Identification of Adducts Formed in the Reaction of 5‘-Acetoxy-N‘-Nitrosonornicotine with Deoxyguanosine and DNA

Abstract

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, adducts 12 and 16 were identified 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.

DNA Adduct Formation from Metabolic 5′-Hydroxylation of the Tobacco-Specific Carcinogen N′-Nitrosonornicotine in Human Enzyme Systems and in Rats

“…The mechanism of formation has been detailed previously. 22, 23 These adducts were not detected in vivo in these studies.…”

“…22 For the reactions with (2′ S )-5′-acetoxyNNN, we observed that ( R )-py-py-dI was formed to a greater extent than ( S )-py-py-dI (Figure 2K), demonstrating that the reaction likely proceeded via an S N 2 mechanism, but with some racemization (Scheme 2). This ratio of py-py-dI formation was the same for the reaction with dGuo as for the reaction with DNA.…”

“…Representative chromatograms of py-py-dI in hydrolysates of DNA from the liver and olfactory mucosa are shown in Figure 2B and C, respectively. As discussed in our previous publication, 22 the samples were treated with NaBH 3 CN because the equilibrium among 8 – 10 interferes with chromatography and makes quantitation difficult. There are 8 separable species present among the diastereomers of 8 , 9 , and 10 , whereas py-py-dI elutes as two easily-separable peaks.…”

Analysis of Pyridyloxobutyl DNA Adducts in F344 Rats Chronically Treated with (R)- and (S)-N‘-Nitrosonornicotine

“…Structures of POB-DNA adducts (10)(11)(12)(13)(14)(15) and DNA adducts derived from 5′-hydroxylation of NNN (16 and 17). dR: 2′-deoxyribosyl.…”

“…DNA pyridyloxobutylation in rats treated with NNN or in organ cultures was demonstrated previously by analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 7)-releasing DNA adducts (8)(9)(10)(11). 5′-Hydroxylation of NNN leads to intermediate 9 which is also DNA reactive, yielding adducts 16 and 17 (Chart 2) (12). 2′-Hydroxylation is believed to be the major bioactivation pathway of NNN in rats based on previous studies in which it was the predominant metabolic pathway in both rat esophagus and nasal cavity, target tissues for NNN tumorigenicity, and POB-DNA adducts were detected in these tissues as HPB-releasing DNA adducts (9)(10)(11)(13)(14)(15)(16)(17) However, individual POB-DNA adducts of NNN have not been previously analyzed in vitro or in vivo.…”