CULTURED HEPATOCYTES AS A MODEL OF ALCOHOL METABOLISM

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In the development of animal models of alcoholic liver disease, little attention has focused on the possibilities of generating an in vitro model using liver cells in primary culture. It is therefore the overall aim of this study to examine the control and regulation of ethanol metabolism in cultured rat hepatocytes. Using this model, we propose to test: (1) Extension of the culture period associated with ethanol exposure. At present, hepatocytes can only be cultured in alcohol containing medium for 7-9 days, whereas we have successfully maintained them in alcohol free medium for up to 1 month. Conditions will be modified to extend the period associated with alcohol containing culture medium in several ways: alterations to the culture medium, 02 and C02 availability, and substrate for cell attachment. (2) To examine the regulation of ethanol and acetaldehyde metabolism by these cells over at least 7 days culture. Studies on th contributions of the three known pathways of ethanol metabolism will be assessed as will be the possibility that ethanol can induce its own metabolism. Metabolic consequences of ethanol metabolism such as the generation of fatty liver and effect on serum protein synthesis and secretion will be evaluated. Regulation of acetaldehyde metabolism and the effect of prolonged exposure to ethanol on alcohol and aldehyde dehydrogenase activities will be investigated. (3) All of the above studies will be carried out using hepatocytes from normal rats. These studies will be paralleled by studies involving cells from chronic alcohol-fed rats cultured in the presence or absence of ethanol, to examine the maintenance/disappearance of tolerance. (4) The induction of and/or maintenance of the alcohol P-450. In addition to ethanol itself, we will use ligands such as pyrazole, 4-methylpyrazole or isoniazid to maintain or induce the alcohol P-450. Cells will also be cultured from animals pretreated with these agents. (5) The possible role of oxygen radicals and lipid peroxidation in the hepatotoxicity associated with ethanol will be evaluated. Generation of oxyradicals, lipid peroxidation and sensitivity to oxidative stress after prolonged exposure to ethanol will be determined. It is anticipated that examination of these parameters under carefully controlled conditions in vitro will lead to a better understanding of some aspects of the pathogenesis of liver disease following prolonged alcohol abuse.