Wilson A M, Sisk R M, O'Brien N M
Department of Nutrition, National Food Biotechnology Centre, University College, Cork, Republic of Ireland.
Br J Nutr. 1997 Sep;78(3):479-92. doi: 10.1079/bjn19970165.
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.
据报道，胆固醇氧化产物（COP）会影响重要的细胞过程，如细胞生长、细胞增殖、膜功能和甾醇从头生物合成。本研究的目的是：（1）建立一种使用新生大鼠肾（NRK）细胞的体外模型来研究COP的作用；（2）研究COP对该模型中细胞活力、内源性抗氧化酶活性（即超氧化物歧化酶（EC 1.15.1.1；SOD）和过氧化氢酶（EC 1.11.1.6；CAT））以及脂质过氧化程度的影响；（3）确定添加100 - 1000 nM的α-生育酚、β-胡萝卜素或丁基羟基甲苯（BHT）是否可以预防COP诱导的细胞毒性。将NRK细胞在存在各种浓度（5 - 50 μM）的胆固醇或胆甾烷-3β,5α,6β-三醇（胆甾三醇）的情况下培养24小时。中性红摄取试验或乳酸脱氢酶（EC 1.1.1.27）释放试验表明，5 - 50 μM范围内的胆固醇未诱导细胞毒性。然而，添加> 10 μM的胆甾三醇会损害细胞活力（P < 0.05）。在添加胆甾三醇的细胞中，添加β-胡萝卜素（100 - 1000 nM）并未显著提高细胞活力。然而，添加α-生育酚（1000 nM）和BHT（1000 nM）显著提高了细胞活力百分比，高于添加胆甾三醇的细胞，但未恢复到对照水平。与胆甾三醇孵育后，NRK细胞中的SOD和CAT活性显著降低（P < 0.05）。添加> 750 nM的α-生育酚、β-胡萝卜素或BHT可使SOD和CAT活性恢复到对照水平。在胆甾三醇存在下，脂质过氧化显著诱导（P < 0.05）。用α-生育酚（250、500或1000 nM）或BHT（750或1000 nM）补充细胞可导致脂质过氧化程度降低（P < 0.05）。与仅暴露于胆甾三醇的细胞相比，在250 - 1000 nM浓度范围内添加β-胡萝卜素并未显著降低脂质过氧化。这些发现表明，添加外源性抗氧化剂可能有助于预防体外COP诱导的毒性。
