Abstract 5550: Identification and quantification of DNA adducts in the oral tissues of mice treated with the environmental carcinogen dibenzo[a,l]pyrene by LC-MS/MS

Abstract

Abstract Oral cancer is the major form of head and neck squamous cell carcinoma, which is the sixth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Our laboratory had developed a novel mouse model of oral cancer induced by DB[a,l]P. The metabolic activation of DB[a,l]P and genotoxic effect in rodent oral cavity have not been examined before. Our working hypothesis is that the stereochemical course of DB[a,l]P metabolism, the conformations of the DNA adducts formed, their removal by mammalian DNA repair enzymes, and their mutagenic properties if not removed in an error-free manner, all play critical roles during the induction of oral carcinogenesis. As an initial study to test our hypothesis, here we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify DB[a,l]PDE-N6-dA adducts in oral tissues of mice treated with DB[a,l]P. (±)-anti-[15N5]-DB[a,l]PDE-N6-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR and CD analysis. After addition of internal standards, DNA was enzymatically hydrolyzed to 2’-deoxyribonucleosides and partially purified by solid-phase extraction. The LC-MS/MS analysis was carried out by monitoring transitions m/z 604 [M+H]+ to m/z 335 [(M+H) +-dA-H2O] for DB[a,l]PDE-N6-dA adducts and m/z 609 to m/z 335 for the internal standards. The detection limit for DB[a,l]PDE-dA adduct is approximately 20 fmol in 100 ug DNA. We have compared the formation and removal of adducts as a function of time following direct application of DB[a,l]P into the oral cavity of B6C3F1 mice (24 nmol, 3 times per week, for 5 weeks). Adducts were measured at 48h, 1, 2 and 4 weeks after the last dose. Only (−)-anti-cis-and (−)-anti-trans- DB[a,l]PDE- N6-dA adducts were detected from oral tissues of DB[a,l]P treated mice, ranging from 1.9± 0.3 to 0.7± 0.1 and from 4.8 ± 0.2 to 2.3 ± 0.3 fmol/ug DNA, respectively. The levels of both adducts decreased in a time-dependent manner; approximately 50% of (−)-anti-cis- and (−)-anti-trans- DB[a,l]PDE- N6-dA adducts were removed within 4 weeks. The results suggest that DB[a,l]P is predominantly metabolized to (−)-anti-DB[a,l]PDE in oral tissues of mice, which is consistent with studies in other preclinical model systems. In this study we developed for the first time a sensitive LC-MS/MS method for the detection and quantification of DNA adducts derived from DB[a,l]P. The persistent (−)-anti-DB[a,l]PDE- N6-dA adducts may account for the initiation of carcinogenesis in the oral tissues of mice treated with DB[a,l]P. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5550. doi:10.1158/1538-7445.AM2011-5550