Adaptive Response in Mammalian Cells

Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1062-1066, February 1984 Biochemistry Adaptive response in mammalian cells: Crossreactivity of different pretreatments on cytotoxicity as contrasted to mutagenicity (DNA repair/nitrosoureas/alkylating agents/O6-methylguanine/rat hepatoma cells) FRANIOISE LAVAL* AND JACQUES LAVALt *Groupe Radiochimie de I'ADN and tUnitd de Biochimie et Enzymologie, Institut Gustave Roussy, 94805 Villejuif Cedex, France Communicated by Heinz Fraenkel-Conrat, November 7, 1983

ABSTRACT Pretreatment of H4 (rat hepatoma) cells for bation of V79 Chinese hamster cells with a subtoxic dose of 48 hr with low nontoxic doses of alkylating agents [methyl MNNG or MNU also modifies the cell sensitivity towards methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguani- these agents (14). dine (MNNG), and N-methyl-N-nitrosoureal renders the cells In mammalian cells, 7-alkylguanine, 3-alkyladenine, and more resistant to the toxic effect of these compounds. Cross- 3-alkylguanine are enzymatically excised through a DNA reactivity for survival is also observed with the different alkyl- glycosylase pathway that creates an apurinic site (15-17), ating agents tested. Pretreatment with MNNG enables the cells whereas O6MeGua is repaired by a transalkylase (18). There- to be less mutated than control cultures during a subsequent fore, mammalian cells contain the potential proteins that challenge with high doses of this compound. However, pre- could mimic the situation observed in adapted E. coli. treatment with MMS does not modify the mutation frequency The aim of our work was to determine whether mammali- of cells challenged with either MMS or MNNG. The adaptive an cells could be adapted for killing and mutagenesis by re- response to mutagenesis is correlated with a faster and more peated treatment with low nontoxic concentrations of alkyl- efficient removal of 06-methylguanine in MNNG-pretreated ating agents and whether these effects could be related to the cells as compared to control cultures, whereas the disappear- type of damage induced by the drugs. We used simple alkyl- ance of this lesion is not modified in MMS-pretreated cells. As ating agents [methyl methanesulfonate (MMS) and MNNG] MMS produces less methylation at the 0' position of guanine that differ in their mutagenicity and give different spectra of and more methylation at the N7 position in comparison to methylated bases (19-21). The results show that adaptation MNNG, the results suggest that: (i) N7-methylguanine is not to killing and mutagenesis are two independent processes implicated in the adaptive response and (ii) adaptation to mu- and that adaptation to mutagenesis, in contrast to carcino- tagenesis can be correlated with the amount of 06-methylgua- genesis, can be correlated with the amount of O6MeGua pro- nine induced during the pretreatment. The effect of pretreat- duced in cellular DNA during the adaptive treatment. ment on other O-alkylated derivatives is not known. MATERIALS AND METHODS When Escherichia coli cells are continuously exposed to Chemical Reagents. Dulbecco's medium and sera were nontoxic levels of a simple alkylating agent, such as N-meth- GIBCO products. MNNG and MNU were purchased from yl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-ni- Sigma. MMS was obtained from Aldrich. MNNG and MNU trosourea (MNU), an adaptative DNA repair pathway is in- were dissolved in spectrophotometric grade acetone. Ali- duced. It protects the bacteria against the killing and muta- quots were placed in test tubes in the dark, and the acetone genic effects of high toxic doses of alkylating agents (1-3). was evaporated under a stream of N2 gas. The tubes were This adaptive response is also observed in Micrococcus lu- kept at -20°C. Prior to use, the tubes were thawed, and the teus (4). mutagen was dissolved (1 mg/ml) in 0.1 M NaOAc (pH 4.0) In Escherichia coli, different processes are implicated in and then diluted in Earle's balanced salt solution (50 ,ug/ml). the adaptive response. Adaptation to killing by alkylating Solutions were used only once. [14C]MNNG (13.9 mCi/ agents is attributed to the induction of high levels of a DNA mmol; 1 Ci = 37 GBq) was obtained from New England Nu- glycosylase, coded by the alk gene (5), which excises 3- clear, and [14C]MMS (58 mCi/mmol), from Amersham. methyladenine, 3-methylguanine (6), and 7-methylguanine