title: Disposition and biological activity of benzo[a]pyrene-7,8-dione. A genotoxic metabolite generated by dihydrodiol dehydrogenase.
abstract: A novel pathway of polycyclic aromatic hydrocarbon metabolism involves the oxidation of non-K-region trans-dihydrodiols to yield o-quinones, a reaction catalyzed by dihydrodiol dehydrogenase (DD). We have recently shown that in isolated rat hepatocytes (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo-[a] pyrene (BP-diol) was oxidized by this route to yield benzo [a] pyrene-7,8-dione (BPQ). We now report the disposition of BPQ and its mutagenic and genotoxic properties. Using [3H]BPQ it was found that 30% of the radioactivity was sequestered by rat hepatocytes into the cell pellet. Isolation of hepatocyte DNA provided evidence for a low level of covalent incorporation of BPQ into DNA (30 +/- 17 adducts/ 10(6) base pairs). Examination of the hepatocellular DNA by agarose gel electrophoresis following treatment with BPQ indicated that extensive fragmentation had occurred. DNA fragmentation was also observed when hepatocytes were treated with BP-diol and this effect was attenuated by indomethacin, a DD inhibitor. Hepatocytes treated with either BP-diol or BPQ were found to produce large quantities of superoxide anion radical (O2.-). The amount of O2.- generated by BP-diol was blocked by DD inhibitors. These data suggest that by diverting BP-diol to BPQ reactive oxygen species (ROS) were generated which caused DNA fragmentation. The ability of BPQ to cause DNA strand scission was further studied using supercoiled phi X174 DNA. It was found that BPQ caused concentration-dependent (0.05-10 microM) strand scission in the presence of 1 mM NADPH (which promoted redox-cycling) provided CuCl2 (10 microM) was present. Complete destruction of the DNA was observed using 10 microM BPQ. This strand scission was prevented by catalase and hydroxyl radical scavengers but not by superoxide dismutase. These data indicate that ROS were responsible for the destruction of the DNA. Using 20 microM (+/-)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene [(+/-)-anti-BPDE] only single nicks in the DNA were observed indicating that BPQ was the more potent chemical nuclease. BPQ was also found to be a direct-acting mutagen in the Ames test using Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102, and TA104, but was 10-5500-fold less efficient as a mutagen than (+/-)-anti-BPDE. Our data indicate that DD suppresses the mutagenicity of (+/-)-anti-BPDE by producing BPQ, but in doing so a potent chemical nuclease is produced which causes extensive DNA fragmentation via the generation of ROS.
authors.affiliation: Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084, USA.
authors.forename: L
authors.initials: L
authors.lastname: Flowers
authors.forename: W F
authors.initials: WF
authors.lastname: Bleczinski
authors.forename: M E
authors.initials: ME
authors.lastname: Burczynski
authors.forename: R G
authors.initials: RG
authors.lastname: Harvey
authors.forename: T M
authors.initials: TM
authors.lastname: Penning
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism; D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism* / Q000633:toxicity*; D016227:Benzoquinones / Q000378:metabolism; D002478:Cells, Cultured; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D053938:DNA Fragmentation; D004278:DNA, Superhelical / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004101:Dihydroxydihydrobenzopyrenes / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D010088:Oxidoreductases / Q000378:metabolism; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D001580:Benzopyrenes; D016227:Benzoquinones; D018736:DNA Adducts; D004278:DNA, Superhelical; D004279:DNA, Viral; D004101:Dihydroxydihydrobenzopyrenes; D009153:Mutagens; D011084:Polycyclic Aromatic Hydrocarbons; D017382:Reactive Oxygen Species; C017228:benzo(a)pyrene 7,8-dihydrodiol; C025232:semiquinone radicals; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C076588:benzo(a)pyrene-7,8-dione; D010088:Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase