STABLE EXPRESSION OF HUMAN CYTOCHROME-P450 1B1 IN V79 CHINESE-HAMSTERCELLS AND METABOLICALLY CATALYZED DNA ADDUCT FORMATION OF DIBENZO[A,L]PYRENE

Chinese hamster V79 cell Lines were constructed for stable expression of human cytochrome P450 1B1 (P450 1B1) in order to study its role in the metabolic activation of chemicals and toxicological consequences. The new V79 cell lines were applied to studies on DNA adduct formation of the polycyclic aromatic hydrocarbon (PAH) dibenzo[alpha,l]pyrene ( DB[alpha,l]P). This compound has been found to be an environmental pol lutant, and in rodent bioassays it is the most carcinogenic PAH yet di scovered. Activation of DB[alpha,l]P in various metabolizing systems o ccurs via fjord region DB[alpha,l]P-11,12-dihydrodiol 13,14-epoxides ( DB[alpha,l]PDE): we found that DB[alpha,l]P is stereoselectively metab olized in human mammary carcinoma MCF-7 cells to the (-)-anti- and (+) -syn-DB[alpha,l]PDE which both bind extensively to cellular DNA. To fo llow up this study and to relate specific DNA adducts to activation by individual P450 isoforms, the newly established V79 cells stably expr essing human P450 1B1 were compared with those expressing human P450 1 A1. DNA adduct formation in both V79 cell lines differed distinctively after incubation with DB[alpha,l]P or its enantiomeric 11,12-dihydrod iols. Human P450 Al catalyzed the formation of DB[alpha,l]PDE-DNA addu cts as well as several highly polar DNA adducts as yet unidentified. T he proportion of these highly polar adducts to DB[alpha,l]PDE adducts was dependent upon both the concentration of DB[alpha,l]P and the time of exposure. In contrast, V79 cells stably expressing human P450 1B1 generated exclusively DB[a,l]PDE-DNA adducts. Differences in the total level of DNA binding were also observed. Exposure to 0.1 mu M DB[a,l] P for 6h caused a significantly higher level of DNA adducts in V79 cel ls stably expressing human P450 1B1 (370 pmol/mg of DNA) compared to t hose with human P450 1A1 (35 pmol/ mg of DNA). A 4-fold higher extent of DNA binding was catalyzed by human P450 1B1 (506 pmol/mg of DNA)com pared to human P450 1A1 (130 pmol/mg of DNA) 6 h after treatment with 0.05 mu M (-)-(11R,12R)-dihydrodiol. In cells stably expressing human P450 1B1 the DNA adducts were derived exclusively from the (-)-anti-DB [alpha,l]PDE. These results indicate that human P450 1B1 and P450 1A1 differ in their regio-and stereochemical selectivity of activation of DB[alpha,l]P with P450 1B1 forming a higher proportion of the highly c arcinogenic (-)-anti-(11R,12S,13S,14R)-DB[alpha,l]PDE metabolite.