Hepatocyte nuclear factor-4alpha (HNF-4alpha), a zinc finger protein, is the most abundant transcription factor in the liver. HNF-4alpha regulates a large number of genes involved in most aspects of hepatocyte functions. The present study was undertaken to determine the role of HNF-4alpha in zinc protection against tumor necrosis factor-alpha (TNF-alpha) hepatotoxicity. Mice were treated with murine TNF-alpha via intravenous injection at 20 mug/kg body wt 30 mins after d-galactosamine (d-Gal) sensitization (800 mg/kg body wt). Two doses of zinc sulfate (5 mg elemental zinc/kg body wt) were administered at 36 and 12 hrs before TNF-alpha treatment via subcutaneous injection. TNF-alpha treatment after sensitization induced liver injury as detected by plasma alanine aminotransferase activity and apoptotic cell death in the liver. Zinc pretreatment attenuated TNF-alpha-induced liver injury. Furthermore, TNF-alpha-induced activations of caspase 3 and caspase 8 in the liver were significantly inhibited by zinc pretreatment. The mRNA and protein levels of HNF-4alpha in the liver were remarkably decreased by TNF-alpha treatment, which was suppressed by zinc. To determine if HNF-4alpha depletion is involved in d-Gal sensitization to TNF-alpha toxicity, mice were administered either d-Gal or TNF-alpha. Immunohistochemistry demonstrated that HNF-4alpha depletion in the liver is associated with d-Gal sensitization but not TNF-alpha treatment. To define the link between HNF-4alpha depletion and TNF-alpha-induced cell death, the effect of silencing the HNF-4alpha gene by siRNA transfection on TNF-alpha cytotoxicity was determined in HepG2 cells. A lactate dehydrogenase cytotoxicity assay showed that neither TNF-alpha nor HNF-4alpha siRNA transfection had a toxic effect, but TNF-alpha treatment after HNF-4alpha siRNA transfection caused HepG2 cell death. These results suggest that zinc protects against TNF-alpha hepatotoxicity, at least partially, through preservation of the zinc finger protein HNF-4alpha.