Tumor necrosis factor-alpha (TNF-alpha) is a mediator of liver injury. The objective of this study was to develop an in vitro model of TNF-mediated liver cell injury using the Hep G2 cell line. Hep G2 cells normally are insensitive to TNF cytotoxicity, but they were rendered susceptible, or sensitized, to TNF cytotoxicity by inhibitors of RNA and protein synthesis. The concentration of TNF required to kill 50% of Hep G2 cells sensitized with 0.8 mumol/L actinomycin D (Act D) was 35 pmol/L compared with 5 pmol/L for LM fibroblasts, a classic target cell used in TNF cytotoxicity bioassays. Similarly, TNF cytotoxicity occurred in Hep G2 cells sensitized with cycloheximide (CHX), and cytotoxicity to both inhibitors was dose dependent. Both protein and RNA synthesis were inhibited in Hep G2 cells by the concentrations of CHX and Act D associated with TNF cytotoxicity. Hep G2 cells pretreated with TNF alone and later exposed to normally toxic concentrations of TNF with DACT did not develop cytotoxicity. Thus, in vitro tolerance to TNF was induced. Cytotoxicity also was more severe at modestly increased temperatures (39 degrees C versus 37 degrees C), which may have clinical relevance to hepatic decompensation during febrile episodes. We suggest that the Hep G2 cell line sensitized by inhibiting RNA and protein synthesis is a useful in vitro model for evaluating mechanism(s) of TNF-mediated liver cell injury.