Identification and Quantification of DNA Adducts in the Oral Tissues of Mice Treated with the Environmental Carcinogen Dibenzo[a,l]pyrene by HPLC-MS/MS

Tobacco smoking is one of the leading causes for oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke constituent, is the most carcinogenic polycyclic aromatic hydrocarbon (PAH) tested to date in several animal models (target organs: skin, lung, ovary, and mammary tissues). We have recently demonstrated that DB [a,l]P is also capable of inducing oral cancer in mice; however, its metabolic activation to the ultimate genotoxic metabolite dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) in mouse oral cavity has not been examined. Here we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method method to detect and quantify (+/-)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [N-15(5)]-(+/-)-anti-DB[a,l]PDE-N-6-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR, and CD analysis. The detection Limit of the method is 8 fmol with 100 mu g of digested DNA as the matrix. Two adducts were detected and identified as (-)-anti-cis and (-)-anti-trans-DB[a,l]PDE-dA in the oral tissues of mice following the direct application of DB[a,l]P (240 nmol per day, for 2 days) into the oral cavity, indicating that DB[a,l]P is predominantly metabolized into (-)-anti-DB[a,l]PDE in this target organ. We also compared the formation and removal of adducts as a function of time, following the direct application of DB[a,l]P (24 nmol, 3 times per week for 5 weeks) into the oral cavity of mice. Adducts were quantified at 48 h, 1, 2, and 4 weeks after the last dose. Maximal levels of adducts occurred at 48 h, followed by a gradual decrease. The levels (fmol/mu g DNA) of (-)-anti-trans adducts (4.03 +/- 0.27 to 1.77 +/- 0.25) are significantly higher than (-)-anti-cis-DB[a,l]PDE-dA adduct (1.63 +/- 0.42 to 0.72 +/- 0.04) at each time point (p < 0.005). The results presented here indicate that the formation and persistence of (-)-anti-DB[a,l]PDE-dA adducts may, in part, contribute to the initiation of DB[a,l]P-induced oral carcinogenesis.