Our previous observations suggest that one of the major physiologic effects of alcohol on the mammalian liver is to inhibit the capacity of the liver to repair itself following a surgical deficit or cellular injury. Indeed, in vitro studies with short term hepatocyte cultures suggest that ethanol inhibits hormone stimulated DNA synthesis and that metabolism of ethanol may be required. There appears to be a critical time in the cell cycle, namely G1, where the hepatocyte may be most susceptible to the action of ethanol. We wish to consolidate and extend these gains and perform studies designed to explore the effect of ethanol at the physiologic, cellular and molecular level. We plan to do the following; 1) perform additional in vitro studies of ethanol on hormone stimulated hepatocyte DNA synthesis. In this regard, we will initiate hepatocyte cultures from animals chronically fed ethanol and determine its long term effect on the hepatocyte proliferative response. We will dispense hepatocyte cultures from rats under the influence of acute ethanol administration in the presence and absence of 4MP. Hepatocyte cultures will be prepared at various times after 2/3 hepatectomy in the setting of acute and chronic ethanol feeding. We are particularly committed to evaluate cells isolated 4 - 12 hours after surgery. 2) Pursue the effect of acute and chronic ethanol feeding on liver regeneration in vivo. We plan to assess the role of alcohol metabolism in relationship to the stage of the cell cycle. We will measure alcohol metabolism in hepatocytes at various intervals following 213 hepatectomy in concord with DNA sythesis and mitosis. Studies are planned to assess ethanol metabolism by hepatocytes in late G1 since these cells appear to be the most susceptible to the inhibitory actions of ethanol. We will use multiparameter flow cytometric analysis for determination of hepatocyte nuclear RNA and DNA content. 3) Examine the possible cellular molecular mechanisms of ethanol on hepatocyte proliferation both in vitro and in vivo. We wish to explore, therefore, ethanol effects on some of earlier events associated with hepatocyte proliferation such as oncogene activation and expression. We will evaluate rat ADH gene expression during various stages of the cell cycle and comparisons will be made to functional studies of alcohol metabolism. Finally, we will measure EGF receptor binding and subsequent autophosphorylation of its receptor and other membrane proteins. We suspect that ethanol may effect the binding and number of EGF receptors particularly prior to and during the G 0 and G 1 transition of the hepatocyte cell cycle. A clinical question to be addressed in this grant proposal. is with ongoing liver injury, the capacity of the liver to repair itself may become important and ethanol consumption may adversely effect this process. For example, continuous cellular injury in the liver that has a diminished capacity to regenerate because of ethanol exposure could promote the development of hepatic failure. Thus, a better understanding of ethanol effects on the hepatic repair process at an experimental level may have important implications with respect to the progression of clinical liver injury induced by acute and chronic alcohol abuse.
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