Quantitation of Pyridyloxobutyl DNA Adducts of Tobacco-Specific Nitrosamines in Rat Tissue DNA by High-Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

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• 作者：Yanbin Lao ; Peter W. Villalta ; Shana J. Sturla ; Mingyao Wang ; and Stephen S. Hecht
• 刊名：Chemical Research in Toxicology
• 出版年：2006
• 出版时间：May 2006
• 年：2006
• 卷：19
• 期：5
• 页码：674 - 682
• 全文大小：185K
• 年卷期：v.19,no.5(May 2006)
• ISSN：1520-5010

文摘

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 bycytochrome P450 enzymes generates a pyridyloxobutylating agent 6, which alkylates DNA to producepyridyloxobutyl (POB)-DNA adducts. POB-DNA adduct formation plays a critical role in NNN andNNK carcinogenicity in rodents. To further investigate the significance of this pathway, we developeda 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. Thecorresponding deuterated analogues were synthesized and used as internal standards. DNA samples, spikedwith 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. DNAsamples 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 thein 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 invivo. 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 comprehensivequantitation of four POB-DNA adducts, support an important role of O⁶-POB-dGuo in NNK lungtumorigenicity in rats, and suggest that O²-POB-dThd may be a useful tobacco-specific DNA biomarkerfor future tobacco carcinogenesis studies.