Mutation Research, 282 (1992) 235-239
235
©. 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 0685
Mutagenicity of methyl methanesulfonate and cyclophosphamide in resting and growing Saccharomyces cerevisiae D7 cells M. Monaco a, R. Dominici b, p. Barisano b, G. Di Palermo b, A. Galli c and G. Bronzetti c a lstituto Superiore per la Prevenzione e la Sicurezza del Lavoro, b Centro Studi di Medicina dei Trasporti F.S., Rome, Italy and c lstituto di Mutagenesi e Differenziamento, C.N.R., Pisa, Italy
(Received 10 January 1992) (Revision received 27 March 1992) (Accepted 1 April 1992)
Keywords: Methyl methanesulfonate; Cyclophosphamide; Saccharomyces cerevisiae, strain D7; Mitotic gene conversion; Reverse
point mutation; Logarithmic phase; Stationary phase
Summary In order to evaluate the optimal experimental conditions and to identify the best growth phase for yeast genotoxicity studies, comparative experiments were performed with stationary and growing cells. Methyl methanesulfonate (MMS) and cyclophosphamide (CP) were used as chemical mutagens and strain D7 of Saccharomyces cerevisiae as detector of induced mitotic gene conversion (trp ÷ convertants) and point reverse mutation (ilv ÷ revertants) in log or stationary phase cells after either 4 or 16 h of treatment. The highest MMS-induced toxicity and genotoxicity were observed after 16 h of exposure in a suspension test with log phase cells, which is consistent with the greater permeability and sensitivity of growing yeast cells. The maximal induction of genetic effects and toxicity by CP was conversely obtained after 16 h of treatment in stationary phase ceils. This may be ascribed to the greater ability of detoxication of growing cells as compared to resting cells. Our results suggest that in evaluating the mutagenicity of chemicals in yeast systems it is important to consider factors such as growth phase and exposure time.
Many published reports on induced genotoxicity in yeast are based on treatments using cells in stationary a n d / o r growth phases. Precise definition of the relative sensitivity of yeast assays
Correspondence: Dr. M. Monaco, Istituto Superiore per la Prevenzione e la Sicurezza del Lavoro, Via Urbana 167, 1-00184 Rome, Italy.
under these two different experimental conditions is important in mutagenicity testing. Methyl methanesulfonate (MMS) and cyclophosphamide (CP) are well known genotoxins and are used as standard mutagens in many systems, including yeasts (Kondo, 1981; IARC, 1987; Zimmermann et al., 1984). The purpose of this study was to establish the best conditions of yeast cell growth for the detec-
236 TABLE 1 RESPONSE OF STRAIN D7 TO TREATMENT WITH MMS MMS (mM)
Survivors %
Convertants per 105 survivors
Revertants per 106 survivors
Stationary phase cells Control 0.56 1.12 1.68 2.24
100 100 100 100 98.1
(2823) (3031) (2940) (2888) (2772)
0.51 2.71 3.59 5.68 8.04
(71) (823) (1057) (1642) (2231)
0.19 0.46 1.13 1.54 2.33
(56) (140) (333) (446) (646)
Logarithmic phase cells Control 0.56 1.12 1.68 2.24
100 (3525) 80.3 (2832) 65.5 (2311) 64.8 (2286) 61.9 (2185)
0.40 (144) 5.01 (1420) 7.50 (1757) 15.60 (3568) 24.36 (5 323)
0.19 (68) 1.26 (359) 2.49 (577) 3.93 (899) 5.25 (1 149)
Treatment conditions: pH 7.4 at 32°C for 4 h. Numbers in parentheses: actual colony from two independent experiments.
tion of MMS and CP genotoxicity in strain D7 of Saccharomyces cerevisiae. The induction of trp + convertants and ilv + revertants was measured in the suspension test using growing and resting cells after 4 and 16 h of treatment.
locus and reverse point mutation at the ilvl locus (Zimmermann et al., 1975). Complete liquid (YEP) and solid (YEPD) media and supplemented minimal media were prepared according to Zimmermann et al. (1975).
Materials and methods
Chemicals MMS (CAS No. 66-27-3) was purchased from Aldrich-Chemie, CP (CAS No. 50-18-0) from Asta Werke. They were dissolved in distilled water immediately prior to use.
