This application requests continuation of aggregate funding for programs involving the enzymology and chemistry of bioactivation and mechanisms of mutagenesis. The research plan is outlined along six major topics. (i) Characterization of structures of cytochrome P-450 (P450) enzymes and their active sites. The initial focus will be on P-450 1A2 enzymes in several animal species and upon a number of human P-450s; techniques of recombinant expression, X-ray crystallography, and affinity labeling are proposed. Some specific studies involve elucidation of the mechanism of allosteric stimulation of P-450 3A4 and computer modeling of the active site of P-450 2D6. (ii) analysis of structure-activity relationships in (human) P450 enzymes. Some specific site-directed mutations are proposes; major effort will be given to random mutagenesis and the use of selective pressure to produce more efficient (human) P- 450s in bacterial vectors, in order to examine the basis of function. (iii) Purification and characterization of human P-450 enzymes. The principal enzymes to be studied initially are human P450 2B6 and the P- 450 (2C) (S)-mephenytoin 4'-hydroxylase. More efficient methods of purifying hepatic and recombinant human P-450s will also be developed. (iv) Elucidation of chemical mechanisms of P450 catalysis. The major topic for consideration is amine oxidation and details involving fates of aminium radicals. Several approaches are to used. Other compounds under consideration include polycyclic hydrocarbons and olefins. (v) Elucidation of the mechanisms of base-pair mutagenesis by ethylene dibromide-DNA adducts. Attention will be focused on the working hypothesis that S-[2N7-guanyl)ethyl]glutathione is the principal mutagenic lesion and that mispairing can be understood only in terms of the polymerase complex. Site-specific mutagenesis, primer extension, and physical studies with synthetic oligonucleotides bearing the above adduct are proposed. Elucidation of chemical mechanisms of formation of DNA- carcinogen adducts. The mechanism of C8-guanyl arylamine adduct formation will be probed, first using C8-methylguanosine as a diagnostic model, utilizing the working hypothesis that the N7 atom is an initial site of attack. Studies on the mechanism of formation of etheno-DNA adducts will be extended to N2,3-ethenoguanine and 1,N2-ethenoguanine ( with reactive epoxides derived from carcinogenic olefins). A multi-faceted approach involving chemical and biological methods will be used toward these goals. All of these studies should enhance our understanding of how industrial chemical and natural products are activated to potentially carcinogenic species and our information regarding the roles of inter-individual variations in susceptibility to such agents.
Showing the most recent 10 out of 288 publications
