217
Mutation Research, 57 (1978) 217-224 @ Elsevier/North-Holland Biomedical Press
COMPARATIVE MUTAGENICITY OF ALKYLSULFATE AND ALKANESULFONATE DERIVATIVES IN CHINESE HAMSTER OVARY CELLS
DAVID B. COUCH, * NANCY L. FORBES and ABRAHAM W. HSIE ** University of Tennessee - Oak Ridge Graduate School of Biomedical Sciences and Biology Division, Oak Ridge National Laboratory, *** Oak Ridge, Tenn. 37830 (U.S.A.) (Received 6 September 1977) (Revision received 29 November 1977) (Accepted 2 December 1977)
Summary Mutation induction and cell killing produced by selected alkylsulfates and alkanesulfonates have been quantitated using the Chinese hamster ovary/hypoxanthinewanine phosphoribosyl transferase (CHO/HGPRT) system. Doseresponse relationships of cytotoxicity and mutagenicity are presented for two alkylsulfates [dimethylsulfate (DMS), diethylsulfate (DES)] and three alkyl alkanesulfonates [methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and isopropyl methanesulfonate (iPMS)] . Under the experimental conditions employed, cytotoxicity decreased with the size of the alkyl group. DMS was more toxic than DES, and MMS was more toxic than EMS and iPMS. All agents produced linear dose-response of mutation induction: DMS was more mutagenic than DES, and MMS was more mutagenic than EMS and iPMS based on mutants induced per unit mutagen concentration. However, the following relative mutagenic potency was observed when comparisons were made at 10% survival: DES > DMS; EMS > MMS > iPMS. By acceptance merit to retain
of this article, a nonexclusive,
* Postdoctoral Investigator, Institute. Present address: 21109 (U.S.A.). ** To whom ***
correspondence
the publisher royalty-free
or recipient acknowledges the right of the U.S. Govemlicense in and to any copyright covering the article.
Carcinogenesls Training Grant No. CA 06296 from the National Cancer Chemical Industry Institute of Toxicology, Research Triangle Park, NC should
be addressed.
Operated by Union Carbide Corporation for the by the Department of Energy, the Environmental Toxicological Research. Abbreviations: CHO, ethyl methanesulfonate; propyl methanesulfonate;
Department Protection
of Energy. Agency,
Research supported jointly and the National Center for
Chinese hamster ovary; DES, diethylsulfate; DMS. HGPRT, hypoxanthine-guanine phosphoribosyl MMS, methyl methanesulfonate: TG. 6-thiowanine.
dimethylsulfate; EMS, transferase; iPMS. iso-
218
Introduction Since the demonstration that alkylating agents react with nucleic acids in vivo [3], there has been considerable interest in correlating the extent of such reactions with biological effects produced by the agents. Different alkylating agents vary in the extent to which they react with various sites in DNA, and attempts have been made to relate alkylation of DNA in vivo and in vitro to cytotoxic and mutagenic effects [11,12,14,20,21]. One idea that has emerged from these studies is that alkylating agents which react by a unimolecular nucleophilic substitution (Snl) mechanism would be expected to react to a greater extent with less nucleophilic sites than agents which follow a bimolecular nucleophihc substitution (S,2) mechanism [12]. Products of nucleic acid alkylation resulting from reaction with various compounds have been identified, and the ability of these agents to react at relatively less nucleophilic sites has been found to be associated with &l-type agents [12]. This difference is potentially relevant to mutagenicity of alkylating agents, since, for example, alkylation of the N-7 position of guanine, a site of relatively high nucleophilicity, does not lead to anomalous base-pairing, whereas alkylation of a reatively less reactive site, the O-6 atom of guanine, does cause mispairing to occur [7]. Although the introcution of mispairing alterations in DNA is not the only mechanism by whch mutagenesis can occur [6], the ability of chemical agents to alkylate sites of relatively low nucleophiclity has been found to correlate with mutagenic potency in some cases. For example, the work of Ehrenberg and co-workers with alkyl alkanesulfonates in barley showed a positive correlation between mutagenic efficiency and SN1 reactivity [18]. Furthermore, the alkylating agents MMS, EMS, methylnitrosourea, and ethylnitrosourea show the same relative ability to react with the oxygen atom sites in DNA and to induce mutations in bacteriophate T4 [13]. The mutagenicity of alkylating agents in mammalian cells has been compared [10,19,20], and in these studies no clear relationship between mutation induction and known chemical reactivity was found. We have been interested in the effect of chemical structure of alkylating agents on their cytotoxicity and mutagenicity to CHO cells. The CHO/HGPRT mutational assay system has been developed, which permits quantitation of mutation induction at the HGPRT locus [9,15] with a variety of chemical and physical mutagens [ 9,15,16]. In the studies presented here, we have compared the mutagenicities of agents which are thought to react largely by the Sn2 mechanism, including alkylsulfates (DMS and DES) and an alkanesulfonate (MMS), those with predominantly Snl character (such as iPMS), and one with intermediate properties (EMS). The latter has been tested previously [ 91, and certain of these results are included for comparison. Materials
and methods
The protocol for determining cell survival and induction to 6thioguanine (TG) resistance has been published [9,15]. Briefly, in all experiments described, a subclone of CHO cells designated CHO-K1-BH4 was employed.
219
These cells were routinely maintained in Ham’s F12 medium supplemented with 10% heat-inactivated (56°C for 30 min) fetal calf serum. For determination of cytotoxicity and mutagenicity, cells were grown in F12 medium supplemented with 10% extensively dialyzed fetal calf serum to a density of approximately 10” tells/60-mm dish. Mutagens were dissolved in Hanks’ phosphate-buffered saline immediately prior to addition to cells. Cells were incubated in mutagen-containing medium for 16 h unless otherwise specified, then washed three times with Puck’s saline G [8]. Cells were removed from dishes by treatment with 0.05% trypsin, counted, and plated on 60-mm dishes for determination of single-cell survival. After incubation in fresh growth medium for 7 days, plates were fixed with 3.7% formalin and stained with dilute crystal violet solution. The effect of mutagen on singlecell survival was expressed as percent survival relative to untreated (solvent) controls. Cells not plated for survival were plated in loo-mm dishes and allowed to express the mutant phenotype for 7 days. Although the optimum expression time for each agent used has not been determined, the 7-day interval used has been found to near optimal for a number of chemical mutagens, including EMS, N-methyl-N’nitro-N-nitrosoguanidine and ICR-191, irrespective of mutagen concentration [ 15,171. At the end of the expression time, cells were plated in F12 medium lacking hypoxanthine but containing 5% extensively dialyzed fetal calf serum and 10 I.~M TG. For each mutagen concentration, 2 X 10’ cells were plated on each of five loo-mm dishes. Cell survival at the time of selection was determined by plating 200 cells in the same medium, but without TG, on each of three 60-mm dishes for each mutagen dose. After a 7-day incubation, plates were fixed, stained, and counted. As with survival plates, a cluster of 50 or more cells was considered a colony. Mutation frequency was calculated as the number of drug-resistant colonies per survivor at the time of selection. Statistical analyses of survival and mutagenicity data were performed as previously described [ 91. DMS and DES were purchased from Aldrich Chemical Company; MMS was obtained from Eastman Kodak Company. For the gift of iPMS we thank G.A. Sega, who obtained it from Koch-Light Laboratories Ltd., England. Results and discussion Dose-response relationships of cell killing, as measured by loss of cellular reproductive capacity, and induced mutation frequency for each alkylating agent are shown in Figs. l-4; certain features of these relationships are summarized in Table 1. Data from individual experiments are shown in the figures, while the values in the table represent the average of at least three determinations. The alkylsulfates varied markedly in cytotoxity. For DMS (Fig. l), doses of mutagen above 30 FM produced exponential cell killing; an average concentration (Table 1) of approximately 90 PM reduced survival to 10% that of untreated controls. In contrast, the ethyl analog of DMS, DES (Fig. 2), was considerably less toxic. Concentrations of 1000 ,uM and greater were required to affect survival appreciably, and about 2800 PM DES reduced survival to 10% (Table 1). Within the alkyl alkanesulfonates, cytotoxicity was also found to
0 0
20
40 OMS CONCN
60 (,uM)
00
”
I
5 E DES CONCN (mM)
4
Fig. 1. Doseiesponse relationships of cytotoxicity and mutagenicity of DMS. The straight-line fit for the survival data (0) Using weighted lea&squares regression analysis is log y = 2.32-0.02x. where 32 < x < 80. in which x is concentration of DMS (@kl) and y is 96 survival. The 95% confidence limits of the slope and intercept are -0.022,--0.014and 2.14.2.6l.respectively. Thestraight-line fit for the mutagenicity data (0) is f(x) = (1.64 + 1.10.x) X 10-e. The 95% confidence limits of the slope and intercept sre 0.97 X 10-6, 1.22 x 10-6 and 3.06 X lo*, 2.98 X 10-6, respectively. All confidence limits were estimated based on the residual sum of squares. Fig. 2. Dose--response relationships of cytotoxicity and mutagenicity of DES. The straight-line fit for the survival data (0) is log y = 2.83-0.94x. where 1.3 d x G 3.9, in which x is the concentration of DES (mM). The 95% confidence limits of the slope and intercept are -1.24. -064 and 2.14. 3.51. respectively. The straight-line fit of the mutagenicity data (0) is f(r) = (1.43 +295x) X 10-e. The 95% confidence X 10W6, 7.6 X 10-6, respectively. limits of the slope and intercept are 240 X 10 *, 350 X 10m6 and 4.8
decrease with size of the alkyl substituent. MMS (Table 1) was the most toxic of this group; a concentration of 95 PM was necessary to reduce relative survival to lo%, compared with concentrations of 4540 PM for iPMS and 3700 PM for EMS (reported previously, see ref. [9]). This relation between size of alkyl group and cytotoxicity to CHO cells has also been observed for certain nitrosamides and nitrosamidines [ 51. All survival curves exhibited a range of concentrations which were without significant effect on survival; MMS (Fig. 3) concentrations of 45 PM were required to appreciably alter survival (Fig. 3), and significant cell killing was produced by iPMS concentrations greater than 720 PM (Fig. 4). Despite the appearance of shoulders on their survival curves, linear relationships between induced mutation frequency and mutagen concentration were observed for all agent8 throughout the range of concentrations used. Considerable differences in mutagenic potency were noted, however. Among the alkylsulfates, DES produced higher mutation frequencies than did equitoxic doses of DMS (Fig. 5; Table 1). Because DES is much less toxic than DMS, however, DES appears to be less mutagenic when the comparison is made per
221
0
50
100
150
200
250 300
MMS CONCN (pM) Fig. 3. Dose-response relationships of cytotoxicity and mutagenicity of MMS. The straight-line fit for the survival data (0) is log y = 2.41-0.013x. where 45 G x G 273, in which x is the MMS concentration (PM). The 95% confidence limits of the slope and intercept are -0.014, 4.012 and 2.28, 2.54, respectively. The straight-line fit for the mutagenicity data (0) is f(x) = (6.5 + 0.9x) X 10-6. The 96% confidence limits of the slope and intercept are 0.3 X 10-6.1.6 X lo* and -4.8 X 10~6,16.0 X lo*, respectively.
