HUMAN METABOLISM OF HALOTHANE-MECHANISMS OF TOXICITY

氟烷的人体代谢-毒性机制

基本信息

批准号：
3420086
负责人：
JAMES Robert TRUDELL
金额：
$ 28.2万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-12-01 至 1990-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3420086
关键词：
NADPH cytochrome c2 reductase anesthesia complication anesthetics carbene chemical addition chemical carcinogen cytochrome P450 cytochrome oxidase density gradient ultracentrifugation drug adverse effect drug metabolism electron microscopy electrophoresis endoplasmic reticulum enflurane environmental toxicology enzyme induction /repression enzyme reconstitution gas chromatography gel filtration chromatography halothane health care personnel hepatotoxin high performance liquid chromatography hospital personnel human subject human tissue hydrocarbons inhalation anesthesia isoniazid laboratory rabbit laboratory rat lipid metabolism liposomes liver cells liver pharmacology liver toxic disorder mass spectrometry membrane proteins microsomes occupational hazard phospholipids protein reconstitution radiotracer spectrometry tissue /cell culture toxin metabolism triiodothyronine

项目摘要

The long term goal of this grant is to understand the mechanism of toxicity of both acute hepatic necrosis and chronic sub-clinical impairment of liver function that result from anesthetic metabolism in humans. We will carry out experiments in two complementary experimental systems: Monolayers of hepatocytes in primary culture will allow study of the effects of metabolism on the integrated systems of the cell as well as attack of hepatocytes by macrophages activated by metabolically-produced chemo attractants or antigens. A synthetic model of a liver cell formed by reconstituting human cytochrome p-450 and NADPH cytochrome P-450 reductase into phospholipid vesicles will allow study of individual steps of anesthetic metabolism, formation of chemo-attractant leukotrienes, and antigenic metabolites on the cell surface. The following experiments with halothane will also contribute to the understanding of how other halogenated hydrocarbons cause liver disease and cancer. 1) We wil continue our studies of the toxicity of halothane metabolism in monolayers of hepatocytes under conditions that mimic those that exacerbate halothane toxicity in vivo, especially the effect of hypoxia. 2) We will use reconstituted human cytochromes P-450 to continue our studies of production of arachidonic acid hydroperoxides during metabolism of halothane and their conversion by specific isozymes of human cytochrome P-450 to the potent chemoattractant leukotriene B-4 (LTB-4) 3) We will measure production and release of LTB-4 from monolayers of hepatocytes in response to injury by halothane metabolism as a function of oxygen concentration. 4) We will isolate both leukocytes and the resident liver macrophages, Kupffer cells, and measure their lysis of hepatocytes as a consequence of activation by leukotrienes produced during halothane metabolism. 5) We will use reconstituted human cytochrome P-450 to study production of N-trifluoroacetyl-phosphatidylethanolamide, a known metabolite of halothane that we suggest may act as an antigen if exposed on the hepatocyte plasma membrane. We will prepare antibodies to this antigen. 6) We will allow monolayers of hepatocytes to metabolize halothane to produce the proposed antigen on the plasma membrane surface, add the specific antibodies, and measure immune complex mediated lysis of hepatocytes by leukocytes or Kupffer cells.

这笔赠款的长期目标是了解该机制急性肝坏死和慢性亚临床毒性的研究麻醉引起的肝功能损害人体的新陈代谢。我们将分两次进行实验补充实验系统：单层肝细胞原代培养将允许研究新陈代谢的影响对细胞的集成系统以及攻击肝细胞被代谢产生的巨噬细胞激活化学引诱剂或抗原。肝细胞的合成模型由重组人细胞色素 p-450 和 NADPH 形成细胞色素 P-450 还原酶进入磷脂囊泡将允许研究麻醉代谢的各个步骤，形成化学引诱剂白三烯和抗原代谢物细胞表面。以下氟烷实验也将有助于理解其他卤代化合物如何碳氢化合物会导致肝病和癌症。 1）我们将继续研究氟烷的毒性条件下单层肝细胞的代谢模仿那些加剧体内氟烷毒性的物质，尤其是缺氧的影响。 2) 我们将使用重组的人细胞色素 P-450 继续研究花生四烯酸的生产氟烷代谢过程中的氢过氧化物及其通过人细胞色素 P-450 的特定同工酶进行转化有效的化学引诱剂白三烯 B-4 (LTB-4) 3) 我们将测量 LTB-4 的生产和释放肝细胞单层对损伤的反应氟烷代谢随氧的变化专注。 4) 我们将分离白细胞和驻留肝脏巨噬细胞、库普弗细胞，并测量它们的裂解白三烯激活的肝细胞氟烷代谢过程中产生。 5) 我们将使用重组的人细胞色素P-450来研究 N-三氟乙酰基-磷脂酰乙醇酰胺的生产我们建议可能充当氟烷的已知代谢物如果暴露在肝细胞质膜上，则为抗原。我们将制备针对该抗原的抗体。 6）我们将允许单层肝细胞代谢氟烷在血浆上产生建议的抗原膜表面，添加特异性抗体，测定免疫复合物介导的肝细胞裂解白细胞或库普弗细胞。