K-ras gene sequence effects on the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-DNA adducts.

The tobacco specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolically activated to electrophilic species that form methyl and pyridyloxobutyl adducts with genomic DNA, including O(6)-methylguanine, N7-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine. If not repaired, these lesions could lead to mutations and the initiation of cancer. Previous studies used ligation-mediated polymerase chain reaction (LMPCR) in combination with PAGE to examine the distribution of NNK-induced strand breaks and alkali labile lesions (e.g., N7-methylguanine) within gene sequences. However, LMPCR cannot be used to establish the distribution patterns of highly promutagenic O(6)-methylguanine and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts of NNK. We have developed methods based on stable isotope labeling HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS) that enable us to accurately quantify NNK-induced adducts at defined sites within DNA sequences. In the present study, the formation of N7-methylguanine, O(6)-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts at specific positions within a K-ras gene-derived double-stranded DNA sequence (5'-G(1)G(2)AG(3)CTG(4)G(5)TG(6)G(7)CG(8)TA G(9)G(10)C-3') was investigated following treatment with activated NNK metabolites. All three lesions preferentially formed at the second position of codon 12 (GGT), the major mutational hotspot for G-->A and G-->T base substitutions observed in smoking-induced lung tumors. Therefore, our data support the involvement of NNK and other tobacco specific nitrosamines in mutagenesis and carcinogenesis.