Cell Culture. H4 cells, which are epithelial cells derived (6, 7). Resistance to mutagenesis has been attributed to the from a rat hepatoma (22) were obtained as a gift from J. B. induction of high levels of an 06-alkylguanine methyltrans- Little (Harvard University). They were grown in Dulbecco's ferase (8-10). medium supplemented with 5% fetal calf serum, 5% horse Evidence for an adaptive response in mammals comes serum, penicillin (50 units/ml), and streptomycin (50 ,ug/ml) from the experiments of Montesano et al. (ref. 11; reviewed in humidified 5% C02/95% air. The doubling time was about in ref. 12), showing that the livers of rats continuously ex- 14 hr. Cultures were periodically checked and found free of posed to low levels of dimethylnitrosamine and then chal- mycoplasma contamination. When H4 cells are cultured with lenged with high doses of dimethylnitrosamine, accumulate daily medium changes, they reach a stable density-inhibited 7-methylguanine but not 06-methylguanine (O6MeGua) in plateau in growth (22). their DNA. Of the several animal species used for such stud- Mutagen Treatment and Survival Measurement. Mutagen ies, only the rat shows an increase in methyltransferase. pretreatment began 24 hr after seeding 5 x 105 cells in 25- In vitro, it has been shown that chronic treatment of Chi- cm2 flasks (Falcon). Every 6 hr the medium was removed nese hamster ovary (CHO) cells with nontoxic doses of and the cells MNNG renders the cells resistant to both killing and forma- were incubated for 1 hr in 5 ml of serum-free tion of sister chromatid exchanges upon further treatment medium that was either 0.27 ,uM in MNNG or 4.85 ,tM in with high toxic levels of MNNG or MNU (13). A single incu- MNU. In the case of MMS, every 12 hr the cells were incu- Abbreviations: MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; The publication costs of this article were defrayed in part by page charge MNU, N-methyl-N-nitrosourea; MMS, methyl methanesulfonate; payment. This article must therefore be hereby marked "advertisement" O6MeGua and N7MeGua, O6- and N7-methylguanine; 6SGua, 6- in accordance with 18 U.S.C. §1734 solely to indicate this fact. thioguanine.

1062 Downloaded by guest on September 26, 2021 Biochemistry: Laval and Laval Proc. NatL. Acad. Sci USA 81 (1984) 1063 bated for 1 hr in 5 ml of serum-free medium that was 25 AuM in MMS. At the end of each incubation, the cells were rinsed twice and reincubated in 5 ml of complete medium. These pretreatments were done over a 48-hr period and correspond to nine incubations with MNNG or MNU and to five incubations with MMS. Control cultures were treated in a similar manner except that the drug was omitted. Six hours after the final adapting dose, pretreated and control cells were incubated for 1 hr with the challenge doses of either MNNG, MNU, or MMS added to 5 ml of serum-free medium. Cells were in exponential phase of growth during the experiment. At the end of the challenging treatment, cells were rinsed twice and trypsinized, and then aliquots were plated in Petri dishes. Colonies were stained and counted after 12 days as MMS, AIM described (23). The plating efficiency of control cells was be- FIG. 1. Effect of varying the adaptive dose of MMS on the sur- tween 80% and 90%. vival of H4 cells challenged with 4 mM MMS. Every 12 hr each Mutation Frequency Determination. Cells were cultured culture was pretreated for 1 hr with a different MMS concentration. and pretreated as described above. Six hours after the final After 48 hr of adaptive treatment, the cells were challenged for 1 hr adaptive dose, control and pretreated cells were challenged with 4 mM MMS and then plated for survival. for 1 hr with MNNG or MMS. They were rinsed, trypsinized, and immediately either plated for survival as described maximal enhancement of survival was observed with 25 /LM above or subcultured in fresh medium. After different ex- MMS. Higher MMS concentrations were less effective in pression times (1-9 days), the cells were plated in 100-mm terms of adaptation because of decreases in the plating effi- Petri dishes (105 viable cells per dish) in Dulbecco's medium ciency. Similar results were obtained with cells pretreated supplemented with 10% dialyzed fetal calf serum and 6-thio- and challenged with MNNG. In the case of this compound, guanine (6SGua; 2.5 ,ug/ml). Colonies were counted after 15 the pretreatment was done every 6 hr for 48 hr, and