Contribution of dihydrodiol dehydrogenase to the metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in fortified rat liver subcellular fractions.
Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) purified to homogeneity from rat liver cytosol will catalyze the NAD(P)(+)-dependent oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-diol) to yield benzo[a]pyrene-7,8-dione (BPQ). To verify that BPQ is a metabolite of B[a]P-diol in rat liver, an S100 fraction was supplemented with NAD+ and NADP+, and the formation of BPQ was followed by reverse-phase HPLC. The identity of BPQ was established by co-chromatography with an authentic standard (under different solvent conditions) and by RP-HPLC using a diode-array detector which established that the metabolite shared spectral identity with BPQ. The formation of BPQ in the S100 fraction was blocked by either a competitive inhibitor (indomethacin) or a suicide substrate [1-(4-nitrophenyl)-propen-1-ol] for DD, indicating that BPQ was being formed by this enzyme. To assess the contribution of DD to the metabolism of [3H]B[a]P-diol, subcellular fractions obtained from uninduced rat liver were fortified with co-factors to optimize the activity of enzymes that would compete for this proximate carcinogen. Under these conditions, S100 fractions fortified with NAD+ and NADP+ metabolized 25% of the B[a]P-diol, producing 731 +/- 154 pmol of BPQ. In contrast, rat liver microsomes fortified with an NADPH generating system metabolize 75% of the B[a]P-diol producing 2614 +/- 379 pmoles of benzo[a]pyrene-tetrahydrotetrols. Rat liver homogenates (S10) fortified with either uridine diphosphoglucuronic acid or phosphoadenosine phosphosulfate produced 180 +/- 56 and 95 +/- 31 pmoles of conjugates respectively, which were recovered as B[a]P-diol after treatment of the aqueous phase with either beta-glucuronidase or aryl sulfatase. Of the metabolites analyzed BPQ was formed in the second largest amount. These studies show that in uninduced rat liver DD may play a significant role in the metabolism of B[a]P-diol. The metabolic fate of BPQ remains to be determined.
Shou
M
M
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
Harvey
R G
RG
Penning
T M
TM
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism; D002384:Catalysis; D004101:Dihydroxydihydrobenzopyrenes / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism* / Q000648:ultrastructure; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions
D001580:Benzopyrenes; D004101:Dihydroxydihydrobenzopyrenes; C017228:benzo(a)pyrene 7,8-dihydrodiol; D009249:NADP; C076588:benzo(a)pyrene-7,8-dione; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C023125:dihydrodiol dehydrogenases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C116788:cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase