Inhibitory effects of isoflavones on nitric oxide- or peroxynitrite-mediated DNA damage in RAW 264.7 cells and φX174 DNA

Abstract

The inhibitory effects of isoflavones (genistin, daidzin and their aglycones genistein, daidzein) on sodium nitroprusside (SNP; nitric oxide donor)- or peroxynitrite-mediated DNA damage in intact cells and in plasmid DNA was investigated. RAW 264.7 cells, a murine macrophage cell line, are capable of producing nitric oxide and superoxide anion. However, macrophages themselves are also shown to be more sensitive to nitric oxide or peroxynitrite, and were therefore used in these studies. Results from single-cell gel electrophoresis (the comet assay) showed that these isoflavones, at the concerning of 25–200 μ m, inhibited the induction of nitric oxide- or peroxynitrite-mediated macrophage genotoxicity, with genistein showing the greatest inhibition. Genistein and daidzein, at a concentration of 1–25 μm, dose-dependently inhibited peroxynitrite-induced φX174 DNA degradation based on the results of agarose gel electrophoretic analysis. Although SNP could increase the cellular GSH level, no significant differences in the glutathione content or the GSH:GSSG ratio were observed for genistein and daidzein in the presence or absence of SNP as compared with SNP-only treated RAW 264.7 cells. Exposure of RAW 264.7 cells to SNP caused the enzyme activities of GSH peroxidase, GSH reductase and catalase decrease to 44, 20 and 34% of that of untreated cells, respectively. On the contrary, exposure of RAW 264.7 cells to SNP in the presence of 100 μm of genistein or daidzein caused the enzyme activities of GSH peroxidase, GSH reductase and catalase decrease to 18, 9 and 12% (genistein) or 13, 9 and 19% (daidzein) of that of untreated cells, respectively. These results suggest that the inhibition by isoflavones of SNP- or peroxynitrite- mediated DNA damage could be attributed to their nitric oxide or peroxynitrite scavenging activities and their prevention of antioxidant enzyme inactivation.