Studies of DNA repair and toxicity mechanism after an oxidative challenge by hydrogen peroxide in Saccharomyces cerevisiae

A reliable and sensitive technique for detection of DNA damage is the Single Cell Gel Assay (comet assay). In this procedure, cells are exposed to an electric field and damaged DNA moves out of the nucleus, which can be visualized by ethidium bromide staining, displaying a comet appearance under fluorescence microscopy. We show that there is a correlation between peroxide concentration and comet tail length and that DNA damage increases as the yeast population enters the post-diauxic phase, which is in agreement with the accumulation of DNA damage with aging. We show also that quercetin, an antigenotoxic natural compound, and a water extract from Ginkgo biloba leaves protect DNA against oxidative stress. DNA repair kinetics was assessed as well by the comet assay after oxidative challenge and a subsequent recovery period. For this, we are using different mutants affected in NER and BER DNA damage repair pathways to determine the repair pathway involved in the oxidative damage response. To investigate the peroxide toxicity mechanism and to assess the protection mechanism of antigenotoxic compounds, we are testing the non-metabolizable glucose-analog 2-deoxyglucose in cell viability and in DNA damage after peroxide treatment. Our results show an increased viability concomitant with decreased tail length, suggesting that peroxide requires an active energetic metabolism for its toxicity