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-N2-dG, as well as (−)-anti-cis- and (−)-anti-trans-DB[a,l]PDE-N6-dA in oral and tongue tissues of mice treated with DB[a,l]P. Levels of (−)-anti-trans-DB[a,l]PDE-N6-dA were ≥2 folds higher than (−)-anti-cis-DB[a,l]PDE-N6-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-N6-dA may, in part, explain the carcinogenicity of DB[a,l]P in the oral tissues but not in the tongue.

Effects of Black Raspberry Extract and Protocatechuic Acid on Carcinogen-DNA Adducts and Mutagenesis, and Oxidative Stress in Rat and Human Oral Cells

“…2D, peaks with RT 26.5 and 29 min are DBPDE-dG adducts; peaks with RT 30.5 and 36.4 are (+)-DBPDE-dA adducts. They are all correlated with the doses of DBPDE, but we only quantified (−)-DBPDE-dA adducts because they are more important to DBP-induced carcinogenesis as reported in our previous publications (21, 29). DBPDE was much more effective at producing adducts than DBP-diol.…”

“…The method used for analysis of the DBPDE-dA adducts has been published (19–21). In brief, DNA was isolated from cells using the Qiagen DNA easy kit as described by the manufacturer.…”

Effects of chronic alcohol consumption on DNA damage and immune regulation induced by the environmental pollutant dibenzo[a,l]pyrene in oral tissues of mice

“…Our previous studies showed that both dibenzo[ a,l ]pyrene (DB[ a,l ]P), a representative example of PAHs, and its diol epoxide (DB[ a,l ]PDE) induced SCC in the oral tissues of mice in a dose-dependent manner. We have also demonstrated the formation of stable covalent DNA adducts derived from DB[ a,l ]PDE in oral tissues of mice treated with DB[ a,l ]P (24). Measurement of these adducts provides an experimental approach to monitor carcinogen exposure and provides a risk assessment for susceptibility to carcinogenesis (25).…”

“…We previously reported that DB[ a,l ]PDE-dA is the major DNA adduct detected in oral tissues of mice treated with DB[ a,l ]P, and thus this adduct was analyzed in this study (30). The method used for the analysis of DB[ a,l ]PDE -dA adduct by LC-MS/MS is identical to our previously published procedure (27, 30, 31).…”

“…The method used for the analysis of DB[ a,l ]PDE -dA adduct by LC-MS/MS is identical to our previously published procedure (27, 30, 31). Briefly, DNA was isolated using the Qiagen genomic DNA isolation procedure from frozen tissues.…”