Unit concentration of mutagen. Similarly, if alkyl alkaneSUlfOnate8 are compared per unit concentration of mutagen, the relative order of mutagenic potency reflects cytotoxicity, with MMS inducing 3.3 and 15-fold more mutations than EMS and iPMS, respectively (Table 1). When induced mutation frequencies produced by equitoxic dose8 are compared without regard to concentration, however, EMS is approximately lo- and 3.5-fold more effective in inducing mutations than MMS and iPMS, respectively. Comparisons of induced mutation frequencies as a function of cell surival (Fig. 5) were made in the mutagen concentration ranges that produce exponential loss of cellular cloning capacity. Similar results have been obtained with certain nitrosamides and nitrosamidines [5]; increasing the size of the alkyl substituent from methyl to ethyl increased mutagenicity, but higher homologs were of lower mutagenic potency than the ethyl derivative. Qualitatively similar results were reported by several investigators who have also compared the mutagenicity of EMS and MMS in other mammalian cell mutation systems. In the P388 mouse lymphoma cell line, EMS induced mutation to thymidine resistance without significant toxicity and was consistently more mutagenic than MMS at each comparable survival level [ 11. Similarly, in Chinese hamster V79 cells, mutation induction to resistance to either 8-aza-
222
r 100
3
600 0
50
0
25
d 2 2 .\”
IO 5
100 IPMS CONCN
50
(mM)
25
10
5
25
% SURVIVAL
Fig. 4. Dose-response relationships of cytotoxicity and mutagenicity of iPMS. The straight-line fit for the survival data (0) is log y = 2.62-0.31x. where 2.9 < x < 7.2, in which x is iPMS concentration (mM). The 96% confidence limits of the slope and intercept are -0.49, -0.22 and 2.06. 3.19. respectively. The straight-line fit for the mutagenicity data (0) is f(x) = (2.8 + 62.2.x) X 10-6. The 95% confidence limits for the slope and intercept are 55.2 X 10-6, 69.3 X 10-6 and -1.4 X 10-6, 7.1 X 10-6. respectively. Fig. 5. Induced mutation to illustrate the relation from ref. [91).
frequency of mutation
as a function frequency
of cell survival. and survival for
Data DMS,
from DES,
Figs. l-4 have been plotted MMS, iPMS, and EMS (data
guanine or ouabain by EMS occurred at nontoxic doses, and EMS was a more effective mutagen than MMS at equitoxic concentrations [2]. On an equirnolar basis, MMS was more cytotoxic and mutagenic than EMS in the V79 cells system when 8azaguanine resistance was used as a genetic marker [ 41. It is difficult to assess the contribution of chemical reactivity of these compounds to their biological effects, as it cannot be determined if equal concentrations of chemicals in cell-culture medium necessarily reflect equivalent
TABLE
1
CYTOTOXICITY Compound
AND
MUTAGENICITY
Concentration to produce
10%
OF ALKYLSULFATES (PM) survival
Mutation At
DMS DES MMS EMS a
89 2760 96 3700 a
iF-MS
4540 a Calculated
from
data
435 191.
frequency
10% survival
92 780 140 1550 a
in ref.
