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Comparison of the formation of stable adducts and apurinic sites in DNA damaged by polycyclic aromatic hydrocarbons

Purdue University
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  • Biology
  • Molecular|Chemistry
  • Biochemistry|Health Sciences
  • Pathology
  • Biology
  • Ecology
  • Geography


Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants and some are potent carcinogens. Carcinogenic member compounds, such as dibenzo[ a,l]pyrene (DB[a,l]P), 7,12-dimethylbenz[ a]anthracene (DMBA) and benzo[a]pyrene (B[ a]P) are metabolically activated to reactive derivates that bind to DNA, and this damage may lead to tumor initiation. To investigate whether PAH exert their carcinogenic activity via formation of stable DNA adducts or apurinic (AP) sites from depurination of unstable DNA adducts, the relative proportions of the stable adducts and AP sites present in DNA after treatment with PAH were determined in cells in culture and mouse epidermis. This was accomplished by exposure of various cell lines and mouse skin to DB[ a,l]P, DMBA or B[a]P and subsequent analysis of stable DNA adducts, by 33P-postlabeling and high-performance liquid chromatography (HPLC) analysis. The induction of AP sites in DNA was analyzed using Southern blotting techniques, or a biotin-tagged aldehyde reactive probe in a microtiter plate or by a slot-blot method. The results obtained revealed that DB[ a,l]P and its fjord-region diol epoxides formed mainly stable DNA adducts in solution and in several cell lines. No evidence for the formation of AP sites was obtained. Exposure to DMBA or B[a]P produced high levels of stable DNA adducts, in a human carcinoma MCF-7 cell line but only low levels of AP sites that remained constant with time. The amount of stable DNA adducts formed in mouse epidermis by DB[a,l]P was significantly higher than that formed by DMBA and B[a]P, whereas B[a]P exhibited lower binding potency. In general, the level of AP sites was significantly lower than the level of stable adducts irrespective of the doses and exposure time. It was demonstrated that cells with high peroxidase activity failed to activate PAH to reactive metabolites that form either stable adducts or AP sites in the human leukemia HL-60 cell line. The results obtained indicate that metabolic activation of PAH to diol epoxides by cytochrome P450 enzymes and subsequent formation of stable DNA adducts, rather than one-electron oxidation catalyzed by either cytochrome P450 enzymes or peroxidases leading to the formation of AP sites, is responsible for the exceptionally high carcinogenic potency of these PAH. ^

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