The repair of methyl methanesulphonate-induced lesions in the DNA of a murine lymphoma cell

The introduction of single-strand breaks into the DNA of a murine lymphoma (L5178Y) cell treated in vivo with methyl methanesulphonate (MMS) and the behaviour of these breaks on post-treatment incubation were studied. A large proportion of single-strand breaks present after MMS treatment could be repaired as shown by sedimentation in alkaline sucrose. Two inhibitors of DNA synthesis, hydroxyurea and cytosine arabinoside affected the repair process differently-hydroxyurea had only a small effect while cytosine arabinoside blocked repair and at some doses allowed further degradation of the DNA. It was also found that the level of ‘repair replication’ in the presence of cytosine arabinoside was lower than that found in the presence of hydroxyurea.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. III. Effect on DNA synthesis in vivo and on DNA polymerase activity assayed in vitro.

A single treatment with dimethylnitrosamine (DMN) but not with methyl methanesulphonate (MMS) induces liver cell carcinoma if given during the period of restorative hyperplasia following partial hepatectomy, a higher incidence of tumours being induced if the carcinogen is given during the period of DNA synthesis (24 h after the operation) than if given early in the prereplicative stage (at 6 h). To study the effect of treatment with DMN and with MMS on the regenerating liver, DNA replication was measured in vivo in partially hepatectomised animals treated with the methylating agents, and DNA polymerase activity was assayed in vitro.     

Heritable translocation test and dominant-lethal assay in mice with methyl methanesulfonate.

A dominant-lethal test and a heritable translocation test were performed with methyl methanesulphonate (MMS) at 40 mg/kg by treating the sensitive periods of post-meiotic spermatogenesis i.e. spermatozoa and spermatids. In the dominant-lethal test 25 to 60% dominant-lethal mutations were obtained depending on the mating intervals. In the heritable translocation test 11% sterile and partially sterile F1 males were observed in 250 offspring of the MMS group. All of the 14 partially sterile and 6 of the 14 sterile F1 males were demonstrated to be translocation carriers. Fertility of the partial steriles was about 40% of normal fertility. The translocation frequencies in the primary spermatocytes of the partially sterile F1 males varied between 2 and 99%. Transmission of partial sterility and translocations was confirmed in the F2 generation. There were no partially sterile or sterile males among the 245 controls.     

Organ-specific effects of DNA methylation by alkylating agents in the inbred Swiss mouse.

Young adult inbred Swiss mice given single or repeated equitoxic doses of N-methyl-N-nitrosourea (MNUA) or methyl methanesulphonate (MMS) develop thymomas and pulmonary adenomas only following MNUA in spite of nearly identical overall alkylation of DNA of tumour target tissues by both agents due mainly to the biologically ineffective product 7-methylguanine. The main difference in DNA alkylation was the production of O6-methylguinine, a known pre-mutagenic product, by MNUA in amounts 10 or more times as large as following MMS. This supports the possibility that somatic mutations are a part of the process of carcinogenesis.     

Single-strand breaks in DNA of hamster and human cells exposed to methyl methanesulfonate and ultraviolet light

The number of single-strand breaks produced in DNA after exposure to UV light or to methyl methanesulfonate (MMS) was additive when cells were exposed to both agents in close succession. Repair of the damage from either agent was partially inhibited by cytosine arabinoside, resulting in higher break frequencies under all conditions of exposure. Exposure to both agents followed by growth in cytosine arabinoside resulted in break frequencies that were approximately the same as the sum of those from each agent individually. These findings contrast with previous results in which pyrimidine dimer excision and repair replication after exposure to UV light were inhibited by MMS. These observations are not due to cell permeability changes after alkylation, but can be explained if the complex of excision-repair proteins is only partially inactivated by alkylation. Initial incisions to start repair would still occur but only limited amounts of repair replication would ensue without actual removal of the pyrimidine dimers.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. IV. Effect on methylase-mediated methylation of DNA.

