EXAMINATION OF DIOLS AND DIOL EPOXIDES OF POLYCYCLIC AROMATIC-HYDROCARBONS AS SUBSTRATES FOR RAT-LIVER DIHYDRODIOL DEHYDROGENASE

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) can suppress the formation of anti-diol epoxides that arise from the metabolic activation of PAH by oxidizing their precursor trans-dihydrodiols to o-quinones [Smithgall, T. E., et al. (1988) J. Biol. Chem. 263,1814-18201. DD has also been found to reduce the mutagenicity of benz[a]anthracene (+/-)-anti-8,9-dihydrodiol 10,11-epoxide [(+/-)-anti-BADE] in the Ames test (Glatt, H. R., et al. (1982) Science 215,1507-1509), suggesting that anti-diol epoxides are substrates for this enzyme. In this study, attempts have been made to demonstrate directly that diol epoxides of PAH are substrates for homogeneous DD. Spectrophotometric assays indicate that high concentrations of the stable anti-diol epoxides, naphthalene (+/-)-anti-1,2-dihydrodiol 3,4-epoxide (10 mM) and (+/-)-anti-BADE (20-mu-M; limit of solubility) were not oxidized by micromolar concentrations of enzyme. By contrast, 20-mu-M (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [(+/-)-trans-BP-diol] was oxidized by 50-fold less enzyme. Using a reverse-phase high-performance liquid chromatography (RP-HPLC)based assay [1,3-H-3]-(+/-)-trans-BP-diol could be almost completely oxidized by DD in the presence of NADP+. Using a similar assay, [1,3-H-3]benzo[a]pyrene (+/-)-anti-7,8-dihydrodiol 9,10-epoxide [(+/-)-anti-BPDE], and unlabeled (+/-)-syn-BPDE and (+/-)-anti-BADE were tested as substrates for DD. Incubations were performed in the presence of 2.3 mM NADP+ in either potassium phosphate (pH 7.0) or glycine (pH 9.0) buffer, and reactions were terminated by the addition of 2-mercaptoethanol (2 mM) to trap unreacted diol epoxides as thiol ether adducts. RP-HPLC analysis of the reaction mixtures using diode array detection showed that they contained only hydrolysis products (tetraols) and thiol ether adducts of diol epoxides. No loss of enzyme activity was found after incubation of the enzyme with the diol epoxides. The results indicate that PAH diol epoxides are not substrates for DD and that they do not inactivate the enzyme. However, when (+/-)-anti-BADE (20-mu-M) was incubated with bovine serum albumin or DD alone, its rate of hydrolysis and formation of its thiol ether adduct were prevented in a concentration-dependent manner. The decrease in mutagenicity of this diol epoxide in the presence of DD, therefore, may be due to its sequestration by protein.