Formation of stable adducts and absence of depurinating DNA adducts in cells and DNA treated with the potent carcinogen dibenzo[ <i>a,l</i> ]pyrene or its diol epoxides

Proceedings of the National Academy of Sciences of the United States of America - Tập 94 Số 25 - Trang 13542-13547 - 1997
Victor J. Melendez-Colon1, Charles A. Smith1, Albrecht Seidel1, Andreas Luch1, Karl L. Platt1, William M. Baird1
1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907; Department of Biological Sciences, Stanford University, Stanford, CA 94305; and Institute of Toxicology, University of Mainz, D-55131 Mainz, Germany

Tóm tắt

Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants, and some are potent carcinogens in rodents. Carcinogenic PAH are activated in cells to metabolites that react with DNA to form stable covalent DNA adducts. It has been proposed [Cavalieri, E. L. & Roger, E. G. (1995) Xenobiotica 25, 677–688] that unstable DNA adducts are also formed and that apurinic sites in the DNA resulting from unstable PAH adducts play a key role in the initiation of cancer. The potent carcinogen dibenzo[ a,l ]pyrene (DB[ a,l ]P) is activated in cells to (+)- syn - and (−)- anti -DB[ a,l ]P-11,12-diol-13,14-epoxide (DB[ a,l ]PDE), which have been shown to form stable adducts with DNA. To evaluate the importance of unstable PAH adducts, we compared stable adduct formation to apurinic site formation. Stable DB[ a,l ]PDE adducts were determined by 33 P-postlabeling and HPLC. To measure apurinic sites they were converted to strand breaks, and these were monitored by examining the integrity of a particular restriction fragment of the dihydrofolate reductase gene. The method easily detected apurinic sites resulting from methylation by treatment of cells or DNA with dimethyl sulfate or from reaction of DNA with DB[ a,l ]P in the presence of horseradish peroxidase. We estimate the method could detect 0.1 apurinic site in the 14-kb fragment examined. However, apurinic sites were below our limit of detection in DNA treated directly with (+)- syn - or (−)- anti -DB[ a,l ]PDE or in DNA from Chinese hamster ovary B11 cells so treated, although in these samples the frequency of stable adducts ranged from 3 to 10 per 14 kb. We also treated the human mammary carcinoma cell line MCF-7 with DB[ a,l ]P and again could not detect significant amounts of unstable adducts. These results indicate that the proportion of stable adducts formed by DB[ a,l ]P activated in cells and its diol epoxides is greater than 99% and suggest a predominant role for stable DNA adducts in the carcinogenic activity of DB[ a,l ]P.

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Proceedings of the National Academy of Sciences of the United States of America

Tập 94 Số 25

13542-13547

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