Division of Molecular Toxicology, LACDR, Vrije Universiteit, Amsterdam, The Netherlands.
Chem Res Toxicol. 2012 Nov 19;25(11):2532-41. doi: 10.1021/tx300334w. Epub 2012 Oct 10.
Use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with serious idiosyncratic hepatotoxicity. Covalent binding of reactive intermediates of DF to proteins is considered to initiate the process leading to this severe side-effect. The aim of this study was to characterize the nature of covalent protein modifications by reactive metabolites of DF which result from bioactivation by cytochrome P450. DF and its major monohydroxylated metabolites 4'-hydroxydiclofenac (4'-OH-DF) and 5-hydroxydiclofenac (5-OH-DF) were bioactivated using a highly active P450 BM3 mutant (CYP102A1M11H) in the presence of the model target protein human glutathione-S-transferase P1-1 (hGST P1-1). Protein-adducts were subsequently identified by LC-MS/MS analysis of tryptic digests of hGST P1-1. In total, 10 different peptide adducts were observed which result from modifications of Cys-47 and Cys-14 of hGST P1-1. The majority of the protein thiol modifications appeared to be derived from 5-OH-DF, which produced seven different peptide adducts with mass increments of 289.0, 309.0, and 339.0 Da. Remarkably, no peptide adducts were observed upon the bioactivation of 4'-OH-DF. Incubations of P450 BM3 with DF also showed the peptide adducts derived from 5-OH-DF and peptide adducts that are not derived from quinone imine. A peptide adduct with a mass increment of 249.0 Da most likely results from the o-imine methide formed by oxidative decarboxylation of DF. In addition, a peptide adduct was observed with a mass increment of 259.0 Da, which corresponds to the substitution of one of the chlorine atoms of DF by protein thiol. A corresponding GSH-conjugate with a similar mass increment was only observed if incubations of DF with P450 and GSH were supplemented by human GST P1-1. The results of this study not only confirm the importance of 5-OH-DF in covalent protein-binding but also suggest that the nature of protein adduction is not necessarily reflected by chemical conjugation with GSH.
使用非甾体抗炎药双氯芬酸 (DF) 与严重的体质特异肝毒性相关。DF 的反应性中间产物与蛋白质的共价结合被认为是导致这种严重副作用的过程的开始。本研究的目的是通过细胞色素 P450 生物转化来表征由 DF 的反应性代谢物引起的共价蛋白质修饰的性质。DF 及其主要的单羟基化代谢物 4'-羟基双氯芬酸 (4'-OH-DF) 和 5-羟基双氯芬酸 (5-OH-DF) 在模型靶蛋白人谷胱甘肽 S-转移酶 P1-1 (hGST P1-1) 的存在下，使用高度活跃的 P450 BM3 突变体 (CYP102A1M11H) 进行生物转化。通过 LC-MS/MS 分析 hGST P1-1 的胰蛋白酶消化产物，随后鉴定蛋白质加合物。总共观察到 10 种不同的肽加合物，这些加合物是由 hGST P1-1 的 Cys-47 和 Cys-14 的修饰引起的。大多数蛋白质巯基修饰似乎来自 5-OH-DF，它产生了七个不同的肽加合物，质量增加了 289.0、309.0 和 339.0 Da。值得注意的是，在 4'-OH-DF 的生物转化过程中没有观察到肽加合物。P450 BM3 与 DF 的孵育也显示了来自 5-OH-DF 的肽加合物和不是来自醌亚胺的肽加合物。质量增加 249.0 Da 的肽加合物很可能是由 DF 的氧化脱羧形成的 o-亚胺甲醚。此外，观察到一个质量增加 259.0 Da 的肽加合物，这对应于 DF 的一个氯原子被蛋白质巯基取代。仅在 DF 与 P450 和 GSH 的孵育中补充人 GST P1-1 时，才观察到具有相似质量增量的相应 GSH 缀合物。本研究的结果不仅证实了 5-OH-DF 在共价蛋白质结合中的重要性，还表明蛋白质加合物的性质不一定反映与 GSH 的化学结合。