AND
ALKANESULFONATES (mutants/l06
survivors)
Per PM mutagen 1.0 0.3 1.5 0.4 a 0.1
223
exposure to relevant intracellular sites. Furthermore, we have found that modifications in experimental protocol or in culture history of cells, such as exponential growth vs. stationary state prior to treatment, can influence the observed cytotoxicity and/or mutagenicity of various agents. Thus optimal conditions for quantitating the biological effects of one agent may not necessarily be those for another. Subject to those difficulties in interpretation, it does not appear that mutagenic potency, as determined in this system, was clearly associted with SN1 reactivity. Of the agents studied, iPMS has been reported to have the greatest SN1 character [12] but was found to be less mutagenic at equitoxic doses than either DES or EMS; if compared on the basis of concentration, iPMS was the least effective mutagen tested. As previously noted, alkylation-induced mutations need not arise from promutagenic alterations in DNA, but may be produced by defective repair of alkylated DNA. In studies on induction of 8-azaguanine resistance in Chinese hamster V79 cells in which no correspondence between chemical reactivty and mutagenic potency of a series of methylating agents was observed, it was concluded that the major cause of mutations was defective repair [20]. This mechanism of mutation induction may also operate for the compounds studied here. Acknowledgements We thank J. Patrick O’Neil and E.I. Shaw for reviewing the manuscript Mayphoon H. Hsie and David G. Gosslee for statistical analyses.
and
References 1 Anderson, D., and M. Fox. The induction of thymidineand IUdR-resistant variants in P388 mouse lymphoma cells by X-rays, W, and monoand bi-functional alkylating agents. Mutation Res.. 25 (1974) 107-122. 2 Arlett, C.F., D. Turnball. S.A. Harcourt, A.R. Lehmann and C.M. Cole&. A comparison of the Sazaguanine and ouabain resistance systems for the selection of induced mutant Chinese hamster cells. Mutation Res.. 33 (1975) 261-278. 3 Brooks, P., and P.D. Lawley, The reaction of mustard gas with nucleic acids in vitro and in viva. Biothem. J.. 77 (1960) 478484. 4 Chu. E.H.Y.. and H.V. Mailing, Chemical induction of specific locus mutations in Chinese hamster cells in vitro, Proc. Natl. Acad. Sci. (U.S.A.). 61 (1968) 1306-1312. 5 Couch, D.B.. and A.W. Hsie, Mutagenicity and cytotoxicity of congeners of two classes of nitroso compounds in Chinese hamster ovary cells, Mutation Res.. 57 (1978) 209-216. 6 Drake, J.W., and R.H. Baltz. The biochemistry of mutagenesis, Annu. Rev. Biochem.. 45 (1976) ll37. 7 Gerchman, L.L.. and D.B. Ludlum, Properties of G6-methylguanine in templates for RNA polymerase. Biochim. Biophys. Acta. 308 (1973) 310-316. 8 Ham, R.G. and T.T. Puck, in: S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology. Vol. 5. Academic Press. New York, 1962. pp. 90-119. 9 Hsie, A.W., P.A. Brimer, T.J. Mitchell and D.G. Gosslee. The dose--response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells, Somat. Cell Genet.. 1 (1975) 247-261. 10 Kao, F.-T., and T.T. Puck, Genetics of somatic mammalian cells, IX. Quantitation of mutagenesis by physical and chemical agents, J. Cell. Physiol., 74 (1969) 245-258. 11 Lawley, P.D., Some aspects of the cellular response to chemical modifications of nucleic acid purines, in: E.D. Bergmann and B. Pullman (Eds.), The Purines: Theory and Experiment, Jerusalem Symposia on Quantum Chemistry and Biochemistry, Vol. IV, Academic Press, New York, 1972. PP. 678-592. 12 Lawley. P.D.. Some chemical aspects of dose--response relationships in alkylation mutagenesis. Mutation Res., 23 (1974) 283-296.
224 13 14 16
16
17 13 19 20
21
Loveless, A., Possible relevance of O-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides. Nature (London), 223 (1969) 206-207. Loveless, A., and C.L. Hampton, Inactivation and mutation of cohphage T2 by N-methyland N-ethyl-N-nitrosourea, Mutation Res., 7 (1969) l-12. O’NeiB, J.P.. P.A. Brimer. R. Machanoff, G.P. Hirsch and A.W. Hsie, A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system, Mutation Res., 45 (1977) 91-101. O’NeiR. J.P.. D.B. Couch, R. Machanoff. J.R. San Sebastian. P.A. Brimer and A. W. Hsie. A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Utilization with a variety of mutagens, Mutation Res.. 46 (1977) 103-109. O’Neill. J.P., J.C. Fuscoe and A.W. Hsie, Mutagenicity of heterocyclic nitrogen mustards (ICR compounds) in cultured mammalian cells. Center Research, in press. Osterman-Golhar. S., L. Ehrenberg and C.A. Wachmeister. Reaction kinetics and biological action in barley of monofunctional methanesulfonic esters, Radiat. Biol., 10 (1970) 303927. Roberts, J.J., and J.E. Sturrock, Enhancement by caffeine of MNU-induced mutation and chromosome aberrations in Chinese hamster cells. Mutation Res.. 20 (1973) 243-265. Roberts, J.J., J.E. Sturrock and K.N. Ward. Enhancement by caffeine of alkylation-induced cell death, mutations and chromosome aberrations in Chinese hamster cells as a result of inhibition of post-replicational DNA repair, Mutation Res., 26 (1974) 129-143. Verley, W.G., Monofunctional alkylating agents and apurinic sites in DNA, Biochem. Pharmacol., 23 (1974) 3-3.
