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

Abstract

The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines <i>N</i>'-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)-<i>N</i>-pyrrolidinyl]-2'-deoxyinosine (Py-Py-dI), 6-[2-(3-pyridyl)-<i>N</i>-pyrrolidinyl]-2'-deoxynebularine (Py-Py-dN), and <i>N</i>⁶-[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (<i>N</i>⁶-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, <i>N</i>⁶-[5-(3-pyridyl)tetrahydrofuran-2-yl]-2'-deoxyadenosine (<i>N</i>⁶-Py-THF-dAdo) and 6-[2-(3-pyridyl)-<i>N</i>-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 <i>N</i>⁶-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 <i>in vitro</i> and <i>in vivo</i>, which was confirmed by its isotopically labeled internal standard [pyridine-<i>d</i>₄]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.