Investigation of 2′-Deoxyadenosine-Derived Adducts Specifically Formed in Rat Liver and Lung DNA by N′-Nitrosonornicotine Metabolism

The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as "carcinogenic to humans"(Group 1). To exert its carcinogenicity, NNN requires metabolic activation to form reactive intermediates which alkylate DNA. Previous studies have identified cytochrome P450-catalyzed 2′-hydroxylation and 5′-hydroxylation of NNN as major metabolic pathways, with preferential activation through the 5′-hydroxylation pathway in some cultured human tissues and patas monkeys. So far, the only DNA adducts identified from NNN 5′-hydroxylation in rat tissues are 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2′-deoxyinosine (Py-Py-dI), 6-[2-(3-pyridyl)-N-pyrrolidinyl]-2′-deoxynebularine (Py-Py-dN), and N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2′-deoxyadenosine (N6-HPB-dAdo) after reduction. To expand the DNA adduct panel formed by NNN 5′-hydroxylation and identify possible activation biomarkers of NNN metabolism, we investigated the formation of dAdo-derived adducts using a new highly sensitive and specific liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Two types of NNN-specific dAdo-derived adducts, N6-[5-(3-pyridyl)tetrahydrofuran-2-yl]-2′-deoxyadenosine (N6-Py-THF-dAdo) and 6-[2-(3-pyridyl)-N-pyrrolidinyl-5-hydroxy]-2′-deoxynebularine (Py-Py(OH)-dN), were observed for the first time in calf thymus DNA incubated with 5′-acetoxyNNN. More importantly, Py-Py(OH)-dN was also observed in relatively high abundance in the liver and lung DNA of rats treated with racemic NNN in the drinking water for 3 weeks. These new adducts were characterized using authentic synthesized standards. Both NMR and MS data agreed well with the proposed structures of N6-Py-THF-dAdo and Py-Py(OH)-dN. Reduction of Py-Py(OH)-dN by NaBH3CN led to the formation of Py-Py-dN both in vitro and in vivo, which was confirmed by its isotopically labeled internal standard [pyridine-d4]Py-Py-dN. The NNN-specific dAdo adducts Py-THF-dAdo and Py-Py(OH)-dN formed by NNN 5′-hydroxylation provide a more comprehensive understanding of the mechanism of DNA adduct formation by NNN.