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Mutagenic action of S-methylmethionine in bacteria
Mutation Research, 119 (1983) 251-254 Elsevier Biomedical Press
251
Mutagenic action of S-methylmethionine in bacteria S. H u s s a i n Wallenberg Laboratory, University of Stockholm (Sweden)
(Accepted 4 November 1982)
Sulfonium compounds are alkylating agents (Ross, 1962; Ingold, 1969); the ubiquitous natural metabolite S-adenosylmethionine (SAM) should therefore be considered a potential endogenous mutagen and carcinogen (N~islund et al., 1983) in similarity with its ethyl homolog, S-adenosylethionine (Lawley, 1978). SAM exhibits the same alkylation pattern as methyl methanesulfonate (MMS), but its reactivity is some 3 orders of magnitude lower than that of MMS (N~islund et al., 1983). Owing to the participation of SAM in regulatory processes, it is difficult to decide whether slight effects on mutation frequency in samples treated with this compound are due to alkylation or to physiological changes. For this reason it was considered important to study the mutagenic potency of S-methylmethionine (SMM), a simple analog of SAM with about the same reactivity. S-Methylmethionine (vitamin U) occurs as a natural constituent of milk, potato, sweet corn, soybean, asparagus and cabbage (cf. Ramirez et al., 1973). It is biosynthesized from S-adenosylmethionine and methionine (Greene and Davis, 1960). In cell-free homogenates prepared from the seeds of plants, SMM serves as a substrate for methyl transferases (Turner and Shapiro, 1961; Mudd et al., 1966). Owing to the possible clinical utility of SMM - it is implicated in the cure of ulcers and hepatic disorders in humans, ulcers of shay in rats and of dietary hypercholesterolemia in rabbits - and its status as a vitamin, it is o f interest to evaluate its genetic toxicity.
Materials and methods Chemical. S-Methylmethioninesulfonium chloride was purchased from Sigma,
St. Louis (U.S.A.). Strains. Salmonella typhimurium TA1535 and TA100, two histidine auxotrophic strains from the Ames test system, were used, and reversion to histidine prototrophy was studied. S. typhimurium SV3, an arabinose-sensitive strain (Ruiz-Vazquez et al., 1978) was used for mutation to arabinose resistance. E. coil Sd-4, a streptomycin-dependent strain, was used for mutation to streptomycin nondependence. 0165-7992/83/0000-0000/$ 03.00 © Elsevier SciencePublishers
252
Media. The media for these strains are described by Ames (1971), Ruiz-V~izquez et al. (1978) and by Hussain and Ehrenberg (1979), in the order they are listed. Treatment. Cells, cultured overnight, were washed twice with 0.1 M phosphate buffer (pH 7), resuspended and then treated in the same buffer for 1 or 2 h at 37°C. The treatment was terminated by adding cold buffer with which the cells were washed twice before being resuspended for plating to determine the surviving and mutant cells.
Results and discussion Treatment of TA1535 and TA100 with S-methylmethioninesulfonium chloride was carried out in the dose range 1-27 mM. h. There was some increase in the actual mutational count in both Salmonella strains (Table 1), but because there was an increase, too, in the number of surviving ceils, it was not clear whether the increase
TABLE 1 MUTAGENIC EFFECT typhimurium STRAINS Dose (mM. h)
0 1 3 9 27
OF
S-METHYLMETHIONINESULFONIUM
IN
S.
TA100
TA1535 Survival
CHLORIDE
(%)
Mutants, mean per petri dish
Mutants per lOs bacteria
Survival (%)
Mutants, mean per petri dish
Mutants per lOs bacteria
100 128 146.7 151.8 153
11 16 21.2 15.4 19.6
6 7 9 6 8
100 145.9 181.1 154.5 128.3
157.4 167 176.4 177 168.5
101 73 62 73 84
TABLE 2 MUTAGENIC EFFECT OF DL-METHYLMETHIONINESULFON1UM C H L O R I D E IN E. coli Sd-4 Dose (mM. h)
Survival (% of control)
Mutants counted
Mean per petri dish
Mutants per 108 bacteria
0 1.9 5.7 17 51 153
100 111.9 107.4 110.8 98.9 93.2
83 55 56 71 67 60
3.6 3.9 4 5.1 4.8 4
2.301 2.375 2.506 3.096 3.286 3.298
Slope with standard error
(0.0057 + 0.001) × 10 -8
In no individual case was the increase in mutation frequency significant.
