| Abstract
| - N‘-Nitrosonornicotine (NNN) is believed to play an important role as a cause of cancer in people whouse tobacco products and is considered to be a human carcinogen. NNN requires metabolism to formDNA adducts, which are absolutely critical to its carcinogenic properties. Previous studies have identifiedcytochrome P450-catalyzed 2‘- and 5‘-hydroxylation of NNN as potential DNA adduct forming metabolicpathways. 5‘-Hydroxylation is the more prevalent of these in monkeys and humans and is known togenerate mutagenic intermediates, but the DNA adducts formed by this pathway have never beencharacterized. In this study, we used 5‘-acetoxyNNN as a stable precursor to 5‘-hydroxyNNN andinvestigated its esterase-catalyzed reactions with deoxyguanosine (dGuo) and DNA. Adducts resultingfrom carbocation and oxonium ion intermediates, produced by the spontaneous decomposition of5‘-hydroxyNNN, were identified. The carbocation pathway resulted in the formation of 2-[2-hydroxy-5-(3-pyridyl)pyrrolidin-1-yl]deoxyinosine (12) which was characterized by comparison to an independentlysynthesized standard. Treatment of 12 with NaBH3CN produced two diastereomers of 2-[2-(3-pyridyl)pyrrolidin-1-yl]deoxyinosine (14), and their absolute configurations at the 2-position were determinedby comparison to synthetic standards. The oxonium ion pathway produced diastereomers of N2[5-(3-pyridyl)tetrahydrofuran-2-yl]dGuo (16), identified by comparison to synthetic standards. The absoluteconfiguration at the 5-position was determined by establishing the stereochemistry of the enantiomers of5-(3-pyridyl)-2-hydroxytetrahydrofuran at the 5-position and allowing these to react individually withdGuo. Treatment of 16 with NaBH3CN produced N2[4-hydroxy-4-(3-pyridyl)but-1-yl]dGuo (18) whichwas also synthesized independently. Using liquid chromatography−electrospray ionization−tandem massspectrometry with selected reaction monitoring, we identified adducts 12 and 16 as products of the reactionsof 5‘-acetoxyNNN with dGuo. Similarly, adducts 14 and 18 were identified as products of the reactionof 5‘-acetoxyNNN with DNA followed by NaBH3CN treatment and enzymatic hydrolysis. These resultsprovide the first structural characterization of DNA adducts that can be formed by 5‘-hydroxylation ofNNN.
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