No-observed effect levels are associated with up-regulation of MGMT following MMS exposure

Abstract

The alkylating agents methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS) have non-linear dose–response curves, with a no-observed effect level (NOEL) and a lowest observed effect level (LOEL) for both gross chromosomal damage and mutagenicity. However, the biological mechanism responsible for the NOEL has yet to be identified. A strong candidate is DNA repair as it may be able to efficiently remove alkyl adducts at low doses resulting in a NOEL, but at higher doses fails to fully remove all lesions due to saturation of enzymatic activity resulting in a LOEL and subsequent linear increases in mutagenicity. We therefore assessed the transcriptional status of N-methylpurine-DNA glycoslase (MPG) and O6-methylguanine DNA methyltransferase (MGMT), which represent the first line of defence following exposure to alkylating agents through the respective enzymatic removal of N7-alkylG and O6-alkylG. The relative MPG and MGMT gene expression profiles were assessed by real-time RT-PCR following exposure to 0–2μg/ml MMS for 1–24h. MPG expression remained fairly steady, but in contrast significant up-regulation of MGMT was observed when cells were treated with 0.5 and 1.0μg/ml MMS for 4h (2.5- and 6.5-fold increases respectively). These doses lie within the NOEL for MMS mutagenicity (LOEL is 1.25μg/ml), thus this boost in MGMT expression at low doses may be responsible for efficiently repairing O6methylG lesions and creating the non-linear response for mutations. However, as the LOEL for MMS clastogenicity is 0.85μg/ml, O6-alkylG is unlikely to be responsible for the clastogenicity observed at these concentrations. Consequently, at low doses N7-methylG is possibly the predominant cause of MMS clastogenicity, while O6-methylG is more likely to be responsible for MMS mutagenicity, with MGMT up-regulation playing a key role in removal of O6-alkylG lesions before they are fixed as permanent point mutations, resulting in non-linear dose–responses for direct acting genotoxins.