Modulation of N-Methyl-N-nitrosourea Mutagenesis in Mouse Embryo Fibroblasts Derived from the gpt Delta Mouse by an Inhibitor of the O⁶-Methylguanine Methyltransferase, MGMT

Abstract

DNA methylating agents are abundant in the environment and are sometimes used in cancer chemotherapy. They react with DNA to form methyl-DNA adducts and byproduct lesions that can be both toxic and mutagenic. Foremost among the mutagenic lesions is O 6 -methylguanine (m6G), which base pairs with thymine during replication to cause GC→AT mutations. The gpt delta C57BL/6J mouse strain of Nohmi et al. (Mol. Mutagen, 1996;28:465-70) reliably produces mutational spectra of many DNA damaging agents. In this work, mouse embryo fibroblasts (MEFs) were made from gpt delta C57BL/6J mice and evaluated as a screening tool to determine the qualitative and quantitative features of mutagenesis by N-methyl-N-nitrosourea (MNU), a direct-acting DNA alkylator that serves as a model for environmental N-nitrosamines, such as Nnitrosodimethylamine and therapeutic agents such as temozolomide. The DNA repair protein MGMT (O 6 -methylguanine DNA methyltransferase) protects against environmental mutagenesis by DNA methylating agents and, by removing m6G, limits the therapeutic potential of temozolomide in cancer therapy. The gpt delta MEFs were treated with MNU to establish dosedependent toxicity. In parallel, MNU mutagenicity was determined in the presence and absence of the MGMT inhibitor AA-CW236 (4-(2-(5-(chloromethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)-3,5-dimethylisoxazole). With and without the inhibitor, the principal mutagenic event of MNU was GC→AT, but more mutations were observed when the inhibitor was present. Evidence that the mutagenic lesion was m6G was based on mass spectral data collected using O 6 -methyl-d 3 -guanine as an internal standard; m6G levels were higher in AA-CW236 treated MEFs by an amount proportional to the higher mutation

Molecular origins of mutational spectra produced by the environmental carcinogen N-nitrosodimethylamine and SN1 chemotherapeutic agents

“…The present work defined the high-resolution mutagenic fingerprints of NDMA, TMZ, STZ and MNU across 96 3-base contexts in the mouse genome. As expected ( 40 , 41 , 46 , 47 ), the mutations of NDMA were primarily GC→AT transitions and were strongly enhanced in cells lacking O 6 -methylguanine-methyltransferase (MGMT), a direct reversal DNA repair protein that removes the methyl group from m6G, restoring undamaged guanine. The detailed mutational landscape across all 3-base contexts was the same in the presence or absence of MGMT, indicating that the repair protein did not favor some contexts over others.…”

“…As one example, DNA repair systems offer defenses against several DNA adducts and the loss or exceeded capacity of these defenses enhances cancer risk ( 37–39 ). Many studies with repair-deficient cells or animals show that the levels of specific methylated DNA adducts typically track with mutagenic and carcinogenic endpoints, pointing to the role that these adducts could have in malignant transformation and the role that specific repair systems have to prevent cancer initiation ( 40 , 41 ). With agents such as NDMA, a second factor that determines risk is the level of expression of enzymatic systems that generate the DNA-interactive metabolites.…”

Dissection of Cancer Mutational Signatures with Individual Components of Cigarette Smoking

“…In order to compare cellular responses to different compounds at concentration levels that induce similar cytotoxic responses, in line with previous in vitro mutational signature studies, , we first determined an IC 50 value for each chemical. Furthermore, since metabolic activation is required for genotoxicity of the majority of the compounds (SI Table 1) and BEAS-2B cells have low P450 expression, S9 mix from rat liver was used.…”

“…We used human lung bronchial BEAS-2B cells because they are noncancerous cells that can become malignant upon chemical exposure, which makes this cell line a relevant model to study aspects of lung carcinogenesis. 14 In order to compare cellular responses to different compounds at concentration levels that induce similar cytotoxic responses, in line with previous in vitro mutational signature studies, 5,7 we first determined an IC 50 value for each chemical. Furthermore, since metabolic activation is required for genotoxicity of the majority of the compounds (SI Table 1) and BEAS-2B cells have low P450 expression, 15 S9 mix from rat liver was used.…”

Next-generation DNA damage sequencing

“…137,[144][145][146] In contrast, recent work from Essigmann and coworkers showed no specific mutational patterns arising from AGT repair in mouse cells treated with the alkylation agent N-methyl-N-nitrosourea (MNU). 147 However, there is little work on AGT accessibility to different chromatin states in mammalian cells. Thus, developing a mapping method for methylation formation and repair processes would help fill this gap.…”