The possibility that carcinogens may affect methylase-mediated methylation of replicating DNA was investigated. A system eminently suitable for this purpose is liver regenerating after partial hepatectomy, as one injection of dimethylnitrosamine (DMN) given during the ensuing period of increased DNA synthesis induces hepatocellular carcinoma. Methylation of DNA by DNA methylase normally occurs only in proportion to DNA synthesis. Therefore simultaneous measurements were made of synthesis (incorporation of [14C]adenine into DNA adenine, or of d[5-3H]cytidine into DNA cytosine), and of methylation (incorporation of [methyl-3H]methionine into 5-methylcytosine of DNA) in liver regenerating after partial hepatectomy. After treatment with DMN, the ratio of methylation: synthesis remained within the normal range. Methyl methanesulphonate (MMS), a compound which damages DNA in regenerating liver in a similar but not identical way to DMN and which does not induce tumors in liver even when given after partial hepatectomy, caused an increase in methylation in relation to synthesis. These experiments therefore do not support the view that altered DNA methylase activity is involved in carcinogenesis.     

Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 chinese hamster cells,independence of cytotoxic and mutagenic responses

The mutagenic and cytotoxic effects of 4 antineoplastic drugs, vinblastine, vincristine, adriamycin and nitrogen mustard and of several monofunctional alkylating agents have been assayed in V79 Chinese hamster cells. Vincristine, vinblastine and nitrogen mustard did not significantly increase the frequency of TG^RHGPRT^? mutants but were all highly cytotoxic. Adriamycin and the monofunctional alkylating agents were all significantly mutagenic even at the lowest doses tested (approx. 70 % survival level). Induced mutant frequency increased linearly with increasing dose whereas dose-response curves for cytotoxicity for these effective mutagens invariably showed a shoulder followed by an exponential decline. At equitoxic doses the relative mutagenic effectiveness was MNU ENU EMS MMS ? DMS. MNU was approx. 20 times more effective than MMS and DMS.Measurement of the total amount of alkylation and the relative amounts of reaction with individual DNA bases at approx. equitoxic doses of MNU and DMS indicated a significantly higher O⁶/N⁷ ratio after MNU (0.15) than after DMS (0.005). However, approx. equal numbers of mutants/10⁵ cells/μM O⁶-Meguanine were induced by these 2 agents. These results support previous conclusions, that mutagenic and cytotoxic responses are independent in V79 cells.     

DNA strand breakage caused by dichlorvos, methyl methanesulphonate and iodoacetamide in Escherichia coli and cultured Chinese hamster cells

The DNA damaging properties of dichlorvos (2,2 dichlorovinyl dimethyl phosphate), methyl methanesulphonate (MMS) and iodoacetamide (IAA) have been studied, using alkaline sucrose sedimentation. In a strain of E. coli deficient in DNA polymerase I (polA) both dichlorvos and MMS caused random strand breakage, MMS being about twice as efficient as dichlorvos on a molar basis. In pol⁺ bacteria, DNA strand breaks or alkali labile bonds were detected following treatment with roughly five-fold higher concentrations of MMS but at similar high concentrations of dichlorvos there was an all or none breakdown of DNA molecules to fragments of very low molecular weight which correlated well with lethality.DNA synthesized after treatment of pol⁺ and polA bacteria with MMS was of low molecular weight, indicating the presence of discontinuities. With dichlorvos, the effect was much smaller.Apparent all-or-none DNA breakdown was also found when the polA strain of E. coli was treated with low concentrations of iodoacetamide, an agent that does not detectably alkylate DNA. At higher concentrations the breakdown was suppressed and random strand breakage occurred instea. These effects did not occurr with pol⁺ bacteria and correlated well with the greater sensitivity to iodoacetamide of the polA strain in survival experiments. We suggest that the major DNA damage resulting from treatment with iodoacetamide and dichlorvos arises indirectly through alkylation of other cellular constituents and consequent uncontrolled nuclease attack on the DNA. Discontinuities in newly synthesized DNA and mutagenesis following dichlorvos treatment, however, presumably result from direct alkylation of DNA.Strand breakage caused by dichlorvos and MMS in Chinese hamster cells tended to correlate with the extent to which these agents alkylate DNA, but survivval tended to correlate with the alkylation of protein.     

