We have discovered a new class of DNA adducts, 3-alkyl uracil residues, which are the product of cytosine alkylation and deamination by a variety of aliphatic epoxides. Aliphatic epoxides are both mutagenic and carcinogenic, but have not been shown to produce any known prematagenic DNA lesion. We will test the hypothesis that 3-alkyl uracil adducts may be the critical premutagenic lesions produced by aliphatic epoxides in vivo. We have found in studies conducted in vitro that cytosine residues initially alkylated by the epoxides ethylene oxide (EO), propylene oxide (PO), and epichlorhydrin (ECH) undergo rapid hydrolytic deamination to form stable 3- alkyl uracil is a potentially mutagenic lesion; it occupies a central Watson-Crick hydrogen-bonding position and is likely to disrupt normal base-pairing. If 3-alkyl uracil is persistent in vivo, it may play an important premutational role in aliphatic-epoxide-induced mutagenesis and carcinogenesis. We will investigate the significance of 3-alkyl uracil in the mutagenic and carcinogenic activity of several environmentally important aliphatic epoxides and epoxide metabolites. EO, PO and ECH are mutagenic and carcinogenic epoxides which have extensive industrial uses and potential for human exposure. Acrylonitrile (AN) and acrylamide (AM) are important industrial chemicals with toxic, mutagenic and carcinogenic activity. Metabolism to the epoxides cyanoethylene oxide (CEO) and glycidamide (GA) are believed to be responsible for their genotoxic properties. In vivo studies with EO, PO and ECH will determine the degree to which 3-alkyl uracil forms in DNA and its persistence. These studies will be conducted in rats using inhalation, the primary route of human exposure to these agents. We will investigate the mispairing potential of specific 3-alkyl uracil lesions induced by EO, PO, ECH and CEO/GA using an in vitro DNA replication system to see if these lesions can be mutagenically bypassed by replicative DNA polymerases and to determine the kinetic mechanisms for insertion of a base opposite and extension past these lesions. We will use the bacteriophage phiX174-based site-directed mutagenesis system to quantitate the mutagenic potential and determine the mutagenic specificity of these 3-alkyl uracil adducts in vivo. This research will provide a clearer understanding of the molecular mechanisms of initiation of carcinogenesis by an environmentally significant class of carcinogens for which the mechanism of somatic mutagenesis in unknown.
