Dataset Information

Simultaneous detection of deoxyadenosine and deoxyguanosine adducts in the tongue and other oral tissues of mice treated with Dibenzo[a,l]pyrene.

ABSTRACT: We were the first to demonstrate that direct application of the environmental pollutant and tobacco smoke constituent dibenzo[a,l]pyrene (DB[a,l]P) into the oral cavity of mice induced squamous cell carcinoma (SCC) in oral tissues but not in the tongue; however, the mechanisms that can account for the varied carcinogenicity remain to be determined. Furthermore, we also showed that not only dA adducts, but also dG adducts can account for the mutagenic activity of DB[a,l]P in the oral tissues in vivo. In this study, we initially focused on DB[a,l]P-induced genotoxic effects in both oral and tongue tissues. Therefore, to fully assess the contribution of these DNA adducts in the initiation stage of carcinogenesis induced by DB[a,l]P, an LC-MS/MS method to simultaneously detect and quantify DB[a,l]PDE-dG and -dA adducts was developed. Mice were orally administered with DB[a,l]P (24 nmole, 3 times per week for 5 weeks) or its fjord region diol epoxide, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE, 12 nmole, single application); animals were sacrificed at 2, 7, 14, and 28 days after the last dose of carcinogen administration. Oral and tongue tissues were obtained and DNA were isolated followed by enzymatic hydrolysis. Following the development of an isotope dilution LC-MS/MS method, we successfully detected (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(2)-dG, as well as (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(6)-dA in oral and tongue tissues of mice treated with DB[a,l]P. Levels of (-)-anti-trans-DB[a,l]PDE-N(6)-dA were ?2 folds higher than (-)-anti-cis-DB[a,l]PDE-N(6)-dA adduct and those of dG adducts in the oral tissues and tongue at all time points selected after the cessation of DB[a,l]P treatment. Levels of dG adducts were comparable in both tissues. Collectively, our results support that DB[a,l]P is predominantly metabolized to (-)-anti-DB[a,l]PDE, and the levels and persistence of (-)-anti-trans-DB[a,l]PDE-N(6)-dA may, in part, explain the carcinogenicity of DB[a,l]P in the oral tissues but not in the tongue.

SUBMITTER: Zhang SM

PROVIDER: S-EPMC4106691 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Simultaneous detection of deoxyadenosine and deoxyguanosine adducts in the tongue and other oral tissues of mice treated with Dibenzo[a,l]pyrene.

Zhang Shang-Min SM Chen Kun-Ming KM Sun Yuan-Wan YW Aliaga Cesar C Lin Jyh-Ming JM Sharma Arun K AK Amin Shantu S El-Bayoumy Karam K

Chemical research in toxicology 20140617 7

We were the first to demonstrate that direct application of the environmental pollutant and tobacco smoke constituent dibenzo[a,l]pyrene (DB[a,l]P) into the oral cavity of mice induced squamous cell carcinoma (SCC) in oral tissues but not in the tongue; however, the mechanisms that can account for the varied carcinogenicity remain to be determined. Furthermore, we also showed that not only dA adducts, but also dG adducts can account for the mutagenic activity of DB[a,l]P in the oral tissues in v ...[more]

PMID: 24911113

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Project description: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 ?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/?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.

Project description:Dibenzo[a,l]pyrene (DB[a,l]P) (dibenzo[def,p]chrysene) is a highly carcinogenic polycyclic aromatic hydrocarbon (PAH) that has been identified in tobacco smoke and is found in our environment due to incomplete combustion of organic matter. Its metabolites are known to form stable DNA adducts in bacteria and mammalian cells, and can lead to tumors in animal models. Glucuronidation of major metabolites of DB[a,l]P by the uridine-5'-diphosphate glucuronosyltransferase (UGT) family of enzymes is an important route of detoxification of this pro-carcinogen. The focus of the current study was to characterize the glucuronidation of the pro-carcinogenic enantiomers DB[a,l]P-(+)-trans-11S,12S-diol and DB[a,l]P-(-)-trans-11R,12R-diol. Glucuronidation assays with HEK293 cell lines overexpressing individual human UGT enzymes demonstrated that UGTs 1A1, 1A4, 1A7, 1A8, 1A9, 1A10, and 2B7 glucuronidated one or both DB[a,l]P-trans-11,12-diol enantiomers. Three glucuronide conjugates were observed in activity assays with UGTs 1A1 and 1A10, while two glucuronides were formed by UGTs 1A7, 1A8, and 1A9, and one glucuronide was made by UGT1A4 and UGT2B7. Enzyme kinetic analysis indicated that UGT1A9 was the most efficient UGT at forming both the (+)-DB[a,l]P-11-Gluc and (-)-DB[a,l]P-11-Gluc products, while UGTs 1A1 and 1A10 were the most efficient at forming the (+)-DB[a,l]P-12-Gluc product (as determined by k(cat)/K(M)). Incubations with human liver microsomes showed the formation of three diastereomeric glucuronide products: (+)-DB[a,l]P-11-Gluc, (+)-DB[a,l]P-12-Gluc, and (-)-DB[a,l]P-11-Gluc, with an average overall ratio of 31:32:37 in four liver specimens. Human bronchus and trachea tissue homogenates demonstrated glucuronidation activity against both DB[a,l]P-trans-11,12-diol enantiomers, with both tissues producing the (+)-DB[a,l]P-11-Gluc and (+)-DB[a,l]P-12-Gluc with little or no formation of (-)-DB[a,l]P-11-Gluc. These results indicate that multiple UGTs are involved in the stereospecific glucuronidation of DB[a,l]P-trans-11,12-diol in a pattern consistent with their expression in respiratory tract tissues and that glucuronidation may be an important first-line detoxification mechanism of DB[a,l]P metabolites.

Dataset Information

Simultaneous detection of deoxyadenosine and deoxyguanosine adducts in the tongue and other oral tissues of mice treated with Dibenzo[a,l]pyrene.

Publications

Simultaneous detection of deoxyadenosine and deoxyguanosine adducts in the tongue and other oral tissues of mice treated with Dibenzo[a,l]pyrene.

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