Opposite Behaviors of Reactive Metabolites of Tienilic Acid and Its Isomer toward Liver Proteins: Use of Specific Anti-Tienilic Acid−Protein Adduct Antibodies and the Possible Relationship with Different Hepatotoxic Effects of the Two Compounds

作者：Eric Bonierbale, Philippe Valadon, Catherine Pons, Bernard Desfosses, Patrick M. Dansette, Daniel Mansuy · 发表于：Chemical Research in Toxicology · 年份：1999 · DOI：10.1021/tx980136z · 被引用次数：58 · 研究领域：Drug-Induced Hepatotoxicity and Protection、Pharmacogenetics and Drug Metabolism、Drug Transport and Resistance Mechanisms

Tienilic acid (TA) is responsible for an immune-mediated drug-induced hepatitis in humans, while its isomer (TAI) triggers a direct hepatitis in rats. In this study, we describe an immunological approach developed for studying the specificity of the covalent binding of these two compounds. For this purpose, two different coupling strategies were used to obtain TA-carrier protein conjugates. In the first strategy, the drug was linked through its carboxylic acid function to amine residues of carrier proteins (BSA-N-TA and casein-N-TA), while in the second strategy, the thiophene ring of TA was attached to proteins through a short 3-thiopropanoyl linker, the corresponding conjugates (BSA-S-5-TA and betaLG-S-5-TA) thus preferentially presenting the 2, 3-dichlorophenoxyacetic moiety of the drug for antibody recognition. The BSA-S-5-TA conjugate proved to be 30 times more immunogenic than BSA-N-TA. Anti-TA-protein adduct antibodies were obtained after immunization of rabbits with BSA-S-5-TA (1/35000 titer against betaLG-S-5-TA in ELISA). These antibodies strongly recognized the 2, 3-dichlorophenoxyacetic moiety of TA but poorly the part of the drug engaged in the covalent binding with the proteins. This powerful tool was used in immunoblots to compare TA or TAI adduct formation in human liver microsomes as well as on microsomes from yeast expressing human liver cytochrome P450 2C9. TA displayed a highly specific covalent binding focused on P450 2C9 which is the main cytochrome P450...