REACTIVE METABOLITES AND DRUG TOXICITY

反应性代谢物和药物毒性

• 批准号: 6518921
• 负责人: ROBERT P HANZLIK
• 金额: $ 21.12万
• 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6518921
• 关键词: SDS polyacrylamide gel electrophoresis; adduct; antibody; antigen antibody reaction; biotransformation; bromobenzenes; cytotoxicity; drug adverse effect; drug metabolism; enzyme activity; hepatotoxin; high performance liquid chromatography; immunological substance; laboratory rabbit; laboratory rat; liver metabolism; mitochondria; protein binding; protein purification; western blottings

Many simple organic molecules containing phenyl substituents or benzene rings become cytotoxic upon biotransformation to reactive electrophilic metabolites. Prime examples include halothane, acetaminophen and bromobenzene (BB). Their hepatotoxicity is correlated with covalent binding of reactive metabolites to cellular proteins. As a start toward elucidating the biochemical mechanism(s) of their cytotoxicity we identified the structures of ten adducts of BB metabolites to protein-SH groups; most arose via quinone metabolites, but we also found that BB-3,4-oxide (BBO), thought to be the primary "toxic" metabolite of BB, alkylates histidine and lysine as well as cysteine residues of rat liver proteins. Key questions concerning the mechanism of cell injury by reactive metabolites include the identity of the proteins they target and the functional consequences of their covalent modification. We recently identified several rat liver proteins targeted by BB metabolites. One was a nonspecific esterase also known to be a target for metabolites of halothane and molinate. Another, surprisingly, was epoxide hydrolase, which is supposed to detoxify BBO. To address the mechanism of BB-induced cytotoxicity it is essential to expand this list by identifying other liver proteins targeted by BB metabolites. In doing so we will emphasize mitochondrial proteins but will continue to explore cytosolic and microsomal proteins. To facilitate recognition of BBO adducts, we raised antibodies to p-bromophenyl-cysteine and demonstrated their utility for western blotting; we will now develop antibodies to p-bromophenyl-histidine and p-bromophenyl-lysine as well. These antibodies, coupled with [C14]-BB, will give us a broad and powerful means for finding and identifying those proteins of greatest toxicological interest. Very little is known about the chemistry and consequences of protein adduction by reactive metabolites. Thus we will elucidate in detail the specific site(s), metabolite(s) and linkage(s) involved in adduct formation for select BB target proteins. For those target proteins which are enzymes, we will evaluate the effect of adduction on catalytic activity to assess its potential contribution to cell injury. Comparing the proteins modified by bromobenzene to those modified by other small bioactivated toxins may reveal the existence, or the lack of, a "common pathway" for chemically-induced cytotoxic responses.

许多含有苯基取代基或苯的简单有机分子 环在生物转化为反应性亲电性后变得具有细胞毒性 代谢物。 主要例子包括氟烷、对乙酰氨基酚和 溴苯（BB）。 它们的肝毒性与共价键相关 反应性代谢物与细胞蛋白质的结合。 作为开始 我们阐明了其细胞毒性的生化机制 鉴定了 BB 代谢物与蛋白质-SH 的十种加合物的结构 团体；大多数是通过醌代谢物产生的，但我们也发现 BB-3,4-氧化物 (BBO)，被认为是 BB 的主要“有毒”代谢物， 烷基化大鼠肝脏的组氨酸和赖氨酸以及半胱氨酸残基 蛋白质。 有关细胞损伤机制的关键问题 反应性代谢物包括其靶向蛋白质的身份 以及它们的共价修饰的功能后果。 我们 最近发现了几种 BB 靶向的大鼠肝脏蛋白 代谢物。 一种是非特异性酯酶，也被认为是目标 用于氟烷和禾草酸盐的代谢物。 另一个令人惊讶的是 环氧化物水解酶，据说可以解毒 BBO。 为了解决 BB 诱导细胞毒性的机制有必要扩大这个列表 通过识别 BB 代谢物靶向的其他肝脏蛋白。 在 这样做我们将强调线粒体蛋白，但将继续 探索细胞质和微粒体蛋白。 为了方便识别 BBO 加合物，我们产生了对溴苯基半胱氨酸的抗体， 证明了它们在蛋白质印迹中的实用性；我们现在将开发 对溴苯基组氨酸和对溴苯基赖氨酸的抗体。 这些抗体与 [C14]-BB 结合，将为我们提供广泛且 寻找和鉴定那些最重要的蛋白质的强大手段 毒理学兴趣。 人们对化学和化学知之甚少 反应性代谢物引起蛋白质加合的后果。这样我们就会 详细阐明特定位点、代谢物和连接 参与选择 BB 靶蛋白的加合物形成。 对于那些 目标蛋白质是酶，我们将评估其效果 催化活性加合以评估其潜在贡献 细胞损伤。比较经溴苯修饰的蛋白质与那些 被其他小型生物活性毒素修饰可能会揭示其存在， 或缺乏化学诱导细胞毒性的“共同途径” 回应。