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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.

SUBMITTER: Lao Y

PROVIDER: S-EPMC2518847 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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

Lao Yanbin Y Yu Nanxiong N Kassie Fekadu F Villalta Peter W PW Hecht Stephen S SS

Chemical research in toxicology 20070201 2

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 adduc ...[more]

PMID: 17305408

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Project description:The tobacco-specific carcinogens N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) require metabolic activation to exert their carcinogenicity. NNN and NNK are metabolized to the same reactive diazonium ions, which alkylate DNA forming pyridyloxobutyl (POB) DNA base and phosphate adducts. We have characterized the formation of both POB DNA base and phosphate adducts in NNK-treated rats and the formation of POB DNA base adducts in NNN-treated rats. However, POB DNA phosphate adducts in NNN-treated rats are still uncharacterized. In this study, we quantified the levels of POB DNA phosphate adducts in tissues of rats chronically treated with ( S)-NNN or ( R)-NNN for 10, 30, 50, and 70 weeks during a carcinogenicity study. The highest amounts of POB DNA phosphate adducts were observed in the esophagus of the ( S)-NNN-treated rats, with a maximum level of 5400 ± 317 fmol/mg DNA at 50 weeks. The abundance of POB DNA phosphate adducts in the esophagus was consistent with the results of the carcinogenicity study showing that the esophagus was the primary site of tumor formation from treatment with ( S)-NNN. Compared to the ( R)-NNN group, the levels of POB DNA phosphate adducts were higher in the oral mucosa, esophagus, and liver, while lower in the nasal mucosa of the ( S)-NNN-treated rats. Among 10 combinations of all isomers of POB DNA phosphate adducts, Ap(POB)C and combinations with thymidine predominated across all the rat tissues examined. In the primary target tissue, esophageal mucosa, Ap(POB)C accounted for ∼20% of total phosphate adducts in the ( S)-NNN treatment group throughout the 70 weeks, with levels ranging from 780 ± 194 to 1010 ± 700 fmol/mg DNA. The results of this study showed that POB DNA phosphate adducts were present in high levels and persisted in target tissues of rats chronically treated with ( S)- or ( R)-NNN. These results improve our understanding of DNA damage during NNN-induced carcinogenesis. The predominant POB DNA phosphate isomers observed, such as Ap(POB)C, may serve as biomarkers for monitoring chronic exposure of tobacco-specific nitrosamines in humans.

Project description:N'-Nitrosonornicotine (NNN) is one of the most important strong carcinogens in tobacco products and is believed to play a significant role in the induction of esophageal cancer in smokers and oral cavity cancer in snuff dippers. NNN is metabolically activated through cytochrome P450-catalyzed alpha-hydroxylation. 2'-Hydroxylation produces a reactive intermediate 4-(3-pyridyl)-4-oxobutanediazohydroxide (7), which alkylates DNA to form pyridyloxobutyl (POB)-DNA adducts. DNA pyridyloxobutylation from NNN treatment, as measured by released 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 8), has been observed in vitro and in vivo. In the present study, we have used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to analyze specific POB-DNA adducts in the nasal olfactory, nasal respiratory, and oral mucosa of F344 rats treated chronically with (R)-NNN or (S)-NNN in the drinking water (10 ppm, 1-20 weeks). Adduct levels in the nasal respiratory mucosa exceeded those in the nasal olfactory and oral mucosa. (R)-NNN treatment generated 2-4 times more adducts in the nasal olfactory and respiratory mucosa than did (S)-NNN at most time points. O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd, 11) predominated in the nasal olfactory and respiratory mucosa, followed by 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 14). Levels of O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O(2)-POB-Cyt, 13) and O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O(6)-POB-dGuo, 12) were significantly lower. In the oral mucosa, the opposite stereoselectivity was observed, with (S)-NNN treatment producing 3-5 times more POB-DNA adducts than did (R)-NNN. O(2)-POB-dThd and 7-POB-dGuo were the two major adducts, and their levels were similar. Overall, POB-DNA adduct formation in the nasal olfactory and nasal respiratory mucosa was similar to that previously observed in the lung, whereas in the oral mucosa, the trend resembled that in the esophagus. These results indicate that different mechanisms are involved in NNN metabolism and tumorigenesis in rat nasal and oral tissues. NNN enters the nasal mucosa through the circulation, and tissue-specific metabolism is important, while in the oral mucosa, direct exposure and local activation both play significant roles. Our results also support the potential importance of NNN as an oral carcinogen in people who use smokeless tobacco products.

