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Induction of point mutations by benomyl in DNA-repair-deficient Aspergillus nidulans
Mutation Research, 91 (1981) 115-118 © Elsevier/North-Holland Biomedical Press
115
I N D U C T I O N OF P O I N T M U T A T I O N S D E F I C I E N T Aspergillus nidulans
BY B E N O M Y L IN D N A - R E P A I R -
A. KAPPAS a and B.A. BRIDGES b
aBiology Department, Nuclear Research Center 'Democritus', Athens (Greece) andbMRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BAIl 9QG (Great Britain) (Received 26 October 1980) (Accepted 28 October 1980)
Non-disjunction in heterozygous diploid strains of Aspergillus nidulans is greatly increased by the systemic fungicide benomyl (methyl-l-(butylcarbamoyl)-2benzimidazole carbamate) (Hastie, 1970; Kappas et al., 1974; Kappas, 1978). No induction of point mutations was, however, observed in DNA-repair-proficient A. nidulans conidia (Hastie, 1970), a result now confirmed with growing cells in the sensitive fluctuation assay (Green et al., 1976) using back mutations from biotin and pyridoxine requirement in UT439 (Table 1) and suppressor mutations (Lilly, 1965) of methG1 in strain methl biA1 (Table 2). Experiments with Escherichia coli indicated that base-pair-substitution mutations were induced by benomyl via the lexA ÷-dependent pathway in excision-deficient bacteria but were not detectable in excision-proficient bacteria (Kappas et al., 1976). Since benzimidazole, the nucleus of benomyl appears to be incorporated into DNA (Seiler, 1975), it was suggested (Kappas et al., 1976) that benomyl itself might, after removal of the butylcarbamoyl moiety be similarly incorporated as a base analogue. If the carbamate group remained at the C-8 position, it might be recognized by the bacterial UVendonuclease and the base excised. An entire replication cycle would elapse between incorporation and subsequent replication allowing ample time for complete excision in UVR ÷ bacteria, thus explaining the apparent absence of mutagenesis. Such a process might also occur in A. nidulans in which case an excision-deficient strain would be expected to show induced mutation. We have, therefore, examined this in 2 strains presumed to differ in the ability to excise pyrimidine dimers. We O'~'~ C - - N - - C 4 H 9 I H
,o,
H
x,C-- N-- C - - O C H 3 N/ H Benomyl
Fig. 1. Chemical structures.
Benzimidazole
116 TABLE 1 M U T A T I O N TO BIOTIN A N D P Y R I D O X I N E I N D E P E N D E N C E IN Aspergillus nidulans UT439 A N D A uvsD53 DERIVATIVE UT517 IN T H E F L U C T U A T I O N TEST IN T H E PRESENCE A N D ABSENCE OF BENOMYL Strain a
Benomyl Number of concentration experiments (#g/ml)
Total number Total number of tubes of tubes tested positive b
bi
Probability c
pyr
UT439
0 0.25 0.30 0.40
5 5 5 5
250 250 250 250
5 3 6 4
5 4 6 5
UT517
0 0.25 0.30 0.40
5 5 5 5
250 250 250 250
4 70 90 100
4 80 85 110
(uvsD53)
< 0.001 < 0.001 < 0.001
aBoth strains require proline, p-aminobenzoic acid, biotin and pyridoxine. Each tube contained 2 ml of conidia suspension (105 conidia/ml) in liquid minimal medium (MM) supplemented with proline (100 ~g/ml) and p-aminobenzoic acid (0.1 t~g/ml) in all cases and with biotin (0.04 #g/ml) and pyridoxine (0.5 ~g/ml) where appropriate. Small aliquots were added to MM stock solutions of benomyl in ethanol to give the required concentrations. The solvent concentration in the medium never exceeded 2% (v/v). bTurbid tubes were scored 10 days after inoculation; incubation temperature 38 °. An increased number of turbid tubes in the treated sets was observed in all 5 Expts. cSignificance by X2 test as described by Green et al. (1976).
