The effect of oxysterols, individually and as a representative mixture from food, on in vitro cultured bovine ovarian granulosa cells.

The cytotoxicity of five oxysterols identified in cooked fish, 7-ketocholesterol, 7beta-hydroxycholesterol, cholesterol 5alpha,6alpha-epoxide, cholestanetriol and 4-cholesten-3-one, was investigated in primary cultures of bovine ovarian granulosa cells. Cells were exposed to the oxysterols individually and to a mixture of the same oxysterols for 24 h. Cell viability as determined by trypan blue exclusion and mitochondrial integrity (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction) was reduced by 0.5 and 2.5 microM 7beta-hydroxycholesterol, cholesterol 5alpha,6alpha-epoxide, cholestanetriol and 4-cholesten-3-one, but not by 0.5 or 2.5 microM 7-ketocholesterol under the same culture conditions. A mixture of 7-ketocholesterol, 7beta-hydroxycholesterol, cholesterol 5alpha,6alpha-epoxide, cholestanetriol and 4-cholesten-3-one 0.5 microM each; 2.5 microM oxysterol in total) did not change cell viability relative to controls. Lipid peroxidation, as determined by thiobarbituric acid reactive substances assay, was unaffected by a 24-hour exposure of granulosa cells to individual oxysterols but was increased slightly by the oxysterol mixture. The specific activities of antioxidant enzymes superoxide dismutase and catalase were increased to different extents (1.17- to 6.43-fold), relative to controls, by the administration of individual oxysterols and the oxysterol mixture. These results indicate that while some individual oxysterols can induce cytotoxic effects and defensive responses in bovine ovarian granulosa cells, administration of the same oxysterols as a mixture does not elicit the same responses. In addition, the oxysterols tested exerted a pro-apoptotic effect on granulosa cells when administered individually at concentrations of 0.5 and 2.5 microM, but not when administered as a 2.5 microM oxysterol mixture. The results suggest that major oxysterols are not universally cytotoxic, they may complete with other oxysterols for receptor sites, and that the simultaneous presence of several different oxysterol species may reduce the adverse effects of individual oxysterols.