There is increasing evidence that cholesterol oxidation products contribute to the progression of a number of diseases. Among these diseases is atherosclerosis where it is hypothesized that lipid peroxidation and attendant cholesterol oxidation in serum lipoproteins and arterial walls contribute to abnormal deposition of cells and lipids. We have shown that several cholesterol oxides are potent and persistent cytotoxic agents. Although cholesterol oxides are recognized as cytotoxic, their mechanism of toxicity remains unknown. We therefore propose to characterize the cytotoxic mechanisms of the major cholesterol oxides. Since evidence to date suggests that cholesterol oxide cytotoxicity is manifested via membrane perturbations, our approach will be to study membrane structural/functional alterations by exposing cultured cells to biologically attainable concentrations of specific cholesterol oxides. Their uptake, distribution and toxicity will be analyzed in rabbit aortic endothelial and V79 cells using confluent and logarithmically dividing cultures. Endothelial cells serve as a model representing the aortic intima which is breached during atherogenesis. Cell growth, DNA synthesis inhibition and DNA damage serve as basic measures of toxicity, however, other specific cell functions will also be assayed. Effects of cholesterol oxides on membrane structure will be studied using artificial membranes and extended to isolated cell membranes and intact cells. Cell lipid composition will be analyzed following exposure to cholesterol oxides and any noted alterations compared to effects on membrane fluidity which serves as a basic measure of structure. The duration and reversibility of lipid or membrane functional changes will be determined during and after cessation of exposures. Basic membrane functions to be analyzed in conjunction with structural measurements are ion fluxes. concentrating on calcium since disturbances in calcium transport appear to be linked to toxicity. The transport of glucose, passive permeability and inhibition of metabolic cooperation represent other membrane effects associated with cytotoxicity, cell retraction, and membrane damage. We will determine if cholesterol oxides affect one or more membrane functions, if common effects are shared, if the basis for common effects is altered membrane structure and if these effects account for cytotoxicity. These studies should better characterize potentially angiotoxic cholesterol oxides laying the groundwork for in vivo studies on their role in atherogenesis.
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