Role of O6-methylguanine-DNA methyltransferase in protecting from alkylating agent-induced toxicity and mutations in mice

The DNA repair protein O6 -methylguanine-DNA methyltransferase (MGMT) protects from toxicity and mutations incurred following alkylating agents by removing O6 -alkylguanine lesions. Using Mgmt −/− mice, we examined MGMT's role in protecting from in vivo mutations induced by three different alkylating agents, temozolomide (TMZ), 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and cyclophosphamide. Mutant frequencies were determined in the hypoxanthine–guanine phosphoribosyltransferase gene of splenic T-lymphocytes from C57BL/6 mice ( Mgmt +/+ and Mgmt −/−) following TMZ, BCNU or cyclophosphamide. Following TMZ, the mutation frequency was significantly greater in Mgmt −/− mice (5.5 and 9.8 × 10 −6 for 7 and 10 mg/kg TMZ, respectively) compared with vehicle-treated mice (1.0 × 10 −6 , P ≤ 0.05). In contrast, TMZ-induced mutations were not increased over vehicle in Mgmt +/+ mice. The mutation frequency of mice treated with BCNU (7.5 mg/kg) was the same regardless of Mgmt status. Similarly, pretreatment of Mgmt +/+ mice with 30 mg/kg O6 -benzylguanine, a potent inactivator of MGMT, prior to BCNU (15 mg/kg) did not result in significantly more mutations than mice treated with BCNU alone. Following cyclophosphamide, mutation frequencies significantly increased from 1.8 × 10 −6 in control-treated mice to 12.9 × 10 −6 in Mgmt +/+ and 18.1 × 10 −6 in Mgmt −/− mice, although the difference in Mgmt −/− compared with Mgmt +/+ was not significant. Acrolein and chloroacetaldehyde, metabolites of cyclophosphamide, were not mutagenic in Mgmt +/+ and Mgmt −/− mice. These results demonstrate that MGMT significantly protects against in vivo TMZ-induced mutations and that MGMT deficiency does not result in greater mutation frequency following cyclophosphamide or BCNU compared with wild-type mice.