MECHANISMS OF HEPATOTOXICITY--ACETAMINOPHEN

尽管止痛药对乙酰氨基酚经常被用作模型 用于研究肝毒性的化学物质，其产生的关键机制 细胞内的毒性尚不清楚。普遍的假设是 有毒代谢物与关键蛋白的共价结合 重要细胞功能的抑制是导致 细胞毒性。尽管多年来人们都知道 共价结合是通过对乙酰氨基酚与半胱氨酸基结合 蛋白质，直到最近才有工具可用于识别特定的 对乙酰氨基酚共价结合的蛋白质。在最近的工作中，我们 开发了针对对乙酰氨基酚的免疫学检测方法 共价结合到蛋白质上。这些检测方法被用来确定 对乙酰氨基酚的肝毒性与其共价结合的关系 亚细胞组分、单个肝细胞和特定蛋白质。在……里面 免疫印迹研究表明，肝脏的主要蛋白质 扑热息痛共价结合的是一个55 kDa的胞浆蛋白。 在为这一提议生成的初步数据中，55 kDa蛋白是 从7个内源性多肽中分离出85个氨基酸，并对其进行了序列测定。 用计算机数据库对序列进行比对表明， 其氨基酸序列与A基因的氨基酸序列同源性为97%。 56 kDa硒结合蛋白的cDNA克隆它是被假设的 成为一种细胞调节蛋白。在这项提案中，其他蛋白质可以 对乙酰氨基酚与哪些共价结合将被分离和表征 包括氨基酸序列分析在内的各种方法。重要的是 与特定蛋白质的共价结合将通过比较确定 对乙酰氨基酚与一种无毒的类似物结合，该类似物也与蛋白质结合。 此外，还将确定其他模型肝毒素是否共价 结合到这个硒结合蛋白上。

Even though the analgesic acetaminophen is frequently used as a model chemical to study hepatotoxicity, critical mechanisms by which it produces toxicity within the cell are unknown. The prevailing hypothesis is covalent binding of a toxic metabolite to crucial proteins and subsequent inhibition of vital cellular functions is responsible for the cytotoxicity. Even though it has been known for a number of years that covalent binding is via acetaminophen bound to cysteine groups on proteins, only recently have tools been available to identify the specific proteins to which acetaminophen covalently binds. In recent work we developed immunological assays which are specific for acetaminophen covalently bound to protein. These assays were utilized to determine the relationship between acetaminophen hepatotoxicity and covalent binding to subcellular fractions, individual hepatocytes, and specific proteins. In Western immunoblot studies it was shown that the principal liver protein to which acetaminophen covalently bound was a 55 kDa cytosolic protein. In preliminary data generated for this proposal the 55 kDa protein was isolated and 85 amino acids were sequenced from 7 internal peptides. Comparison of the sequence using computer data bases indicated that the protein had a 97% homology with the deduced amino acid sequence from a cDNA clone of a 56 kDa Selenium Binding Protein. It has been hypothesized to be a cellular regulatory protein. In this proposal other proteins to which acetaminophen covalently binds will be isolated and characterized by various methods including amino acid sequence analysis. The importance of covalent binding to specific proteins will be determined by comparing acetaminophen binding to a nontoxic analog which also binds to protein. In addition, it will be determined if other model hepatotoxins covalently bind to this Selenium Binding Protein.