Analysis of pyridyloxobutyl DNA adducts in F344 rats chronically treated with (R)- and (S)-N'-nitrosonornicotine

Chem Res Toxicol. 2007 Feb;20(2):246-56. doi: 10.1021/tx060208j.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Administration, Oral
  • Animals
  • Chromatography, High Pressure Liquid / methods
  • DNA Adducts / analysis*
  • DNA Adducts / metabolism
  • Dose-Response Relationship, Drug
  • Esophagus / chemistry
  • Esophagus / drug effects
  • Esophagus / metabolism
  • Liver / chemistry
  • Liver / drug effects
  • Liver / metabolism
  • Lung / chemistry
  • Lung / drug effects
  • Lung / metabolism
  • Lung Neoplasms / chemically induced*
  • Male
  • Molecular Structure
  • Nitrosamines / administration & dosage
  • Nitrosamines / chemistry
  • Nitrosamines / toxicity*
  • Pyridines / analysis*
  • Pyridines / metabolism
  • Rats
  • Rats, Inbred F344
  • Sensitivity and Specificity
  • Spectrometry, Mass, Electrospray Ionization / methods
  • Stereoisomerism
  • Time Factors


  • DNA Adducts
  • Nitrosamines
  • Pyridines
  • N'-nitrosonornicotine