Yeast strain and media • The D7 diploid strain of S. cerevisiae was used to measure mitotic gene conversion at the trp5 TABLE 2 RESPONSE OF STRAIN D7 TO TREATMENT WITH MMS MMS (mM)
Survivors %
Convertants per 105 survivors
Revertants per 106 survivors
100 (3268) 100 (3706) 86.8 (3150) 82.0 (2978) 78.6 (2 854)
0.27 (101) 9.17 (3599) 18.01 (5 675) 29.61 (8818) 38.06 (10 863)
0.22 (81) 1.24 (460) 3.92 (1235) 9.16 (2728) 11.85 (3 383)
100 (4043) 31.6 (1280) 23.7 (961) 14.2 (576) 12.2 (496)
0.31 27.91 30.33 69.91 71.06
0.20 (83) 11.59 (1484) 11.77 (1132) 29.89 (1722) 24.35 (1208)
Stationary. phase cells Control 0.56 1.12 1.68 2.24
Logarithmicphasecells Control 0.56 1.12 1.68 2.24
(127) (3573) (2915) (4027) (3525)
Treatment conditions: pH 7.4 at 32°C for 16 h. Numbers in parentheses: actual colony counts from two independent experiments.
237 TABLE 3 RESPONSE OF STRAIN D7 TO TREATMENT WITH CP CP (mM)
Survivors %
Convertants per 105 survivors
Revertants per 106 survivors
(3085) (3100) (3 081) (3 063) (2895)
0.21 (66) 0.49 (152) 0.82 (254) 1.40 (429) 2.16 (628)
0.18 (58) 1.12 (349) 1.72 (533) 1.89 (579) 2.81 (814)
Logarithmic phase cells with $9 Control 100 (3473) 0.31 100 (3531) 0.62 93.3 (3 242) 1.25 95.0 (3 302) 2.50 99.4 (3 453)
0.38 (132) 0.52 (184) 0.84 (274) 1.10 (365) 1.14 (489)
0.18 (64) 0.39 (141) 0.62 (203) 0.88 (292) 1.03 (359)
Stationary phase cells with $9 Control 100 0.31 100 1.62 99.8 1.25 99.2 2.50 93.8
Treatment conditions: pH 7.4 at 32°C for 4 h. Numbers in parentheses: actual colony counts from two independent experiments.
Suspension test procedure From a culture with low spontaneous gene conversion and reverse point mutation frequencies, the cells were pelleted and resuspended in sterile potassium phosphate buffer (0.1 M; pH 7.4) and checked microscopically. For tests with stationary phase cells, the cell density was 2-2.2
× 108 cells/ml. For tests in the logarithmic growth phase, cells were resuspended in liquid YEP at a density of 4.5 x 107 and 9 × 107 cells/ml for 16 and 4 h treatments, respectively. $9 mix was prepared according to Ames et al. (1975), and contained liver $9 fractions from phenobarbital and /3-naphthoflavone-induced
TABLE 4 RESPONSE OF STRAIN D7 TO TREATMENT WITH CP CP (mM)
Survivors %
Convertants per 10 s survivors
Revertants per 106 survivors
Stationary phase cells with $9 Control 100 (3 374) 0.31 77.9 (2 940) 0.62 72.2 (2 728) 1.25 61.2 (2 311) 2.50 16.5 (624)
0.23 (87) 16.95 (4 984) 23.35 (6 371) 59.22 (3 686) 69.16 (4316)
0.20 (78) 8.49 (2 998) 14.58 (3 978) 29.55 (6 830) 51.07 (3187)
Logarithmic phase cells with $9 Control 100 (3902) 0.31 107.2 (4183) 0.62 84.8 (3 309) 1.25 57.7 (2251) 2.50 39.6 (1545)
0.31 (124) 5.53 (2317) 10.16 (3 362) 18.26 (4111) 36.02 (5 690)
0.18 (72) 4.22 (1767) 7.42 (2457) 14.45 (3253) 27.79 (4 294)
Treatment conditions: pH 7.4 at 32°C for 16 h. Numbers in parentheses: actual colony counts from two independent experiments.
238
Sprague-Dawley rats. The incubation mixture was composed of: cell suspension 0.9 ml, test compound or solvent control 0.1 ml and phosphate buffer or $9 mix 1.0 ml. The mixtures were incubated horizontally at 32°C for 4 or 16 h. Then cells were plated in selective and complete media as previously described (Bronzetti et al., 1981, 1985).
effects obtained in stationary phase cells. However, in the highly permeable growing cells, the CP-induced genotoxicity is likely to be mainly influenced by the detoxicating metabolism. The present study provided further evidence that, in genotoxicity assays using yeast as the detector microorganism, the choice of treatment conditions may be critical in determining the genotoxic power of chemicals.
Results and discussion Acknowledgements
Under all treatment conditions MMS and CP induced a significant dose-dependent increase of mitotic gene conversion and point reverse mutation (Tables 1-4). When comparing the results shown in Tables 1 and 2, it is evident that the highest MMS-induced genotoxicity was observed in log-phase cells. This is probably due to the higher permeability of growing cells, as compared to resting cells, which is also supported by the higher toxicity of MMS in log than in stationary phase. It was previously reported that log-phase cells are more sensitive to potassium dichromate and chromic chloride (Galli et al., 1985). In particular, permeability of bacterial and yeast cells to trivalent chromium, which normally does not cross cell membranes, could only be achieved after incubation in liquid medium with a high phosphate molarity (De Flora and Wetterhahn, 1989). The situation was different for CP. In fact, the greatest CP-induced genetic activity was obtained in stationary phase cells (Tables 3 and 4). It is known that, in order to exibit a genotoxic effect, CP has to be biotransformed by the cytochrome P-450-dependent monooxygenase system (Struck et al., 1984). The active metabolites are then detoxicated by enzymatic systems and excreted from the cell. A number of enzymatic activities are higher in growing ceils than in stationary phase cells (Zimmermann et al., 1984). In addition, growing cells of strain D7 of S. cerevisiae were able to detoxicate direct mutagens, such as 4-nitroquinoline 1-oxide (Del Carratore et al., 1986). Accordingly, our results may be explained by assuming that yeast log phase cells are more able to detoxicate the CP metabolites produced by the $9 fraction. This is also confirmed by the toxic
The authors wish to thank Mrs. Grazia Cecchi for typing the manuscript. This work is supported by grants from CNR, Comitato Ambiente and Habitat No. 88.03.600.13 and Progetto Finalizzato ' FATMA'. References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31,347-369. Bronzetti, G., C. Bauer, C. Corsi, C. Leporini, R. Nieri and R. Del Carratore (1981) Genetic activity of vinylidene chloride in yeast, Mutation Res., 89, 179-185. Bronzetti, G., C. Bauer, C. Corsi, R. Del Carratore, A. Galli, R. Nieri and M. Paolini (1983) Genetic and biochemical studies on perchloroethylene in vitro and in vivo, Mutation Res., 116, 323-331. De Flora, S., and K.E. Wetterhahn (1989) Mechanisms of chromium metabolism and genotoxicity, Life Chem. Rep., 7, 169-244. Del Carratore, R., E. Cundari, R. Vellosi, A. Galli and G. Bronzetti (1986) Specific inhibitors of the monooxygenase system of Saccharomyces cerevisiae modified the mutagenic effect of 4-nitroquinoline 1-oxide and the deethylation activity of the yeast, Carcinogenesis, 7, 1127-1130. Galli, A., P. Boccardo, R. Del Carratore, E. Cundari and G. Bronzetti (1985) Conditions that influence the genetic activity of potassium dichromate and chromium chloride in Saccharomyces cerevisiae, Mutation Res., 144, 165-169. International Agency for Research on Cancer (1987) Monographs on the evaluation of carcinogenic risks to humans: genetic and related effects: an updating of selected IARC Monographs, Volumes 1-42, Suppl. 6, pp. 196-205. Kondo, S. (1981) Comparative mutagenicity of methylmethanesulfonate and ethylmetanesulfonate, in: F.J. de Serres and M.D. Shelby (Eds.), Comparative Chemical Mutagenesis, Plenum, New York, pp. 743-785. Struck, R.F., P. Karl, J. Kalin, J.A. Montgomery, A.J. Marinello, J. Love, S.K. Bansal and H.L. Gurtoo (1984) Metabolism of cyclophosphamide by purified cytochrome
239 P-450 from microsomes of phenobarbital-treated rats, Biochem. Biophys. Res. Commun., 120, 390-396. Zimmermann, F.K., R. Kern and H. Rasenberger (1975) A yeast strain for simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381-388. Zimmermann, F.K., R.C. von Borstel, E.S. von Halle, J.M.
