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In November 2004, The International Agency for Research on Cancer concluded that exposure to the tobacco-related nitrosamines NNK and N’-nitrosonornicotine is carcinogenic to humans. Metabolic activation of NNK leads to the formation of DNA adducts, which play a critical role in NNK carcinogenesis. Adducts specific to NNK result from covalent linkage of a pyridyloxobutyl (POB-1-yl) group to DNA. Furthermore, some such adducts are unstable, releasing the degradation product 4-hydroxy-1-(3-pyridyl)-1-butanone (4-HPB). Previous qualitative reports from our laboratory have established the chemical structures of the major (POB-1-yl)-DNA adducts, but quantitation was not available. In this study, we have determined the levels of each of these adducts in DNA, as well as their contribution to the biomarker of DNA pyridyloxobutylation, 4-HPB. Standards for the POB-DNA adducts O6-(POB-1-yl)-dGuo, 7-(POB-1-yl)-Gua, O2-(POB-1-yl)-dThd, and O2-(POB-1-yl)-Cyt were synthesized and used to calibrate their relative responses by reverse phase High Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS). DNA was incubated with varying amounts of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) in the presence of an esterase, conditions favorable to the formation of an active pyridyloxubutylating agent. After enzymatic and/or neutral thermal hydrolysis, isolation, and purification, the modified DNA was analyzed by HPLC-ESI-MS to quantify the relative level of each of these four adducts. O2-(POB-1-yl)-dThd was one of the two least abundant adducts, produced at levels of 0.7-2.6 times the levels of the mutagenic adduct O6-(POB-1-yl)-dGuo. Ranges in levels reflected increases in relative adduct formation at increased NNKOAc concentration. The highest adduct levels measured were those of nucleosides resulting from loss of deoxyribose upon neutral thermal hydrolysis, namely 7-(POB-1-yl)Gua and O2-(POB-1-yl)Cyt, which were formed at levels 3.6-10.7 and 1.8-6.3 times higher than O6-(POB-1-yl)-dGuo, respectively. These data support the view that these adducts are the major source of 4-HPB released from (POB-1-yl)-DNA. Furthermore, the relative quantitative importance of pyridyloxobutylation sites in DNA contributes to our understanding of the mechanism of carcinogenesis of the tobacco-specific nitrosamine NNK.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]