METABOLIC DETERMINANTS OF SPONTANEOUS MUTAGENESIS

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DESCRIPTION: The goal of this proposal is to evaluate the ability of the antioxidants Vitamin C (ascorbate), Vitamin E (a-tocopherol) and metallothionein (MT) to reduce the frequency and spectrum of spontaneous mutations at the HPRT locus. Ascorbate is anticipated to reduce DNA damage resulting from direct oxidation of the nitrogenous bases, a-tocopherol by blocking DNA adduct formation by lipid peroxidation products and metallothionein by scavenging cellular free radicals. The spectra of spontaneous mutations and pattern of DNA damage will be evaluated in three human cell lines: HCT116, a mismatch repair deficient colon carcinoma cell line; HCT116/ch3, and HCT116 derivative into which an intact human chromosome 3 has been introduced, and SW480, a mismatch repair proficient colon carcinoma cell line. Cells will be incubated with one of the two antioxidants or transfected with a metallothionein expression vector and incubated with Zn, HPRT mutants collected and the mutational spectra and DNA damage profile determined. In addition, the mismatch repair deficient cell line will be evaluated for an undescribed panel of microsatellite markers for determination of whether oxidative damage is a principal cause of microsatellite mutations.