Mutation Research, 57 (1978) 217-224 @ Elsevier/North-Holland Biomedical Press
COMPARATIVE MUTAGENICITY OF ALKYLSULFATE AND ALKANESULFONATE DERIVATIVES IN CHINESE HAMSTER OVARY CELLS
DAVID B. COUCH, * NANCY L. FORBES and ABRAHAM W. HSIE ** University of Tennessee - Oak Ridge Graduate School of Biomedical Sciences and Biology Division, Oak Ridge National Laboratory, *** Oak Ridge, Tenn. 37830 (U.S.A.) (Received 6 September 1977) (Revision received 29 November 1977) (Accepted 2 December 1977)
Summary Mutation induction and cell killing produced by selected alkylsulfates and alkanesulfonates have been quantitated using the Chinese hamster ovary/hypoxanthinewanine phosphoribosyl transferase (CHO/HGPRT) system. Doseresponse relationships of cytotoxicity and mutagenicity are presented for two alkylsulfates [dimethylsulfate (DMS), diethylsulfate (DES)] and three alkyl alkanesulfonates [methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and isopropyl methanesulfonate (iPMS)] . Under the experimental conditions employed, cytotoxicity decreased with the size of the alkyl group. DMS was more toxic than DES, and MMS was more toxic than EMS and iPMS. All agents produced linear dose-response of mutation induction: DMS was more mutagenic than DES, and MMS was more mutagenic than EMS and iPMS based on mutants induced per unit mutagen concentration. However, the following relative mutagenic potency was observed when comparisons were made at 10% survival: DES > DMS; EMS > MMS > iPMS. By acceptance merit to retain
of this article, a nonexclusive,
* Postdoctoral Investigator, Institute. Present address: 21109 (U.S.A.). ** To whom ***
correspondence
the publisher royalty-free
or recipient acknowledges the right of the U.S. Govemlicense in and to any copyright covering the article.
Carcinogenesls Training Grant No. CA 06296 from the National Cancer Chemical Industry Institute of Toxicology, Research Triangle Park, NC should
be addressed.
Operated by Union Carbide Corporation for the by the Department of Energy, the Environmental Toxicological Research. Abbreviations: CHO, ethyl methanesulfonate; propyl methanesulfonate;
Department Protection
of Energy. Agency,
Research supported jointly and the National Center for
Chinese hamster ovary; DES, diethylsulfate; DMS. HGPRT, hypoxanthine-guanine phosphoribosyl MMS, methyl methanesulfonate: TG. 6-thiowanine.
dimethylsulfate; EMS, transferase; iPMS. iso-
218
Introduction Since the demonstration that alkylating agents react with nucleic acids in vivo [3], there has been considerable interest in correlating the extent of such reactions with biological effects produced by the agents. Different alkylating agents vary in the extent to which they react with various sites in DNA, and attempts have been made to relate alkylation of DNA in vivo and in vitro to cytotoxic and mutagenic effects [11,12,14,20,21]. One idea that has emerged from these studies is that alkylating agents which react by a unimolecular nucleophilic substitution (Snl) mechanism would be expected to react to a greater extent with less nucleophilic sites than agents which follow a bimolecular nucleophihc substitution (S,2) mechanism [12]. Products of nucleic acid alkylation resulting from reaction with various compounds have been identified, and the ability of these agents to react at relatively less nucleophilic sites has been found to be associated with &l-type agents [12]. This difference is potentially relevant to mutagenicity of alkylating agents, since, for example, alkylation of the N-7 position of guanine, a site of relatively high nucleophilicity, does not lead to anomalous base-pairing, whereas alkylation of a reatively less reactive site, the O-6 atom of guanine, does cause mispairing to occur [7]. Although the introcution of mispairing alterations in DNA is not the only mechanism by whch mutagenesis can occur [6], the ability of chemical agents to alkylate sites of relatively low nucleophiclity has been found to correlate with mutagenic potency in some cases. For example, the work of Ehrenberg and co-workers with alkyl alkanesulfonates in barley showed a positive correlation between mutagenic efficiency and SN1 reactivity [18]. Furthermore, the alkylating agents MMS, EMS, methylnitrosourea, and ethylnitrosourea show the same relative ability to react with the oxygen atom sites in DNA and to induce mutations in bacteriophate T4 [13]. The mutagenicity of alkylating agents in mammalian cells has been compared [10,19,20], and in these studies no clear relationship between mutation induction and known chemical reactivity was found. We have been interested in the effect of chemical structure of alkylating agents on their cytotoxicity and mutagenicity to CHO cells. The CHO/HGPRT mutational assay system has been developed, which permits quantitation of mutation induction at the HGPRT locus [9,15] with a variety of chemical and physical mutagens [ 9,15,16]. In the studies presented here, we have compared the mutagenicities of agents which are thought to react largely by the Sn2 mechanism, including alkylsulfates (DMS and DES) and an alkanesulfonate (MMS), those with predominantly Snl character (such as iPMS), and one with intermediate properties (EMS). The latter has been tested previously [ 91, and certain of these results are included for comparison. Materials
and methods
The protocol for determining cell survival and induction to 6thioguanine (TG) resistance has been published [9,15]. Briefly, in all experiments described, a subclone of CHO cells designated CHO-K1-BH4 was employed.
