title: 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 O(6)-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 O(6)-alkylG. The relative MPG and MGMT gene expression profiles were assessed by real-time RT-PCR following exposure to 0-2 microg/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 1.0 microg/ml MMS for 4h (2.5- and 6.5-fold increases respectively). These doses lie within the NOEL for MMS mutagenicity (LOEL is 1.25 microg/ml), thus this boost in MGMT expression at low doses may be responsible for efficiently repairing O(6)methylG lesions and creating the non-linear response for mutations. However, as the LOEL for MMS clastogenicity is 0.85 microg/ml, O(6)-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 O(6)-methylG is more likely to be responsible for MMS mutagenicity, with MGMT up-regulation playing a key role in removal of O(6)-alkylG lesions before they are fixed as permanent point mutations, resulting in non-linear dose-responses for direct acting genotoxins.
authors.affiliation: Institute of Life Science, School of Medicine, Swansea University, Singleton Park, Swansea, Wales, UK. s.h.doak@swansea.ac.uk
authors.forename: Shareen H
authors.initials: SH
authors.lastname: Doak
authors.forename: Katja
authors.initials: K
authors.lastname: Brüsehafer
authors.forename: Ed
authors.initials: E
authors.lastname: Dudley
authors.forename: Emma
authors.initials: E
authors.lastname: Quick
authors.forename: George
authors.initials: G
authors.lastname: Johnson
authors.forename: Russell P
authors.initials: RP
authors.lastname: Newton
authors.forename: Gareth J S
authors.initials: GJ
authors.lastname: Jenkins
mesh_terms: D002478:Cells, Cultured; D018736:DNA Adducts / Q000032:analysis; D045647:DNA Glycosylases / Q000235:genetics; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016158:Genes, p53 / Q000187:drug effects; D006801:Humans; D008741:Methyl Methanesulfonate / Q000633:toxicity*; D019055:No-Observed-Adverse-Effect Level; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D018736:DNA Adducts; D025521:Tumor Suppressor Proteins; D008741:Methyl Methanesulfonate; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045647:DNA Glycosylases; C076131:DNA-3-methyladenine glycosidase II; D045643:DNA Repair Enzymes