Toxicity of cholesterol oxidation products to Caco‐2 and HepG2 cells: modulatory effects of α‐ and γ‐tocopherol

Abstract

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), 7beta-hydroxycholesterol (7beta-OHC), cholesterol-5beta,6beta-epoxide (beta-epox) and cholesterol-5alpha,6alpha-epoxide (alpha-epox), in two human cell culture models. Further, the ability of 10 and 100 micro m alpha- and gamma-tocopherol (alpha-TOC and gamma-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, 7beta-OHC, beta-epox and alpha-epox (0-25 micro g 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% (ic(50) 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 beta-epox and alpha-epox were more toxic to HepG2 cells than to Caco-2 cells after the 48-h exposure. Pretreatment of cells with either alpha- or gamma-TOC did not protect against oxysterol-induced cytotoxicity. The caco-2 cells treated with the high concentration (100 micro m) of gamma-TOC were found to be more susceptible to oxysterol-induced toxicity under the conditions employed in this study.

Evidence of cell damage induced by major components of a diet-compatible mixture of oxysterols in human colon cancer CaCo-2 cell line

“…Studies from O'Sullivan and colleagues [24] have shown that 25-hydroxycholesterol, a-epoxy, b-epoxy or 7b-OH added individually to undifferentiated CaCo-2 cells determined a reduction in cell viability. The cytotoxicity exerted on undifferentiated CaCo-2 cells by 24 h treatment was assessed by the neutral red uptake assay and showed IC50 values with increasing concentrations of b-epoxy and 7b-OH (0e50 mM) [24].…”

“…Studies from O'Sullivan and colleagues [24] have shown that 25-hydroxycholesterol, a-epoxy, b-epoxy or 7b-OH added individually to undifferentiated CaCo-2 cells determined a reduction in cell viability. The cytotoxicity exerted on undifferentiated CaCo-2 cells by 24 h treatment was assessed by the neutral red uptake assay and showed IC50 values with increasing concentrations of b-epoxy and 7b-OH (0e50 mM) [24]. Similar results were obtained in Caco-2 cells treated with 30 mM 7b-OH that inhibited cell growth by 50% at 32 h incubation and induced apoptosis, but in this case caspase-3 activity remained within control range during the whole observation time (72 h) [25].…”

TGFβ1 expression in colonic mucosa: modulation by dietary lipids

“…Different types of oxysterols may influence cell signaling in various ways. 25-Hydroxycholesterol or 7-beta-hydroxycholesterol have been found cytotoxic for Caco-2 human colon carcinoma cell line [78], whereas lower concentrations of 25-hydroxycholesterol associated with IL-1beta induced production of inflammatory IL-8 in the same cells [6]. On the other hand, a mixture of oxysterols, obtained by cholesterol heating, that mimics the concentration of these compounds in cooked foods, increased the activation of liver X receptors (LXRs), which are important regulators of cholesterol catabolism and lipid metabolism, thus negatively influencing inflammatory cell response [85,120].…”

“…Dietary lipids have an important pathophysiological impact on mucosa function: they influence membrane fluidity, which may alter cell signaling from the receptor system, immune response, inflammatory reactions and cell death [70,78]. A large body of evidence from epidemiological studies indicates that dietary lipids, such as polyunsaturated fatty acids (PUFAs) or cholesterol oxidation products, and intestinal microflora, are the main responsible for the production of oxidized species in the colon [78,115].…”

“…A large body of evidence from epidemiological studies indicates that dietary lipids, such as polyunsaturated fatty acids (PUFAs) or cholesterol oxidation products, and intestinal microflora, are the main responsible for the production of oxidized species in the colon [78,115]. Excessive oxidative cell reactions together with reduced antioxidant tissue defenses are prominent features in the pathogenesis of several chronic gastrointestinal diseases [13,46].…”