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 premutagenic DNA lesion such as 06-alkyl G or 04-alkyl T. Our proposal is designed to test the hypothesis that 3-alkyl uracil adducts may be the critical premutagenic lesions produced by aliphatic epoxides in vivo. Aliphatic epoxides are an environmentally important class of carcinogens, and are also implicated as the ultimate carcinogenic form of such compounds as vinyl chloride and acrylonitrile. The simple volatile epoxides, especially ethylene oxide (EO), propylene oxide (PO) and epichlorohydrin (ECH) have extensive industrial uses and the potential for human exposure in the workplace. These compounds are direct-acting mutagens and carcinogens in animals, often producing tumors at the site of administration. Eu is Classified by the International Agency for Research on Cancer as a probable human carcinogen. We have found in studies conduced in vitro that cytosine residues initially alkylated by EO, PO, and ECH subsequently undergo hydrolytic deamination to form 3-alkyl uracil adducts. The deamination is catalyzed by the hydroxyl group which forms on the alkyl side chain when the epoxide reacts at N-3 of cytosine. 3-Alkyl uracil is stable in DNA in vitro, and is a potentially mutagenic lesion. It occupies a central Watson-Crick hydrogen-bonding position and is likely to disrupt normal base-pairing. The proposed research will investigate the biological consequences of 3-alkyl U damage. We will determine whether EO, PO, and ECH can form 3-alkyl-dU adducts in vivo. We will study both formation and persistence of these adducts in rats after inhalation exposure, the primary route of human contact. The mutational spectrum induced by EO and PO will be determined in the lacZ(alpha) gene in single-strained M13mp2. Finally, we will use the (phi)Xl74-based site-directed mutagenesis system to quantitate the mutagenic potential and determine the mutagenic specificity of 3-alkyl U adducts produced by EO and PO in biologically active DNA. It is our hope that 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 is unknown.
