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

• Published in: Biochemistry (Easton) 1998-05, Vol.37 (19), p.6781-6790
• Main Authors: Burczynski, Michael E, Harvey, Ronald G, Penning, Trevor M
• Format: Article
• Language: English
• Subjects: Benzopyrenes - chemistry; Benzopyrenes - metabolism; Biotransformation; Carcinoma, Hepatocellular; Catalysis; Dihydroxydihydrobenzopyrenes - metabolism; DNA, Complementary - isolation & purification; Escherichia coli - enzymology; Escherichia coli - genetics; Genetic Vectors - metabolism; Glycine - chemistry; Humans; Isoenzymes - biosynthesis; Isoenzymes - genetics; Isoenzymes - isolation & purification; Kinetics; Liver - enzymology; Oxidation-Reduction; Oxidoreductases - biosynthesis; Oxidoreductases - genetics; Oxidoreductases - isolation & purification; Phospholipid Ethers - chemistry; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Stereoisomerism; Tumor Cells, Cultured
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi972725u

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 3α-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3α-HSD/DD), a member of the aldo−keto reductase (AKR) superfamily, oxidizes PAH trans-dihydrodiols to redox-cycling 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 3α-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.