MURINE HEPATOCYTE APOPTOSIS INDUCED IN-VITRO AND IN-VIVO BY TNF-ALPHAREQUIRES TRANSCRIPTIONAL ARREST

Freshly isolated mouse hepatocytes were essentially insensitive to TNF -alpha cytotoxicity. However, TNF-alpha induced a concentration-depend ent cell death in hepatocytes that had been pretreated with the transc riptional inhibitors actinomycin D (ActD), D-galactosamine, or cr-aman itin. Unlike RNA synthesis inhibition, a translational block in the pr esence of cycloheximide (CHX) or puromycin did not sensitize hepatocyt es to TNF. On the contrary, these agents prevented hepatocytotoxicity induced by ActD/TNF. Pretreatment with peroxides or glutathione deplet ors had no significant influence on TNF cytotoxicity. In vivo treatmen t of mice with ActD/TNF caused hepatic failure, which was significantl y reduced by co-treatment with CHX. These findings demonstrate that pr otein synthesis is required for this mechanism of cell death. To test whether TNF may trigger an endogenous suicide program in hepatocytes, we examined whether DNA fragmentation preceded cell death. In the cult ure system, hepatocellular DNA fragmentation in the presence of ActD/T NF was observed several hours before lactate dehydrogenase release and was inhibited by CHX. Similar results were obtained in vivo. Chromati n condensation and the formation of apoptotic bodies were observed in livers from mice treated with ActD/TNF and significant DNA fragmentati on was detected as early as 4 h after challenge. At this time, organ t otal glutathione content and plasma transaminase levels were not signi ficantly different from those of untreated controls. The findings of t his study demonstrate that direct hepatotoxicity of TNF-alpha is assoc iated with an apoptotic mechanism that becomes manifest under the meta bolic condition of arrested transcription and functional translation.