Detection of hydrogen peroxide and hydroxyl radicals in murine skin fibroblasts under UVB irradiation

Abstract

The purpose of this study was to identify the active oxygen species generated in murine skin fibroblasts under UVB irradiation. When fibroblasts were exposed to UV light (UVA + UVB), hydroxyl radicals were detectable by ESR-spin trapping using DMPO as a spin trapping agent. Cell damage induced by UVB irradiation was prevented by addition of DMSO, a typical hydroxyl radical scavenger. Fibroblasts treated with a catalase inhibitor 3-amino-1H-1,2,4-triazole were more sensitive to UVB irradiation than untreated-cells, but the sensitivity was improved by addition of DMSO. To find the origin of the hydroxyl radicals in fibroblasts during UVB irradiation, H2O2 was determined by dihydrorhodamine 123 and it was found that the formation of H2O2 increased under UVB irradiation. These results suggest that UVB-irradiated skin fibroblasts produce H2O2 and were followed by the formation of hydroxyl radicals, probably derived from the Fenton-like reaction (H2O2 + Fe2+ --> .OH + OH- + Fe3+), which in turn damages the cells.