BENZO(A)PYRENE MUTAGENIC MECHANISMS

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Benzo [a] pyrene is a potent mutagen/carcinogen and reacts with DNA via its (+)-anti-7, 8-diol-9, 10-epoxide ( (+)-anti-B{a}PDE) to give DNA adducts, principally at N2 -Gua and N6 -Ade. The mechanisms of mutagenesis by (+)-anti-B{a}PDE was investigated using both site-specific and random mutagenesis approaches. (10 An E. coli plasmid was constructed that contained (+)-anti-B{a}P-N2-Gua in a 5'-TGC-3' sequence context and was used to determine the G->T mutations were virtually exclusively induced. (2) random mutagenesis by (+)-anti-B{a}PDE was studied in a system we developed, where a plasmid a supF target gene was adducted in vitro and transformed into E. coli. G->T mutations predominated in 5'-TG-3" sequence contexts, which correlated with the site-specific studies and suggested that (+)-anti-B{a}P-N2-Gua was responsible. In contrast, 5'-GG-3', 5'-CG-3' and 5'-AG-3' sequence contexts showed a significant fraction of GC->AT and GC->CG mutations. G115 was the major base pairing hot-spot. Some mutational complexity suggested either: (1) that there are two possible conformations for an adduct at G115 with different mutational outcomes; or (2) that there is a labile adduct at G115, which is converted to an AP-site. Preliminary data suggests that the former is more likely. (+)-anti-B{a}PDE generates labile adducts, which are probably not N7-Gua adducts. It is more likely that N2-Gua adducts can adopt multiple conformations, where one is stable, while the other is labile. This may relate to the evidence for multiple conformations obtained in mutagenesis studies at G115. The following unifying hypothesis will be tested. A single adduct can adopt multiple conformations, each of which can cause a different kind of mutation (s). Furthermore, adduct conformation can be influenced by DNA sequence context, as well as environmental factors, such as heating.