Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants that may cause lung cancer. They require metabolic activation to either diol-epoxides or radical cations to exert their carcinogenic effects. A third mechanism of activation may involve dihydrodiol dehydrogenases (DDs). These enzymes divert PAH trans-dihydrodiols to form highly reactive PAH o-quinones which enter into futile redox cycles and produce o-semiquinone radicals and reactive oxygen species (ROS) (superoxide anion, H2O2 and hydroxyl radical) multiple times. PAH o-quinones cause cell death, are frame-shift mutagens, form stable DNA adducts, and act as potent nucleases. They may also form depurinating DNA adducts and oxidatively damage bases within DNA offering several mechanisms by which tumor proto-oncogenes (ras) and tumor suppressor (p53) genes can be mutated. These studies will extend the relevance of the DD pathway to PAH metabolism/activation. The regio- and stereo-specificity of recombinant human DD isoforms for PAH trans-dihydrodiols is unknown. Identification of the isoform(s) that metabolize non-K region trans-dihydrodiols will demonstrate that reactions catalyzed by rat DD are mediated by its human homologues. cDNAs for rat and human DDs are being stably transfected into either human cells that activate PAH but are DD deficient (MCF7 cells) or into human bronchiolo-alveolar cell lines that are sites of PAH activation. The consequences of rat and human DDs converting trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) to benzo[a]pyrene7,8-dione (BPQ) will be assessed in the transfectants using several cytotoxic [production of ROS; increases in redox state; decreased cell viability] and genotoxic [formation of BPQ-deoxyguanosine (BPQ-dG) adducts, 8'-hydroxy-dG and DNA strand breaks] end points. Calf thymus DNA and bronchiolo-alveolar cells will be treated with PAH o-quinones and the formation of stable and depurinating adducts and 8'-hydroxydG will be measured. Structures of covalent adducts are sought using NMR and LC/ESMS techniques. The specificity of PAH o-quinones to cleave [32P]-labeled-RNA, -ssDNA and -dsDNA encoding ras and p53 fragments will be determined. Base-specific cleavage sites will be identified by DNA footprinting. Sites of bulky adduct formation or depurination in ras and p53 will be determined by DNA polymerase fingerprint analysis. Sites of cleavage or base modification will be compared to mutational hot spots observed in human tumors. The transforming potential of BPQ treated ras will be measured by foci formation in NIH/3T3 cells.
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