The long-term goal of this proposal is to understand the chemistry of the key reactive intermediates that mediate the biological effects of mutagenic and carcinogenic nitrosamines and the effects of nitrosamine and reactive intermediate structure that impact their biological activity. Nitrosamines are a large class of structurally diverse carcinogens to which there is significant human exposure. Classically, the deleterious effects of nitrosamines have been ascribed to metabolic activation by P450 enzymes that yield unstable alpha-hydroxynitrosamines that decompose to diazoic acids. These yield diazonium ions and carbocations that alkylate DNA, ultimately producing mutations. This is well established for methyl and ethyl-substituted compounds, but in contrast the activity of more complex nitrosamines may be only partly or poorly described by the classical pathway. This proposal seeks to: a) delineate the non classical aqueous chemistry of more complex alpha-hydroxynitroamines; and b) to determine the extent of formation and biological implications, with respect to human polymerase interactions and human mutagenesis, of branched alkyl group adducts of DNA. There are 5 specific aims: 1) synthesis and study of the aqueous chemistry of alpha hydroxynitrosamines and related derivatives of N-nitrosomorpholine, nitrosonornicotine, and nitrosoanabasine; 2) quantitation of the formation of isopropyl adducts of A, G and T bases from reactions with DNA of isopropyl cations derived from the iso-propyl or n-propyl diazonium ions; 3) synthesis of isopropylated exocyclic oxygen and nitrogen DNA base phosphoramidites and incorporation into synthetic oligonucleotides containing adducts at defined positions; 4) analysis of coding and extension opposite the adducts by human DNA polymerases; and 5) determination of the frequency and spectrum of mutations in human fibroblasts arising from the adducted oligos, by means of the shuttle plasmid pLS189.
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