Identification of Adducts Formed by Pyridyloxobutylation of Deoxyguanosine and DNA by 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, a Chemically Activated form of Tobacco Specific Carcinogens

Document Type


Publication Date



The tobacco specific carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are metabolically activated to 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide (7), which is known to pyridyloxobutylate DNA. A substantial proportion of the adducts in this DNA releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 11) under various hydrolysis conditions, including neutral thermal hydrolysis. These HPB-releasing DNA adducts have been detected in target tissues of animals treated with NNK and NNN as well as in lung tissue from smokers. Although their presence in pyridyloxobutylated DNA was conclusively demonstrated 15 years ago, their structures have not been previously determined. We investigated this question in the present study by determining the structures of products formed in reactions with dGuo and DNA of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH(2)OAc, 3), a stable precursor to 7. Reaction mixtures from NNKCH(2)OAc and dGuo were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) with selected ion monitoring at m/z 415. A major peak was detected and identified as 7-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (37) by its ESI-MS fragmentation pattern and by neutral thermal hydrolysis, which converted it to 11 and 7-[4-oxo-4-(3-pyridyl)but-1-yl]Gua (26). The latter was identified by comparison to synthetic 26 using LC-ESI-MS with selected ion monitoring at m/z 299, M + 1 of 26. Further evidence was obtained by NaBH(4) reduction of 26 to 7-[4-hydroxy-4-(3-pyridyl)but-1-yl]Gua, which was also matched with a standard. Adduct 37 was similarly identified in enzyme hydrolysates of DNA reacted with NNKCH(2)OAc, accounting for 30-35% of the HPB-releasing adducts in this DNA. Several other adducts resulting from pyridyloxobutylation of the N(2)- and O(6)-positions of Gua were also identified as products in the dGuo or DNA reactions by comparison to standards; their concentrations were considerably less than that of 37. These adducts were N(2)-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (23), N(2)-[4-oxo-4-(3-pyridyl)but-2-yl]dGuo (25), N(2)-[2-(3-pyridyl)tetrahydrofuran-2-yl]dGuo (31a) (or its open chain tautomer 31b), and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (10). Adducts 23, 25, and 10 did not release HPB upon neutral thermal hydrolysis. The results of this study provide the first structural identification of an HPB-releasing DNA adduct of the tobacco specific nitrosamines NNK and NNN.