MODULATION OF CHOLESTAN-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 v ital cellular processes such as cell growth, cell proliferation, membr ane function and de novo sterol biosynthesis. The objectives of the pr esent study were: (1) to develop an in vitro model using newborn rat k idney (NRK) cells to investigate the actions of COP; (2) to investigat e 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-tocophero l, beta-carotene or butylated hydroxytoluene (BHT) could protect again st COP-induced cytotoxicity. NRK cells were cultured in the presence o f various concentrations (5-50 mu M) of cholesterol or cholestan-3 bet a,5 alpha,6 beta-triol (cholestantriol) for a period of 24 h. Choleste rol over the range 550 mu M did not induce cytotoxicity as indicated b y the neutral-red-uptake assay or the lactate dehydrogenase (EC 1.1.1. 27)-release assay. However, cell viability was compromised by the addi tion of > 10 mu M-cholestantriol (P < 0.05). The addition of beta-caro tene (100-1000 nM) did not increase cell viability significantly in ch olestantriol-supplemented cells. However, the addition of alpha-tocoph erol (1000 nM) and BHT (1000 nM) significantly increased percentage ce ll viability above that of the cholestantriol-supplemented cells but n ot back to control levels, SOD and CAT activities in NRK cells signifi cantly decreased (P < 0.05) following incubation with cholestantriol. The addition of >750 nM-alpha-tocopherol, beta-carotene or BHT returne d SOD and CAT activities to that of the control. Lipid peroxidation wa s significantly induced (P < 0.05) in the presence of cholestantriol. Supplementation of the cells with ar-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 conce ntration range of 250-1000 nM did not reduce lipid peroxidation signif icantly compared with cells exposed to cholestantriol alone. These fin dings suggest that addition of exogenous antioxidants may be beneficia l in the prevention of COP-induced toxicity in vitro.