Quantitation of Pyridyloxobutyl-DNA Adducts in Tissues of Rats Treated Chronically with (R)- or (S)-N′-Nitrosonornicotine (NNN) in a Carcinogenicity Study

Abstract

We quantified DNA adducts resulting from 2’-hydroxylation of enantiomers of the tobacco-specific nitrosamine N’-nitrosonornicotine (NNN) in tissues of male F-344 rats after 10, 30, 50, and 70 weeks of treatment with 14 ppm in the drinking water. These rats were in subgroups of a carcinogenicity study in which (S)-NNN was highly tumorigenic in the oral cavity and esophagus while (R)-NNN was relatively weakly active. DNA adducts were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry in six tissues – oral mucosa, esophageal mucosa, nasal respiratory mucosa, nasal olfactory mucosa, liver, and lung. O2-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 7) and 7-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (7-POB-dGuo, 8), the latter as 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 11), were detected at each time point in each tissue. In the target tissues for carcinogenicity, oral mucosa and esophageal mucosa, levels of 7-POB-Gua (11) and O2-POB-dThd (7) were similar, or 11 predominated, while in all other tissues at all time points for both enantiomers, 7 was clearly present in greater amounts than 11. Total measured DNA adduct levels in esophageal mucosa and oral mucosa were higher in rats treated with (S)-NNN than (R)-NNN. The highest adduct levels were found in the nasal respiratory mucosa. DNA adducts generally persisted in all tissues without any sign of substantial decreases throughout the 70 week time course. The results of this study suggest that inefficient repair of 7-POB-dGuo (8) in the rat oral cavity and esophagus may be important in carcinogenesis by NNN and support the development of these DNA adducts as potential biomarkers of NNN metabolic activation in people who use tobacco products.

Analysis of O6-[4-(3-Pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine and Other DNA Adducts in Rats Treated with Enantiomeric or Racemic N′-Nitrosonornicotine

“…4–9 In a metabolism study of enantiomeric NNN, cultured rat esophagus metabolized ( S )-NNN extensively through 2′-hydroxylation but ( R )-NNN was metabolized almost equally through the 2′ and 5′-hydroxylation pathways. 10 This is consistent with the observation that ( S )-NNN produces 3–5 times more POB-DNA adducts than ( R )-NNN in esophagus and oral mucosa 8, 9, 11 and with the carcinogenicity data, 3 but DNA adduct formation from enantiomeric and racemic NNN has never been compared previously.…”

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

“…O 2 -POB-thymidine is generally the major POB adduct in rats treated with NNN under these conditions, 10 and it was the only quantifiable adduct in these experiments. The limit of detection was 500 zmol for a dilute standard in H 2 O.…”

“…10 For samples where py-py-dI and POB adducts were both quantified, the DNA was dissolved in 2 mL buffer and half of the solution was analyzed for py-py-dI as described above, while half was analyzed for POB adducts. Briefly, the POB analysis requires heating to 100 °C for 30 min prior to enzyme hydrolysis, no NaBH 3 CN was added, and the solid-phase extraction wash step was only 10% (v/v) MeOH, while the elution step was 95% MeOH.…”

“…The 2′-hydroxylation pathway has been extensively studied in vitro and in target tissues of rats, and it is believed to be the more mutagenic and carcinogenic pathway. 5–10 However, data suggest that 5′-hydroxylation of NNN is the more prevalent metabolic pathway in non-human primates. 11 Also, in vitro data demonstrate that human liver microsomes and human P450s preferentially 5′-hydroxylate NNN, showing 3-fold to 40-fold selectivity over 2′-hydroxylation.…”

Exposure and Metabolic Activation Biomarkers of Carcinogenic Tobacco-Specific Nitrosamines

“…These amounts are up to 1000 times greater than those found in human lung and esophagus, and are comparable to those reported in tissues of rats treated with NNN or NNK. 66 The relatively high levels of HPB-releasing DNA adducts found in oral cells compared to other tissues could be a reflection of the fact that the oral mucosa is the first site of contact of tobacco smoke in cigarette smokers. It is also possible that there are other compounds in tobacco smoke, so far unidentified, that could produce HPB-releasing DNA adducts.…”