Evaluation of antigenotoxic effects of phytochemicals: DNA protection from oxidative stress and improved DNA repair ability

Abstract

In recent years, plant extracts and their phytochemicals have received increasing attention, especially in the field of pharmaceutical sciences and medicine, due to the potential prevention activity of a number of chronic and degenerative diseases including cancer and cardiovascular diseases. Most of the health-promoting effects have been attributed to antioxidant action including the ability to scavenge reactive oxygen species and to chelate metal ions. The yeast Saccharomyces cerevisiae is perhaps the best studied eukaryotic organism and exhibits striking similarities in molecular mechanisms such as transcription, translation, replication, and DNA repair with higher eukaryotes. For these reasons, it is a good model for studying the DNA damage and search for antigenotoxic effects of plant compounds. We aim to study the protective effect of the flavonoids quercetin, isoquercitrin, quercitrin, rutin and hyperoside, in DNA and the potential inducing activity of DNA damage repair. Experiments involved pre-treatment, co-treatment and post-treatment of S.cerevisiae cells with phytochemicals (regarding the order of incubation with phytochemicals in relation to oxidative stress imposed by H2O2 or KMnO4) and DNA damage was subsequently assessed by the yeast comet assay, using tail length as the parameter for DNA damage. The results obtained from pre-treatment experiments with quercetin, isoquercitrin, quercitrin, rutin and hyperoside show that tail lengths are shorter, suggesting that these compounds protect cells against oxidative DNA damage caused by 10 mM H2O2 and 500 µM KMnO4. Furthermore, we have observed quercetin-mediated DNA protection against oxidative stress, which has improved with the time of incubation. In co-incubation experiments with 10 mM H2O2, quercetin promoted decreased DNA damage in cells, suggesting reactive oxygen species scavenging activity. In assays of pre and post-incubation with different concentrations of quercetin, we have observed a decrease of DNA damage in a dose-dependent manner. This suggests that quercetin stimulates the DNA repair machinery besides direct protection of DNA from oxidation. The observed improvement in DNA repair depends on the DNA repair machinery since post-treatments of mutants affected in Base Excision Repair (BER) with quercetin showed no reduction of comet tails, indicating that quercetin presumably could activate DNA repair enzymes from BER pathways, which repair oxidative damage.