The metabolism of benzo(a)pyrene and other polynuclear aromatic hydrocarbon carcinogens, which are ubiquitous environmental pollutants, to highly reactive diol epoxide derivatives is thought to be an important step in the process of carcinogenesis. Diol epoxides bind covalently to DNA both in vitro and in vivo, forming adducts which differ in chemical structure and in their conformation in native DNA. As a result of these differences in structure, different adducts have varying biological potencies. Furthermore, differences in DNA sequence and in chromatin structure probably influence the sites of adduct formation by these bulky chemicals. The long range goals of this lab are to gain a better understanding of factors which affect the ability of carcinogens to bind covalently to DNA in cells and of factors which affect the conversion of such damage into the observed biological effects of mutagenesis and carcinogenesis. During the last grant period, a large difference in the mutagenic efficiency of the ultimate carcinogenic derivative of benzo(a)pyrene, BPDE-I, has been found in DNA-repair-deficient Chinese hamster ovary (CHO) cells at two genetic loci, adenosine phosphoribosyl transferase (aprt) and hypoxanthine phosphoribosyl transferase (hprt). This difference is not found with ultraviolet light as the mutagen and is not explicable by trivial effects such as target size or chromosome loss. It is proposed that differences in primary sequence and/or chromatin structure between the two genes result in differential binding of BNA and in differential repair of the lesions. This hypothesis will be tested by comparing the transcriptional activity, chromatin structure and level of BPDE-I modification o the aprt and hprt genes in vivo. These experiments will utilize recombinant DNA methods and powerful laser-induced strand scission techniques to detect BPDE-I adducts in these single copy genes. Rates of removal of adducts from the two genes will be measured in several CHO cell lines which differ in their overall rates of repair. Cloned fragments of the two genes will be used to determine the distribution of in vitro covalent binding sites within each gene. Finally, the mutagenic efficiencies of a series of isomeric diol epoxides will be determined in the same cell lines for comparison to the BPDE-I results.