Pre-replication recovery from induced chromosomal damage

Barley seeds were treated with diethyl sulphate (DES) or methyl methane-sulphonate (MMS) and stored at 30% water content for 3–28 days, i.e. under conditions artificially prolonging the period between the mutagenic treatment and the onset of DNA synthesis and favourable for the repair of induced DNA single-strand breaks. Storage of seed led to a marked decrease in the frequency of induced chromosomal structural changes (aberrant metaphases) as compared with treatments without storage. The recovery involved mainly chromatid-type aberrations, whereas chromosome-type aberrations induced by MMS were recovered to a lesser extent. As no, or only a negligible, DNA synthesis took place in the course of seed storage at 30% water content, the recovery from the induced chromosomal aberrations (more exactly the repair of DNA lesions responsible for aberrations) may probably be ascribed to the pre-replication type of repair. No similar recovery was observed after exposure of seeds to X-rays.     

Protoplasmic Incompatibility and Cell Lysis in PODOSPORA ANSERINA. I. Genetic Investigations on Mutations of a Novel Modifier Gene That Suppresses Cell Destruction

The distribution of chromosomal aberrations between and within chromosomes of male D, melanogaster somatic cells after treatment with UV has been analyzed. -- Distribution of the breaks between chromosomes was largely nonrandom since we found a higher aberration frequency than that expected on the Y chromosome. Moreover, within the chromosomes the aberrations are clustered in the pericentromeric heterochromatic regions. The above distribution is compared with that of the breaks induced by X rays and methyl-methane-sulphonate (MMS) which were distributed in a different pattern.     

Hypersensitivity of ataxia telangiectasia skin fibroblasts to DNA alkylating agents

3 ataxia telangiectasia (AT) fibroblast cell strains, AT4BI, AT5BI and AT2BE (CRL1343) were studied for their colony-forming ability after treatment with various concentrations of 4 different DNA alkylating agents. The results were compared to the response of fibroblast strains from 3 normal individuals. None of the AT strains were abnormally sensitive to N-methyl-N'-nitro-N-nitrosoguanidine. 1 strain (AT5BI) was significantly more sensitive to treatment with methyl methanesulfonate (MMS) based on a survival curve D0 value of 0.29 mM vs. the normal average D0 of 0.38 mM (P less than 0.02) and a D10 value of 0.85 mM vs. the normal average D10 of 1.2 mM (P less than 0.025). Strain AT4BI was also significantly more sensitive to MMS treatment when D10 values were compared (0.73 mM, P less than 0.01). All 3 AT cell strains were significantly more sensitive to treatment with ethyl methanesulfonate when D10 values were the criterion of sensitivity, AT4BI 16 mM, AT5BI 13 mM and AT2BE 15 mM vs. the normal human fibroblast average D10 value of 28 mM (P less than 0.01 for all 3 AT strains). 2 of the 3 AT cell strains (AT4BI and AT2BE) were abnormally sensitive to treatment with 4-nitroquinoline-1-oxide; the D0 values were 0.045 microM and 0.05 microM, respectively, vs. the normal average D0 value of 0.11 microM (P less than 0.01 for both AT strains). The corresponding D10 values were 0.08 microM and 0.11 microM, respectively, vs. the normal average D10 value of 0.27 microM (P less than 0.01 for AT4BI and P less than 0.025 for AT2BE). These results indicate that there is a heterogeneity in the response of AT fibroblast cell strains to treatment with DNA alkylating agents, except possibly in the case of ethylating compounds.     

Increased urinary excretion of pyrimidine and nicotinamide derivatives in rats treated with methyl methanesulphonate

Rats treated with methyl methanesulphonate (MMS) excreted significantly higher quantities of deoxycytidine, thymidine, uracil, 1-methylnicotinamide (1-meNmd) and 1-methyl-6-pyridone-3-carboxylamide (6-pyr-1-meNmd) in their urine 0–24 h after MMS injection (100 $\text{mg}\text{kg}$). Excretion of thymidine, which was not detectable in untreated rats, was dose-dependent. No increase in urinary 7-methylguanine was found, and creatinine excretion was decreased by MMS treatment. Experiments with methyl-¹⁴C-labelled MMS showed transfer of ¹⁴C-label to 7-methylguanine and 1-meNmd. X-Irradiation (500 rad) caused increased excretion of pyrimidines, like MMS, but did not increase excretion of the nicotinamide derivatives.     

