CONTROL OF DRUG AND ETHANOL METABOLISM

这个项目的三个具体目标源于一个长期存在的 申请者对乙醇对肝脏影响的兴趣 氧气代谢。这个项目的长期目标是 肝毒性机制的确定和最终的发展 预防人类酗酒者肝脏损害的方法--一种病理学 起源于肝小叶中心周围区域。我们 已经发现氧气在肝脏中的自然分布 小叶调节关键肝脏的代谢区划 生化过程；因此，我们计划系统地 评估氧气通过调节摄氧量的假说 产生唯一秒的生理负反馈系统 改变肝脏细胞内钙离子的信使分子。 具体地说，这一目标将通过研究 氧对新鲜沙棘细胞内游离钙离子和呼吸作用的影响 从门静脉周围和中心周围区域分离的组织 肝小叶。接下来，我们计划确定氧气的影响 二十烷基类化合物和肌醇磷酸生产的紧张 参与该过程的第二个信使。此外，我们计划 确定Km值高的酶产品的影响 氧对细胞内游离钙的影响，我们将阐明 细胞内钙离子对线粒体的作用机制 从肝门周围和中心周围区域分离 小叶。在这些实验中，我们将偶联特定的微探针 本实验室开发的探测器系统可用于摄氧量和 门静脉周围和门静脉周围其他重要的肝脏代谢事件 大鼠肝脏灌流后肝小叶中心周围区域的变化 正常大鼠和乙醇处理大鼠。 我们最近证实了过氧化氢酶-过氧化氢可以参与 灌流对大鼠肝脏乙醇代谢的影响 鹿鼠肝脏，如果有足够的底物 白蛋白结合的脂肪酸。由于乙醇会导致脂质 在肝脏中积累，我们计划评估这一假设 乙醇通过以下途径激活乙醇代谢的过氧化氢酶途径 为过氧化氢的产生提供脂肪酸的方法是比较 ADH~鹿小鼠肝脏灌流的乙醇氧化作用 通过评估上述指标，在体外进行过氧化物体的β-氧化 ADH~鹿小鼠乙醇代谢昼夜变化的途径 在体内，并通过研究急性和慢性治疗的效果 乙醇在这条重要但以前被忽视的途径上。 我们最近还发现，脂肪酸-白蛋白复合体可以 在灌流中以高速率产生有毒的氧气物种(例如，过氧化氢) 肝脏。由于脂肪最初在心脏中心周围积聚 我们计划评估以过氧化物酶体为主的肝小叶 酒精性肝毒性是由局部原因引起的假设 细胞内还原氧物种的产生 中央静脉。首先，将开发研究酒精的模型- 对ADH~-鹿小鼠造成肝损伤。接下来，方法将是 是为了研究脂肪的积累和 过氧化氢在门静脉周围和周围的动态变化 基于荧光的肝小叶中心周围区域 亲油性染料的性质及其吸附特性 利用灌流的大鼠和鹿鼠肝脏对过氧化氢复合体的研究 分别进行了分析。最后，还原的氧物种和脂类自由基 将被捕获并由肝脏中的电子自旋共振决定 来自酒精处理的大鼠和鹿小鼠。一旦我们理解了 肝脂和氧分压在慢性酒精性肝损伤中的作用 疾病，我们可以提供合理治疗的第一个关键步骤 这种广为流传的疾病。

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.