Mutation Research, 228 (1990) 97-103

97

Elsevier MUT 04824

Differences between survival, mutagenicity and D N A replication in MMS- and MNU-treated V79 hamster cells D. Slamefiovfi, M. Du~insk~t, T. Bastlovh and A. Ghbelovh Department of Mutagenesis and Chemical Carcinogenesis, CancerResearch Institute, Slovak Academy of Sciences, Ceskoslooenskej arm~dy 21, 812 32 Bratislava (Czechoslovakia)

(Received 1 May 1989) (Revision received 1 August 1989) (Accepted 4 September1989)

Keywords: Methyl methanesulfonate; N-Methyl-N-nitrosourea;Differencein effect

Summary After treatment with methyl methanesulfonate (MMS) or N-methyl-N-nitrosourea (MNU), the mutagenicity and survival of Chinese hamster V79 cells were investigated, as well as the inhibition of daughter D N A synthesis and, using the DNA unwinding technique and hydroxylapatite chromatography, the character of the newly synthesized DNA was studied. It was found that different cytotoxicity and mutagenicity of MMS and M N U was accompanied by different types of D N A synthesis inhibition. The treatment with the former compound resulted in a longer inhibition of D N A synthesis, while the treatment with the latter showed that as early as 2 h after exposure the percentage of nascent D N A increased. Shortly after the exposure to both alkylating agents, the newly synthesized D N A contained a higher number of gaps than control D N A , in dependence on the concentration used. During culturing after treatment, the character of nascent D N A in MMS-treated cells gradually returned to that of control DNA, while MNU-treated cells, for the whole time of our study, synthesized DNA with a larger number of gaps than control DNA. We suggest that the character of nascent daughter D N A reflects the occurrence of lesions in parental DNA. These are repaired within a shorter time in MMS- than in MNU-treated cells. The long-term persistence of lesions in the DNA of MNU-treated ceils might be one of the factors responsible not only for the higher cytotoxic but also for the many times higher mutagenic effect of this alkylating agent.

Methyl methanesulfonate (MMS) and Nmethyl-N-nitrosourea (MNU) markedly differ from each other in their mutagenic and carcinogenic effects. M N U is a strong mutagen and Correspondence: Dr. D. Siame~tovh,Department of Mutagenesis and Chemical Carcinogenesis, Cancer Research Institute, Slovak Academy of Sciences, (~eskoslovenskejarmhdy 21, 812 32 Bratislava (Czechoslovakia).

carcinogen, while MMS is a compound with very weak mutagenic and carcinogenic activity. Despite the fact that M N U reacts with D N A and other nucleophils by the so-called SN 1 mechanism (which is a monomolecular reaction) and MMS by the SN 2 mechanism (a bimolecular reaction), the nature of changes caused by them in D N A is, to a certain extent, similar (Montesano, 1981). The main differences are in the extent of methylation

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98 of N 7 of guanine (MNU: 66-70%, MMS: 81-85%

of total DNA methylation), 0 6 of guanine (MNU: 5.9-7.7%, MMS: 0.31% of total DNA methylation), and of phosphated groups (MNU: 12.18%, MMS: 0.8% of total D N A methylation). The main products with mutagenic activity are thought to be O6-methylguanine and O4-methylthymidine, but it can be expected that other methylated bases (first of all N3-methyladenine) may also exhibit promutagenic activity (Lawley, 1976; Todd et al., 1981; Singer and Kusmierek, 1982; Safhill et al., 1985). The repair of methylated bases generally must therefore be an important factor which influences the level of induced mutations. In this respect, the degree of repair of parental D N A in MMS- and MNU-treated V79 cells was assessed on the basis of analysis of daughter D N A synthesized within several hours of the treatment. Material and methods

