REACTIVE METABOLITES AND DRUG TOXICITY

反应性代谢物和药物毒性

• 批准号: 2173789
• 负责人: ROBERT P HANZLIK
• 金额: $ 18.07万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-08-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2173789
• 关键词: SDS polyacrylamide gel electrophoresis; adduct; autoradiography; biotransformation; bromobenzenes; chemical hydration; chemical structure function; chemical synthesis; covalent bond; cytochrome P450; drug adverse effect; drug metabolism; electrospray ionization mass spectrometry; enzyme activity; epoxides; hepatotoxin; high performance liquid chromatography; hydrolysis; laboratory rat; mass spectrometry; oxygenases; protein sequence; scintillation counter

Reactive chemicals re often cytotoxic because they covalently modify (i.e. denature) important cellular macromolecules. Chemically reactive metabolites of otherwise innocuous compounds also covalently modify cellular macromolecules, in most cases with toxic consequences. Bromobenzene (BB) exemplifies the latter situation, and is an important model compound for studying biochemical mechanisms of chemically-induced hepatotoxicity. The applicant's laboratory recently identified the structure of ten protein-S-adducts and one protein-histidine adduct; these derived from two epoxide- and four quinone metabolites of BB and represented ca. 10% of total covalent binding. The applicant now proposes to utilize this background to pursue two major objectives. One is the elucidation of structures which account for the majority of radioactivity covalently bound to liver proteins of rats treated with [14C]-BB. Although adducts have been released by acid hydrolysis, the use of milder enzymatic hydrolysis will be explored, and systematic separation and elucidation of adduct structures will be carried out. The second major objective is to isolate and identify specific rat hepatocyte target proteins adducted by reactive metabolites of BB, to determine the nature of the adduct(s) formed to each, to evaluate whether metabolite adduction of a given protein contributes to the hepatotoxicity of BB, and to determine (via inter-laboratory collaboration) whether the same proteins are targeted by reactive metabolites of other hepatotoxic chemicals (e.g. from acetaminophen or halothane). Polyclonal anti-hepten antibodies capable of selective recognition of protein adducts of BB- epoxide vs. -quinone metabolites in ELISA assays have been raised. However, in Western blotting applications, these antibodies cross-reacted with some non-adducted liver proteins was detected. Therefore, monoclonal (i.e. monospecific) anti-hapten antibodies will also be raised and evaluated. In addition, autoradiography has been used successfully to detect proteins adducted with [14C]-BB on PAGE gels. These methods will be used to detect and monitor the purification of BB-adducted proteins formed in vitro and in vivo. Target proteins will be analyzed using combinations of peptide mapping, N-terminal and internal peptide sequencing and electrospray LC-MS/MS. It is anticipated that identifying target proteins will help bridge a major knowledge-gap between early cellular events which specifically involve bromobenzene or its metabolites, and late-phase events which may be common cytotoxic mechanisms activated by many different cytotoxic chemicals. Conceivably, better understanding of toxic mechanisms could lead to more effective ways of managing clinical toxicities resulting from chemicals and/or drugs which are bioactivated to chemically reactive metabolites.

反应性化学物质通常具有细胞毒性，因为它们会共价修饰 （即变性）重要的细胞大分子。 化学反应性 其他无害化合物的代谢物也会共价修饰 细胞大分子，在大多数情况下会产生毒性后果。 溴苯（BB）就是后一种情况的例证，并且是一种重要的 用于研究化学诱导的生化机制的模型化合物 肝毒性。 申请人的实验室最近鉴定出 十种蛋白质-S-加合物和一种蛋白质-组氨酸加合物的结构； 这些衍生自 BB 的两种环氧化物和四种醌代谢物 代表约。总共价结合的 10%。 现申请人 建议利用这一背景来实现两个主要目标。 一 是对占大多数的结构的阐明 放射性与经处理的大鼠的肝蛋白共价结合 [14C]-BB。 尽管酸水解已释放出加合物，但 将探索使用温和的酶水解，并系统地 将进行加合物结构的分离和阐明。 这 第二个主要目标是分离和鉴定特定的大鼠肝细胞 BB 反应性代谢物加合的靶蛋白，以确定 每种形成的加合物的性质，以评估代谢物是否 给定蛋白质的加合会导致 BB 的肝毒性，并且 确定（通过实验室间合作）是否相同 蛋白质是其他肝毒性物质的反应性代谢物的靶标 化学品（例如对乙酰氨基酚或氟烷）。 多克隆抗庚蛋白 能够选择性识别 BB- 蛋白质加合物的抗体 ELISA 检测中环氧化物与 β-醌代谢物的比较有所提高。 然而，在蛋白质印迹应用中，这些抗体会发生交叉反应 检测到一些非加合的肝脏蛋白。 所以， 单克隆（即单特异性）抗半抗原抗体也将产生 并进行了评价。 此外，放射自显影术已成功使用 在 PAGE 凝胶上检测与 [14C]-BB 加合的蛋白质。 这些方法 将用于检测和监测 BB 加合的纯化 蛋白质在体外和体内形成。 将分析目标蛋白质 使用肽图谱、N 末端和内部肽的组合 测序和电喷雾 LC-MS/MS。 预计识别靶蛋白将有助于弥合 早期细胞事件之间的主要知识差距，特别是 涉及溴苯或其代谢物，以及可能的后期事件 是由许多不同的细胞毒性物质激活的常见细胞毒性机制 化学品。 可以想象，更好地了解毒性机制可以 导致更有效的方法来管理由此产生的临床毒性 从生物活性到化学反应性的化学品和/或药物 代谢物。