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

Abstract

NNN (1) is an esophageal carcinogen in rats. 2'-Hydroxylation of NNN is believed to be the major bioactivation pathway for NNN tumorigenicity. (S)-NNN is preferentially metabolized by 2'-hydroxylation in cultured rat esophagus, whereas there is no preference for 2'-hydroxylation versus 5'-hydroxylation in the metabolism of (R)-NNN. 2'-Hydroxylation of NNN generates the reactive intermediate 4-oxo-4-(3-pyridyl)butanediazohydroxide (8), resulting in the formation of pyridyloxobutyl (POB)-DNA adducts. On the basis of these observations, we hypothesized that (S)-NNN treatment would produce higher levels of POB-DNA adducts than that by (R)-NNN in the rat esophagus. We tested this hypothesis by treating male F344 rats with 10 ppm of (R)-NNN or (S)-NNN in drinking water. After 1, 2, 5, 10, 16, or 20 weeks of treatment, POB-DNA adducts in esophageal, liver, and lung DNA were quantified by HPLC-ESI-MS/MS. In the rat esophagus, (S)-NNN treatment generated levels of POB-DNA adducts 3-5 times higher than (R)-NNN treatment, which supports our hypothesis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 14) was the major adduct detected, followed by O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 11) and O2-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (POB-Cyt, 15). O6-[4-(3-Pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-POB-dGuo, 10) was not detected. The total POB-DNA adduct levels in the esophagus were 3-11 times higher than those in the liver for (R)-NNN and 2-6 times higher than those for (S)-NNN. In contrast to the esophagus and liver, (R)-NNN treatment produced more POB-DNA adducts than (S)-NNN treatment in the rat lung, which suggested an important role for cytochrome P450 2A3 in NNN metabolism in the rat lung. In both the liver and lung, O2-POB-dThd was the predominant adduct and accumulated during the experiment. The results of this study demonstrate that individual POB-DNA adducts form and persist in the esophagi, livers, and lungs of rats chronically treated with NNN enantiomers and demonstrate that (S)-NNN produces higher levels of POB-DNA adducts in the esophagus than (R)-NNN, suggesting that (S)-NNN is more tumorigenic than (R)-NNN to the rat esophagus.

Levels of (S)-N'-Nitrosonornicotine in U.S. Tobacco Products

“…The 2′-hydroxylation pathway, which is the dominant metabolic activation pathway for NNN carcinogenicity in rat target tissues, is more favored in (S)-NNN metabolism . This, along with the results of studies on the metabolism and carcinogenicity of NNN enantiomers demonstrate that (S)-NNN is more tumorigenic than (R)-NNN to the rat esophagus and oral mucosa (Balbo et al, 2012;Lao, Yu, Kassie, Villalta, & Hecht, 2007;Zhang et al, 2009).…”

“…In vivo, the urine of rats treated with (S)-NNN contained higher levels of metabolites formed via 2′-hydroxylation than the urine of rats treated with (R)-NNN . Furthermore, treatment of rats with (S)-NNN produced two to six times higher levels of DNA adducts in the esophagus and three to five higher levels of DNA adducts in oral tissue, compared with treatment with (R)-NNN (Lao et al, 2007;Zhang et al, 2009). In agreement with these results, the treatment of rats with (S)-NNN in our recent study produced a 100% incidence of oral and esophageal tumors, compared with only 5 and 3 out of 24 rats developing oral and esophageal tumors, respectively, upon treatment with (R)-NNN (Balbo et al, 2012).…”

Quantitation of Pyridylhydroxybutyl-DNA Adducts in Liver and Lung of F-344 Rats Treated with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of Its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

“…As a control for the possible interconversion of pyridyloxobutylating and pyridylhydroxybutylating agents or the corresponding DNA adducts, two DNA samples, one each from the livers of rats treated with either enantiomer of N'-nitrosonornicotine (NNN, 16, Scheme 2) in a previous study (23), were analyzed for PHB-DNA adducts. None were detected.…”

Versatility of Y-family Sulfolobus solfataricus DNA Polymerase Dpo4 in Translesion Synthesis Past Bulky N2-Alkylguanine Adducts

“…Crotonaldehyde-and acetaldehyde-derived 1,N 2 -propanodeoxyguanosine adducts have been detected in DNA from human tissues (8). An N 2 -guanyl adduct has been detected in urine of rats treated with N-nitrosopyrrolidine (17), and pyridyloxobutyl-derived N 2 -guanyl adducts have been found in rats treated with the tobacco-specific nitrosamine N ' -nitrosonornicotine (18). The nature of the mutations produced during replication is not only dependent on the specific damage in the DNA but also on the DNA polymerases involved (19,20).…”