ACETAMINOPHEN COVALENT BINDING AND HEPATOXICITY

急性过量使用解热镇痛药对乙酰氨基酚（APAP）， 会导致严重的致命的肝坏死 肝毒性是APAP的结果 活化成共价结合肝蛋白的亲电体。 尽管APAP的蛋白共价结合和 毒性尚不清楚，58 kDa APAP结合蛋白的选择性芳基化 （58-ABP）与毒性的相关性优于总共价结合。 58-ABP是抗APAP抗体检测到的最突出的靶标， 小鼠和人类肝脏在APAP肝毒性。 在小鼠中，58-ABP 芳基化与靶组织损伤（肝， 肺和肾）。 芳基化蛋白质是 也检测到小鼠血浆后，有毒的APAP暴露，这可能 反映在人血浆中APAP-蛋白加合物的检测， 中毒 我们的工作假设是58-ABP的芳基化是 对APAP诱导的靶器官毒性很重要。 无论是扮演一个 启动或保护作用目前无法解决。 最近的结果 提出了新的问题，这些问题是拟议研究的基础。 第一章 是否存在58-ABP结合的阈值，如果超过该阈值， 毒性？ 2)58-ABP是否也被其他药物靶向， 共价连接到蛋白质上 3)什么是毒理学意义的 血浆中是否出现芳基化58-ABP？ 拟议的研究将 广泛使用抗APAP和抗58，一种新的抗 58-ABP。 这将允许定量评估是否存在 共价结合阈值 抗体将针对结合的 溴苯，以允许比较其与APAP的结合， 溴苯毒性 用抗58抗体进行免疫沉淀 通过其它异生物质检测58-ABP结合。 体外研究将 确定其他试剂是否在58-ABP和APAP上的相同位点结合，和 培养和体内研究将揭示这种结合是否会导致毒性 同时暴露时的相互作用。我们还将确定 APAP或其他外源性物质后血浆中58-ABP的出现反映了 芳基化蛋白质的细胞分泌或由于 细胞死亡 使用结合但不引起毒性的药物进行的研究将 以确定这些试剂是否靶向与靶向蛋白质相同的蛋白质， 有毒化合物。 总的来说，这些研究将提供新的见解 通过APAP和其他有毒物质进行选择性58-ABP芳基化的重要性 靶器官毒性的化学物质。 他们还将确定， 血浆中58-ABP的出现可以成为一个有用的诊断标志物， 外源性物质引起的靶器官损伤。

Acute overdosage with the antipyretic analgesic, acetaminophen (APAP) can cause severe, fatal hepatic necrosis. Hepatotoxicity is the result of APAP activation to an electrophile which covalently binds to liver proteins. Although, the relationship between APAP's protein covalent binding and toxicity is not clear, selective arylation of a 58 kDa APAP binding protein (58-ABP) is better associated with toxicity than is total covalent binding. The 58-ABP is the most prominent target detected with anti-APAP antibody in mouse and human liver during APAP hepatotoxicity. In mice, 58-ABP arylation was most closely associated with target tissue damage (liver, lung and kidney) in varied experimental paradigms. The arylated protein is also detected in mouse plasma after toxic APAP exposures, and this may mirror the detection of APAP-protein adducts in human plasma during poisoning. Our working hypothesis is that arylation of the 58-ABP is important for APAP-induced target organ toxicity. Whether is plays an initiating or protective role cannot presently be resolved. Recent results have raised new questions which are the basis of the proposed research. 1) Is there a threshold for 58-ABP binding which, if exceeded, is associated with toxicity? 2) Is the 58-ABP also targeted by other agents which bind covalently to proteins? 3) What is the toxicological significance of the appearance of arylated 58-ABP in plasma? The proposed research will extensively use anti-APAP and anti-58, a new antibody prepared against the 58-ABP. This will permit quantitative assessment of the existence of a covalent binding threshold. Antibodies will be developed against bound bromobenzene to permit comparison of its binding with APAP's and with bromobenzene toxicity. Immunoprecipitation with anti-58 will facilitate detection of 58-ABP binding by other xenobiotics. In vitro studies will determine if other agents bind at the same site on the 58-ABP and APAP, and culture and in vivo studies will reveal if such binding can cause toxic interactions during simultaneous exposures. We will also determine if the appearance of 58-ABP in plasma after APAP or other xenobiotics reflects cellular secretion of arylated protein or incidental loss as a result of cell death. Studies with agents which bind but do not cause toxicity will also be included to determine if such agents target the same proteins as the toxic compounds. Collectively such studies will provide new insight into the importance of selective 58-ABP arylation by APAP and other toxic chemicals for target organ toxicity. They will also determine if the appearance of 58-ABP in plasma can become a useful diagnostic marker for xenobiotic-induced target organ damage.