Matthews A M, Hinson J A, Roberts D W, Pumford N R
Division of Toxicology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Toxicol Lett. 1997 Jan 15;90(1):77-82. doi: 10.1016/s0378-4274(96)03831-3.
The hepatotoxicity of the analgesic acetaminophen has been previously attributed to metabolic activation by cytochrome P450 to the reactive intermediate N-acetyl-p-benzoquinone imine. At therapeutic doses this species is detoxified by reaction with glutathione; however, following a hepatotoxic dose, liver glutathione levels are depleted and the metabolite covalently binds primarily to cysteine groups on proteins as 3-(cystein-S-yl)acetaminophen adducts. Altered function of critical proteins has been postulated to be the mechanism of hepatotoxicity. Covalent binding has been studied by both radiochemical methods and immunochemical methods. Utilizing Western blot analysis with an antiserum which recognizes acetaminophen we have previously shown that covalent binding occurs on a number of proteins in various hepatic fractions. In an effort to better understand the role of covalent binding in the toxicity, others have studied the non-hepatotoxic isomer 3'-hydroxyacetanilide. Administration of large doses of radiolabeled acetaminophen or 3'-hydroxyacetanilide resulted in similar levels of covalent binding to proteins. To better understand the role of covalent binding in toxicity we have administered mice 3'-hydroxyacetanilide and acetaminophen, and analyzed liver fractions for protein adducts using anti-3-(cystein-S-yl)acetaminophen and anti-arylacetamide antisera in Western blot assays. Analysis of liver fractions from acetaminophen-treated mice, with both antisera showed, as has been previously reported, that acetaminophen covalently binds to a number of hepatic proteins. In liver from 3'-hydroxyacetanilide-treated mice, covalent adducts were detected with an anti-arylacetamide antiserum only. A major 3'-hydroxyacetanilide protein adduct was observed in microsomes at 50 kDa. Minor adducts were observed at 47 kDa in microsomes and 56 kDa in cytosol. 3'-Hydroxyacetanilide protein adducts were not observed in the 10,000 x g pellet. Densitometric analysis of a time course of 3'-hydroxyacetanilide protein adducts indicated that peak levels of the 50 kDa microsomal protein adduct occurred at 1 h and subsequently decreased.
镇痛剂对乙酰氨基酚的肝毒性先前被认为是由细胞色素P450代谢活化生成反应性中间体N - 乙酰 - 对 - 苯醌亚胺所致。在治疗剂量下，该物质通过与谷胱甘肽反应而解毒；然而，在肝毒性剂量之后，肝脏谷胱甘肽水平会耗尽，代谢产物会主要以3 -（半胱氨酰 - S - 基）对乙酰氨基酚加合物的形式与蛋白质上的半胱氨酸基团共价结合。关键蛋白质功能的改变被认为是肝毒性的机制。共价结合已通过放射化学方法和免疫化学方法进行了研究。利用对乙酰氨基酚识别抗血清的蛋白质印迹分析，我们先前已表明共价结合发生在各种肝组分中的多种蛋白质上。为了更好地理解共价结合在毒性中的作用，其他人研究了非肝毒性异构体3'-羟基乙酰苯胺。给予大剂量的放射性标记对乙酰氨基酚或3'-羟基乙酰苯胺导致与蛋白质的共价结合水平相似。为了更好地理解共价结合在毒性中的作用，我们给小鼠给予3'-羟基乙酰苯胺和对乙酰氨基酚，并在蛋白质印迹分析中使用抗3 -（半胱氨酰 - S - 基）对乙酰氨基酚和抗芳基乙酰胺抗血清分析肝脏组分中的蛋白质加合物。用两种抗血清对对乙酰氨基酚处理的小鼠肝脏组分进行分析，如先前报道的那样，表明对乙酰氨基酚与多种肝脏蛋白质共价结合。在3'-羟基乙酰苯胺处理的小鼠肝脏中，仅用抗芳基乙酰胺抗血清检测到共价加合物。在微粒体中观察到一种主要的3'-羟基乙酰苯胺蛋白质加合物，分子量为50 kDa。在微粒体中观察到次要加合物，分子量为47 kDa，在胞质溶胶中为56 kDa。在10,000×g沉淀中未观察到3'-羟基乙酰苯胺蛋白质加合物。对3'-羟基乙酰苯胺蛋白质加合物时间进程的光密度分析表明，50 kDa微粒体蛋白质加合物的峰值水平在1小时出现，随后下降。