253 300
200
10C
~
,
J
J
~
I 10
i
i
i
I
I 20
mM.h
Fig. 1. The mutagenic effect of S-methylmethioninesulfoniumchloride in Salmonella typhimurium SV3. The points bear 95% confidence limits. Mutations to arabinose resistance were studied.
in the mutational count was due to induction o f mutations or to an increase in the number of cells. Mutation to arabinose resistance in SV3 indicated that an actual increase in the mutational count might be at hand in the dose range 0.6-49.5 m M . h (see Fig. I), but a slight increase in the number of cells complicated the situation in this experiment, too, When an increase in the number o f surviving ceils makes the results equivocal, it may be expedient to use the strain E. coli Sd-4, since, unless streptomycin is provided, the cells usually do not increase in number. Two tests were carried out and the results were pooled. At no individual dose did S-methylmethioninesulfonium chloride produce a significant increase in mutation frequency (Table 2). However, a calculation o f the slope by the least squares method revealed that it was positive and significantly different from zero: (0.0057 _ 0.001) × 10- 8 mutants per cell and per m M . h. (This slight mutagenic action, of course, cannot be distinguished f r o m the physiological action of this agent.) The following equation was obtained with the aid of a linear regression model: l0 s Y = 2.59 + 0.0057 D ( P < 0 . 0 0 1 )
254 A comparison o f the mutagenic effectiveness of S-methylmethionine and methyl methanesulfonate indicates that the former may be 500-1500 times less effective in E. coli Sd-4, in agreement with expectation from the low reactivity of compounds o f this class (Ingold, 1969). Therefore, it is possible that naturally occurring sulfonium ions, to the extent that they are not separated from D N A by cellular compartmentalization, could contribute to 'spontaneous' mutation. This is a tentative conclusion in view of the preliminary nature o f the data presented. According to the present state of our knowledge, critical D N A methylations are effectively repaired. It should then be remembered that, in principle, any of the S ligands may generate the alkyl group, e.g. in the case of methylmethionine (and SAM) a 3-amino-3-carboxypropyl group may also be generated (Ramirez et al., 1973).
Acknowledgement Valuable suggestions from Prof. L. Ehrenberg, Docent S. Osterman-Golkar and Dr. C.J. Calleman are gratefully acknowledged. The investigation was supported financially by the Swedish Natural Science Research Council,
References Ames, B.N. (1971) The detection of chemical mutagens with enteric bacteria, in: A. Hollaender (Ed.), Chemical Mutagens: Principles and Methods for their Detection, Vol. 1, Plenum, New York, pp. 267-282. Greene, R.C., and N.B. Davis (1960) Biosynthesis of S-methylmethionine in the jack bean, Biochim. Biophys. Acta, 43, 360-362. Hussain, S., and L. Ehrenberg (1979) Mutagenicity of radiations at low doses, Hereditas, 91, 111-116. Ingold, C.K. (1969) Organic Chemistry, Cornell University Press, Ithaca, Chapt. 5. Lawley, P.D. (1976) Carcinogenesis by alkylating agents, in: L. Prakash et al. (Eds.), Chemical Carcinogens, Monograph 173, Am. Chem. Soc., Washington, pp. 83-244. Mudd, S.H., W.A. Klee and P.D. Ross (1966) Enthalpy changes accompanyingthe transfer of a methyl group from S-adenosylmethionineand other sulfonium compounds to homocysteine, Biochemistry, 5, 1653-1660. N~islund, M., D. Segerb/ick and A. Kolman (1983) S-Adenosylmethionine, an endogenous alkylating agent, Mutation Res., 119, 229-232. Ramirez, F., J.L. Finnan and M. Carlson (1973) In vitro decomposition of S-methylmethioninesulfonium salts, J. Org. Chem., 38, 2597-2600. Ross, W.C.J. (1962) Biological Alkylating Agents, Butterworths, London, Chap. 1. Ruiz-Viizquez, R., C. Pueyo and E. Cerda-Olmedo (1978) A mutagen assay detecting forward mutations in an arabinose-sensitive strain of Salmonella typhimurium, Mutation Res., 54, 121-129. Turner, J.E., and S.K. Shapiro (1961) S-Methylmethione- and S-adenosylmethionine-homocysteine transmethylase in higher plant seeds, Biochim. Biophys. Acta, 51, 581-584.
