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 ra diation-induced apoptosis. Incubating mouse thymocytes with 25 mu M eb selen immediately after Co-60 gamma-radiation exposure (5 Gy) inhibite d morphological changes associated with apoptosis. Treatment of thymoc ytes with ebselen before, during, or after irradiation completely bloc ked internucleosomal DNA fragmentation, a biochemical marker for apopt osis. We measured peroxides formed in cells during and after irradiati on, using the oxidation-sensitive fluorescent probe 2',7'-dichlorofluo rescin diacetate. By 2 min postirradiation, levels of peroxides in irr adiated thymocytes were approximately 10-11 times greater than those i n the same cells before irradiation, and levels continued to increase with time. We also measured membrane lipid peroxidation using cis-pari naric acid, a naturally fluorescent polyunsaturated fatty acid that re adily incorporates into cell membranes. The oxidation of cis-parinaric acid also began soon after irradiation and increased with time. Perox ide generation and membrane lipid peroxidation preceded both internucl eosomal 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 sc avenging 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 cel ls.