Elsevier

Toxicology Letters

Volume 195, Issues 2–3, 2 June 2010, Pages 135-141
Toxicology Letters

Direct toxicity effects of sulfo-conjugated troglitazone on human hepatocytes

https://doi.org/10.1016/j.toxlet.2010.03.010Get rights and content

Abstract

Troglitazone (TGZ), an orally active hypoglycemic agent, was found to be associated with severe drug-induced liver failure and was withdrawn from the market in 2000. Although the exact mechanism is not clear, it has been postulated that the formation of its major sulfo-conjugated metabolite (TGZS) plays an important role in its toxicity. TGZS inhibits bile salt export pump (BSEP) that causes accumulation of bile salts in liver. High concentration of bile salts causes cell death and mitochondrial dysfunction via detergent properties. One question arises whether TGZS has direct toxicity effect on human liver cells in addition to BSEP inhibition. In this study, both TGZ and chemically synthesized TGZS were incubated with normal human hepatocytes (THLE-2 cells) for measuring their cytotoxicity in vitro using the MTT assay. Glutathione (GSH) and protein carbonyl (PC) assays were further performed to measure the oxidative stress generated by these two compounds during incubation with THLE-2 cells. The results from this study indicated that TGZS (EC50 = 21.74 ± 5.38 μM) was more toxic than TGZ (EC50 = 41.12 ± 4.3 μM) in THLE-2 cells. The GSH and PC data further confirmed that TGZS produced greater oxidative stress in THLE-2 cells as compared to TGZ. In conclusion, our study demonstrated for the first time that TGZS has direct toxicity effect on human liver cells and may be partially responsible for the hepatotoxicity of TGZ.

Introduction

Troglitazone (TGZ, Fig. 1), a 2,4-thiazolidinedione (TZD) antidiabetic drug, was developed and used for the treatment of type II diabetes mellitus (non-insulin dependent diabetes mellitus) (Parker, 2002). It lowers the glucose concentration in blood by exerting partial agonistic action on the peroxisome proliferator activated receptor gamma (PPARγ) (Henry, 1997). Upon PPARγ activation, TGZ increases serum triglycerides level, alters thyroid metabolism (Miller et al., 1997, Sherman et al., 1999, Rizvi et al., 1999) and inhibits phosphoenolpyruvate carboxylase enzyme which is mainly responsible for hepatic gluconeogenesis (Camp et al., 2000). Several cases of fulminant hepatic failure were reported with the clinical use of TGZ, leading to its withdrawal from the market in 2000 (Chojkier, 2005). The exact nature of TGZ hepatotoxicity has not yet been completely established but several mechanisms had been proposed (Smith, 2003). These include the formation of electrophilic reactive intermediates, binding to PPARγ, causation of mitochondrial injury, inhibition of bile salt export pump (BSEP) by TGZ and/or TGZS and a combination of multiple mechanisms and varying host factors (Kawai et al., 1997, Kostrubsky et al., 2000). In vitro and in vivo experiments suggested that TGZ is mainly metabolized by cytochrome 3A4 (CYP3A4) and 2C8 (CYP2C8) enzymes (Sahi et al., 2000, Ramachandran et al., 1999, He et al., 2001). The metabolism of TGZ in liver includes oxidation (phase I metabolism), sulfation and glucuronidation (phase II metabolism). The glucuronide metabolite of TGZ is found in human plasma at very low concentration while the major metabolites of TGZ in humans are the sulphate (TGZS, 70%, Fig. 1) and quinone (10%) conjugates, respectively (Loi et al., 1997, Loi et al., 1999).

While it was traditionally perceived that the phase II TGZS metabolite is a detoxifying species, recent evidence suggested that it might have toxicological potential through the binding and inhibition of BSEP (Funk et al., 2001). As BSEP plays an important role in removing bile salts from liver cells using energy in the form of ATP (Kullak-Ublick et al., 2000), the accelerated accumulation of bile salts may lead to cholestasis and subsequent hepatocyte apoptosis. High levels of bile salt have been shown to induce cell death and mitochondrial dysfunction due to their detergent properties (Delzenne et al., 1992, Gores et al., 1998). The cholestatic potential of TGZ and TGZS had been demonstrated previously using in vitro and in vivo rat models (Funk et al., 2001).

Although TGZS has been demonstrated to inhibit BSEP, its direct toxic effect on human liver cells has not been investigated. It is important to ascertain this property of TGZS because it is a major metabolite of TGZ and its mechanism of direct hepatocyte toxicity is still unclear. While it had been determined that TGZS showed no cytotoxicity in human hepatoma cell lines such as HepG2, HLE, HLF and HuH-7 cells (Yamamoto et al., 2001), its effect on physiologically relevant normal human hepatocytes has not been established. In this study, TGZ and chemically synthesized TGZS were evaluated for their in vitro toxicity and oxidative stress potentials using metabolically active normal human hepatocytes, THLE-2 cells (Andrea et al., 1993, Saha et al., 2010). It is demonstrated for the first time that TGZS has direct toxic effect on normal human hepatocytes.

Section snippets

Chemicals and reagents

HPLC-grade acetonitrile (ACN) was purchased from Tedia Company Inc., (Fairfield, OH, USA). Fibronectin and collagen were purchased from BD Biosciences (Woburn, MA, USA). TGZ was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Formic acid (99% purity) and glacial acetic acid were purchased from VWR International Ltd., (Leicestershire, UK). Dimethyl sulfoxide (DMSO, ACS grade) was obtained from Panreac Quimica SA (Barcelona, Spain). Ethylenediaminetetraacetic acid disodium salt

Sulfotransferase enzyme inhibition assay

LC/MS/MS analysis of THLE-2 cells incubation with TGZ revealed the presence of TGZS using MRM at m/z 520.0  440.0. The MRM experiment confirmed the presence of metabolically generated TGZS (retention time [RT] 4.92 min) which was absent in “0 h TGZ-THLE-2” and negative control samples but present in “24 h TGZ-THLE-2” incubation. TGZS is the sulfo-conjugate of TGZ where the conjugation with bi-sulphate (–OSO3H) moiety occurred at the 6-hydroxyl (–OH) group of TGZ (Fig. 2A). TGZS was found to be

Discussion

TGZ was withdrawn from the market in 2000 due to its idiosyncratic liver toxicity (Chojkier, 2005). While several mechanisms of toxicity had been proposed for TGZ (Smith, 2003), it remains unclear if the sulfo-conjugated metabolite (TGZS) of TGZ has direct toxic effect on human liver cells. In the current project, the aim was to ascertain and establish the role of TGZS in causing direct toxicity in human hepatocytes.

To achieve this goal, TGZS was synthesized where 6-hydroxyl (–OH) group of TGZ

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the National University of Singapore (NUS) grants R-148-050-088-101/133 and R-148-000-100-112 to E.C.Y.C. The UPLC system was kindly supported by NUS grant R-279-000-249-646. S.S. is supported by the NUS graduate scholarship.

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