Cell culture We used near-diploid V79 hamster cells (obtained from Dr. A. Abbondandolo, Laboratory of Mutagenesis, Pisa, Italy). The cells were cultivated in M E M medium supplemented with bovine serum (10%) and fetal bovine serum (3%), a mixture of non-essential amino acids, 0.1 g sodium pyruvate per 1000 mi, penicillin (200 U / m l ) , streptomycin (100 /~g/ml) and kanamycin (100 /zg/ml) in a humidified 5% CO 2 atmosphere at 3 7 ° C in glass petri dishes. The cells were checked under an electron microscope for the presence of PPLO; no contamination was noted. Chemicals The alkylating agents MMS (Eastman Kodak) and M N U (ICN) were dissolved and diluted in phosphate-buffered saline (PBS) immediately before use. Cytotoxicity and mutagenicity testing 1 x 10 6 cells were inoculated into each of a number of petri dishes (diameter 10 cm). After incubation for 24 h MMS (15 or 30 rain in PBS buffer) and M N U (15 or 30 min in PBS buffer) treatments were performed. V79 cells kept for 15 or 30 min in PBS buffer were used as controls. Treated and thoroughly washed cells as well as

control cells were trypsinized and plated on 5-8 petri dishes, diameter 5 cm, at 5 x 102 cells/dish for the estimation of cytotoxicity (colony-forming ability) after treatment. The assay for the detection of 6-TG ~ mutations was carried out as described previously (Slamefaovh and Ghbelovh, 1980; Slame~aovh et al., 1983). Essentially, mutagenized (treated) cells, after triple subcultivation, were used (a) for detection of 6-TG ~ mutations on the 6th day of expression, (b) for estimation of plating efficiency (necessary for the calculation of the frequency of 6-TG ~ mutations), and (c) for the next expression time testing on the 8th day of expression. For the detection of 6-TG ~ mutations the cells were plated on 5 dishes, diameter 10 cm (2 x l0 s cells/dish). After the cells had attached, 6-thioguanine (5/~g/ml) was added. Colonies were stained with methylene blue on the 7th day after plating (colony-forming ability of 6-TG ~cells) and on the 10th day after plating (6-TG r mutations). The numbers of 6-TG r mutations per 1 x l0 s viable cells were scored.

DNA inhibition test This test, originally described by Painter (1977a), has been adapted to suit the conditions of our laboratory (Slamefaovfi et al., 1980). Briefly, V79 cells were cultivated for 24 h in the presence of radioactive [~4C]thymidine (Tdr), 0.02 /~Ci/ml = 0.8 kBq/ml, in complete medium. Prelabelled cells were treated with MMS or M N U in PBS buffer for 30 min. After rinsing they were cultivated in fresh inactive medium. At various intervals after treatment a part of the samples from single groups was pulse-labelled (10 min) with radioactive [3H]Tdr, 10 / x C i / m l = 4 0 0 kBq/ml. The cells were washed with cold SSC buffer (0.15 M sodium chloride + 0.015 M sodium citrate) and the acid-insoluble fraction of cells was precipitated by 5% trichloroacetic acid (TCA). The 1 4 C ' 3 H ratio in treated samples was related to a 1 4 C : 3 H ratio in controls and the percentage inhibition of nascent D N A synthesis was calculated. Alkaline DNA unwinding technique and hydroxylapatite chromatography The number of DNA-strand breaks was determined by alkaline unwinding and subsequent separation of single-stranded and double-stranded