observed using the fluctuation protocol (Green et al., 1976) that benomyl (pure, duPont) induced back mutations from both biotin and pyridoxin requirement in a dose-dependent manner in the presumed excision-deficient UT517 (Fortuin, 1971) whereas there was no detectable mutagenic effect in the repair-proficient parent UT439 (Table 1). In agreement with these results we have also observed induction of back mutations from adenine requirement in another UV-sensitive strain UT540 (Jansen, 1967) (Table 2). With all 3 mutation systems showing mutation induction by benomyl in UVsensitive strains, the dose-response was markedly non-linear (i.e., not extrapolating back to the spontaneous level at zero dose) with a plateau or near-plateau at the higher concentrations tested. This had also been noted in comparable experiments with bacteria (Kappas et al., 1976). In these experiments benomyl was dissolved in ethanol, the concentration of which never exceed 2070 (v/v) in the medium. There was no detectable e.ffect of this concentration on either growth or mutation rate in control experiments. The highest concentration of benomyl used in these experiments (0.4 #g/ml) reduced viability to about 40 or 50070 equally in UV-resistant and UV-sensitive
117 TABLE 2 M U T A G E N I C I T Y OF BENOMYL IN UV-SENS1TIVE A N D -RESISTANT STRAINS OF Aspergillus nidulans IN T H E F L U C T U A T I O N T E S T Strain
Benomyl concentration (/zg/ml)
Number of experiments
Total number of tubes tested a
Total n u m b e r of tubes positive b
methG1 biA1 d
0 0.25 0.30 0.40
5 5 5 5
250 250 250 250
5 4 3 4
UT540e (adD3 uvsAlO1)
0 0.25 0.30 0.40
5 5 5 5
250 250 250 250
5 90 120 100
Probability c
< 0.001 < 0.001 < 0.001
aEach tube contained 2 ml o f conidia suspension (105 conidia/ml) in liquid minimal medium (MM), supplemented in the case of the methG1 biAl strain with biotin (0.01 #g/ml). From stock solutions of benomyl in ethanol small aliquots were added to MM to give the appropriate concentrations. The solvent concentration in the medium never exceeded 2% (v/v). bTurbid tubes were scored 10 days after inoculation; incubation temperature 38 °. An increased number of turbid tubes in the treated sets was observed in all 5 Expts. cSignificance by X2 test as described by Green et al. 0976). dSuppressor mutations of methG1 scored. eReversions of adD3 scored.
strains suggesting that direct reaction with DNA was not involved in the toxic action of benomyl. Although the possibility cannot be completely excluded that benomyl might be mutagenic by a carbamoylation reaction with DNA, this seems unlikely in view of the similar weak mutagenic activity of methyl-2-benzimidazole carbamate (the hydrolysis product of benomyl) and benzimidazole (J.P. Seiler, personal communication). We therefore prefer to explain both the A. nidulans and E. coli results on the hypothesis that a benomyl metabolite is incorporated into DNA and once incorporated is recognized by a UV-endonuclease as if it were a non-pairing purine with a large alkyl or aryl group attached. ACKNOWLEDGEMENT
The authors thank Drs. B.R. Scott and B. Bainbridge for providing the strains of A. nidulans.
118 REFERENCES Fortuin, J.J.H. (1971) Another two genes controlling mitotic intragenic recombination and recovery from UV damage in Aspergillus nidulans, I. UV sensitivity, complementation and location of six mutants, Mutation Res., 11, 149-162. Green, M.H.L., W.J. Muriel and B.A. Bridges (1976) Use of a simplified fluctuation test to detect low levels of mutagens, Mutation Res., 38, 33-42. Hastie, A.C. (1970) Benlate-induced instability of Aspergillus diploids, Nature (London), 226, 771. Jansen, G.J.O. (1967) Some properties of the uvsl mutant of Aspergillus nidulans, Aspergillus Newsl., 8, 20-21. Kappas, A. (1978) On the mechanisms of induced somatic recombination by certain fungicides in Aspergillus nidulans, Mutation Res., 51, 189-197. Kappas, A., S.G. Georgopoulos and A.C. Hastie (1974) On the genetic activity of benzimidazole and thiophanate fungicides on diploid Aspergillus nidulans, Mutation Res., 26, 17-27. Kappas, A., M.H.L. Green, B.A. Bridges, A.M. Rogers and W.J. Muriel (1976) Benomyl - a novel type of base analogue mutagen? Mutation Res., 40, 379-382. Lilly, L.J. (1965) An investigation of the suitability of the suppressors of methl in Aspergillus nidulans for the study of induced and spontaneous mutation, Mutation Res., 2, 192-195. Seiler, J.P. (1975) Toxicology and genetic effects of benzimidazole compounds, Mutation Res., 32, 151-167.