the opti- days. Mutation frequency is expressed as the number of col- mal pretreatment concentration was found to be 0.27 /iM onies in selective medium divided by (the number of cells (data not shown). Less increase of cell survival was ob- plated x the viable fraction) (22). served when H4 cells were incubated fewer than five times Analytical Methods. Cells were grown in 75-cm2 flasks, with MMS or fewer than nine times with MNNG (data not with or without pretreatment with low concentrations of al- shown). Therefore, the survival increase is a function of the kylating agents under the conditions described above, and drug concentration and of the number of incubations with then exposed either to [14C]MMS (1 mM, 58 mCi/mmol) or the drug used during the pretreatment. to [14C]MNNG (1.7 ,uM, 13.9 mCi/mmol) in serum-free me- The survival of H4 cells pretreated with 25 ,M MMS and dium. After a 30-min incubation period, the cells were washed, then challenged with different MMS concentrations (Fig. 2A) trypsinized, and lysed by the addition of NaDodSO4, and the was higher in pretreated than in control cells. When the cells cellular DNA was separated by centrifugation in CsCl densi- were pretreated with MNNG and then challenged with dif- ty gradients as described (22). The DNA was recovered, dia- ferent concentrations of this compound, an enhanced surviv- lyzed against potassium phosphate (1 mM, pH 7.6), and then al also was observed (Fig. 2B). hydrolyzed to purine bases and oligodeoxyribonucleotides In order to investigate the crossreactivity with different as described by Frei et al. (24). DNA hydrolysates were ana- alkylating agents, the survival of cells pretreated with MMS lyzed by using Waters HPLC equipment (25) as described by and then challenged with MNNG was measured. Results Frei et al. (24). The amount of DNA P was calculated as (Fig. 2C) also show an increased survival with this pretreat- described by Medcalf and Lawley (26), and the amount of ment. Other crossreactivity studies were performed with dif- alkylated bases was determined by liquid scinitillation count- ferent alkylating agents. Table 1 summarizes the results of ing. these experiments, expressed as the Do values. In all proto- Measurements of total acid-soluble thiols was as follows. cols of adaptation described, the pretreatment increased the Six hours after the end of the adaptive treatment, pretreated cell survival, although this increment was slightly, but repro- and control cells (3 x 107 cells) were washed, harvested by ducibly, higher when the cells were pretreated and chal- trypsinization, centrifuged, and resuspended in 1 ml of 5% lenged with the same agent. trichloroacetic acid. After centrifugation, aliquots of the su- pernatant were added to 1.4 ml of dithiobis(2-nitrobenzoic -r- C-- acid) (200 mg/ml in 0.2 M sodium phosphate buffer, pH 7.6) 100 C. as described by Ellman (27). The absorption was measured &I at 410 nm, and the thiol concentration was obtained from a standard curve constructed by using reduced glutathione. 10 Protein and DNA concentrations were measured as de- .. -+ scribed (22). .5 1 ST RESULTS by 0.1 _1I- L Cell Survival. The influence of a pretreatment with increasing MMS concentrations on the sensitivity of H4 cells 0 2 4 6 0 10 20 30 40 0 10 20 30 40 to this drug was first determined. Every 12 hr the cells were MMS, mM MNNG, AM MNNG, kLM incubated for 1 hr with different MMS doses. This pretreat- FIG. 2. Survival curves of H4 cells challenged with increasing ment was carried out during a 48-hr period. Six hours later doses of MMS or MNNG under nonadapted (o, A, z) or adapted (*, the cells were challenged with 4 mM MMS. The lack of tox- A, .) conditions. (A) Cells pretreated with 25 AM MMS and chal- icity was due to the pretreatment alone. Results show that lenged with MMS. (B) Cells pretreated with 0.27 AuM MNNG and the survival of challenged cells increased with the dose of challenged with MNNG. (C) Cells pretreated with 25 /LM MMS and alkylating agent used during the pretreatment (Fig. 1). The challenged with MNNG. Downloaded by guest on September 26, 2021 1064 Biochemistry: Laval and Laval Proc. NatL Acad ScL USA 81 (1984) Table 1. Crossreactivity of the adaptive response with different pretreated cultures, the number of mutants was determined mutagens at different times after the incubation with each challenge Adaptive drug Challenge drug Do, dose of mutagen (1-9 days). In