99 D N A by hydroxylapatite chromatography according to Ab_nstriSm and Edvardsson (1974) and AhnstriSm and Erixon (1981). V79 cells were plated on a series of glass scintillation vials (2.5 × 104 cells/cm2). The day after plating, the cells were treated with MMS and M N U in PBS buffer for 15 min. At 0, 2, 4, 6 and 8 h post-MMS or postMNU-treatment a part of the samples from single groups was pulse-labelled (30 min) with 2 p C i / m l = 80 k B q / m l radioactive [3H]Tdr. The cells were rinsed with ice-cold PBS and then lysed at 0 ° C in 1 ml of 0.03 M NaOH, 0.15 M NaC1 for 30 min in the dark and neutralized with 2 ml of 0.02 M N a H 2 P O 4 (Erixon and AhnstriSm, 1979). The molecular weight of the D N A was further reduced by ultrasonic treatment by 30 W for 10 s and then 0.1% of sodium dodecyl sulfate (SDS) was added to each sample. Single-stranded D N A was separated by hydroxylapatite chromatography with 5 ml of 0.1 M potassium phosphate buffer (pH 6.8) at 60 ° C. Double-stranded D N A was separated with 5 ml of 0.25 M potassium phosphate buffer (pH 6.8) at 60 ° C. Eluates were mixed with TCA at a final concentration of 5% and filtered through SYNPOR 6 membrane filters (porosity 0.44 pm) as previously described in detail (Slamefaovh et al., 1980). After drying of the membranes their activity was measured in a liquid scintillation counter (Beckman LS 1801) and the percentage of singlestranded D N A was counted in each sample. Results and discussion

Inhibition of D N A synthesis in mammalian cells treated with genotoxic factors can be caused mainly by inhibition of the shift of the replication fork which is brought about by the presence of D N A lesions, or by the inhibition of initiation of D N A replication. The inhibition of initiation of D N A replication is expressed either at the level of single replicons or at the level of clusters of replicons. Painter et al. (1982) on the basis of comparative studies showed that factors which cause direct D N A breaks (X- and y-radiation) inhibit D N A replication at the level of clusters of replicons, while the majority of chemical mutagens inhibit D N A replication at the level of single replicons. With respect to D N A repair, the authors suggest the former type of inhibition to be more advanta-

geous than the latter. Effects similar to those of Xand "t-radiation on D N A replication were also described with MMS (Painter, 1977b). However, monofunctional alkylating agents, such as MMS and MNU, are not capable of causing direct breaks in native D N A (Fornace et al., 1986). Singlestranded DNA breaks detected under alkaline conditions are almost exclusively breaks which arise as a consequence of incision of D N A by repair nucleases, or alkali-labile sites arising after chemical or enzymatic depurination or depyrimidination. It is probable that if this type of D N A break inhibited the initiation of replication in clusters of replicons, the character of inhibition of D N A synthesis would be similar in MMS- and MNU-treated cells. However, our study of the inhibitory effects of lower and higher concentrations of MMS and M N U on D N A synthesis did not confirm this assumption (Figs. 1 and 2). Lower concentrations of M N U (0.05-1.6 mM), which allowed 78-10% survival (Fig. 3), decreased the percentage of D N A synthesis during the first 2 h, then a gradual increase followed (Fig. 2). On the other hand, at the same concentrations of MMS, which allowed 100-42% survival (Fig. 3), maximal inhibition of D N A synthesis was observed immediately after treatment, which persisted approximately at the same level for 4 - 6 h (Fig. 1). With respect to the similar character of D N A damages caused by MMS and M N U in cell D N A (Montesano, 1981), we assume that the longer inhibition of DNA synthesis in MMS-treated cells was not caused by single-strand breaks which could inhibit the initiation of D N A synthesis in clusters of replicons as suggested by Painter (1977b), but resides in the specific inhibitory effect of MMS on cellular D N A polymerases, as shown by Norman et al. (1986). The above-mentioned differences in the character of inhibition of D N A synthesis in cells treated with MMS and M N U as well as great differences in toxic (Fig. 3) and mutagenic (Table 1) effects of the 2 alkylating agents led us to compare the character of D N A repair in MMS- and MNU-treated V79 cells. If it is right that long-term inhibition of D N A synthesis is favourable for the repair of D N A lesions, while a rapid restoration of D N A synthesis is less favorable (Tolmach et al., 1980), then the MMS-induced damage should be repaired

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