Expression and characterization of four recombinant human dihydrodiol dehydrogenase isoforms:: Oxidation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene to the activated o-quinone metabolite benzo[a]pyrene-7,8-dione

The bioactivation of polycyclic aromatic hydrocarbons (PAHs) to their ultimate carcinogenic forms proceeds via the formation of proximate carcinogen trans-dihydrodiols. Previous studies demonstrated that rat liver 3 alpha-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3 alpha-HSD/DD), a member of the aldo-keto reductase (AKR) superfamily, oxidizes PAH trans-dihydrodiols to redoxcycling o-quinones, Multiple closely related AKRs exist in human liver; however, it is unclear which, if any, participate in PAH activation by catalyzing the NADP(+)-dependent oxidation of PAH trans-dihydrodiols. In this study, cDNAs encoding four human DD isoforms were isolated from HepG2 cells using isoform-selective RT-PCR. The recombinant proteins were overexpressed in Escherichia coli, purified to homogeneity, and kinetically characterized, Calculation of K-M and k(cat) values of each isoform for model substrates revealed that they possessed enzymatic activities assigned to native human liver DD1, DD2, DD4, and type 2 3 alpha-HSD (DDX) proteins. The ability of human DDs to oxidize the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-diol) was then examined. A reverse phase HPLC radiochemical assay demonstrated that all four isoforms oxidize (+/-)-BP-diol in the following rank order: DD2 > DD1 > DD4 > DDX. Each DD consumed the entire racemic BP-diol mixture, indicating that both the minor (+)-S,S-and major (-)-R,R-stereoisomers formed in vivo are substrates. First-order decay plots showed that DD1 and DD2 displayed preferences for one of the stereoisomers, and circular dichroism spectroscopy indicated that this isomer was the (+)-7S,8S-enantiomer. The products of these reactions were trapped as either glycine or thiol ether conjugates of benzo[a]pyrene-7,8-dione (BPQ), indicating that the initial oxidation product was the reactive BPQ. Thus, human liver possesses multiple AKRs which contribute to PAH activation by catalyzing the NADP(+)-dependent oxidation of PAH trans-dihydrodiols to redox-active o-quinones.