N'-Nitrosonornicotine (NNN) is believed to play an important role as a cause of
cancer in people who use tobacco products and is considered to be a human
carcinogen. NNN requires metabolism to form
DNA adducts, which are absolutely critical to its carcinogenic properties. Previous studies have identified
cytochrome P450-catalyzed 2'- and 5'-hydroxylation of NNN as potential
DNA adduct forming metabolic pathways. 5'-Hydroxylation is the more prevalent of these in monkeys and humans and is known to generate mutagenic intermediates, but the
DNA adducts formed by this pathway have never been characterized. In this study, we used
5'-acetoxyNNN as a stable precursor to 5'-hydroxyNNN and investigated its
esterase-catalyzed reactions with
deoxyguanosine (dGuo) and
DNA. Adducts resulting from carbocation and
oxonium ion intermediates, produced by the spontaneous decomposition of 5'-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 independently synthesized standard. Treatment of 12 with
NaBH(3)CN produced two diastereomers of 2-[2-(3-pyridyl)pyrrolidin-1-yl]
deoxyinosine (14), and their absolute configurations at the 2-position were determined by comparison to synthetic standards. The
oxonium ion pathway produced diastereomers of N(2)[5-(3-pyridyl)
tetrahydrofuran-2-yl]dGuo (16), identified by comparison to synthetic standards. The absolute configuration at the 5-position was determined by establishing the stereochemistry of the enantiomers of 5-(3-pyridyl)-2-hydroxytetrahydrofuran at the 5-position and allowing these to react individually with dGuo. Treatment of 16 with
NaBH(3)CN produced N(2)[4-hydroxy-4-(3-pyridyl)but-1-yl]dGuo (18) which was also synthesized independently. Using liquid chromatography-electrospray ionization-tandem mass spectrometry with selected reaction monitoring, we identified adducts 12 and 16 as products of the reactions of
5'-acetoxyNNN with dGuo. Similarly, adducts 14 and 18 were identified as products of the reaction of
5'-acetoxyNNN with
DNA followed by
NaBH(3)CN treatment and enzymatic hydrolysis. These results provide the first structural characterization of
DNA adducts that can be formed by 5'-hydroxylation of NNN.