Treatment of human cells with N-nitroso(acetoxymethyl)methylamine: Distribution patterns of piperidine-sensitive DNA damage at the nucleotide level of resolution are related to the sequence context

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) presen t in tobacco smoke is a major carcinogen involved in tobacco-induced lung c ancer. Its complex bioactivation along two pathways, which leads to methyla tion and pyridyloxobutylation of DNA, makes the study of NNK-induced DNA da mage difficult. We selected two nitroso compounds, N-methyl-N-nitrosourea ( MNU) and N-nitroso(acetoxymethyl)methylamine (NDMAOAc), with which to map N NK-induced DNA methylation frequency at every nucleotide position. We addre ss the issue of how sequence context and complex chromatin structures, pres ent in living cells, regulate the formation of modified purines through met hylation generated by MNU and NDMAOAc. For comparison purposes, purified DN A was treated with dimethyl sulfate (DMS). We used ligation-mediated polyme rase chain reaction to map and conduct a high-resolution footprinting analy sis of the DNA damage along the p53 gene (exons 5-8), the ras gene family ( exons 1 and 2 of H-, K-, and N-ras genes), and the c-jun promoter in living cells. The distribution of piperidine-sensitive DNA damage induced in cell ular DNA and purified DNA by MNU or NDMAOAc was identical. MNU and NDMAOAc methylate more frequently the central guanines in a run of guanines, sugges ting a regioselective mechanism for DNA methylation. In contrast, DMS methy lates more frequently guanines at the 5'-end of a guanine run; this frequen cy decreased from the 5'- to the 3'-end. While the presence of adenines in a guanine run does not affect the distribution pattern, the presence of pyr imidines does change said pattern. Our data lead us to suggest that NNK wou ld also methylate DNA sequences in a way similar to that of MNU or NDMAOAc. Footprinted areas of DNA methylated with MNU or NDMAOAc correspond to a co nsensus sequence for transcription factors AP-1, NF-Jun, CCAAT box, SP-1, a nd RSRF, as observed in c-jun promoters. Our results are in line with the f act that NNK metabolites, generated through the alpha-hydroxylation pathway s, could potentially be mutagenic, since these activated metabolites can me thylate guanines. In p53 and ras genes, the frequency of methylation of gua nines parallels the frequency of mutations of those same guanines in lung c ancer.