The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats

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Abstract

To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague–Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3–6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.

Introduction

Drug-induced hepatotoxicity is a serious issue for new drug candidates as well as for marketed products. Tienilic acid was initially launched as a diuretic hypotensive drug in 1979, but was withdrawn from the market because of fulminant hepatic failure. Although the specific mechanisms of the hepatotoxicity are unclear, the pathway of tienilic acid metabolism has been well documented based on in vitro studies using microsomal fractions (Dansette et al., 1990, Dansette et al., 1991, Bonierbale et al., 1999). Tienilic acid is metabolized by the hepatic drug-metabolizing enzyme cytochrome P450 (CYP) 2C9 and its electrophilic reactive intermediates bind covalently to macromolecules including the metabolizing enzyme (Lopez-Garcia et al., 1994). Based on the covalently bound complex, immune-mediated mechanisms have been proposed for tienilic acid hepatotoxicity, being supported by the presence of the anti-liver and -kidney microsomal type 2 antibody directed to CYP2C9 in the serum of patients with hepatotoxicity (Lopez-Garcia et al., 1994). However, clinical signs characteristic of immune-mediated mechanisms, such as peripheral blood eosinophilia, fever and rash, were observed in only 15% of patients with tienilic acid-associated hepatic injury (Zimmerman et al., 1984). In fact, direct hepatocyte injury by tienilic acid, which is presumably associated with lipid peroxidation and production of the toxic metabolites, has also been shown on primarily cultured rat hepatocytes (Takagi et al., 1991) and isolated perfused rat liver (Zimmerman et al., 1982). Furthermore, tienilic acid has been demonstrated to decrease the hepatic glutathione content and also to covalently bind to several proteins other than CYP2C11, a rat orthologue of human CYP2C9, in rat primary hepatocyte culture in vitro (Lopez-Garcia et al., 2005). The above in vitro reports have led to the hypothesis that tienilic acid could cause hepatotoxicity by oxidative stress and the formation of electrophilic metabolites binding covalently with vital macromolecules, accompanied by decreased hepatic GSH levels. Most recently, it has been reported in an in vivo study using microarrays that tienilic acid induces gene expression changes related to cellular stress including oxidative stress in rat liver (Pacitto and Uetrecht, 2007). This study, however, was only performed using a low dose and no overt hepatotoxicity was demonstrated in rats.

The purpose of the present study is to investigate the hepatotoxic potential of tienilic acid in rats. In the first experiment (Experiment 1), tienilic acid was orally administered once to rats and the hepatic expression of thousands of genes was examined by Affymetrix GeneChip as well as hepatic reduced glutathione (GSH) content, serum biochemistry and histopathology of the liver. In Experiment 1, we expected that a comprehensive gene expression analysis together with other toxicological examinations and their dose relationship could provide information on the key responses associated with the hepatotoxicity in rats. As a result, we identified the early upregulation of some genes related to glutathione metabolism including glutathione synthetase (Gss) and glutamate-cysteine ligase (Gcl), oxidative stress response, phase II drug metabolism and organic ion transport along with altered hepatic GSH levels at dose levels, but this was only accompanied by very mild changes in serum biochemistry and liver morphology. Therefore, in the second experiment (Experiment 2), we clarified the toxicological significance of these gene expression changes in tienilic acid-induced liver toxicity. In this case, the chemical was administered to rats pretreated with buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl, and then the modification of hepatotoxicity was examined by serum biochemistry and histopathology. Based on the results of the above studies, the possible mechanism of serious hepatic injury induced by tienilic acid in humans is discussed.

Section snippets

Test substance

Tienilic acid was synthesized at DAIICHI SANKYO Co., Ltd. (Tokyo, Japan) and was suspended in 1% methylcellulose (Wako Pure Chemical Industries, Osaka, Japan) aqueous solution. The high purity (99.7%) of tienilic acid was confirmed by an HPLC analysis. BSO was purchased from Sigma-Aldrich (MO, USA). All other chemicals and reagents were from commercial sources.

Animal treatment and sample preparation

Male Sprague–Dawley (SD) rats aged 7 weeks were obtained from Japan SLC (Hamamatsu, Japan). They were housed in wire-mesh cages in an

Serum biochemistry and liver histopathology after tienilic acid treatment

Following a single oral dosing of tienilic acid at 300 and 1000 mg/kg, the total bilirubin and/or bile acid levels increased over 3 to 24 h, whereas ALT and ALP were not changed throughout the experimental period (Fig. 1). Histopathologically, single hepatocyte deaths were sporadically seen at 300 and 1000 mg/kg from 3 to 9 h in 1 to 3 of 5 animals, respectively, and mononuclear cell infiltration in the centrilobular region was noted at 9 and 24 h in 1 and 1 of 5 animals, respectively.

Discussion

A single oral administration of tienilic acid did not induce apparent hepatic injury at doses up to 1000 mg/kg, although very mild hepatocyte changes with mononuclear cell infiltration in the centrilobular region, and slight increases in the serum total bilirubin and bile acid levels were observed after dosing at 300 and/or 1000 mg/kg. Thus, the hepatotoxicity of tienilic acid itself was shown to be very mild even at the high dose 1000 mg/kg, which was consistent with the previous report that

Acknowledgments

We would like to thank Chikako Maru, Takami Suzuki and Kousuke Nozaki for their excellent technical assistance in the animal treatments and sample preparations, as well as Michiyuki Kato for his thoughtful comments.

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