Ebselen inhibition of apoptosis by reduction of peroxides

We investigated the capacity of ebselen [2-phenyl-1,2-benzisoselenazol-3 (2H)-one], a glutathione peroxidase mimic, to protect cells from radiation-induced apoptosis. Incubating mouse thymocytes with 25 mu M ebselen immediately after Co-60 gamma-radiation exposure (5 Gy) inhibited morphological changes associated with apoptosis. Treatment of thymocytes with ebselen before, during, or after irradiation completely blocked internucleosomal DNA fragmentation, a biochemical marker for apoptosis. We measured peroxides formed in cells during and after irradiation, using the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescin diacetate. By 2 min postirradiation, levels of peroxides in irradiated thymocytes were approximately 10-11 times greater than those in the same cells before irradiation, and levels continued to increase with time. We also measured membrane lipid peroxidation using cis-parinaric acid, a naturally fluorescent polyunsaturated fatty acid that readily incorporates into cell membranes. The oxidation of cis-parinaric acid also began soon after irradiation and increased with time. Peroxide generation and membrane lipid peroxidation preceded both internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis. Treatment of cells with ebselen reduced peroxide levels and appeared to protect thymocytes from radiation-induced apoptosis by scavenging peroxides generated during and after irradiation. The results suggest that peroxide generation and membrane lipid peroxidation may be important signaling events that trigger apoptosis in irradiated cells.