Altered integrity and decreased expression of hepatocyte tight junctions in rifampicin-induced cholestasis in mice

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Abstract

Rifampicin is a well-known hepatotoxicant, but little is known about the mechanism of rifampicin-induced hepatotoxicity. The aim of this study was to characterize the expression and localization of hepatocyte tight junctions in rifampicin-induced cholestasis in mice. Cholestasis was induced by administration of rifampicin (200 mg/kg) for 7 consecutive days or treatment with a single dose of rifampicin (200 mg/kg) by gastric intubation. The expression of mRNA for hepatic zonula occludens (ZO)-1, ZO-2, ZO-3, occludin and claudin-1 was determined using RT-PCR. Localization of ZO-1 and occludin was detected using immunofluorescence. Results showed that there was an 82-fold increase in the conjugated bilirubin in serum in rifampicin-treated mice. In addition, an 8-fold increase in total bile acid in serum was observed after a seven-day administration of rifampicin. The integrity of hepatocyte ZO-1 and occludin was altered by a seven-day administration of rifampicin. Importantly, the integrity and intensity of hepatocyte tight junctions were altered as early as 30 min after a single dose of rifampicin. The expression of hepatic ZO-1 and ZO-2 mRNA was significantly decreased, beginning as early as 30 min and remaining a lower level 12 h after a single dose of rifampicin. Taken together, these results suggest that the altered integrity and internalization of hepatocyte tight junctions are associated with rifampicin-induced cholestasis.

Introduction

Drug-induced liver injury is an important clinical problem with significant morbidity and mortality. Drug-induced liver injury can be classified into hepatocellular, cholestatic, and mixed types of liver damage depending on serum biochemical parameters. Hepatocellular injury is mainly characterized by the elevation of serum aminotransferases, whereas cholestatic liver damage is reflected by increased levels of alkaline phosphatase, γ-glutamyltranspeptidase, and conjugated bilirubin in serum (Mohi-ud-din and Lewis, 2004). Although the consequences of cholestatic injury are generally regarded as being far less serious than those of acute parenchymal injury with its potential risk of fulminant hepatitis, the significance of drug-induced cholestasis is revealed by its broad spectrum of acute and chronic liver diseases. Well-known drugs known to cause cholestasis include estrogens and anabolic steroids, chlorpromazine, erythromycin (Crocenzi et al., 2008, Buchweitz et al., 2002, Westphal et al., 1994). In addition, some structurally similar congeners of above drugs, such as tamoxifen and azithromycin, may also cause cholestasis (Mazokopakis et al., 2007, Chandrupatla et al., 2002). Some recent case reports showed that more and more drugs, including ticlopidine, terbinafine, nimesulide, irbesartan, ciprofloxacin, and atorvastatin, are associated with cholestasis (Hirata et al., 2008, Perveze et al., 2007, Giannattasio et al., 2006, Andrade et al., 2002, Okan et al., 2008, Ridruejo and Mandó, 2002). Interestingly, even some herbal medicines, such as greater celandine, may also result in cholestatic hepatitis (Benninger et al., 1999).

Rifampicin, one of the most commonly used anti-tubercular drugs, has been known to be hepatotoxic, but little is known about the mechanism of rifampicin-induced hepatotoxicity. Several studies showed that rifampicin caused a direct toxic injury to the hepatocytes (Meng et al., 2006, Shen et al., 2009). Some earlier studies found that rifampicin significantly increased the level of serum bile acids in man (Galeazzi et al., 1980, Berg et al., 1984). Rifampicin-induced inhibition of organic anion transporting polypeptide (Oatp)2-mediated taurocholate uptake has also been demonstrated in primary rat hepatocytes (Fattinger et al., 2000). Moreover, a recent study showed that rifampicin repressed bile salt export pump (BSEP)-mediated taurocholate transport in insect cells expressing human BSEP (Mita et al., 2006). However, it is obscure whether in vivo treatment with rifampicin induces cholestasis in mice.

Hepatocyte tight junctions (TJs) are composed of multiple proteins that are anchored directly or indirectly to the actin-based cytoskeleton. Integral membrane proteins forming TJs include occludin, claudins and junction adhesion molecule. Zonula occludens (ZO)-1, a protein associated with TJ formation, interacts with cytoplasmic tails of occludin and claudins (Mitic and Anderson, 1998). In addition, ZO-1 interacts with two additional membrane-associated proteins, ZO-2 and ZO-3 (Wittchen et al., 1999). The altered integrity of hepatocyte tight junctions (TJs) was observed in several types of cholestasis (Mottino et al., 2007; Kawaguchi et al., 2000, Kawaguchi et al., 1999). Therefore, it is interesting whether the altered integrity of hepatocyte TJs is associated with rifampicin-induced hepatotoxicity.

The aim of the present study was to establish a model of rifampicin-induced cholestasis and to detect the expression and localization of hepatic TJs in rifampicin-induced cholestasis. We found that in vivo administration of rifampicin significantly increased the levels of serum conjugated bilirubin and total bile acids in a time-dependent manner.

Section snippets

Chemicals

Rifampicin was purchased from Sigma Chemical Co. (St. Louis, MO). All the other reagents were from Sigma or as indicated in the specified methods.

Animals and treatments

Female CD-1 mice (6–8 week-old, 24–26 g;) were purchased from Beijing Vital River (Beijing, China) whose foundation colonies were introduced from Charles River Laboratories, Inc. The animals were allowed free access to food and water at all times and were maintained on a 12-h light/dark cycle in a controlled temperature (20–25 °C) and humidity (50 ± 5%)

Rifampicin-induced liver injury

As shown in Fig. 1, administration of rifampicin for 7 consecutive days induced liver injury, as indicated by an increase of serum ALT, AST and ALP. Hepatic histology showed predominantly steatosis, associated with mild necrosis and inflammation (Fig. 2). In another experiment, mice were given with a single dose of rifampicin (200 mg/kg) by gastric intubation. As shown in Fig. 3, serum ALT, AST and ALP were slightly increased, beginning as early as 30 min and remained elevated up to 12 h after

Discussion

According to the consensus conference of the Council for International Organization of Medical Sciences, drug-induced liver injury can be classified into hepatocellular, cholestatic, and mixed types of liver damage depending on serum biochemistry markers. Cholestatic liver damage is reflected by increased levels of ALP, γ-glutamyltranspeptidase and the conjugated bilirubin in serum, whereas hepatocellular injury is mainly characterized by the elevation of serum ALT (Benichou, 1990, Pauli-Magnus

Acknowledgments

This project was supported by National Natural Science Foundation of China (30371667, 30572223) and Natural Science Foundation of Anhui province (090413142).

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    These authors contributed equally to this work.

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