Dihydrodiol dehydrogenase (DD;EC 1.3.1.20) can suppress the formation of anti-diol epoxides of polycyclic aromatic hydrocarbons (PAH), which are ultimate carcinogens, by oxidizing their precursor transdihydrodiols to yield PAH o-quinones. For example, DD will catalyze the oxidation of trans-7,8-dihydroxy-7,8-dihydrobenzoa[a]pyrene (B[a]P-diol) to yield 7,8- dihydroxybenzo[a]pyrene which then autooxidizes to benzo[a]pyrene-7,8- dione (BPQ. BPQ and other PAH o-quinones produced by this enzyme form conjugates will cellular nucleophiles, enter le minus redox-cycles and generate presumptive superoxide anion and o-semiquinone radicals and are cytotoxic in hepatoma cells. BPQ can also form BPQ-deoxyguanosine (dG) adducts with DNA. Since PAH o-quinones may be cyto- and genotoxic, DD may initiate a new pathway of PAH metabolism and carcinogen activation. This pathway of PAH metabolism will be assessed in isolated rat hepatocytes and hamster embryo fibroblasts by measuring the conversion of [3H]-b[a]P-diol into BPQ and its conjugates. These metabolites will be identified by co-chromatography with authentic synthetic standard on RP-HPLC. To verify that DD catalyzes the formation of these metabolites studies will be replicated in the presence of selective inhibitors for this enzyme. To amplify this new pathway of PAH metabolism, mammalian expression vectors containing the cDNA for DD in the sense (pRcCMV/DD) and anti-sense (pRcCMV/DD anti-sense) direction, will be stably transfected into cells which metabolize PAH but display only basal levels of DD expression (Hep-G2 and MCF7 cells). As a result stable transfectants should divert [3H]-B[a]P-diol into 7,8- dihydroxybenzo[a]pyrene and BPQ. Metabolites that arise from the intermediate hydroquinone and quinone can then be identified. The metabolic fate of [1,3-3H-BPQ will also be elucidated in hepatoma cells and isolated rat hepatocytes. To determine if DD initiates a pathway of PAH activation, the formation of transient superoxide anion radicals during the enzyme catalyzed oxidation of PAH trans-dihydrodiols will be inferred by measuring oxygen uptake and H2O2 formation. Intermediate o-semiquinone radicals will be spin-trapped with 5,5-dimethyl-1-pyrroline N-oxide for ESR spectroscopy. o-Semiquinone radicals produced during the activation of PAH o-quinones by enzymatic le minus redox-cycling will be detected by spin- stabilization. The genotoxicity of BPQ will be measured by incubating hepatoma cells with [3H]-BPQ and detecting BPQ-dG adducts. The genotoxicity of ten unlabeled PAH o-quinones generated by DD will be examined in cell free systems (calf thymus DNA in the presence and absence of le minus redox-cycling), and in hepatoma cells. The formation of covalent adducts will be measured by [32P]-post-labeling and oxidative DNA damage will be measured by detecting increases in 8-hydroxy-2'-dG levels. The mutagenicity of these PAH o-quinones will be assessed using Salmonella tester strains (Ta102 and TA104) which are sensitive to mutagens that cause oxidative DNA damage.
Showing the most recent 10 out of 72 publications