A comparison of mutation induction by 14 MeV fast neutrons and 137 Cs -rays in meiotic spermatocytes in the silkworm

Barley seeds were treated with methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), stored at 15% water content and washed for 16–24 h. These treatments resulted in an increase of toxic and genetic effects. In teh DNA of embryos of such stored MMS- and EMS-treated seeds, a strong enhancement of the amount of single-strand breaks and/or alkali-labile sites took place. In contrast, the amount of alkylated sites, particularly of 7-methylguanine, was somewhat lower. It can be that the depurination and/or backbone breakage, which proceeds during the storage period, is responsible for the enhancement of toxic and genetic effects, whereas the influence of the alkylation of DNA during the storage period by the unreacted residual mutagen is negligible.     

Effect of methyl methanesulphonate (MMS) and methylene dimethanesulphonate (MDMS) on the template activity of DNA isolated from MDMS-sensitive and -resistant Yoshida cells.

Both methylene dimethanesulphonate (MDMS) and methyl methanesulphonate (MMS) cause the template activity of Yoshida cell DNA to decrease in a dose-dependent manner. The MDMS-resistant subline of Yoshida tumour is less sensitive in terms of DNA template activity than the MDMS-sensitive subline towards both agents. Although the difference in sensitivity is not reflected in the survival data after each agent, it does suggest that the DNA from each cell line differs in its capacity to function as an efficient template.     

Mutagenicity of dichlorvos and methyl methanesulphonate for Escherichia coli WP2 and some derivatives deficient in DNA repair

The mutagenic and lethal action of methyl methanesulphonate (MMS) and dichlorvos (DDVP) has been studied on Escherichia coli WP2 and some derivatives deficient in DNA repair genes. The exrA⁺ and recA⁺ alleles were necessary for significant mutagenesis by either compound, and the uvrA gene affected neither the lethal nor mutagenic responses. Increased sensitivity to both compounds was shown by the exrA and uvrAexrA strains and in a more pronounced way by the uvrApolA, recA, and uvrAexrApolA strains.Bacteria deficient at the polA locus were 2 and 3 times more mutable by DDVP and MMS respectively, consistent with the hypothesis that the absence of the polA system for the repair of single-strand gaps results in a greater proportion of the total repair being channelled through the error-prone exrA⁺/recA⁺-dependent system. Single-strand breaks were detectable by alkaline sucrose gradient centrifugation after both MMS and DDVP treatment of polA bacteria. Thus in all the tests carried out, both compounds showed similar patterns of activity, and the results are consistent with their known ability to alkylate DNA. The chief differences were quantitative; sensitivity increases were far more pronounced with MMS which was also a far more potent mutagen than DDVP.     

Methyl methanesulphonate mutagenesis in L5178Y mouse lymphoma cells.

The alkylating agent MMS was toxic to mouse lymphoma L5178Y cells and decreased their growth rate. A dose-dependent induction of thioguanine- and thymidine- but not ouabain-resistant variants was observed. The prolonged period for expression of thioguanine-resistant variants observed with other mutagens was also found in these studies. A comparison of MMS and EMS showed that MMS on a molar basis was approximately 10 times more toxic than EMS. With mutation, however, when evaluated at equal levels of cell killing MMS and EMS induced the same number of thymidine-resistant variants. For thioguanine-resistant variants MMS was approximately 10-fold less efficient than EMS, while for ouabain-resistance MMS, unlike EMBS, produced no variants at all. The ouabain results were further compared with positive results obtained using a modified Luria--Delbrück fluctuation test.     

Unscheduled DNA synthesis in human leucocytes

Polymorphonuclear leucocytes have been induced to synthesize new DNA by exposure to UV light. Preliminary observations (not included) also indicate that 6-MeV electrons and incubation with the radiomimetic agent methyl methanesulfonate (MMS) are effective agents for inducing unscheduled DNA synthesis (UDS). A study of the kinetics of UV-induced DNA synthesis suggests that there are at least two processes operating, one fast and essentially complete within the first 1–2 h and the second lasting at least 8 h.     

Radiation equivalent of genotoxic chemicals: Validation in cultured mammalian cell lines

Published data on mutations induced by ionizing radiation and 6 monofunctional alkylating agents, namely EMS, MMS, ENNG, MNNG, ENU and MNU, in different cell lines (Chinese hamster ovary, Chinese hamster lung V79, mouse lymphoma L5178 and human cells) were analysed so that radiation-equivalent chemical (REC) values could be calculated.REC values thus obtained for a given alkylating agent with different cell lines fall within a narrow range suggesting its validation in cultured mammalian cell systems including human.     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.