Dehydrogenase of PAH Activation Catalyzed by Human Dihydrodiol and Oxidative Stress: Implications for the Alternative Pathway by Polycyclic Aromatic Hydrocarbons (PAHs), Electrophiles, Isoform-specific Induction of a Human Aldo-Keto Reductase

Human dihydrodiol dehydrogenase (DD) isoforms are aldo-keto reductases (AKRs) that activate polycyclic aromatic hydrocarbons (PAHs) by oxidizing trans-dihydrodiol proximate carcinogens to reactive and redox-active ortho-quinones. Of these, human AKR1C1 (DD1) and AKR1C2 (DD2) oxidize trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene to the cytotoxic and genotoxic metabolite benzo[a]pyrene-7,8-dione (BPQ) with the highest catalytic efficiency. Exposure of HepG2 cells to a panel of inducers revealed that mRNA encoding one or more human AKR1C member(s) was induced (3to 10-fold) by benzo[a]pyrene and other polycyclic aromatic compounds (bifunctional inducers), electrophilic Michael acceptors and phenolic antioxidants (monofunctional inducers), and reactive oxygen species (ROS). The induction of AKR1C mRNA by bifunctional inducers was delayed with respect to the induction of CYP1A1 mRNA, and AKR1C mRNA was not induced by the nonmetabolizable aryl hydrocarbon receptor ligand 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). These data suggest that, in contrast to the CYPs, induction of AKR1C member(s) by PAHs and other bifunctional inducers is mediated indirectly via an antioxidant response element rather than a xenobiotic response element. Immunoblot and enzymatic assays confirmed that the increases in AKR1C mRNA were faithfully translated into functional AKR1C protein(s). The increased DD activity in HepG2 lysates was inhibited only by high concentrations of ursodeoxycholate, which suggested that AKR1C2 (DD2, bile-acid-binding protein) was not the isoform induced. RNase protection assays identified AKR1C1 (DD1) mRNA as the transcript which was up-regulated by monoand bi-functional inducers and ROS in both human hepatoma (HepG2) and colon carcinoma (HT29) cells. BPQ, the electrophilic and redox-cycling product of the AKR1C1 reaction, also induced AKR1C1 expression. Thus, BPQ formation by AKR1C1 results in both a chemical (redox-cycling) and a genetic (AKR1C1 induction) amplification of ROS in PAH-exposed cells. Because ROS have been implicated in both tumor initiation and tumor promotion, the amplification of ROS by this pathway may play a significant role in PAH carcinogenesis. INTRODUCTION The AKRs are a growing superfamily of monomeric cytosolic NADP(H)-dependent oxidoreductases (Mr ;34,000) that catalyze the interconversion of aldehydes and ketones to alcohols on drugs and xenobiotics. Their ability to generate alcohols for conjugation reactions on a variety of endogenous (steroids and prostaglandins) and exogenous substrates (aromatic aldehydes and ketones; Refs. 2–6) suggest that they play a central role in the metabolism of xenobiotics in a manner similar to the microsome-delimited CYPs. Several AKR superfamily members are involved in carcinogen metabolism. The rat ethoxyquin-inducible aflatoxin aldehyde reductase (AKR7A1) catalyzes the reduction of aflatoxin dialdehyde to its corresponding diol and represents an important detoxification route for aflatoxin (7). In contrast, rat 3a-HSD/DD (AKR1C9) catalyzes the oxidation of PAH trans-dihydrodiols (proximate carcinogens) to reactive PAH o-quinones (8, 9) with the concomitant production of deleterious ROS (Ref. 10; Fig. 1). The propensity of PAH o-quinones to enter into futile redox cycles and amplify ROS and o-semiquinone radicals is in part responsible for the cytotoxic and genotoxic properties of the parent quinones (11–13). Such reactive and redox-active o-quinone metabolites may contribute to the complete carcinogenic potential of PAH. The diversion of PAH trans-dihydrodiols from diol-epoxides to o-quinones may thus provide an alternative pathway of PAH activation. Multiple human DD isoforms (AKR1C1-AKR1C4) were recently cloned from HepG2 cells and the recombinant proteins were expressed in Escherichia coli and characterized with respect to their native proteins in human liver (14). All of the four human AKR1C subfamily members were capable of oxidizing the potent proximate carcinogen B[a]P-diol to BPQ in the following rank order: AKR1C2 . AKR1C1 . AKR1C4 . AKR1C3 (Table 1). These studies verified that multiple human homologues of rat 3a-HSD/DD are also capable of catalyzing this alternative pathway of PAH activation in human cells. Genes that encode enzymes involved in PAH metabolism are often inducible by one or more classes of xenobiotics. One class of xenobiotics, monofunctional inducers (e.g., t-BHQ, EA), are electrophilic compounds that signal via an unclear mechanism to activate the expression of genes (GST Ya, NQO1, gGCS) carrying cis-elements termed EpRE/ARE(s) (for a review, see Ref. 15). Although it is likely that the ability of chemicals to signal via the EpRE/ARE is related to their electrophilicity (16), the actual signal transduction pathway may be mediated by an indirect effect such as alteration in intracellular redox status and/or oxidative stress (17). Polycyclic aromatic compounds (B[a]P, 3-MC, and b-NF), on the other hand, are classified as bifunctional inducers (16) because they can induce gene expression via two distinct mechanisms: (a) unmetabolized PAHs bind directly to Received 10/2/98; accepted 11/24/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grant CA55711 (to T. M. P.) and a Pharmaceutical Research and Manufacturers of America Foundation Advanced Predoctoral Fellowship (to M. E. B.). A preliminary account of this work was presented at the 19th Annual Meeting of the American Association for Cancer Research, New Orleans, LA, March 28–April 1, 1998. 2 To whom requests for reprints should be addressed, at Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Phone: (215) 898-9445; Fax: (215) 573-2236; E-mail: penning@pharm. med.upenn.edu. 3 The abbreviations used are: AKR, aldo-keto reductase; AhR, aryl hydrocarbon receptor; anti-BPDE, (6)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; ARE, anti-oxidant response element; b-NF, b-naphthoflavone; B[a]P, benzo[a]pyrene; B[a]Pdiol, (6)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; BPQ, benzo[a]pyrene-7,8-dione; CYP, cytochrome P450(s); DD, dihydrodiol dehydrogenase [trans-1,2-dihydrobenzene-1,2diol dehydrogenase (EC 1.3.1.20)]; DMBA, 7,12-dimethylbenz[a]anthracene; DMNQ, dimethoxy-1,4-naphthoquinone; EA, ethacrynic acid; EpRE, electrophilic response element; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GSH, glutathione; GST, glutathione S-transferase; 3a-HSD, 3a-hydroxysteroid dehydrogenase (EC 1.1.1.213: A-face specific); 3-MC, 3-methylcholanthrene; NQO1, NADPH quinone oxidoreductase; 89-OH-dG, 89hydroxy-deoxyguanosine; PAH, polycyclic aromatic hydrocarbon(s); ROS, reactive oxygen species; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; t-BHQ, tert-butylhydroquinone; XRE, xenobiotic response element. 4 The nomenclature for the AKR superfamily was proposed by Jez et al. (1) and adopted at the 8th International Symposium on Enzymology and Molecular Biology of Carbonyl Metabolism, held in Deadwood, SD, June 24–July 3, 1996.