Modulation of cholestane-3 beta,5 alpha,6 beta-triol toxicity by butylated hydroxytoluene, alpha-tocopherol and beta-carotene in newborn rat kidney cells in vitro.

Cholesterol oxidation products (COP) have been reported to influence vital cellular processes such as cell growth, cell proliferation, membrane function and de novo sterol biosynthesis. The objectives of the present study were: (1) to develop an in vitro model using newborn rat kidney (NRK) cells to investigate the actions of COP; (2) to investigate the effect of COP on cell viability, endogenous antioxidant enzymes activities, i.e. superoxide dismutase (EC 1.15.1.1; SOD) and catalase (EC 1.11.1.6; CAT), and the extent of lipid peroxidation in this model; (3) to determine whether the addition of 100-1000 nM-alpha-tocopherol, beta-carotene or butylated hydroxytoluene (BHT) could protect against COP-induced cytotoxicity. NRK cells were cultured in the presence of various concentrations (5-50 microM) of cholesterol or cholestane-3 beta,5 alpha,6 beta-triol (cholestantriol) for a period of 24 h. Cholesterol over the range 5-50 microM did not induce cytotoxicity as indicated by the neutral-red-uptake assay or the lactate dehydrogenase (EC 1.1.1.27)-release assay. However, cell viability was compromised by the addition of > 10 microM-cholestantriol (P < 0.05). The addition of beta-carotene (100-1000 nM) did not increase cell viability significantly in cholestantriol-supplemented cells. However, the addition of alpha-tocopherol (1000 nM) and BHT (1000 nM) significantly increased percentage cell viability above that of the cholestantriol-supplemented cells but not back to control levels. SOD and CAT activities in NRK cells significantly decreased (P < 0.05) following incubation with cholestantriol. The addition of > 750 nM-alpha-tocopherol, beta-carotene or BHT returned SOD and CAT activities to that of the control. Lipid peroxidation was significantly induced (P < 0.05) in the presence of cholestantriol. Supplementation of the cells with alpha-tocopherol (250, 500 or 1000 nM) or BHT (750 or 1000 nM) resulted in a reduction in the extent of lipid peroxidation (P < 0.05). The addition of beta-carotene over the concentration range of 250-1000 nM did not reduce lipid peroxidation significantly compared with cells exposed to cholestantriol alone. These findings suggest that addition of exogenous antioxidants may be beneficial in the prevention of COP-induced toxicity in vitro.