Project description:The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are potent pulmonary carcinogens in rats. NNK and NNAL require metabolic activation to express their carcinogenicity. Cytochrome P450-catalyzed alpha-hydroxylation at the methyl position of NNK or NNAL generates reactive intermediates, which alkylate DNA to form pyridyloxobutyl (POB)-DNA adducts or pyridylhydroxybutyl (PHB)-DNA adducts. NNK is metabolized to NNAL in a reversible and stereoselective manner, and the tissue-specific retention of (S)-NNAL is believed to be important to the carcinogenicity of NNK. In the present study, we investigated the formation of POB- and PHB-DNA adducts in extrahepatic tissues of F344 rats treated chronically with NNK and (R)- and (S)-NNAL (10 ppm in the drinking water, 1-20 weeks). POB- and PHB-DNA adducts were quantified in nasal olfactory mucosa, nasal respiratory mucosa, oral mucosa, and pancreas of treated rats. Adduct formation in the nasal respiratory mucosa exceeded that in the other tissues. O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd) or O(2)-[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine (O(2)-PHB-dThd) was the major adduct, followed by 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua) or 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]guanine (7-PHB-Gua). There was a remarkable similarity in adduct formation between the NNK and the (S)-NNAL groups, both of which were distinctively different from that in the (R)-NNAL group. For example, in the nasal olfactory mucosa, POB-DNA adduct levels in the NNK and (S)-NNAL groups were not significantly different from each other, while (R)-NNAL treatment generated 6-33 times lower amounts of POB-DNA adducts than did NNK treatment. In contrast, (R)-NNAL treatment produced significantly higher levels of PHB-DNA adducts than did NNK or (S)-NNAL treatment. Similar trends were observed in the nasal respiratory mucosa, oral mucosa, and pancreas. These results suggest extensive retention of (S)-NNAL in various tissues of NNK-treated rats and support a mechanism in which the preferential metabolism of NNK to (S)-NNAL, followed by sequestration of (S)-NNAL in the target tissues and reoxidation to NNK, is important to NNK tumorigenesis.

Project description:The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN, 1) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2) are potent carcinogens in rodents. Bioactivation of NNN and NNK by cytochrome P450 enzymes generates a pyridyloxobutylating agent 6, which alkylates DNA to produce pyridyloxobutyl (POB)-DNA adducts. POB-DNA adduct formation plays a critical role in NNN and NNK carcinogenicity in rodents. To further investigate the significance of this pathway, we developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantitative analysis of four POB-DNA adducts with known structures. The corresponding deuterated analogues were synthesized and used as internal standards. DNA samples, spiked with internal standards, were subjected to neutral thermal hydrolysis followed by enzymatic hydrolysis. The hydrolysates were partially purified by solid phase extraction prior to HPLC-ESI-MS/MS analysis. The method was accurate and precise. Excellent sensitivity was achieved, especially for O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 11) with a detection limit of 100 amol per mg DNA. DNA samples treated with different concentrations of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3) were subjected to HPLC-ESI-MS/MS analysis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 12) was the most abundant adduct, followed by O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-POB-dGuo, 8), O2-POB-dThd, and O2-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O2-POB-Cyt, 13). Lung and liver DNA isolated from NNK-treated rats were analyzed. Consistent with the in vitro data, 7-POB-Gua was the major POB-DNA adduct formed in vivo. However, levels of O6-POB-dGuo were the lowest of the four adducts analyzed, suggesting efficient repair of this adduct in vivo. In contrast to the other three adducts, O6-POB-dGuo was more abundant in lung than in liver. O2-POB-dThd appeared to be poorly repaired in vivo, and its levels were comparable to those of 7-POB-Gua. The results of this study provide a sensitive HPLC-ESI-MS/MS method for comprehensive quantitation of four POB-DNA adducts, support an important role of O6-POB-dGuo in NNK lung tumorigenicity in rats, and suggest that O2-POB-dThd may be a useful tobacco-specific DNA biomarker for future tobacco carcinogenesis studies.

Project description:O(6)-POB-dG (O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O(6)-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O(6)-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O(6)-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O(6)-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI(+)-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O(6)-POB-dG adducts. In our approach, synthetic DNA duplexes containing O(6)-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D(4)-O(6)-POB-dG internal standard and mild acid hydrolysis to release O(6)-POB-guanine (O(6)-POB-G) and D(4)-O(6)-POB-guanine (D(4)-O(6)-POB-G), samples are purified by solid phase extraction (SPE), and O(6)-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI(+)-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O(6)-POB-G-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O(6)-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI(+)-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O(6)-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5'-AATAGTATCT[O(6)-POB-G]GAGCC-3') opposite either C or T. Faster rates of alkyl transfer were observed when O(6)-POB-dG was paired with T rather than with C (k = 1.74 × 10(6) M(-1) s(-1) vs 1.17 × 10(6) M(-1) s(-1)).

Dataset Information

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

Publications

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

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