Parry, D. Siebert, G. Zetterberg, R. Barale and N. Loprieno (1984) Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 133, 199-244. Communicated by S. De Flora
235
©. 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 0685
Mutagenicity of methyl methanesulfonate and cyclophosphamide in resting and growing Saccharomyces cerevisiae D7 cells M. Monaco a, R. Dominici b, p. Barisano b, G. Di Palermo b, A. Galli c and G. Bronzetti c a lstituto Superiore per la Prevenzione e la Sicurezza del Lavoro, b Centro Studi di Medicina dei Trasporti F.S., Rome, Italy and c lstituto di Mutagenesi e Differenziamento, C.N.R., Pisa, Italy
(Received 10 January 1992) (Revision received 27 March 1992) (Accepted 1 April 1992)
Keywords: Methyl methanesulfonate; Cyclophosphamide; Saccharomyces cerevisiae, strain D7; Mitotic gene conversion; Reverse
point mutation; Logarithmic phase; Stationary phase
Summary In order to evaluate the optimal experimental conditions and to identify the best growth phase for yeast genotoxicity studies, comparative experiments were performed with stationary and growing cells. Methyl methanesulfonate (MMS) and cyclophosphamide (CP) were used as chemical mutagens and strain D7 of Saccharomyces cerevisiae as detector of induced mitotic gene conversion (trp ÷ convertants) and point reverse mutation (ilv ÷ revertants) in log or stationary phase cells after either 4 or 16 h of treatment. The highest MMS-induced toxicity and genotoxicity were observed after 16 h of exposure in a suspension test with log phase cells, which is consistent with the greater permeability and sensitivity of growing yeast cells. The maximal induction of genetic effects and toxicity by CP was conversely obtained after 16 h of treatment in stationary phase ceils. This may be ascribed to the greater ability of detoxication of growing cells as compared to resting cells. Our results suggest that in evaluating the mutagenicity of chemicals in yeast systems it is important to consider factors such as growth phase and exposure time.
Many published reports on induced genotoxicity in yeast are based on treatments using cells in stationary a n d / o r growth phases. Precise definition of the relative sensitivity of yeast assays
Correspondence: Dr. M. Monaco, Istituto Superiore per la Prevenzione e la Sicurezza del Lavoro, Via Urbana 167, 1-00184 Rome, Italy.
under these two different experimental conditions is important in mutagenicity testing. Methyl methanesulfonate (MMS) and cyclophosphamide (CP) are well known genotoxins and are used as standard mutagens in many systems, including yeasts (Kondo, 1981; IARC, 1987; Zimmermann et al., 1984). The purpose of this study was to establish the best conditions of yeast cell growth for the detec-
236 TABLE 1 RESPONSE OF STRAIN D7 TO TREATMENT WITH MMS MMS (mM)
Survivors %
Convertants per 105 survivors
Revertants per 106 survivors
Stationary phase cells Control 0.56 1.12 1.68 2.24
100 100 100 100 98.1
(2823) (3031) (2940) (2888) (2772)
0.51 2.71 3.59 5.68 8.04
(71) (823) (1057) (1642) (2231)
0.19 0.46 1.13 1.54 2.33
(56) (140) (333) (446) (646)
Logarithmic phase cells Control 0.56 1.12 1.68 2.24
100 (3525) 80.3 (2832) 65.5 (2311) 64.8 (2286) 61.9 (2185)
0.40 (144) 5.01 (1420) 7.50 (1757) 15.60 (3568) 24.36 (5 323)
0.19 (68) 1.26 (359) 2.49 (577) 3.93 (899) 5.25 (1 149)
Treatment conditions: pH 7.4 at 32°C for 4 h. Numbers in parentheses: actual colony from two independent experiments.
tion of MMS and CP genotoxicity in strain D7 of Saccharomyces cerevisiae. The induction of trp + convertants and ilv + revertants was measured in the suspension test using growing and resting cells after 4 and 16 h of treatment.
locus and reverse point mutation at the ilvl locus (Zimmermann et al., 1975). Complete liquid (YEP) and solid (YEPD) media and supplemented minimal media were prepared according to Zimmermann et al. (1975).
Materials and methods
Chemicals MMS (CAS No. 66-27-3) was purchased from Aldrich-Chemie, CP (CAS No. 50-18-0) from Asta Werke. They were dissolved in distilled water immediately prior to use.
