Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore.
Toxicol Lett. 2010 Feb 1;192(2):141-9. doi: 10.1016/j.toxlet.2009.10.014. Epub 2009 Oct 23.
Troglitazone (TGZ) is an orally active hypoglycemic agent which is used for the treatment of non-insulin-dependent diabetes mellitus. It had been associated with severe drug-induced liver failure which resulted in its withdrawal from the market in 2000. While the exact mechanism of its toxicity remains unknown, it has been postulated that the formation of toxic reactive metabolites (RMs) may play an important role in the hepatotoxicity of TGZ. The purpose of this study is to investigate the role of sulfur moiety of thiazolidinedione (TZD) nucleus in inducing liver toxicity via the formation of RMs. An analogue of TGZ, trosuccinimide (TSN), was synthesized chemically where the sulfur moiety of thiazolidinedione ring was replaced by a methylene group. Both compounds were incubated independently with human liver microsomes enriched with glutathione (GSH) and normal human hepatocytes (THLE-2 cell lines) to profile GSH-adducts using ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS). Four RM-GSH conjugates of TGZ were identified during the profiling experiments of which three were related to the sulfur moiety of the TZD ring, whereas no RM of TSN was detected in both microsomes and hepatocytes. MTT, GSH and protein carbonyl (PC) assays were performed using THLE-2 hepatocytes to measure the levels of toxicity of TGZ and TSN in vitro. Finally, peroxisome proliferator activated receptor gamma (PPAR(gamma)) binding activity was measured to check the binding affinities of both TGZ and TSN. The calculated binding affinities of TGZ and TSN were 332.2 and 1106.0 microM, respectively. Our results indicated collectively that TSN (EC(50)=138.5+/-7.32 microM) was less toxic than TGZ (EC(50)=27.2+/-4.8 microM) in THLE-2 hepatocytes. As both compounds were shown to bind to PPAR(gamma), the substitution of the TZD moiety may be beneficial from a drug design perspective. In conclusion, our study confirmed that the TZD ring of TGZ may be partially responsible for its liver toxicity in humans via the formation of RMs.
曲格列酮（TGZ）是一种口服活性降糖药，用于治疗非胰岛素依赖型糖尿病。它曾与严重的药物诱导性肝衰竭有关，导致其于 2000 年退出市场。虽然其确切的毒性机制尚不清楚，但据推测，有毒的反应性代谢物（RMs）的形成可能在 TGZ 的肝毒性中起重要作用。本研究旨在通过形成 RMs 来研究噻唑烷二酮（TZD）核中硫部分在诱导肝毒性中的作用。合成了 TGZ 的类似物，琥珀酰亚胺（TSN），其中噻唑烷二酮环的硫部分被亚甲基取代。将这两种化合物分别与富含谷胱甘肽（GSH）的人肝微粒体和正常人肝细胞（THLE-2 细胞系）孵育，使用超高效液相色谱串联质谱法（UPLC/MS/MS）对 GSH 加合物进行分析。在 TGZ 的谱图实验中鉴定了 4 种 RM-GSH 缀合物，其中 3 种与 TZD 环的硫部分有关，而 TSN 在微粒体和肝细胞中均未检测到 RM。使用 THLE-2 肝细胞进行 MTT、GSH 和蛋白质羰基（PC）测定，以测量 TGZ 和 TSN 在体外的毒性水平。最后，测量过氧化物酶体增殖物激活受体γ（PPAR(gamma))结合活性，以检查 TGZ 和 TSN 的结合亲和力。计算得到 TGZ 和 TSN 的结合亲和力分别为 332.2 和 1106.0 μM。我们的结果表明，TSN（EC(50)=138.5+/-7.32 μM）在 THLE-2 肝细胞中的毒性低于 TGZ（EC(50)=27.2+/-4.8 μM）。由于这两种化合物均与 PPAR(gamma)结合，因此从药物设计的角度来看，TZD 部分的取代可能是有益的。总之，我们的研究证实，TGZ 的 TZD 环可能通过形成 RMs 部分导致其在人体内的肝毒性。
