To understand the machanism by which polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BP), are able to induce mutations, three questions must be answered. (1) What adducts induce what mutations? (2) How do these adducts induce these mutations (i.e., directly or as intermediates)? (3) Why do these adducts induce the mutations that they do? The work described in this proposal is primarily focused on answering the first question for N2-Guanine, N(7)-Guanine and N6-Adenine adducts derived from polycyclic aromatic hydrocarbons. The major adduct formed from BP in vivo (i.e., BP-N2-Gua) was built into a double standed, E. coli, viral vector using a combination of chemical synthetic and recombinant DNA techiques. Following transformation of repair proficient and repair deficient E. coli cells, no mutants were found to have arisen from the BP-N2-Gua lesion at the level of 1%. A new vector was constructed and BP-N2-Gua was built into it, made single stranded and transformed into E. coli cells. The search for BP-N2-Gua derived mutants is in progress. By similar methods BP-N2-Gua will be built into a shuttle vector, and transfected into human cells in order to study the mutagenic consequences of this lesion in human cells. Adenine adducts appear to be important in the mutagenic activation of oncogenes. The N6-Adenine adduct of 7-bromomethy1-12- methylbenz(a)antracene (BrMBA) will be built into a related viral vector and its mutagenic consequences studied in E. coli cells. N(7)-Gua adducts of BPDE are chemically unstable, which probably precludes their incorporation into vectors utilizing recombinant DNA and chemical synthetic techniques. An alternative method for studying the mutagenic consequences of unstable adducts is proposed.
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