both control and pretreated tuM cells, the optimal expression time was found to be 5 days. None MMS 1100. Therefore, this hypothesis cannot account for the decrease MMS MMS 2000. of mutation frequency in adapted cells. MNNG MMS 1800. When the cells were pretreated with MMS and then challenged with the same compound, the mutation frequency None MNNG 8.5 was not significantly modified under our experimental condi- MNNG MNNG 16.6 tions (Fig. 3B). The same result was obtained in a cross-ex- MMS MNNG 13.7 periment, with cells pretreated with low MMS doses and then challenged with MNNG (Fig. 3C): adapted and non- None MNU 78. adapted cells produced an equal number of 6SGua-resistant MNU MNU 139. mutants. Therefore, a pretreatment with MMS enhances the MNNG MNU 125. survival of cells challenged with either MMS or MNNG as The cells were pretreated with either MMS, MNNG, or MNU and shown in Fig. 2 but is unable to modify the mutation frequen- then challenged with increasing concentrations of these compounds. cy induced by these two compounds. Do values (drug concentration needed to reduce the surviving frac- Kinetics of 06MeGua Removal. As mutagenesis has been tion by 1/e in the exponential portion of the survival curves) were related in a number of experiments to the persistence of obtained by regression analysis of data from three separate survival O6MeGua in cellular DNA, experiments were designed to curves. Adaptative protocol and details are given in the text. measure the mutation frequency in control and adapted cells as a function of the kinetics of O6MeGua removal. In order Different parameters that could modify the cell sensitivity to avoid dilution of bases by DNA synthesis, an appropriate towards MNNG were studied. No modification of the cell number of cells (5 x 106 cells in 75-cm2 flasks) was seeded cycle occurred during the pretreatment period because in that allowed the cultures to reach confluency at the end of adapted cells the doubling time (14 ± 1 hr) and the mitotic the pretreatment period. Under these conditions, the adap- index (1.5%) were identical to those determined in control tive response was similar to that described above. Control cultures. During the pretreatment, there was no modification cells and cells pretreated with either MMS or MNNG were of the intracellular thiol level: the thiol concentration (ex- incubated for 30 min with [14C]MNNG (0.27 mM). The cells pressed as pug of glutathione per Ag of DNA) was 0.28 ± 0.02 were washed and harvested after 0, 2, 3.5, or 24 hr of subse- and 0.29 ± 0.03 in adapted and control cells, respectively. quent incubation. The cellular DNA was then purified and Furthermore, alkylation of the cellular DNA was not modi- hydrolyzed, and the O6MeGua was separated by HPLC and fied by the pretreatment: after incubation of the cells with determined. [14C]MNNG (0.1 mM, 11.3 mCi/mmol) for 30 min, the same Fig. 4 shows the kinetics of O6MeGua removal. The initial amount of radioactivity corresponding to 2.86 ± 0.05 pmol of amount of induced O6MeGua had the same value in control MNNG per Ag of DNA was incorporated in both control and cells and in cells with and without adaptation with MMS or adapted cells. MNNG. In control and MMS-pretreated cells, O6MeGua Mutation Frequency. The influence of pretreatment with was removed with the same slow biphasic kinetics: during alkylating agents on cell mutagenesis was tested by measur- the early postincubation times, about 1.7 ,umol of 06MeGua ing the appearance of 6SGua-resistant cells. When H4 cells per mol of DNA P was removed per hr, whereas later the were adapted with MNNG and then challenged with differ- rate of removal decreased to 0.23 ,mol/hr. In MNNG-pre- ent concentrations of the same agent, the mutation frequen- treated cells, O6MeGua was also removed with two-compo- cy, measured 5 days after the treatment, was markedly reduced compared to control cultures (Fig. 3A). Because this low mutation frequency in adapted cells could be due to a modification of the expression time in the

z 0 a ] I-> 80 0 2 ° 60 - u 0 3 u 40 C C:3 4 u 20 0_