219
These cells were routinely maintained in Ham’s F12 medium supplemented with 10% heat-inactivated (56°C for 30 min) fetal calf serum. For determination of cytotoxicity and mutagenicity, cells were grown in F12 medium supplemented with 10% extensively dialyzed fetal calf serum to a density of approximately 10” tells/60-mm dish. Mutagens were dissolved in Hanks’ phosphate-buffered saline immediately prior to addition to cells. Cells were incubated in mutagen-containing medium for 16 h unless otherwise specified, then washed three times with Puck’s saline G [8]. Cells were removed from dishes by treatment with 0.05% trypsin, counted, and plated on 60-mm dishes for determination of single-cell survival. After incubation in fresh growth medium for 7 days, plates were fixed with 3.7% formalin and stained with dilute crystal violet solution. The effect of mutagen on singlecell survival was expressed as percent survival relative to untreated (solvent) controls. Cells not plated for survival were plated in loo-mm dishes and allowed to express the mutant phenotype for 7 days. Although the optimum expression time for each agent used has not been determined, the 7-day interval used has been found to near optimal for a number of chemical mutagens, including EMS, N-methyl-N’nitro-N-nitrosoguanidine and ICR-191, irrespective of mutagen concentration [ 15,171. At the end of the expression time, cells were plated in F12 medium lacking hypoxanthine but containing 5% extensively dialyzed fetal calf serum and 10 I.~M TG. For each mutagen concentration, 2 X 10’ cells were plated on each of five loo-mm dishes. Cell survival at the time of selection was determined by plating 200 cells in the same medium, but without TG, on each of three 60-mm dishes for each mutagen dose. After a 7-day incubation, plates were fixed, stained, and counted. As with survival plates, a cluster of 50 or more cells was considered a colony. Mutation frequency was calculated as the number of drug-resistant colonies per survivor at the time of selection. Statistical analyses of survival and mutagenicity data were performed as previously described [ 91. DMS and DES were purchased from Aldrich Chemical Company; MMS was obtained from Eastman Kodak Company. For the gift of iPMS we thank G.A. Sega, who obtained it from Koch-Light Laboratories Ltd., England. Results and discussion Dose-response relationships of cell killing, as measured by loss of cellular reproductive capacity, and induced mutation frequency for each alkylating agent are shown in Figs. l-4; certain features of these relationships are summarized in Table 1. Data from individual experiments are shown in the figures, while the values in the table represent the average of at least three determinations. The alkylsulfates varied markedly in cytotoxity. For DMS (Fig. l), doses of mutagen above 30 FM produced exponential cell killing; an average concentration (Table 1) of approximately 90 PM reduced survival to 10% that of untreated controls. In contrast, the ethyl analog of DMS, DES (Fig. 2), was considerably less toxic. Concentrations of 1000 ,uM and greater were required to affect survival appreciably, and about 2800 PM DES reduced survival to 10% (Table 1). Within the alkyl alkanesulfonates, cytotoxicity was also found to
0 0
20
40 OMS CONCN
60 (,uM)
00
”
I
5 E DES CONCN (mM)
4
Fig. 1. Doseiesponse relationships of cytotoxicity and mutagenicity of DMS. The straight-line fit for the survival data (0) Using weighted lea&squares regression analysis is log y = 2.32-0.02x. where 32 < x < 80. in which x is concentration of DMS (@kl) and y is 96 survival. The 95% confidence limits of the slope and intercept are -0.022,--0.014and 2.14.2.6l.respectively. Thestraight-line fit for the mutagenicity data (0) is f(x) = (1.64 + 1.10.x) X 10-e. The 95% confidence limits of the slope and intercept sre 0.97 X 10-6, 1.22 x 10-6 and 3.06 X lo*, 2.98 X 10-6, respectively. All confidence limits were estimated based on the residual sum of squares. Fig. 2. Dose--response relationships of cytotoxicity and mutagenicity of DES. The straight-line fit for the survival data (0) is log y = 2.83-0.94x. where 1.3 d x G 3.9, in which x is the concentration of DES (mM). The 95% confidence limits of the slope and intercept are -1.24. -064 and 2.14. 3.51. respectively. The straight-line fit of the mutagenicity data (0) is f(r) = (1.43 +295x) X 10-e. The 95% confidence X 10W6, 7.6 X 10-6, respectively. limits of the slope and intercept are 240 X 10 *, 350 X 10m6 and 4.8
decrease with size of the alkyl substituent. MMS (Table 1) was the most toxic of this group; a concentration of 95 PM was necessary to reduce relative survival to lo%, compared with concentrations of 4540 PM for iPMS and 3700 PM for EMS (reported previously, see ref. [9]). This relation between size of alkyl group and cytotoxicity to CHO cells has also been observed for certain nitrosamides and nitrosamidines [ 51. All survival curves exhibited a range of concentrations which were without significant effect on survival; MMS (Fig. 3) concentrations of 45 PM were required to appreciably alter survival (Fig. 3), and significant cell killing was produced by iPMS concentrations greater than 720 PM (Fig. 4). Despite the appearance of shoulders on their survival curves, linear relationships between induced mutation frequency and mutagen concentration were observed for all agent8 throughout the range of concentrations used. Considerable differences in mutagenic potency were noted, however. Among the alkylsulfates, DES produced higher mutation frequencies than did equitoxic doses of DMS (Fig. 5; Table 1). Because DES is much less toxic than DMS, however, DES appears to be less mutagenic when the comparison is made per
221
0
50
100
150
200
250 300
MMS CONCN (pM) Fig. 3. Dose-response relationships of cytotoxicity and mutagenicity of MMS. The straight-line fit for the survival data (0) is log y = 2.41-0.013x. where 45 G x G 273, in which x is the MMS concentration (PM). The 95% confidence limits of the slope and intercept are -0.014, 4.012 and 2.28, 2.54, respectively. The straight-line fit for the mutagenicity data (0) is f(x) = (6.5 + 0.9x) X 10-6. The 96% confidence limits of the slope and intercept are 0.3 X 10-6.1.6 X lo* and -4.8 X 10~6,16.0 X lo*, respectively.
