to be incorporated into DNA b u t once incorporated, seen by the cell as a non-pairing purine with a large alkyl or aryl group attached. References 1 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 w i t h the 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 . 2 B r i d g e s , B . A . , R . E . D e n n i s a n d R . J . M u n s o n , D i f f e r e n t i a l i n d u c t i o n and repair o f u l t r a v i o l e t d a m a g e l e a d i n g t o t r u e reversions and e x t e r n a l suppressor m u t a t i o n s o f an o c h r e c o d o n i n Escherichia coli B / r W P 2 , G e n e t i c s , 57 ( 1 9 6 7 ) 8 9 7 - - 9 0 8 . 3 B r i d g e s , B . A . , R . P . M o t t e r s h e a d , M . A . R o t h w e l l a n d M . H . L . G r e e n , R e p a i r - d e f i c i e n t bacterial strains suitable for m u t a g e n i c i t y screening: t e s t s w i t h t h e f u n g i c i d e c a p t a n , C h e m . Biol. I n t e r a c t i o n s 5 (1972) 77--84. 4 D o u b l e d a y , O . P . , B . A . Bridges and M . H . L . G r e e n , M u t a g e n i c D N A r e p a i r in Escherichia coll. II. F a c t o r s a f f e c t i n g loss o f p h o t o r e v e r s i b i l i t y o f U V - i n d u c e d m u t a t i o n s , M o l e c . G e n . G e n e t . , 1 4 0 ( 1 9 7 5 ) 221--230. 5 G r e e n , M . H . L . a n d W . J . M u r i e l , M u t a t i o n t e s t i n g using Trp ÷ reversion in Escherichia coli, M u t a t i o n Res., 38 (1976) 3--32.
382 6 G r e e n , M . H . L . , W . J . M u r i e l a n d B . A . B r i d g e s , Use o f a s i m p l i f i e d f l u c t u a t i o n t e s t t o d e t e c t l o w levels of mutagens, Mutation Res. 38 (1976) 33--42. 7 Hastie, A.C., B e n l a t e - i n d u c e d i n s t a b i l i t y o f A s p e r g i l l u s d i p l o i d s , N a t u r e 2 2 6 ( 1 9 7 0 ) 7 7 1 . 8 Hill, R . F . , U l t r a v i o l e t - i n d u c e d l e t h a l i t y a n d r e v e r s i o n t o p r o t o t r o p h y in E s c h e r i c h i a coli s t r a i n s w i t h normal and reduced dark repair ability, Photochem. Photobiol. 4 (1965) 563--568. 9 K a p p a s , A., S . G . G e o r g o p o u l o s a n d A . C . H a s t i e , O n t h e g e n e t i c a c t i v i t y o f b e n z i m i d a z o l e a n d t h i o p h a n a t e f u n g i c i d e s o n d i p l o i d A s p e r g i l l u s n i d u l a n s , M u t a t i o n Res., 2 6 ( 1 9 7 4 ) 1 7 - - 2 7 . 1 0 P i e t r z y k o w s k a , I., O n t h e m e c h a n i s m o f b r o m o u r a c i l i n d u c e d m u t a g e n e s i s ~ M u t a t i o n R e s . , 19 ( 1 9 7 3 ) 1--9. 11 R u p p , W . D . a n d P. H o w a r d - F l a n d e r s , D i s c o n t i n u i t i e s in t h e D N A s y n t h e s i z e d in a n e x c i s i o n - d e f e c t i v e s t r a i n o f E s c h e r i c h i a coli f o l l o w i n g u l t r a v i o l e t i r r a d i a t i o n , J. M o l . Biol., 31 ( 1 9 6 8 ) 2 9 1 - - 3 0 4 . 1 2 Seiler, J . P . , T h e m u t a g e n i c i t y o f b e n z i m i d a z o l e a n d b e n z i m i d a z o l e d e r i v a t i v e s . I. F o r w a r d a n d reverse m u t a t i o n s in S a l m o n e l l a t y p h i m u r i u m c a u s e d b y b e n z i m i d a z o l e a n d s o m e o f its d e r i v a t i v e s , M u t a t i o n Res. 15 (1972) 273--276. 1 3 Seiler, J . P . , T o x i c o l o g y a n d g e n e t i c e f f e c t s o f b e n z i m i d a z o l e c o m p o u n d s , M u t a t i o n R e s . 3 2 ( 1 9 7 5 ) 161--168. 1 4 T a r m y , E . M . , S. V e n i t t a n d P. B r o o k e s , M u t a g e n i c i t y o f t h e c a r c i n o g e n 7 - b r o r n o m e t h y l b e n z (a) a n t h r a c e n e ; a q u a n t i t a t i v e s t u d y in r e p a i r - d e f i c i e n t s t r a i n s o f E s c h e r i c h i a coli, M u t a t i o n R e s . , 19 (1973) 153--166. 1 5 W i t k i n , E . M . , M u t a t i o n - p r o o f a n d m u t a t i o n - p r o n e m o d e s o f survival in d e r i v a t i v e s o f E s c h e r i c h i a coli B d i f f e r i n g in s e n s i t i v i t y t o u l t r a v i o l e t l i g h t , B r o o k h a v e n S y m p . Biol., 2 0 ( 1 9 6 7 ) 1 7 - - 5 5 . 1 6 W i t k i n , E . M . a n d E . C . Parisi, B r o m o u r a c i l m u t a g e n e s i s : m i s p a i r i n g o r m i s r e p a i r ? M u t a t i o n R e s . , 2 5 (1974) 407--409.