O 0 10 200 1.0 2.0 2.50 10 20 MNNG, ,uM MMS, mM MNNG, ,uM Time, hr FIG. 3. Mutation frequency of H4 cells challenged with increas- FIG. 4. Kinetics of O6MeGua removal by control and pretreated ing doses of MMS or MNNG under nonadapted (A, o, o) or adapted cells. Control H4 cells (5 x 107 cells) and cells (5 x 107 cells) pre- (A, *, *) conditions. (A) Cells pretreated with 0.27 AM MNNG and treated with either MMS or MNNG were incubated for 30 min with challenged with MNNG. (B) Cells pretreated with 25 ,M MMS and [14C]MNNG. The amounts of O6MeGua were determined after dif- challenged with MMS. (C) Cells pretreated with 25 ,uM MS and ferent postincubation times by using aliquots of the purified cellular challenged with MNNG. The surviving fractions corresponding to DNA. The amount of radioactivity corresponding to O6MeGua at the drug concentrations used are given in Fig. 2 and are not less than zero time was 310 dpm. A, Control cells; *, cells pretreated with 25 -10%. The cells were incubated with 6SGua 5 days after the chal- A.tM MMS for 48 hr; A, cells pretreated with 0.27 ,AM MNNG for 48 lenge dose of the alkylating agent. hr. Downloaded by guest on September 26, 2021 Biochemistry: Laval and Laval Proc. NatL. Acad. Sci USA 81 (1984) 1065 nent kinetics. However, this lesion was removed with a the loss of the shoulder that is a characteristic of the survival much higher efficiency than in control or in MMS-adapted curves obtained with this drug (29). However, the survival cells. In this case, the rate of removal was about 5 ,tmol of enhancement observed in adapted cells cannot be related to O6MeGua per mol of DNA P per hr, and no O6MeGua was such modifications, as the cell cycle was not modified during detectable 24 hr after the challenging treatment. The kinetics the pretreatment period under our experimental conditions. of N7-methylguanine (N7MeGua) removal was determined It also has been shown that MNNG is activated by thiol also and found to be at variance with that observed for groups (30). Therefore, a modification of the intracellular thi- O6MeGua because N7MeGua was removed at the same rate ol level during the pretreatment could explain the enhanced in control and in MMS- or MNNG-pretreated cells, with a cell survival observed with this compound. This hypothesis half-life of -25 hr (data not shown). was ruled out, as the total thiol level was not modified in Comparative Alkylation of Cellular DNA by MMS or adapted cells. It also was determined that the alkylation of MNNG. In order to explain the different cell responses ob- cellular DNA by MNNG was not modified after the pretreat- served with MMS and MNNG with respect to mutation fre- ment period. quency and kinetics of O6MeGua removal, the amount of An adaptive response to the killing effect of alkylating O6MeGua induced in H4 cells by these two agents was mea- agents (MNNG and MNU) has been reported in CHO cells sured. N7MeGua also was measured as an internal control. (13) and in V79 cells (14) with different adaptive protocols. H4 cells (nonpretreated) were incubated for 1 hr with equi- Therefore, adaptation to the toxic effect of alkylating agents toxic doses of [14C]MMS (1 mM) or [14C]MNNG (1.7 4uM). occurs in different cell lines, but the survival enhancement The cellular DNA was purified and hydrolyzed, and then the varies among the cell lines studied. The adaptive response in amounts of O6MeGua and N7MeGua residues were deter- mammalian cells is of lesser magnitude than that reported to mined by HPLC analysis. Equitoxic concentrations of MMS be in bacteria, although it should be recalled that in E. coli, or MNNG yielded about the same amount of O6MeGua and adaptation is less effective in the K12 strain than in B/r (31). induced the same number of 6SGua-resistant mutants (Table However, controversy exists about the adaptive response 2). MMS induced about 25-fold more N7MeGua than did of mammalian cells to mutagenicity. After 48 hr of chronic MNNG, suggesting that this lesion is not involved in cell kill- pretreatment with MNNG, the mutation frequency in CHO ing. These results also show that a 588-fold higher MMS than cells is not modified when the cells are challenged with the MNNG concentration is required to induce the same amount same agent (32). Pretreatment with MNU increases the cell of O6MeGua. survival but does not modify the mutagenicity of V79 cells The concentrations of MMS (25 ,uM) and MNNG (0.27 (33). Opposite results were obtained by Kaina et al. (14), ,uM) used during the pretreatment did not allow accurate de- who showed that a single exposure of V79 cells to a subtoxic termination of the amounts of O6MeGua by analysis of cellu- dose of MNU or MNNG renders the cells resistant to the lar DNA. By assuming that these amounts are proportional mutagenic effect of these agents given 6 hr later. to the concentration of the alkylating agent, they can be ex- Under our experimental conditions, pretreatment with trapolated from the data reported in Table 2: in order to ob- MNNG renders the cells resistant to the mutagenic effects of tain with MMS the same amount of O6MeGua as that formed high concentrations of this compound. However, pretreat- with 0.27 ,uM MNNG in the cellular DNA, one needs 158 ment with MMS is unable to induce mutagenic resistance in ,M MMS (0.27 x 588). However, as shown in Fig. 1, this cells challenged with either MMS or MNNG. The crossreac- MMS concentration is much too toxic and does not induce tivity observed for cell survival does not exist for mutagenic- adaptive response in the cells. ity. This suggests that adaptive responses to killing and mutagenesis are at least partially independent processes. Among the different lesions induced in cellular DNA by DISCUSSION alkylating agents, at least six, including 06-alkylguanine are mutagenic (20). In the case of 06-alkylguanine, the lesion is When H4 cells are exposed to low (nontoxic) concentrations repaired by an alkyl transferase (18, 34), which is present in of alkylating agents, they become less sensitive to the toxic limited amounts in the cells (9). A possible pathway leading effect of these compounds. We observed this phenomenon to mutation resistance could be a more efficient O6MeGua with MMS, MNNG, and MNU, and crossreactivity of the removal in adapted cells. Although mammalian cells differ in adaptive response towards cell survival was also observed their ability to repair O6MeGua (35, 36), it has been shown with these drugs. that in vivo rat liver cells were able to remove this lesion It has been shown that the cell sensitivity towards MNNG (37). Under our experimental conditions, cultured rat hepa- could fluctuate throughout the cell cycle (28) and that non- toma cells remove O6MeGua, and the results show a correla- proliferating cells were more sensitive to MMS than expo- tion between the efficiency of 06MeGua removal and the nentially growing cells, the increased sensitivity arising from decrease of mutagenicity in MNNG-pretreated cells. Fur- thermore, the MMS-pretreatment, which is not effective to Table 2. Comparison of cell survival, mutation frequency, and adapt to mutagenesis, does not modify the disappearance of amounts of O6MeGua and N7MeGua induced by MMS or MNNG O6MeGua in H4 cells. in H4 cells As reported for other cell lines (36), the rate of O6MeGua Alkylating agent removal in control or MMS-adapted cells decreases with time. This suggests that in both cases the protein involved is Variable MMS MNNG consumed by the reaction and, therefore, is present in a lim- Concentration, AM 1000 1.7 ited amount in the cells. Because the removal of O6MeGua is Cell survival, % 70 75 faster and more efficient in MNNG-pretreated cells, these Mutation frequency per 105 survivors 9 + 2 10 + 2 cells could contain more O6MeGua transferase than do con- O6MeGua, pmol/mol of Gua 4.5 5 trol cells. An increase of the amount of acceptor protein after N7MeGua, ,umol/mol of Gua 1660 67 multiple exposures to MNNG occurs also in HeLa cells (38), rate in Control cells were incubated for 1 hr with [14C]MMS (1 mM) or but this result has not been correlated to the mutation [14C]MNNG (1.7 FLM), and the amounts of 06MeGua and N7MeGua the cells. were determined. Parallel cultures were incubated with nonradioac- N7MeGua was removed at the same rate in control and in tive drugs and then seeded for survival and mutagenicity determina- adapted cells, with a half-life of -25 hr, which is in agree- tions. ment with data obtained with different cell lines (26), and Downloaded by guest on September 26, 2021 1066 Biochemistry: Laval and Laval Proc. NatL Acad Sci. USA 81 (1984) rules out the possibility that 06MeGua loss in adapted cells 3. Cairns, J., Robins, P., Sedgwick, B. & Talmud, P. (1981) Prog. could be due to cell death or division. Nucleic Acid Res. Mol. Biol. 26, 237-243. MNNG results in a 3-fold increase in 4. Riazzuddin, S., Ather, A. & Ahmed, Z. (1983) J. Cell. Bio- The pretreatment by chem. Suppl. B, 7, 194. the rate of O6MeGua removal, although it renders the cells 5. Evensen, G. & Seeberg, E. (1982) Nature (London) 296, 773- resistant to the mutagenic effect of this compound under our 775. experimental conditions. This discrepancy could be related 6. Karran, P., Hjelmgren, T. & Lindahl, T. (1982) Nature (Lon- to the involvement of other lesions in the adaptive process- don) 296, 770-773. for instance, 04-methylthymine, which is removed by a 7. Laval, J., Pierre, J. & Laval, F. (1981) Proc. Natl. Acad. Sci. transalkylase in E. coli (unpublished data). USA 78, 852-855. Because different responses were obtained with MMS and 8. Olsson, M. & Lindahl, T. (1980) J. Biol. Chem. 255, 10569- MNNG as adaptive drugs, the amounts of two derivatives 10571. and 9. Foote, R. S., Mitra, S. & Pal, B. C. (1980) Biochem. Biophys. produced by these compounds, O6MeGua N7MeGua, Res. Commun. 