251
Mutagenic action of S-methylmethionine in bacteria S. H u s s a i n Wallenberg Laboratory, University of Stockholm (Sweden)
(Accepted 4 November 1982)
Sulfonium compounds are alkylating agents (Ross, 1962; Ingold, 1969); the ubiquitous natural metabolite S-adenosylmethionine (SAM) should therefore be considered a potential endogenous mutagen and carcinogen (N~islund et al., 1983) in similarity with its ethyl homolog, S-adenosylethionine (Lawley, 1978). SAM exhibits the same alkylation pattern as methyl methanesulfonate (MMS), but its reactivity is some 3 orders of magnitude lower than that of MMS (N~islund et al., 1983). Owing to the participation of SAM in regulatory processes, it is difficult to decide whether slight effects on mutation frequency in samples treated with this compound are due to alkylation or to physiological changes. For this reason it was considered important to study the mutagenic potency of S-methylmethionine (SMM), a simple analog of SAM with about the same reactivity. S-Methylmethionine (vitamin U) occurs as a natural constituent of milk, potato, sweet corn, soybean, asparagus and cabbage (cf. Ramirez et al., 1973). It is biosynthesized from S-adenosylmethionine and methionine (Greene and Davis, 1960). In cell-free homogenates prepared from the seeds of plants, SMM serves as a substrate for methyl transferases (Turner and Shapiro, 1961; Mudd et al., 1966). Owing to the possible clinical utility of SMM - it is implicated in the cure of ulcers and hepatic disorders in humans, ulcers of shay in rats and of dietary hypercholesterolemia in rabbits - and its status as a vitamin, it is o f interest to evaluate its genetic toxicity.
Materials and methods Chemical. S-Methylmethioninesulfonium chloride was purchased from Sigma,
St. Louis (U.S.A.). Strains. Salmonella typhimurium TA1535 and TA100, two histidine auxotrophic strains from the Ames test system, were used, and reversion to histidine prototrophy was studied. S. typhimurium SV3, an arabinose-sensitive strain (Ruiz-Vazquez et al., 1978) was used for mutation to arabinose resistance. E. coil Sd-4, a streptomycin-dependent strain, was used for mutation to streptomycin nondependence. 0165-7992/83/0000-0000/$ 03.00 © Elsevier SciencePublishers
252
Media. The media for these strains are described by Ames (1971), Ruiz-V~izquez et al. (1978) and by Hussain and Ehrenberg (1979), in the order they are listed. Treatment. Cells, cultured overnight, were washed twice with 0.1 M phosphate buffer (pH 7), resuspended and then treated in the same buffer for 1 or 2 h at 37°C. The treatment was terminated by adding cold buffer with which the cells were washed twice before being resuspended for plating to determine the surviving and mutant cells.
Results and discussion Treatment of TA1535 and TA100 with S-methylmethioninesulfonium chloride was carried out in the dose range 1-27 mM. h. There was some increase in the actual mutational count in both Salmonella strains (Table 1), but because there was an increase, too, in the number of surviving ceils, it was not clear whether the increase
TABLE 1 MUTAGENIC EFFECT typhimurium STRAINS Dose (mM. h)
0 1 3 9 27
OF
S-METHYLMETHIONINESULFONIUM
IN
S.
TA100
TA1535 Survival
CHLORIDE
(%)
Mutants, mean per petri dish
Mutants per lOs bacteria
Survival (%)
Mutants, mean per petri dish
Mutants per lOs bacteria
100 128 146.7 151.8 153
11 16 21.2 15.4 19.6
6 7 9 6 8
100 145.9 181.1 154.5 128.3
157.4 167 176.4 177 168.5
101 73 62 73 84
TABLE 2 MUTAGENIC EFFECT OF DL-METHYLMETHIONINESULFON1UM C H L O R I D E IN E. coli Sd-4 Dose (mM. h)
Survival (% of control)
Mutants counted
Mean per petri dish
Mutants per 108 bacteria
0 1.9 5.7 17 51 153
100 111.9 107.4 110.8 98.9 93.2
83 55 56 71 67 60
3.6 3.9 4 5.1 4.8 4
2.301 2.375 2.506 3.096 3.286 3.298
Slope with standard error
(0.0057 + 0.001) × 10 -8
In no individual case was the increase in mutation frequency significant.