Unit concentration of mutagen. Similarly, if alkyl alkaneSUlfOnate8 are compared per unit concentration of mutagen, the relative order of mutagenic potency reflects cytotoxicity, with MMS inducing 3.3 and 15-fold more mutations than EMS and iPMS, respectively (Table 1). When induced mutation frequencies produced by equitoxic dose8 are compared without regard to concentration, however, EMS is approximately lo- and 3.5-fold more effective in inducing mutations than MMS and iPMS, respectively. Comparisons of induced mutation frequencies as a function of cell surival (Fig. 5) were made in the mutagen concentration ranges that produce exponential loss of cellular cloning capacity. Similar results have been obtained with certain nitrosamides and nitrosamidines [5]; increasing the size of the alkyl substituent from methyl to ethyl increased mutagenicity, but higher homologs were of lower mutagenic potency than the ethyl derivative. Qualitatively similar results were reported by several investigators who have also compared the mutagenicity of EMS and MMS in other mammalian cell mutation systems. In the P388 mouse lymphoma cell line, EMS induced mutation to thymidine resistance without significant toxicity and was consistently more mutagenic than MMS at each comparable survival level [ 11. Similarly, in Chinese hamster V79 cells, mutation induction to resistance to either 8-aza-
222
r 100
3
600 0
50
0
25
d 2 2 .\”
IO 5
100 IPMS CONCN
50
(mM)
25
10
5
25
% SURVIVAL
Fig. 4. Dose-response relationships of cytotoxicity and mutagenicity of iPMS. The straight-line fit for the survival data (0) is log y = 2.62-0.31x. where 2.9 < x < 7.2, in which x is iPMS concentration (mM). The 96% confidence limits of the slope and intercept are -0.49, -0.22 and 2.06. 3.19. respectively. The straight-line fit for the mutagenicity data (0) is f(x) = (2.8 + 62.2.x) X 10-6. The 95% confidence limits for the slope and intercept are 55.2 X 10-6, 69.3 X 10-6 and -1.4 X 10-6, 7.1 X 10-6. respectively. Fig. 5. Induced mutation to illustrate the relation from ref. [91).
frequency of mutation
as a function frequency
of cell survival. and survival for
Data DMS,
from DES,
Figs. l-4 have been plotted MMS, iPMS, and EMS (data
guanine or ouabain by EMS occurred at nontoxic doses, and EMS was a more effective mutagen than MMS at equitoxic concentrations [2]. On an equirnolar basis, MMS was more cytotoxic and mutagenic than EMS in the V79 cells system when 8azaguanine resistance was used as a genetic marker [ 41. It is difficult to assess the contribution of chemical reactivity of these compounds to their biological effects, as it cannot be determined if equal concentrations of chemicals in cell-culture medium necessarily reflect equivalent
TABLE
1
CYTOTOXICITY Compound
AND
MUTAGENICITY
Concentration to produce
10%
OF ALKYLSULFATES (PM) survival
Mutation At
DMS DES MMS EMS a
89 2760 96 3700 a
iF-MS
4540 a Calculated
from
data
435 191.
frequency
10% survival
92 780 140 1550 a
in ref.