97, 654-659. were determined in order to ascertain whether either deriva- 10. Demple, B., Jacobsson, A., Olsson, P., Robins, P. & Lindahl, tive could be correlated with the two biological end points. T. (1982) J. Biol. Chem. 257, 13776-13780. At equitoxic concentrations, MMS and MNNG produce an 11. Montesano, R., Bresil, H. & Margison, G. P. (1979) Cancer equal number of mutants and the same amount of O6MeGua, Res. 39, 1798-1802. but MMS produces about 25-fold more N7MeGua. Similar 12. Montesano, R. (1981) J. Cell. Biochem. 17, 259-273. results were obtained in hamster embryo fibroblasts (39). 13. Samson, L. & Schwartz, J. L. (1980) Nature (London) 287, This result suggests that N7MeGua is implicated neither in 861-863. cell killing nor in adaptive response. However, these equi- 14. Kaina, B. (1982) Mutat. Res. 93, 195-211. greatly, the MMS/MNNG con- 15. Singer, B. & Brent, T. P. (1981) Proc. Natl. Acad. Sci. USA toxic concentrations differ 78, 856-860. centration ratio being about 588. 16. Margison, G. P. & Pegg, A. E. (1981) Proc. Natl. Acad. Sci. If we assume that this ratio is also true when the cells are USA 78, 861-865. treated with low concentrations of alkylating agents (40), this 17. Thomas, L., Yang, C. H. & Goldthwait, D. A. (1982) Bio- means that the MMS concentration of 25 tsM, which adapts chemistry 21, 1162-1169. the cell for survival but not for mutagenesis, induces much 18. Renard, A. & Verly, W. G. (1980) FEBS Lett. 74, 122-271. less 06MeGua than does the MNNG dose (0.27 gM) used 19. Singer, B. (1975) Prog. Nucleic Acid Res. Mol. Biol. 15, 219- during the pretreatment. However, higher MMS concentra- 284, 330-332. tions cannot be used during the pretreatment because of the 20. Singer, B. & Ku~mierek, J. T. (1982) Annu. Rev. Biochem. 52, toxicity of this compound. Therefore, if 06MeGua is in- 655-693. not the 21. Singer, B. & Grunberger (1983) Molecular Biology of Muta- volved in inducing killing resistance, it is probably gens and Carcinogens (Plenum, New York), pp. 45-96. only damage involved in this process. However, O6MeGua 22. Laval, F. (1980) Proc. Natl. Acad. Sci. USA 77, 2702-2705. appears to be essential to induce mutagenesis resistance, and 23. Laval, F. & Little, J. B. (1977) Radiat. Res. 71, 571-578. adaptation to mutagenesis is probably related to the amount 24. Frei, J. V., Swenson, D. H., Warren, W. & Lawley, P. D. of 06MeGua produced during the adaptive period, although (1978) Biochem. J. 174, 1031-1044. the role of other alkylated bases cannot be excluded. 25. Leblanc, J. P., Martin, B., Cadet, J. & Laval, J. (1982) J. Biol. Variations in the amount of 06MeGua induced in the pre- Chem. 257, 3477-3483. treated cells are a probable explanation for the failure of oth- 26. Medcalf, A. S. C. & Lawley, P. D. (1982) Nature (London) 289, 796-798. er investigators to find adaptation to mutagenesis. Adaptive 27. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. treatments might produce enough damage to induce killing 28. Grisman, J. W., Greenberg, D. S., Kaufman, D. G. & Smith, resistance but not enough 06MeGua to render the cells re- G. J. (1980) Proc. Natl. Acad. Sci. USA 77, 4813-4817. sistant to mutagenesis. It should be recalled that the extent 29. Crathorn, A. R. & Shackleton, J. (1976) Mutat. Res. 15, 117- of alkylation varies with the nature of the drug (19) and the 130. drug concentration (40) used during the pretreatment. In the 30. Lawley, P. D. & Thatcher, C. J. (1970) Biochem. J. 116, 693- case of MNNG, the intracellular thiol concentration is also 707. critical in the generation of the methylation species (30). 31. Sedgwick, B. & Robins, P. (1980) Mol. Gen. Genet. 180, 85- It should be pointed out that most of the drugs used during 90. to agents. 32. Schwartz, J. 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