Toxicology zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ in Vitro 10 (1996) 557-566 Inhibition of Protein Thiol Modification in Hepatocytes Isolated from Rats Supplemented with Vitamin E under Oxidative Stress zyxwvutsrqponmlk C.-K. LII*, H.-W. CHEN and S.-T. WANG Department of Nutrition, Chung-Shan Medical College, 113, Sec. 2, Ta-Chien St, Taichung, Taiwan 40203, Republic of China (Accepted 11 June 1996) Ahstrati-This study examined the effect of vitamin E on maintaining the protein reactive thiols under oxidative stress. Hepatocytes were prepared from male Sprague-Dawley rats fed diets containing three levels of vitamin E (0, 100 and 15,000 mg/kg) for 12 wk. Cells were isolated by collagenase perfusion and treated with 0.5 mM tert-butyl hydroperoxide (t-BuOOH) after 24 hr in culture. Carbonic anhydrase III (CA III) having two reactive thiols that can react with GSH under oxidative stress was chosen as the study subject. CA III S-glutathionation was measured by isoeiectric focusingjimmunoblotting. Results indicated that thiol modification of CA III was induced by t-BuGGH and the pattern of modification was dependent on the vitamin E status. With t-BuOOH treatment, CA III S-glutathionation was quickly induced and the maximum modification was achieved at 3 min in cells isolated from rats fed high levels of vitamin E; however, modification was continuously increased and reached the maximum at 9 min of vitamin E-normal or -deficient cells. Following the maximum modification, a reversion occurred (dethiolation); the rate of reversion was also related to vitamin E status. As shown by image analysis, twofold more (40 v. 20%) CA III was modified in vitamin E-deficient hepatocytes than in cells from rats fed high vitamin E. Glutathione was also abruptly converted to the oxidized state at 3 min in all cells, then gradually reverted to the reduced state. As with the dethiolation of CA III, the rate of glutatbione disulfide reduction was correlated to vitamin E status. The production of thiobarbituric acid-reactive substances corresponded to vitamin E status as well and was significantly inhibited in cells from rats fed high vitamin E. These results suggest that vitamin E not only inhibits lipid peroxidation but also plays a role in maintaining the protein thiols under oxidative stress. Copyright 0 1996 Elsevier Science Ltd. INTRODUCDON Vitamin E, a natural lipophilic antioxidant, is primarily found in cells and organelle membranes and plays a critical role in preventing lipid peroxidation. Vitamin E can donate a hydrogen atom to a lipid peroxyl radical to generate lipid hydroperoxide, thus terminating the progression of lipid peroxidation (Niki, 1991). Other functions of vitamin E have also been proposed, for example in maintaining membrane protein thiols (Reed et al., 1987; Takenaka ef al., 1991), in stabilizing membrane structure (Urano et al., 1992) and in preventing certain diseases (Public Health Service, 1988). Although the role of vitamin E is primarily focused on the lipid bilayer membrane, the interaction of vitamin E with other components of the antioxidant defence system has attracted much attention. *Author for correspondence. Abbreuiations: BSA = bovine serum albumin, CA III = carbonic anhydrase III; GSH = glutathione: GSSG = glutathione disuIBde; IEF = isoelectric focusing; t- BuOGH = rerr-butyl hydroperoxide; TBARS = thiob- art&uric acid-reactive substances. Glutathione (GSH), the major intracellular low molecular weight thiol and antioxidant, has been demonstrated to be closely related to vitamin E. For instance, the regeneration of vitamin E has been shown to be dependent on a GSH-dependent labile factor (Leedle and Aust, 1990; Murphy et al., 1992; Palamanda and Kehrer, 19931, and vitamin E may facilitate GSH biosynthesis as well (Pascoe et al., 1987a). Other than GSH, membrane and soluble protein thiols exhibit the ability of sparing vitamin E by offering antioxidant capacity (Murphy et al., 1992; Takenaka et al., 1991). Additionally, recent investi- gations have demonstrated that either lipid-soluble or water-soluble antioxidants could also act to scavenge radicals generated in the aqueous phase and/or in the phospholipid membrane (Barclay and Vinquist, 1994; Takenaka et al., 1991). In view of this, the interaction between vitamin E and intracellular thiols enables us to study the effect of vitamin E on intracellular protein thiols, especially under oxidative stress. Oxidative stress refers to the disturbance of the equilibrium between antioxidants and pro-oxidants towards oxidants (Sies, 1985). During oxidative stress, many adverse effects are triggered, including 0887-2333/96/815.00 + 0.00 Copyright 0 1996 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII SOSS7.2333(96)00048-3