USE OF TRANSFECTED LIVER-CELLS TO EVALUATE POTENTIAL MECHANISMS OF ALCOHOL-INDUCED LIVER-INJURY

There is increased activity of the proinflammatory cytokine, tumor nec rosis factor (TNF) in alcoholic liver disease (ALD). Hepatic neutrophi l 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 a ctivates and chemotactically attracts neutrophils to the liver where t hey release oxidizing substances, Patients with ALD also have decrease d protective factors for cellular oxidative injury. Manganous superoxi de dismutase (MnSOD) is an antioxidant protective factor. The objectiv es 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 r eporter constructs were used to transiently transfect a derivative (MV h2E1-9) of the HepG2 cell line which expresses P-4502E1 and metabolize s ethanol. Inactivation of the NF-kappa B and 3'NF-IL-6 DNA binding si tes decreased IL-8 gene transcriptional activation in response to TNF while inactivation of the 5'NF-IL-6 binding site increased IL-8 gene t ranscriptional activity in response to TNF. This system may be useful to assess the effects of ethanol on TNF-induced hepatocyte IL-8 produc tion. In the second set of experiments, HepG2 cells were cultured in 2 5 to 100 mmol concentrations of ethanol. Both TNF and ethanol increase d HepG2 cell MnSOD activity in short-term (72 hr) cultures with ethano l. 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 cul ture on HepG2 cell susceptibility to TNF cytotoxicity. We conclude tha t transfected liver cell lines can be used to evaluate mechanisms for increased injurious factors and decreased protective factors in alcoho lic liver injury.