Alpha-tocopherol and inhibition of cytolysis in glutathione-depleted hepatocytes in primary culture.

Treatment of cultured rat hepatocytes with 10 microM 1-chloro-2,4-dinitrobenzene (CDNB) resulted in an acute loss of cellular glutathione (GSH) within 30 min and a marked increase in spontaneous lactic dehydrogenase (LDH) leakage to the culture medium after 24 h, with obvious cellular degeneration as viewed by phase-contrast microscopy. Simultaneous treatment of the cells with alpha-tocopherol markedly protected the cells not only against LDH leakage but cellular degeneration in a dose-dependent manner. The EC50 of alpha-tocopherol was 0.1 microM, ca. 200 times less than normal plasma levels in the rat. In response to the inhibitory effects of alpha-tocopherol on the cytolysis as measured by LDH leakage, GSH biosynthesis was stimulated by CDNB, and cellular GSH levels returned to control levels. The recovery was inhibited by 0.2 mM buthionine-SR-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. However, the stimulation of GSH biosynthesis apparently was not essential for the protection from cytolysis by GSH depletion during the experimental period, because treatment with 0.2 mM BSO and 20 microM tocopherol completely protected the cells against the lysis induced by BSO up to 32 h without cellular GSH recovery. The results suggest that alpha-tocopherol may be a primary natural inhibitor of the cytolysis induced by xenobiotics which consume the cellular GSH in vivo.