ELSEVIER Toxicology Letters 89 (1996) 91-98 Toxicology Letters Vitamin I: protection of cell morphology and protein thiols in rat hepatocytes treated with tert-butyl hydroperoxide Shih-Tsung Wang”, Jyh-Huang Kuo”, Rong-Ghi R. Choub, Chong-Kuei LiPI* zyxwvutsrqp aDepartment of Nutrition, Chung-Shan Medical College, 113 Ta-Chien St., Sec. 2, Taichung 40203, Taiwan bl)epartment of Animal Science, National Chia- Yi Institute of Agriculture, Chai- Yi, Taiwan Received 26 April 1996; revised 18 June 1996; accepted 20 June 1996 zyxwvutsrqponmlkjihgfedcbaZYX Abstract Effects of vitamin E on cell morphology and cellular protein thiols under oxidative stress was investigated in cultured rat hepatocytes with different vitamin E status. Hepatocytes were incubated in the presence or absence of 100 uM a-tocopherol succinate for 24 h then treated with 1.5 mM t-butyl hydroperoxide (t-BH) for different time intervals. Lipid peroxidation, as determined by thiobarbituric acid-reactive substances, was completely inhibited over 60 min of treatment in cells incubated with u-tocopherol. The change of cell morphology, as determined by surface blebs formation, was correlated with cellular vitamin E status. Surface blebs were formed in 25.1 f 5.2 min in the presence of a-tocopherol in contrast to 11.1 _+ 2.9 min in its absence. In cells with cc-tocopherol, surface blebs were induced even though lipid peroxidation was inhibited. Comparing the depletion of membrane protein thiols with t-BH treatment, twice as many (40%) thiols were lost over 60 min in the absence of a-tocopherol whereas 20% were lost in the presence of a-tocopherol. In addition, the extent of thiol modification of carbonic anhydrase III, as determined by combining isoelectric focusing analysis with immunoblotting, further demonstrated that a-tocopherol helps maintain protein th:iols in the reduced state. Results indicate that vitamin E protects cell morphology and prevents the loss of protein thiols with t-BH treatment, and on cell morphology protection is associated with protein thiols rather than membrane lipids. Keywords: Vitamin E; Cell surface blebs; Protein thiol modification; Hepatocytes 1. Introduction Reactive oxygen species (ROS), e.g. singlet oxy- gen, superoxide anion, hydrogen peroxide, and hydroxyl radical * Corresponding author. drug metabolism. 0378-4274/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO378-4274(96)03793-9 are usually generated during When these ROS cannot be