Use of transfected liver cells to evaluate potential mechanisms of alcohol-induced liver injury.

There is increased activity of the proinflammatory cytokine, tumor necrosis factor (TNF) in alcoholic liver disease (ALD). Hepatic neutrophil infiltration is a principal injurious manifestation of ALD. TNF can induce cellular oxidative injury directly, and indirectly by inducing neutrophil chemotactic factor (IL-8) production by hepatocytes. IL-8 activates and chemotactically attracts neutrophils to the liver where they release oxidizing substances. Patients with ALD also have decreased protective factors for cellular oxidative injury. Manganous superoxide dismutase (MnSOD) is an antioxidant protective factor. The objectives of these studies were to investigate mechanisms for induction of an injurious factor (IL-8) and a protective factor (MnSOD) in the HepG2 human hepatoma cell line. In the first set of experiments, IL-8 gene reporter constructs were used to transiently transfect a derivative (MVh2E1-9) of the HepG2 cell line which expresses P-4502E1 and metabolizes ethanol. Inactivation of the NF-kappaB and 3'NF-IL-6 DNA binding sites decreased IL-8 gene transcriptional activation in response to TNF while inactivation of the 5'NF-IL-6 binding site increased IL-8 gene transcriptional activity in response to TNF. This system may be useful to assess the effects of ethanol on TNF-induced hepatocyte IL-8 production. In the second set of experiments, HepG2 cells were cultured in 25 to 100 mmol concentrations of ethanol. Both TNF and ethanol increased HepG2 cell MnSOD activity in short-term (72 hr) cultures with ethanol. However, after long-term (10 weeks) culture with ethanol, there was no induction of MnSOD by ethanol and there was a diminished induction of MnSOD in response to TNF. Further studies are needed to assess the effect of this diminished induction of MnSOD with chronic ethanol culture on HepG2 cell susceptibility to TNF cytotoxicity. We conclude that transfected liver cell lines can be used to evaluate mechanisms for increased injurious factors and decreased protective factors in alcoholic liver injury.