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O6-Methylguanine-DNA methyltransferase (MGMT) is responsible for removal of O6-alkylguanine from DNA induced by alkylating mutagens/carcinogens. To analyze the involvement of O6-alkylguanine in the generation and MGMT in avoidance of various genotoxic effects of alkylating agents, we transfected Chinese hamster ovary (CHO) cells that lack MGMT activity with human MGMT cDNA cloned into a mammalian expression vector (pSV2MGMT). A high proportion (60-80%) of transfectants selected for a cotransfected neo gene survived treatment with high doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU). Parallel transfections with an expression vector containing the bacterial ada gene (pSV2ada) showed the human MGMT to be more effective than the ada expression vector in mediating alkylation resistance. Various clonal CHO cell lines have been established stably transfected with the human MGMT cDNA. The transfectants expressed human MGMT at levels ranging from 8600 to 210,000 molecules per cell. The high MGMT expressors became strongly resistant to the killing effects of MNNG, HeCNU, N-methyl-N-nitrosourea (MNU) and, to a significant lesser degree, methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). No killing resistance was observed to N-ethyl-N-nitrosourea (ENU), though the MGMT and ada transfectants showed reduction in mutation frequency induced by this agent. Protection from mutation induction by MGMT (and ada) expression was also demonstrated for MNNG. The transfectants were also protected from the sister chromatid exchange (SCE) inducing and, to a lesser degree, clastogenic effect of MNNG and MNU, and slightly to EMS and MMS. Again no protection was observed towards ENU. Correlations between MGMT activity and resistance to a given end point suggest that, for MNNG, O6-methylguanine is the preponderant toxic, mutagenic and SCE inducing lesion. About 90% of MNNG (and MNU) induced SCEs and nearly all of the MNNG-induced gene mutations seem to be due to this adduct. For alkylation-induced chromosomal aberrations, however, and for cell killing and SCEs induced by MMS, EMS and ENU, other lesions than O6-alkylguanine appear to be of major importance. The data strongly support the view that O6-methylguanine is a genotoxic lesion and MGMT a function decisively involved in avoidance of genotoxic effects in cells exposed to MNNG and related compounds. They indicate also that it is important to take into account the property and mode of action of any given alkylating agent in assessing the protective role of MGMT against alkylation-induced genotoxicity.

We have studied the influence of anoxia and respiratory deficiency (RD) in yeast on the cytotoxic and recombinogenic effects of 5 direct-acting alkylating agents, namely N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). We found that the effects of both conditions parallel each other for MMS, MNNG, MNU and ENU. Both anoxia and RD did not modify the effects of MMS to any significant extent. On the other hand, anoxic and respiratory-deficient cells were found to be more resistant than euoxic and respiratory-proficient cells respectively for MNNG, MNU and ENU. In the case of EMS, which is similar to MMS in its chemical reaction with DNA, the respiratory-deficient cells were found to be more sensitive than the respiratory-proficient ones. These studies indicate that the response of anoxic and respiratory-deficient cells cannot be predicted solely on the basis of the chemical reactivity pattern of the alkylating agents. The physiological state which exists under these conditions may exert considerable influence on the cellular response.