Mgmt deficiency alters the in vivo mutational spectrum of tissues exposed to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Abstract

It has been proposed that O(6)-methylguanine DNA methyltransferase (MGMT) gene silencing in premalignant lesions and cancers of the lung might result in the acquisition of a 'mutator' phenotype. Previously, however, we found that Mgmt(-/-) mouse DNA failed to show an increase in spontaneous mutations. We thus hypothesized that only during exposure to specific environmental carcinogens would the consequences of MGMT deficiency become evident. Metabolism of the tobacco-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) generates alkylating species that can react with the O(6) position of deoxyguanine, thereby yielding substrates for MGMT-mediated repair. To investigate how MGMT might regulate the mutational effects of NNK, Mgmt(-/-) mice were crossed with a lacI-based transgenic reporter line (Big Blue) thus enabling an assessment of the in vivo mutagenic effects of this agent. We observed the induction of a complex spectrum of NNK-dependent lacI mutations in both control and Mgmt(-/-) tissues, but only a trend in the mutant frequency increases that could be attributed to MGMT deficiency. The mutational spectra of NNK-treated Mgmt(-/-) lungs revealed an increase in the absolute number of G:C to A:T changes accompanied by a shift in these from CpG to GpG sites, consistent with an S(N)1 alkylation mechanism. In keeping with the high levels of MGMT expressed in the liver, more pronounced mutagenic effects and greater differences in O(6) position of deoxyguanosine adduct levels following NNK were observed in Mgmt(-/-) versus wild-type mice. Extrapolating to humans, MGMT-deficient cells would likely exhibit an increased mutational burden, but only following exposures to specific environmental mutagens such as NNK.

Mutagenicity of furan in female Big Blue B6C3F1 mice

“…Previously, a 6-week course of this dosing schedule induced cell proliferation in mouse liver [17], indicating that this treatment would result in sufficient cell turnover for the accumulation of mutations. The second dosing regimen was modeled after one used for the genotoxic carcinogen, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) [43]. This protocol employed single weekly doses of the carcinogen with the sacrifice occurring 1 week after the last dose.…”

Comparative Levels of O6-Methylguanine, Pyridyloxobutyl-, and Pyridylhydroxybutyl-DNA Adducts in Lung and Liver of Rats Treated Chronically with the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

“…Tissues were harvested and stored at Ϫ80°C until DNA isolation. Sandercock et al (2008) and Mijal et al (2004), we quantified O 6 -methyl-G instead of O 6 -methyl-dG. DNA was isolated from the liver and lung of three rats per group as described previously (Lao et al, 2007).…”

Context Matters: Contribution of Specific DNA Adducts to the Genotoxic Properties of the Tobacco-Specific Nitrosamine NNK

“…When the AGT activity was knocked out in this mouse strain, a modest increase in GC to AT mutations were observed in the lacI gene in the lungs of NNK-treated mice. 114 These data indicate that NNK is capable of generating mutations at both GC and AT base pairs in mouse lungs, indicating that multiple mutagenic adducts are formed from NNK.…”