METABOLIC DETERMINANTS OF SPONTANEOUS MUTAGENESIS

描述：这项提案的目标是评估 抗氧化剂维生素C(抗坏血酸)、维生素E(a-生育酚)和 金属硫蛋白(MT)降低自发频率和频谱 HPRT基因座的突变。抗坏血酸有望减少DNA损伤 由于含氮碱的直接氧化，α-生育酚通过 抑制脂质过氧化产物和DNA加合物的形成 清除细胞内自由基的金属硫蛋白。分子的光谱 自发突变和DNA损伤模式将在三个月内进行评估 人结肠癌细胞株：错配修复缺陷细胞HCT116 HCT116/CH3和HCT116衍生物 3号染色体已经被引入，错配修复专家SW480 结肠癌细胞株。细胞将与两者中的一种孵化 抗氧化剂或转金属硫蛋白表达载体，以及 用锌孵育HPRT突变体及其突变谱和DNA 已确定损坏情况。此外，错配修复缺陷细胞 LINE将被评估一组未描述的微卫星标记 以确定氧化损伤是否是 微卫星突变。

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.