Oxygen radicals have been implicated as endogenous initiators of degenerative processes leading to cancer, heart disease and aging. They can be produced during normal cellular functions such as oxygen metabolism and as byproducts of reduction/oxidation reactions. Many exogeneous mutagens and carcinogens act through the generation of oxygen radicals as does ionizing radiation. In the presence of oxygen, various hydroperoxides are formed in DNA exposed to ionizing radiation. These hydroperoxides undergo gradual decomposition yielding more stable derivatives. The hydroperoxides of thymine moiety are formed most readily and have been studied quite extensively. The mutagenic activities of some of the hydroperoxides were significantly increased in the presence of certain transitional metal ions. These metal ions catalyze hydrolysis of hydroperoxides with the formation of reactive oxygen species which potentially can react with bases in DNA. Hence hydroperoxides, which themselves are formed by the action of oxygen radicals, could in turn become sources of such radicals and, therefore, potentiate the damage to DNA, especially when present in DNA. The main objectives of this proposal are to elucidate the mechanism(s) by which transition metal ions, metalloproteins and peroxidases influence the mutagenic activity of 2'-deoxyribonucleoside hydroperoxides, to determine what type of DNA base damage is a result of oxygen radicals produced by the decomposition of hydroperoxides and to establish the identity of a putative repair activity which recognizes one of the decomposition products in DNA. The effects of copper and iron ions (either free or in ferritin, transferrin and ceruloplasmin) and of horseradish and glutathione peroxidases on 5-hydroperoxymethyl-2""""""""-deoxyuridine (HPMdU)-induced modification of bases in [3H]DNA will be determined using HPLC analysis and micro-derivatization. Genetic effects resulting from treatment with HPMdU will be evaluated using lambda prophage induction in the E. coli WP2(lambda) strain and gene mutations in several S. typhimurium strains and V79 Chinese hamster cells. Furthermore, the effects of two HPMdU decomposition products [5-hydroxymethyl-2'-deoxyuridine (HMdU) and 5-formyl-2'-deoxyuridine (FdU) and one potential DNA oxidation product [8-hydroxy-2'-deoxyguanosine (89HdG)] on conformation of DNA will be established using synthetic oligomers, as well as mutagenicity of FdU and 80 HdG using cell lines listed above. In addition, a putative repair activity reducing 5-hydroxymethyluracil to thymine in DNA that is present in S.typhimurium cells will be isolated and characterized.
