The three specific aims of this project arise from a long-standing interest of the applicant in the effects of ethanol on hepatic oxygen metabolism. The long-range goals of this project are to determine mechanic of hepatotoxicity and ultimately to develop methods to prevent liver damage in human alcoholics, a pathology that originates in pericentral regions of the liver lobule. We have found that the natural distribution of oxygen across the liver lobule regulates metabolic compartmentation of key hepatic biochemical processes; therefore, we plan to systematically evaluate the hypothesis that oxygen regulates oxygen uptake via a physiological negative feedback system by producing unique second messenger molecules which alter intracellular calcium in the liver. Specifically, this goal will be achieved by studying the effect of O2 on intracellular free Ca++ and respiration of fresh plugs of tissue isolated from periportal and pericentral regions of the liver lobule. Next, we plan to identify the effect of oxygen tension on the production of eicosanoids and inositol phosphate second messengers involved in the process. Moreover, we plan to identify the effects of products of enzymes with high Km's for oxygen on intracellular free calcium and we will elucidate the mechanism of action of intracellular calcium on mitochondria isolated from periportal and pericentral regions of the liver lobule. In these experiments, we will couple specific microprobe detector systems developed in this laboratory to oxygen uptake and other important hepatic metabolic events in periportal and pericentral regions of the liver lobule in perfused livers from normal and ethanol-treated rats. We demonstrated recently that catalase-H2O2 can participate significantly in hepatic ethanol metabolism in perfused rat and deermouse livers if provided with adequate substrate in the form of albumin-bound fatty acids. Since ethanol causes lipid to accumulate in the liver, we plan to evaluate the hypothesis that ethanol activates the catalase pathway of ethanol metabolism by providing fatty acids for H2O2 generation by comparing rates of alcohol oxidation by perfused livers, from ADH~ deermice with peroxisomal beta-oxidation in vitro, by evaluating the above pathway in diurnal variation in ethanol metabolism in ADH~ deermice in vivo, and by studying the effect of acute and chronic treatment with ethanol on this important but previously overlooked pathway. We also recently discovered that fatty acid-albumin complexes can produce toxic O2 species (e.g., H2O2) at high rates in the perfused liver. Since fat accumulates initially in pericentral regions of the liver lobule where peroxisomes predominate, we plan to evaluate the hypothesis that ethanol-induced hepatotoxicity is due to local production of reduced oxygen species in cells localized around the central vein. First, models will be developed to study alcohol- induced liver damage in the ADH~ deermouse. Next, methods will be developed to study the accumulation of lipid and the production of hydrogen of hydrogen peroxide dynamically in periportal and pericentral regions of the liver lobule based on the fluorescent properties of lipophilic dyes and the absorption characteristics of the H2O2-complex using perfused rat and deermouse livers respectively. Finally, reduced oxygen species and lipid radicals will be trapped and determined by electron spin resonance in livers from ethanol treated rats and deermice. Once we understand the role of hepatic lipid and oxygen tension in chromic alcoholic liver disease, we can provide the first critical step in rational therapy for this widespread disease.
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