HUMAN METABOLISM OF HALOTHANE--MARKERS OF TOXIC EXPOSURE

氟烷的人体代谢——有毒暴露的标志

基本信息

批准号：
3302736
负责人：
JAMES Robert TRUDELL
金额：
$ 23.78万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-12-01 至 1993-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3302736
关键词：
NADPH cytochrome c2 reductase anesthesia complication anesthetics carbene carbon chemical addition chemical carcinogen cytochrome P450 cytochrome oxidase density gradient ultracentrifugation drug adverse effect drug interactions 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 radionuclides 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机制急性肝坏死和慢性亚行的毒性麻醉症引起的肝功能损害人类的代谢。我们将在两个中进行实验互补实验系统：肝细胞的单层在原发性文化中，将允许研究代谢的影响在单元的集成系统上以及攻击由代谢生产激活的巨噬细胞的肝细胞化学吸引剂或抗原。肝细胞的合成模型通过重建人细胞色素P-450和NADPH形成细胞色素P-450还原酶将磷脂囊泡还原为研究麻醉代谢的各个步骤，形成化学吸引白细胞和抗原代谢物上的抗原代谢物细胞表面。氟烷的以下实验也将有助于理解其他卤素烃会导致肝病和癌症。 1）我们将继续研究氟烷的毒性条件下的肝细胞单层代谢那些在体内加剧氟烷毒性的那些模仿特别是缺氧的影响。 2）我们将使用重构的人类细胞色素P-450 继续我们的蛛网膜酸生产研究氟烷及其代谢期间的氢过氧化物及其通过人细胞色素P-450的特定同工酶转化到有效的趋化剂白三烯B-4（LTB-4） 3）我们将测量LTB-4的生产和释放肝细胞的单层响应损伤氟烷代谢作为氧气的函数专注。 4）我们将隔离白细胞和常驻肝脏巨噬细胞，库普弗细胞，并测量其裂解肝细胞是白细胞激活的结果在氟烷代谢期间产生。 5）我们将使用重构的人类细胞色素P-450进行研究 N-三氟乙酰基磷脂酰乙醇胺的产生，A 我们建议的已知代谢产物的代谢产物可能充当如果暴露在肝细胞质膜上的抗原。我们将准备该抗原的抗体。 6）我们将允许肝细胞的单层代谢氟烷在等离子体上产生所提出的抗原膜表面，添加特定抗体并测量免疫复合物介导的肝细胞裂解白细胞或库普弗细胞。