253 300
200
10C
~
,
J
J
~
I 10
i
i
i
I
I 20
mM.h
Fig. 1. The mutagenic effect of S-methylmethioninesulfoniumchloride in Salmonella typhimurium SV3. The points bear 95% confidence limits. Mutations to arabinose resistance were studied.
in the mutational count was due to induction o f mutations or to an increase in the number of cells. Mutation to arabinose resistance in SV3 indicated that an actual increase in the mutational count might be at hand in the dose range 0.6-49.5 m M . h (see Fig. I), but a slight increase in the number of cells complicated the situation in this experiment, too, When an increase in the number o f surviving ceils makes the results equivocal, it may be expedient to use the strain E. coli Sd-4, since, unless streptomycin is provided, the cells usually do not increase in number. Two tests were carried out and the results were pooled. At no individual dose did S-methylmethioninesulfonium chloride produce a significant increase in mutation frequency (Table 2). However, a calculation o f the slope by the least squares method revealed that it was positive and significantly different from zero: (0.0057 _ 0.001) × 10- 8 mutants per cell and per m M . h. (This slight mutagenic action, of course, cannot be distinguished f r o m the physiological action of this agent.) The following equation was obtained with the aid of a linear regression model: l0 s Y = 2.59 + 0.0057 D ( P < 0 . 0 0 1 )
254 A comparison o f the mutagenic effectiveness of S-methylmethionine and methyl methanesulfonate indicates that the former may be 500-1500 times less effective in E. coli Sd-4, in agreement with expectation from the low reactivity of compounds o f this class (Ingold, 1969). Therefore, it is possible that naturally occurring sulfonium ions, to the extent that they are not separated from D N A by cellular compartmentalization, could contribute to 'spontaneous' mutation. This is a tentative conclusion in view of the preliminary nature o f the data presented. According to the present state of our knowledge, critical D N A methylations are effectively repaired. It should then be remembered that, in principle, any of the S ligands may generate the alkyl group, e.g. in the case of methylmethionine (and SAM) a 3-amino-3-carboxypropyl group may also be generated (Ramirez et al., 1973).
Acknowledgement Valuable suggestions from Prof. L. Ehrenberg, Docent S. Osterman-Golkar and Dr. C.J. Calleman are gratefully acknowledged. The investigation was supported financially by the Swedish Natural Science Research Council,
References Ames, B.N. (1971) The detection of chemical mutagens with enteric bacteria, in: A. Hollaender (Ed.), Chemical Mutagens: Principles and Methods for their Detection, Vol. 1, Plenum, New York, pp. 267-282. Greene, R.C., and N.B. Davis (1960) Biosynthesis of S-methylmethionine in the jack bean, Biochim. Biophys. Acta, 43, 360-362. Hussain, S., and L. Ehrenberg (1979) Mutagenicity of radiations at low doses, Hereditas, 91, 111-116. Ingold, C.K. (1969) Organic Chemistry, Cornell University Press, Ithaca, Chapt. 5. Lawley, P.D. (1976) Carcinogenesis by alkylating agents, in: L. Prakash et al. (Eds.), Chemical Carcinogens, Monograph 173, Am. Chem. Soc., Washington, pp. 83-244. Mudd, S.H., W.A. Klee and P.D. Ross (1966) Enthalpy changes accompanyingthe transfer of a methyl group from S-adenosylmethionineand other sulfonium compounds to homocysteine, Biochemistry, 5, 1653-1660. N~islund, M., D. Segerb/ick and A. Kolman (1983) S-Adenosylmethionine, an endogenous alkylating agent, Mutation Res., 119, 229-232. Ramirez, F., J.L. Finnan and M. Carlson (1973) In vitro decomposition of S-methylmethioninesulfonium salts, J. Org. Chem., 38, 2597-2600. Ross, W.C.J. (1962) Biological Alkylating Agents, Butterworths, London, Chap. 1. Ruiz-Viizquez, R., C. Pueyo and E. Cerda-Olmedo (1978) A mutagen assay detecting forward mutations in an arabinose-sensitive strain of Salmonella typhimurium, Mutation Res., 54, 121-129. Turner, J.E., and S.K. Shapiro (1961) S-Methylmethione- and S-adenosylmethionine-homocysteine transmethylase in higher plant seeds, Biochim. Biophys. Acta, 51, 581-584.