Vitamin E protection of cell morphology and protein thiols in rat hepatocytes treated with tert-butyl hydroperoxide.

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 microM alpha-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 alpha-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 +/- 5.2 min in the presence of alpha-tocopherol in contrast to 11.1 +/- 2.9 min in its absence. In cells with alpha-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 alpha-tocopherol whereas 20% were lost in the presence of alpha-tocopherol. In addition, the extent of thiol modification of carbonic anhydrase III, as determined by combining isoelectric focusing analysis with immunoblotting, further demonstrated that alpha-tocopherol helps maintain protein thiols 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.