Toxicity of cholesterol oxidation products to caco-2 and HepG2 cells: Modulatory effects of α-and γ-tocopherol

Cholesterol can be oxidized to form a variety of cholesterol oxidation products also known as oxysterols. The aims of the present study were to compare the cytotoxic effects of four oxysterols, namely 25-hydroxycholesterol (25-OHC), 7β-hydroxycholesterol (7β-OHC), cholesterol-5β,6β-epoxide (β-epox) and cholesterol-5α,6α-epoxide (α-epox), in two human cell culture models. Further, the ability of 10 and 100 μM α- and γ-tocopherol (α-TOC and γ-TOC, respectively) to protect against oxysterol-induced cytotoxicity was also assessed. Human colonic adenocarcinoma Caco-2 and human hepatoma HepG2 cells were supplemented with increasing concentrations of 25-OHC, 7β-OHC, β-epox and α-epox (0-25 μg ml^-1) for 24, 48 or 96 h. Following 24-h and 48-h exposure, test media were replaced with normal growth media and the cells were maintained for 72 and 48 h, respectively. The 96-h exposure represented a constant challenge to the cells. Cytotoxicity was assessed using the neutral red uptake assay. The concentration of compound that inhibited cell viability by 50% (IC₅₀ value) was calculated. All four oxysterols investigated induced the greatest cytotoxic effects following 96 h of exposure. 25-Hydroxycholesterol exhibited the greatest cytotoxicity in both cell lines. Both β-epox and α-epox were more toxic to HepG2 cells than to Caco-2 cells after the 48-h exposure. Pretreatment of cells with either α- or γ-TOC did not protect against oxysterol-induced cytotoxicity. The Caco-2 cells treated with the high concentration (100 μM) of γ-TOC were found to be more susceptible to oxysterol-induced toxicity under the conditions employed in this study.