Yeast strain and media • The D7 diploid strain of S. cerevisiae was used to measure mitotic gene conversion at the trp5 TABLE 2 RESPONSE OF STRAIN D7 TO TREATMENT WITH MMS MMS (mM)
Survivors %
Convertants per 105 survivors
Revertants per 106 survivors
100 (3268) 100 (3706) 86.8 (3150) 82.0 (2978) 78.6 (2 854)
0.27 (101) 9.17 (3599) 18.01 (5 675) 29.61 (8818) 38.06 (10 863)
0.22 (81) 1.24 (460) 3.92 (1235) 9.16 (2728) 11.85 (3 383)
100 (4043) 31.6 (1280) 23.7 (961) 14.2 (576) 12.2 (496)
0.31 27.91 30.33 69.91 71.06
0.20 (83) 11.59 (1484) 11.77 (1132) 29.89 (1722) 24.35 (1208)
Stationary. phase cells Control 0.56 1.12 1.68 2.24
Logarithmicphasecells Control 0.56 1.12 1.68 2.24
(127) (3573) (2915) (4027) (3525)
Treatment conditions: pH 7.4 at 32°C for 16 h. Numbers in parentheses: actual colony counts from two independent experiments.
237 TABLE 3 RESPONSE OF STRAIN D7 TO TREATMENT WITH CP CP (mM)
Survivors %
Convertants per 105 survivors
Revertants per 106 survivors
(3085) (3100) (3 081) (3 063) (2895)
0.21 (66) 0.49 (152) 0.82 (254) 1.40 (429) 2.16 (628)
0.18 (58) 1.12 (349) 1.72 (533) 1.89 (579) 2.81 (814)
Logarithmic phase cells with $9 Control 100 (3473) 0.31 100 (3531) 0.62 93.3 (3 242) 1.25 95.0 (3 302) 2.50 99.4 (3 453)
0.38 (132) 0.52 (184) 0.84 (274) 1.10 (365) 1.14 (489)
0.18 (64) 0.39 (141) 0.62 (203) 0.88 (292) 1.03 (359)
Stationary phase cells with $9 Control 100 0.31 100 1.62 99.8 1.25 99.2 2.50 93.8
Treatment conditions: pH 7.4 at 32°C for 4 h. Numbers in parentheses: actual colony counts from two independent experiments.
Suspension test procedure From a culture with low spontaneous gene conversion and reverse point mutation frequencies, the cells were pelleted and resuspended in sterile potassium phosphate buffer (0.1 M; pH 7.4) and checked microscopically. For tests with stationary phase cells, the cell density was 2-2.2
× 108 cells/ml. For tests in the logarithmic growth phase, cells were resuspended in liquid YEP at a density of 4.5 x 107 and 9 × 107 cells/ml for 16 and 4 h treatments, respectively. $9 mix was prepared according to Ames et al. (1975), and contained liver $9 fractions from phenobarbital and /3-naphthoflavone-induced
TABLE 4 RESPONSE OF STRAIN D7 TO TREATMENT WITH CP CP (mM)
Survivors %
Convertants per 10 s survivors
Revertants per 106 survivors
Stationary phase cells with $9 Control 100 (3 374) 0.31 77.9 (2 940) 0.62 72.2 (2 728) 1.25 61.2 (2 311) 2.50 16.5 (624)
0.23 (87) 16.95 (4 984) 23.35 (6 371) 59.22 (3 686) 69.16 (4316)
0.20 (78) 8.49 (2 998) 14.58 (3 978) 29.55 (6 830) 51.07 (3187)
Logarithmic phase cells with $9 Control 100 (3902) 0.31 107.2 (4183) 0.62 84.8 (3 309) 1.25 57.7 (2251) 2.50 39.6 (1545)
0.31 (124) 5.53 (2317) 10.16 (3 362) 18.26 (4111) 36.02 (5 690)
0.18 (72) 4.22 (1767) 7.42 (2457) 14.45 (3253) 27.79 (4 294)
Treatment conditions: pH 7.4 at 32°C for 16 h. Numbers in parentheses: actual colony counts from two independent experiments.
238
Sprague-Dawley rats. The incubation mixture was composed of: cell suspension 0.9 ml, test compound or solvent control 0.1 ml and phosphate buffer or $9 mix 1.0 ml. The mixtures were incubated horizontally at 32°C for 4 or 16 h. Then cells were plated in selective and complete media as previously described (Bronzetti et al., 1981, 1985).
effects obtained in stationary phase cells. However, in the highly permeable growing cells, the CP-induced genotoxicity is likely to be mainly influenced by the detoxicating metabolism. The present study provided further evidence that, in genotoxicity assays using yeast as the detector microorganism, the choice of treatment conditions may be critical in determining the genotoxic power of chemicals.