AND
ALKANESULFONATES (mutants/l06
survivors)
Per PM mutagen 1.0 0.3 1.5 0.4 a 0.1
223
exposure to relevant intracellular sites. Furthermore, we have found that modifications in experimental protocol or in culture history of cells, such as exponential growth vs. stationary state prior to treatment, can influence the observed cytotoxicity and/or mutagenicity of various agents. Thus optimal conditions for quantitating the biological effects of one agent may not necessarily be those for another. Subject to those difficulties in interpretation, it does not appear that mutagenic potency, as determined in this system, was clearly associted with SN1 reactivity. Of the agents studied, iPMS has been reported to have the greatest SN1 character [12] but was found to be less mutagenic at equitoxic doses than either DES or EMS; if compared on the basis of concentration, iPMS was the least effective mutagen tested. As previously noted, alkylation-induced mutations need not arise from promutagenic alterations in DNA, but may be produced by defective repair of alkylated DNA. In studies on induction of 8-azaguanine resistance in Chinese hamster V79 cells in which no correspondence between chemical reactivty and mutagenic potency of a series of methylating agents was observed, it was concluded that the major cause of mutations was defective repair [20]. This mechanism of mutation induction may also operate for the compounds studied here. Acknowledgements We thank J. Patrick O’Neil and E.I. Shaw for reviewing the manuscript Mayphoon H. Hsie and David G. Gosslee for statistical analyses.
and
References 1 Anderson, D., and M. Fox. The induction of thymidineand IUdR-resistant variants in P388 mouse lymphoma cells by X-rays, W, and monoand bi-functional alkylating agents. Mutation Res.. 25 (1974) 107-122. 2 Arlett, C.F., D. Turnball. S.A. Harcourt, A.R. Lehmann and C.M. Cole&. A comparison of the Sazaguanine and ouabain resistance systems for the selection of induced mutant Chinese hamster cells. Mutation Res.. 33 (1975) 261-278. 3 Brooks, P., and P.D. Lawley, The reaction of mustard gas with nucleic acids in vitro and in viva. Biothem. J.. 77 (1960) 478484. 4 Chu. E.H.Y.. and H.V. Mailing, Chemical induction of specific locus mutations in Chinese hamster cells in vitro, Proc. Natl. Acad. Sci. (U.S.A.). 61 (1968) 1306-1312. 5 Couch, D.B.. and A.W. Hsie, Mutagenicity and cytotoxicity of congeners of two classes of nitroso compounds in Chinese hamster ovary cells, Mutation Res.. 57 (1978) 209-216. 6 Drake, J.W., and R.H. Baltz. The biochemistry of mutagenesis, Annu. Rev. Biochem.. 45 (1976) ll37. 7 Gerchman, L.L.. and D.B. Ludlum, Properties of G6-methylguanine in templates for RNA polymerase. Biochim. Biophys. Acta. 308 (1973) 310-316. 8 Ham, R.G. and T.T. Puck, in: S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology. Vol. 5. Academic Press. New York, 1962. pp. 90-119. 9 Hsie, A.W., P.A. Brimer, T.J. Mitchell and D.G. Gosslee. The dose--response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells, Somat. Cell Genet.. 1 (1975) 247-261. 10 Kao, F.-T., and T.T. Puck, Genetics of somatic mammalian cells, IX. Quantitation of mutagenesis by physical and chemical agents, J. Cell. Physiol., 74 (1969) 245-258. 11 Lawley, P.D., Some aspects of the cellular response to chemical modifications of nucleic acid purines, in: E.D. Bergmann and B. Pullman (Eds.), The Purines: Theory and Experiment, Jerusalem Symposia on Quantum Chemistry and Biochemistry, Vol. IV, Academic Press, New York, 1972. PP. 678-592. 12 Lawley. P.D.. Some chemical aspects of dose--response relationships in alkylation mutagenesis. Mutation Res., 23 (1974) 283-296.
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21
Loveless, A., Possible relevance of O-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides. Nature (London), 223 (1969) 206-207. Loveless, A., and C.L. Hampton, Inactivation and mutation of cohphage T2 by N-methyland N-ethyl-N-nitrosourea, Mutation Res., 7 (1969) l-12. O’NeiB, J.P.. P.A. Brimer. R. Machanoff, G.P. Hirsch and A.W. Hsie, A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system, Mutation Res., 45 (1977) 91-101. O’NeiR. J.P.. D.B. Couch, R. Machanoff. J.R. San Sebastian. P.A. Brimer and A. W. Hsie. A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Utilization with a variety of mutagens, Mutation Res.. 46 (1977) 103-109. O’Neill. J.P., J.C. Fuscoe and A.W. Hsie, Mutagenicity of heterocyclic nitrogen mustards (ICR compounds) in cultured mammalian cells. Center Research, in press. Osterman-Golhar. S., L. Ehrenberg and C.A. Wachmeister. Reaction kinetics and biological action in barley of monofunctional methanesulfonic esters, Radiat. Biol., 10 (1970) 303927. Roberts, J.J., and J.E. Sturrock, Enhancement by caffeine of MNU-induced mutation and chromosome aberrations in Chinese hamster cells. Mutation Res.. 20 (1973) 243-265. Roberts, J.J., J.E. Sturrock and K.N. Ward. Enhancement by caffeine of alkylation-induced cell death, mutations and chromosome aberrations in Chinese hamster cells as a result of inhibition of post-replicational DNA repair, Mutation Res., 26 (1974) 129-143. Verley, W.G., Monofunctional alkylating agents and apurinic sites in DNA, Biochem. Pharmacol., 23 (1974) 3-3.
