N-Methylpurine DNA Glycosylase Plays a Pivotal Role in the Threshold Response of Ethyl Methanesulfonate–Induced Chromosome Damage

Abstract

Genotoxic tolerance to low-level exposure of monofunctional alkylating agents is compound specific, with the mechanism pertaining to alkyl-induced genotoxic threshold response as yet unknown. N-methylpurine DNA glycosylase (MPG), an initiator glycosylase of the base excision repair (BER) pathway, typically repairs alkyl-induced DNA adducts, many of which are associated with genomic instability and tumorigenic risk. Here we demonstrate the involvement of MPG in modulating the genotoxic threshold response induced by the Sn2 alkylating agent ethyl methanesulfonate (EMS) and not the Sn1 alkylating agent N-ethyl-N-nitrosourea (ENU) in human lymphoblastoid cells and suggest the lack of N7-ethylguanine adduct repair as a key factor attributable to an observed increase in EMS-induced chromosome damage. Moreover, an increase in MPG messenger RNA expression levels in response to EMS and not ENU doses administered below the low-observed effect level substantiates the proposed specific involvement of MPG in relation to EMS-induced genotoxicity. We further report an unexpected dose-dependent decrease in the mutation frequency of the MPG-deficient cell line M09B when challenged with ENU, a response deemed consequential to a pronounced dose-dependent increase in the number of apoptotic cells relative to wild type. Collectively, these findings implicate the differential involvement of MPG-directed BER as a primary mechanism of action for the chromosome damage threshold response and cytotoxicity induced by alkane sulfonates and N-nitrosourea compounds, respectively.

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents

“…Manipulation of DNA repair activities in cells with molecular biology approaches resulted in a shift in the PoD values following treatment with alkylating agents, thus demonstrating a key role for DNA repair in PoDs for mutational/genotoxic effects [61, 82]. …”

“…Gene expression analysis has been used in cell lines with inhibited DNA repair activity to investigate the potential links between specific DNA repair pathways and shifts in PoDs, such as NOGELs, for gene mutation and chromosome damage endpoints. This integrated methodology has linked methylpurine glycosylase (MPG)/AAG DNA glycosylase to a shift in the NOGEL for MN induction in the human-derived lymphoblastoid AHH-1 cell line following exposure to EMS [82]; a causal role has not been documented for any specific EMS-induced DNA adduct in MN induction. An increase in MPG/AAG glycosylase gene expression occurred above and below NOGEL for chromosome damage as measured through MN induction at certain time points, but not for the HPRT gene mutation dose-response [82].…”

Theoretical considerations for thresholds in chemical carcinogenesis

“…It is 27 worth noting that such cytoprotective mechanisms will be 28 dependent upon the mode of action of the genotoxicant. Recent 29 evidence suggests that a PoD for mutation is influenced by the DNA 30 adduct spectra and DNA repair proficiencies [4][5][6], as well as 31 chemical clearance through detoxification for reactive oxygen 32 species (ROS) [7]. Given that mutagenesis is an early event in the 33 exposure-to-tumor scenario (the critical steps are summarized in 34 Fig.…”

Assessment of mechanisms driving non-linear dose–response relationships in genotoxicity testing

“…The 237 benefits of MPG are poorly defined and its role in cytoprotection is 238 confused perhaps explaining such a modest difference. As an 239 initiating enzyme in DNA repair, one would expect deficiency to 240 confer such a sensitizing effect [46]; and additional data supports 241 this [47]. however, the opposite has also been reported where 242 Aag À/À (i.e.…”

“…Coupled with 42 advancements in endpoint specificity, in particular the develop- 43 ment of methods and model systems for the detection and organ 44 specific evaluation of mutations (e.g. Muta TM Mouse, Big Blue 1 45 transgenic rodents [6,7]), and also in high-throughput technologies 46 (flow cytometry [8,9]) the statistical power and robustness of 47 genotoxicity assays was greatly increased. This led researchers to 48 question the reliability of the general assumption of dose-49 response linearity at low doses for numerous genotoxicants with 50 diverse modes of action (MoA).…”