Results and discussion Acknowledgements
Under all treatment conditions MMS and CP induced a significant dose-dependent increase of mitotic gene conversion and point reverse mutation (Tables 1-4). When comparing the results shown in Tables 1 and 2, it is evident that the highest MMS-induced genotoxicity was observed in log-phase cells. This is probably due to the higher permeability of growing cells, as compared to resting cells, which is also supported by the higher toxicity of MMS in log than in stationary phase. It was previously reported that log-phase cells are more sensitive to potassium dichromate and chromic chloride (Galli et al., 1985). In particular, permeability of bacterial and yeast cells to trivalent chromium, which normally does not cross cell membranes, could only be achieved after incubation in liquid medium with a high phosphate molarity (De Flora and Wetterhahn, 1989). The situation was different for CP. In fact, the greatest CP-induced genetic activity was obtained in stationary phase cells (Tables 3 and 4). It is known that, in order to exibit a genotoxic effect, CP has to be biotransformed by the cytochrome P-450-dependent monooxygenase system (Struck et al., 1984). The active metabolites are then detoxicated by enzymatic systems and excreted from the cell. A number of enzymatic activities are higher in growing ceils than in stationary phase cells (Zimmermann et al., 1984). In addition, growing cells of strain D7 of S. cerevisiae were able to detoxicate direct mutagens, such as 4-nitroquinoline 1-oxide (Del Carratore et al., 1986). Accordingly, our results may be explained by assuming that yeast log phase cells are more able to detoxicate the CP metabolites produced by the $9 fraction. This is also confirmed by the toxic
The authors wish to thank Mrs. Grazia Cecchi for typing the manuscript. This work is supported by grants from CNR, Comitato Ambiente and Habitat No. 88.03.600.13 and Progetto Finalizzato ' FATMA'. References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31,347-369. Bronzetti, G., C. Bauer, C. Corsi, C. Leporini, R. Nieri and R. Del Carratore (1981) Genetic activity of vinylidene chloride in yeast, Mutation Res., 89, 179-185. Bronzetti, G., C. Bauer, C. Corsi, R. Del Carratore, A. Galli, R. Nieri and M. Paolini (1983) Genetic and biochemical studies on perchloroethylene in vitro and in vivo, Mutation Res., 116, 323-331. De Flora, S., and K.E. Wetterhahn (1989) Mechanisms of chromium metabolism and genotoxicity, Life Chem. Rep., 7, 169-244. Del Carratore, R., E. Cundari, R. Vellosi, A. Galli and G. Bronzetti (1986) Specific inhibitors of the monooxygenase system of Saccharomyces cerevisiae modified the mutagenic effect of 4-nitroquinoline 1-oxide and the deethylation activity of the yeast, Carcinogenesis, 7, 1127-1130. Galli, A., P. Boccardo, R. Del Carratore, E. Cundari and G. Bronzetti (1985) Conditions that influence the genetic activity of potassium dichromate and chromium chloride in Saccharomyces cerevisiae, Mutation Res., 144, 165-169. International Agency for Research on Cancer (1987) Monographs on the evaluation of carcinogenic risks to humans: genetic and related effects: an updating of selected IARC Monographs, Volumes 1-42, Suppl. 6, pp. 196-205. Kondo, S. (1981) Comparative mutagenicity of methylmethanesulfonate and ethylmetanesulfonate, in: F.J. de Serres and M.D. Shelby (Eds.), Comparative Chemical Mutagenesis, Plenum, New York, pp. 743-785. Struck, R.F., P. Karl, J. Kalin, J.A. Montgomery, A.J. Marinello, J. Love, S.K. Bansal and H.L. Gurtoo (1984) Metabolism of cyclophosphamide by purified cytochrome
239 P-450 from microsomes of phenobarbital-treated rats, Biochem. Biophys. Res. Commun., 120, 390-396. Zimmermann, F.K., R. Kern and H. Rasenberger (1975) A yeast strain for simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381-388. Zimmermann, F.K., R.C. von Borstel, E.S. von Halle, J.M.
Parry, D. Siebert, G. Zetterberg, R. Barale and N. Loprieno (1984) Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 133, 199-244. Communicated by S. De Flora
