Mutation Research 648 (2008) 9–14 Contents lists available at ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres No-observed effect levels are associated with up-regulation of MGMT following MMS exposure Shareen H. Doak a,∗ , Katja Brüsehafer a , Ed Dudley b , Emma Quick a , George Johnson a , Russell P. Newton b , Gareth J.S. Jenkins a a Institute of Life Science, School of Medicine, Swansea University, Singleton Park, Swansea, SA2 8PP, Wales, UK b Biomolecular Analysis Mass Spectrometry Facility, Department of Environmental and Molecular Biosciences, School of the Environment and Society, Swansea University, Singleton Park, Swansea, SA2 8PP, Wales, UK article info Article history: Received 7 May 2008 Received in revised form 31 July 2008 Accepted 4 September 2008 Available online 17 October 2008 Keywords: O 6 -methylguanine DNA methyltransferase N-methylpurine-DNA glycoslase Thresholds Methyl methanesulphonate DNA adducts DNA repair 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 respon- sible 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 muta- genicity. 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 expo- sure to 0–2 g/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 g/ml MMS for 4 h (2.5- and 6.5-fold increases respectively). These doses lie within the NOEL for MMS mutagenicity (LOEL is 1.25 g/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 g/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. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The shapes of dose–response curves provide important infor- mation for risk assessments to enable the safety evaluation of chemical agents. These curves may have a variety of shapes [1] that all have different implications as they are dependent upon multiple factors ranging from specific chemical characteris- tics (such as mode of action), to cellular barriers including DNA repair, metabolic activation or membranes that enclose and pro- tect organelles [2]. However, for genotoxins that react directly with DNA the classical assumption (for carcinogen classification and regulatory purposes) has been that a linear relationship prevails ∗ Corresponding author. Tel.: +44 1792 295388; fax: +44 1792 602147. E-mail address: s.h.doak@swansea.ac.uk (S.H. Doak). with permanent genetic aberrations arising at any given level of exposure [3]. This linear model has largely prevailed because for most compounds the dose–response at low concentrations has not been considered, so as a precautionary measure linear extrapo- lations have been the default. We have recently challenged this theory and demonstrated that certain alkylating agents, a classi- cal group of electrophilic DNA reactive genotoxins, have non-linear dose–responses with respect to both gross chromosomal damage and mutagenicity [4]. Both methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS) were shown to lack any signifi- cant biological consequence below a critical dose, resulting in a no-observed effect level (NOEL) and a lowest observed effect level (LOEL). MMS and EMS therefore demonstrated a LOEL for chromo- somal damage at 0.85 and 1.4 g/ml respectively, and for point mutations at 1.25 and 1.4 g/ml. Identification of the biological mechanism that is responsible for the NOEL is necessary to jus- 0027-5107/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2008.09.016