Vitamin C Prevents DNA Mutation Induced by Oxidative Stress

Abstract

The precise role of vitamin C in the prevention of DNA mutations is controversial. Although ascorbic acid has strong antioxidant properties, it also has pro-oxidant effects in the presence of free transition metals. Vitamin C was recently reported to induce the decomposition of lipid hydroperoxides independent of metal interactions, suggesting that it may cause DNA damage. To directly address the role of vitamin C in maintaining genomic integrity we developed a genetic system for quantifying guanine base mutations induced in human cells under oxidative stress. The assay utilized a plasmid construct encoding the cDNA for chloramphenicol acetyl transferase modified to contain an amber stop codon, which was restored to wild type by G to T transversion induced by oxidative stress. The mutation frequency was determined from the number of plasmids containing the wild type chloramphenicol acetyl transferase gene rescued from oxidatively stressed cells. Cells were loaded with vitamin C by exposing them to dehydroascorbic acid, thereby avoiding transition metal-related pro-oxidant effects of ascorbic acid. We found that vitamin C loading resulted in substantially decreased mutations induced by H 2 O 2 . Depletion of glutathione led to cytotoxicity and an increase in H 2 O 2 -induced mutation frequency; however, mutation frequency was prominently decreased in depleted cells preloaded with vitamin C. The mutation results correlated with a decrease in total 8-oxo-guanine measured in genomic DNA of cells loaded with vitamin C and oxidatively stressed. These findings directly support the concept that high intracellular concentrations of vitamin C can prevent oxidation-induced mutations in human cells.DNA damage caused by reactive oxygen species such as H 2 O 2 , O 2 . , and ⅐ OH radicals has been implicated in mutagenesis, oncogenesis, and aging (1). Oxidative lesions in DNA include base modifications, sugar damage, strand breaks, and abasic sites. In vitro studies suggest that the hydroxyl radical is highly reactive toward DNA (2). One of the most common oxidized adducts in human cells and tissues is 8-oxo-7,8-dihydro-2Ј-deoxyguanosine (8-oxo-dG) 1 (3). This adduct results from exposure to oxidizing agents as well as from ␥ irradiation of DNA (4), and quantitation of 8-oxo-dG has been used as a marker of DNA damage (5). 8-oxo-dG "mis-pairs" with adenine during replication (6), resulting in G to T transversions in 50% of the replicated DNA (7).The role of vitamin C in protecting against oxidatively induced DNA mutations is controversial. Although numerous studies demonstrate the antioxidant effects of vitamin C (8, 9), in vitro studies are often confounded by the pro-oxidant effects of ascorbic acid in the presence of free transition metals (10). We circumvented this problem using dehydroascorbic acid to load cells with vitamin C. Vitamin C is transported into most cells in the oxidized form, dehydroascorbic acid (DHA), via facilitative glucose transporters (11, 12) and as ascorbic acid in specialized cells by sodium-dependen...

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