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 un der oxidative stress was investigated in cultured rat hepatocytes with different vitamin E status. Hepatocytes were incubated in the presenc e or absence of 100 mu M alpha-tocopherol succinate for 24 h then trea ted with 1.5 mM t-butyl hydroperoxide (t-BH) for different time interv als. Lipid peroxidation, as determined by thiobarbituric acid-reactive substances, was completely inhibited over 60 min of treatment in cell s incubated with alpha-tocopherol. The change of cell morphology, as d etermined by surface blebs formation, was correlated with cellular vit amin E status. Surface blebs were formed in 25.1 +/- 5.2 min in the pr esence of alpha-tocopherol in contrast to 11.1 +/- 2.9 min in its abse nce. In cells with alpha-tocopherol, surface blebs were induced even t hough lipid peroxidation was inhibited. Comparing the depletion of mem brane protein thiols with t-BH treatment, twice as many (40%) thiols w ere lost over 60 min in the absence of alpha-tocopherol whereas 20% we re lost in the presence of alpha-tocopherol. In addition, the extent o f thiol modification of carbonic anhydrase III, as determined by combi ning isoelectric focusing analysis with immunoblotting, further demons trated that alpha-tocopherol helps maintain protein thiols in the redu ced state. Results indicate that vitamin E protects cell morphology an d prevents the loss of protein thiols with t-BH treatment, and on cell morphology protection is associated with protein thiols rather than m embrane lipids.