Influence of Tumor Necrosis Factor-α and Silibin on the Cytotoxic Action of α-Amanitin in Rat Hepatocyte Culture

Tumor necrosis factor-α is assumed to play a role in toxic liver damage. We examined whether exogenous tumor necrosis factor-α must be present for α-amanitin cytotoxicity in rat hepatocyte culture. α-Amanitin at a concentration of 0.1 μM, which is close to that found in intoxicated patients, inhibits RNA and protein synthesis within 12 h but cytotoxicity only occurs after a latency period and is pronounced at 36 h after the start of treatment. Tumor necrosis factor-α is not indispensable for the development of cytotoxicity but aggravates it and leads to a time shift towards earlier times. Lipid peroxidation is low with α-amanitin alone even at 36 h but markedly increased by cotreatment with tumor necrosis factor-α. The antioxidant silibin prevents the effect of tumor necrosis factor-α, indicating an involvement of reactive oxygen species. α-Amanitin alone does not increase but dose-dependently inhibits the expression of the antioxidant enzyme manganous superoxide dismutase and decreases the inducing effect of TNF-α on the expression of this enzyme. The gene expression of endogenous tumor necrosis factor-α in the hepatocytes is not increased but rather inhibited by α-amanitin treatment. The results suggest that α-amanitin causes delayed cytotoxicity following rapid inhibition of RNA and protein synthesis and that tumor necrosis factor-α shortens the latency period and aggravates the cytotoxicity by a mechanism which may involve reactive oxygen species.