115
I N D U C T I O N OF P O I N T M U T A T I O N S D E F I C I E N T Aspergillus nidulans
BY B E N O M Y L IN D N A - R E P A I R -
A. KAPPAS a and B.A. BRIDGES b
aBiology Department, Nuclear Research Center 'Democritus', Athens (Greece) andbMRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BAIl 9QG (Great Britain) (Received 26 October 1980) (Accepted 28 October 1980)
Non-disjunction in heterozygous diploid strains of Aspergillus nidulans is greatly increased by the systemic fungicide benomyl (methyl-l-(butylcarbamoyl)-2benzimidazole carbamate) (Hastie, 1970; Kappas et al., 1974; Kappas, 1978). No induction of point mutations was, however, observed in DNA-repair-proficient A. nidulans conidia (Hastie, 1970), a result now confirmed with growing cells in the sensitive fluctuation assay (Green et al., 1976) using back mutations from biotin and pyridoxine requirement in UT439 (Table 1) and suppressor mutations (Lilly, 1965) of methG1 in strain methl biA1 (Table 2). Experiments with Escherichia coli indicated that base-pair-substitution mutations were induced by benomyl via the lexA ÷-dependent pathway in excision-deficient bacteria but were not detectable in excision-proficient bacteria (Kappas et al., 1976). Since benzimidazole, the nucleus of benomyl appears to be incorporated into DNA (Seiler, 1975), it was suggested (Kappas et al., 1976) that benomyl itself might, after removal of the butylcarbamoyl moiety be similarly incorporated as a base analogue. If the carbamate group remained at the C-8 position, it might be recognized by the bacterial UVendonuclease and the base excised. An entire replication cycle would elapse between incorporation and subsequent replication allowing ample time for complete excision in UVR ÷ bacteria, thus explaining the apparent absence of mutagenesis. Such a process might also occur in A. nidulans in which case an excision-deficient strain would be expected to show induced mutation. We have, therefore, examined this in 2 strains presumed to differ in the ability to excise pyrimidine dimers. We O'~'~ C - - N - - C 4 H 9 I H
,o,
H
x,C-- N-- C - - O C H 3 N/ H Benomyl
Fig. 1. Chemical structures.
Benzimidazole
116 TABLE 1 M U T A T I O N TO BIOTIN A N D P Y R I D O X I N E I N D E P E N D E N C E IN Aspergillus nidulans UT439 A N D A uvsD53 DERIVATIVE UT517 IN T H E F L U C T U A T I O N TEST IN T H E PRESENCE A N D ABSENCE OF BENOMYL Strain a
Benomyl Number of concentration experiments (#g/ml)
Total number Total number of tubes of tubes tested positive b
bi
Probability c
pyr
UT439
0 0.25 0.30 0.40
5 5 5 5
250 250 250 250
5 3 6 4
5 4 6 5
UT517
0 0.25 0.30 0.40
5 5 5 5
250 250 250 250
4 70 90 100
4 80 85 110
(uvsD53)
< 0.001 < 0.001 < 0.001
aBoth strains require proline, p-aminobenzoic acid, biotin and pyridoxine. Each tube contained 2 ml of conidia suspension (105 conidia/ml) in liquid minimal medium (MM) supplemented with proline (100 ~g/ml) and p-aminobenzoic acid (0.1 t~g/ml) in all cases and with biotin (0.04 #g/ml) and pyridoxine (0.5 ~g/ml) where appropriate. Small aliquots were added to MM stock solutions of benomyl in ethanol to give the required concentrations. The solvent concentration in the medium never exceeded 2% (v/v). bTurbid tubes were scored 10 days after inoculation; incubation temperature 38 °. An increased number of turbid tubes in the treated sets was observed in all 5 Expts. cSignificance by X2 test as described by Green et al. (1976).
observed using the fluctuation protocol (Green et al., 1976) that benomyl (pure, duPont) induced back mutations from both biotin and pyridoxin requirement in a dose-dependent manner in the presumed excision-deficient UT517 (Fortuin, 1971) whereas there was no detectable mutagenic effect in the repair-proficient parent UT439 (Table 1). In agreement with these results we have also observed induction of back mutations from adenine requirement in another UV-sensitive strain UT540 (Jansen, 1967) (Table 2). With all 3 mutation systems showing mutation induction by benomyl in UVsensitive strains, the dose-response was markedly non-linear (i.e., not extrapolating back to the spontaneous level at zero dose) with a plateau or near-plateau at the higher concentrations tested. This had also been noted in comparable experiments with bacteria (Kappas et al., 1976). In these experiments benomyl was dissolved in ethanol, the concentration of which never exceed 2070 (v/v) in the medium. There was no detectable e.ffect of this concentration on either growth or mutation rate in control experiments. The highest concentration of benomyl used in these experiments (0.4 #g/ml) reduced viability to about 40 or 50070 equally in UV-resistant and UV-sensitive
117 TABLE 2 M U T A G E N I C I T Y OF BENOMYL IN UV-SENS1TIVE A N D -RESISTANT STRAINS OF Aspergillus nidulans IN T H E F L U C T U A T I O N T E S T Strain
Benomyl concentration (/zg/ml)
Number of experiments
Total number of tubes tested a
Total n u m b e r of tubes positive b
methG1 biA1 d
0 0.25 0.30 0.40
5 5 5 5
250 250 250 250
5 4 3 4
UT540e (adD3 uvsAlO1)
0 0.25 0.30 0.40
5 5 5 5
250 250 250 250
5 90 120 100
Probability c
< 0.001 < 0.001 < 0.001
aEach tube contained 2 ml o f conidia suspension (105 conidia/ml) in liquid minimal medium (MM), supplemented in the case of the methG1 biAl strain with biotin (0.01 #g/ml). From stock solutions of benomyl in ethanol small aliquots were added to MM to give the appropriate concentrations. The solvent concentration in the medium never exceeded 2% (v/v). bTurbid tubes were scored 10 days after inoculation; incubation temperature 38 °. An increased number of turbid tubes in the treated sets was observed in all 5 Expts. cSignificance by X2 test as described by Green et al. 0976). dSuppressor mutations of methG1 scored. eReversions of adD3 scored.
strains suggesting that direct reaction with DNA was not involved in the toxic action of benomyl. Although the possibility cannot be completely excluded that benomyl might be mutagenic by a carbamoylation reaction with DNA, this seems unlikely in view of the similar weak mutagenic activity of methyl-2-benzimidazole carbamate (the hydrolysis product of benomyl) and benzimidazole (J.P. Seiler, personal communication). We therefore prefer to explain both the A. nidulans and E. coli results on the hypothesis that a benomyl metabolite is incorporated into DNA and once incorporated is recognized by a UV-endonuclease as if it were a non-pairing purine with a large alkyl or aryl group attached. ACKNOWLEDGEMENT
The authors thank Drs. B.R. Scott and B. Bainbridge for providing the strains of A. nidulans.
118 REFERENCES Fortuin, J.J.H. (1971) Another two genes controlling mitotic intragenic recombination and recovery from UV damage in Aspergillus nidulans, I. UV sensitivity, complementation and location of six mutants, Mutation Res., 11, 149-162. Green, M.H.L., W.J. Muriel and B.A. Bridges (1976) Use of a simplified fluctuation test to detect low levels of mutagens, Mutation Res., 38, 33-42. Hastie, A.C. (1970) Benlate-induced instability of Aspergillus diploids, Nature (London), 226, 771. Jansen, G.J.O. (1967) Some properties of the uvsl mutant of Aspergillus nidulans, Aspergillus Newsl., 8, 20-21. Kappas, A. (1978) On the mechanisms of induced somatic recombination by certain fungicides in Aspergillus nidulans, Mutation Res., 51, 189-197. Kappas, A., S.G. Georgopoulos and A.C. Hastie (1974) On the genetic activity of benzimidazole and thiophanate fungicides on diploid Aspergillus nidulans, Mutation Res., 26, 17-27. Kappas, A., M.H.L. Green, B.A. Bridges, A.M. Rogers and W.J. Muriel (1976) Benomyl - a novel type of base analogue mutagen? Mutation Res., 40, 379-382. Lilly, L.J. (1965) An investigation of the suitability of the suppressors of methl in Aspergillus nidulans for the study of induced and spontaneous mutation, Mutation Res., 2, 192-195. Seiler, J.P. (1975) Toxicology and genetic effects of benzimidazole compounds, Mutation Res., 32, 151-167.