title: 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.
abstract: 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 to detect and quantify (±)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [(15)N(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 μg 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/μg 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.
authors: Department of Biochemistry and Molecular Biology, Pennsylvania State College of Medicine , Hershey, Pennsylvania 17033, United States.
authors: Shang-Min
authors: SM
authors: Zhang
authors: Kun-Ming
authors: KM
authors: Chen
authors: Cesar
authors: C
authors: Aliaga
authors: Yuan-Wan
authors: YW
authors: Sun
authors: Jyh-Ming
authors: JM
authors: Lin
authors: Arun K
authors: AK
authors: Sharma
authors: Shantu
authors: S
authors: Amin
authors: Karam
authors: K
authors: El-Bayoumy
mesh_terms: D000818:Animals; D001580:Benzopyrenes / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D002274:Carcinogens, Environmental / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis* / Q000302:isolation & purification; D004852:Epoxy Compounds / Q000737:chemistry / Q000633:toxicity; D005260:Female; D051379:Mice; D009061:Mouth Mucosa / Q000187:drug effects* / Q000378:metabolism; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001580:Benzopyrenes; D002274:Carcinogens, Environmental; D018736:DNA Adducts; D004852:Epoxy Compounds; C087596:11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo(a,l)pyrene; D004247:DNA; C041517:dibenzo(a,l)pyrene