alpha-tocopherol inhibits oxidative stress induced by cholestanetriol and 25-hydroxycholesterol in porcine ovarian granulosa cells

The cytotoxicity of oxysterols including 7-ketocholesterol, alpha-epoxide, cholestanetriol and 25-hydroxycholesterol and the possible protecting effec t of alpha-tocopherol on cholestanetriol and 25-hydroxycholesterol-induced cytotoxicity were investigated in primary cultures of porcine ovarian granu losa cells. Cell viability as determined by % trypan blue staining and mito chondrial function as determined using 3-[4,5-dimethylthiazol-2-yl]-2,5- di phenyltetrazolium bromide (MTT) reduction were decreased significantly afte r 24 h exposure to 2.5 mu M alpha-epoxide, cholestanetriol and 25-hydroxych olesterol. 7-ketocholesterol (2.5 mu M) did not affect cell viability or mi tochondrial function under the same culture conditions. The specific activi ties of catalase and superoxide dismutase, two antioxidant defense enzymes were increased significantly (p < 0.01) following 24 h exposure to 2.5 mu M concentrations of cholestanetriol while only superoxide dismutase was incr eased in 25-hydroxycholesterol-treated cells (p < 0.001). Specific activity of glutathione peroxidase was unchanged relative to control cells. Levels of thiobarbituric acid reactive substances remained unchanged after exposur e to 7-ketocholesterol, alpha-epoxide, cholestanetriol, 25-hydroxycholester ol and cholesterol. Administration of 1 mu M alpha-tocopherol to the cultur e medium significantly improved cell viability and restored both superoxide dismutase and catalase activities to control levels in cholestanetriol -tr eated cells and only superoxide dismutase in 25-hydroxycholesterol-treated cells. These studies suggest that the cytotoxic nature of physiologically r elevant concentrations of cholestanetriol and 25-hydroxycholesterol in gran ulosa cells is in part due to oxidative stress, but it may be reduced in th e presence of a-tocopherol.