Bile salt export pump inhibitors are associated with bile acid-dependent drug-induced toxicity in sandwich-cultured hepatocytes

doi:10.1016/j.bbrc.2011.11.032

Biochemical and Biophysical Research Communications

Volume 416, Issues 3–4, 16 December 2011, Pages 313-317

https://doi.org/10.1016/j.bbrc.2011.11.032 Get rights and content

Abstract

Drug-induced liver injury (DILI) is a major reason for the dropout of candidate compounds from drug testing and the withdrawal of pharmaceuticals from clinical use. Among the various mechanisms of liver injury, the accumulation of bile acids (BAs) within hepatocytes is thought to be a primary mechanism for the development of DILI. Although bile salt export pump (BSEP) dysfunction is considered a susceptibility factor for DILI, little is known about the relationship between drug-induced BSEP dysfunction and BA-dependent hepatotoxicity. Furthermore, few methods are at hand for the systematic and quantitative evaluation of BA-dependent DILI. This study aimed to construct a model of DILI by employing sandwich-cultured hepatocytes (SCHs). SCHs can be used to assess functions of canalicular transporters such as BSEP and the activity of metabolic enzymes. Here, the impact of 26 test compounds (ritonavir, troglitazone, etc.) was investigated on BA-dependent cytotoxicity in SCHs. SCHs were exposed to each compound for 24 h with or without BAs (glycochenodeoxycholic acid, deoxycholic acid, etc.). As a result, BA-dependent toxicity was observed for 11 test compounds in SCHs treated in the presence of BAs, while no signs of toxicity were observed for SCHs treated in the absence of BAs. Of the 11 compounds, nine were known BSEP inhibitors. Moreover, for some compounds, an increase in the severity of BA-dependent toxicity was observed in SCHs that were co-treated with 1-aminobenzotriazole, a non-selective inhibitor of cytochrome P450 (CYP450)-mediated drug metabolism. These results indicate that the SCH-based model is likely to prove useful for the evaluation of BA-dependent DILI, including the effects of drug metabolism and BSEP inhibition on liver injury.

Highlights

► Drug-induced toxicity is triggered by bile acids in SCHs. ► SCH-based model is useful for the evaluation of BA-dependent DILI. ► SCH-based model can reflect the effect of drug metabolism on BA-dependent DILI.

Introduction

Drug-induced liver injury (DILI) is the most common adverse event leading to the dropout of candidate compounds from drug testing and the withdrawal of pharmaceuticals from clinical use [1], [2]. Therefore, elucidating the mechanisms of DILI and constructing useful evaluation methods are critically important issues. Recently, inhibition of bile acid (BA) transport was suggested to be an underlying cause of DILI due to accumulation of BAs within hepatocytes [3], [4], [5], [6], [7].

BAs are synthesized from cholesterol in the liver and are crucial for absorption of fat-soluble vitamins and lipids. Thus, BAs play a key role in digestion and nutritional processes. However, certain BAs, such as glycochenodeoxycholic acid, lithocholic acid, and deoxycholic acid, induce hepatotoxicity as a result of apoptosis and mitochondrial disorder [8], [9], [10], [11]. Therefore, hepatic BA levels must be strictly regulated by various mechanisms.

The bile salt export pump (human BSEP/rat Bsep) is located on the canalicular membrane of hepatocytes and participates in the excretion of BAs from the liver into the bile [12]. BSEP is thus indispensable for the regulation of hepatic BA content. Several genetic mutations of BSEP are associated with progressive familial intrahepatic cholestasis type 2 [13], which results in liver failure stemming from hepatocytic accumulation of BAs. Therefore, BSEP dysfunction is likely related to liver injury. Morgan et al. reported that 25% of 200 benchmark compounds inhibited BSEP-mediated taurocholate excretion, with an IC₅₀ of under 100 μM [7]. In addition, troglitazone (an antidiabetic drug) and nefazodone (an antidepressant drug) were withdrawn from the market due to severe liver injury; these drugs are also potent BSEP inhibitors [6], [7], [14], [15]. As such, a number of methods for the determination of BSEP inhibition (e.g., the membrane vesicle assay) have been developed. However, few reports indicate that drug-induced BSEP dysfunction actually leads to hepatotoxicity, and the relationship between drug-induced BSEP dysfunction and liver injury risk is yet to be determined.

Many endogenous and exogenous substances, including BAs and drugs, undergo vectorial transport and metabolism in the liver. Drug metabolites such as N-acetyl-p-benzoquinone imine (NAPQI, the metabolite of acetaminophen) are integral players in the development of hepatotoxicity [16]. Therefore, the aim of this study was to develop an experimental model reflecting the hepatic functions of transport and drug metabolism for the evaluation of BSEP dysfunction-related/BA-dependent DILI. The model employed sandwich-cultured hepatocytes (SCHs), which are widely used in toxicologic and pharmacokinetic assays [17]. SCHs are distinctive in that they maintain cell polarity and the expression of transporters and metabolic enzymes [17], enabling in vitro studies of vectorial transport via BSEP and drug metabolism.

The current study focused on BA-dependent hepatotoxicity and explored the hypothesis that BA accumulation due to BSEP dysfunction may be a causative factor for the development of DILI. In addition, this study examined whether the SCH model could reflect the effect of drug metabolism on BA-dependent DILI.

Section snippets

Animals

Sprague Dawley rats (SLC Japan Inc., Tokyo, Japan), 7–8-weeks-old, were used throughout the experiments. Animals were treated humanely in accordance with the guidelines issued by the National Institutes of Health. In addition, all procedures were approved by the Animal Care Committee of Chiba University.

Materials

BAs and test compounds were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan), Sigma–Aldrich (St. Louis, MO), and Calbiochem (Darmstadt, Germany). Dulbecco’s modified Eagle’s

Determination of cytotoxicity of BAs and test compounds

Some of the BAs employed in this study (e.g., glycochenodeoxycholic acid, lithocholic acid, etc.) are known to induce hepatotoxicity [8], [9], [10], [11]. Similarly, some of the test compounds are reported to be hepatotoxic at high concentrations [22], [23], [24]. Hence, treatment of SCRHs with high concentrations of BAs or test compounds may directly cause cytotoxicity. However, the goal of this study was not to evaluate the direct effects of BAs and test compounds, but rather to evaluate the

Discussion

This study shows that test compounds including ritonavir, cyclosporin A, simvastatin, and troglitazone were not cytotoxic to SCRHs in the absence of BAs. However, as illustrated in Fig. 3 and Table 2 and 11 compounds were cytotoxic in the presence of BAs. The majority of these compounds are inhibiters of BSEP/Bsep-mediated taurocholate transport [7], [21]. Therefore, the current results suggest that DILI might be induced by accumulation of BAs within hepatocytes due to BSEP/Bsep dysfunction.

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Bile salt export pump inhibitors are associated with bile acid-dependent drug-induced toxicity in sandwich-cultured hepatocytes

Abstract

Highlights

Introduction

Section snippets

Animals

Materials

Determination of cytotoxicity of BAs and test compounds

Discussion

Gastroenterology

Gastroenterology

Am. J. Pathol.

Gastroenterology

J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci.

Chem. Biol. Interact.

Toxicol. In Vitro

Toxicol. Appl. Pharmacol.

Comp. Biochem. Physiol. B.

Biochim. Biophys. Acta

Drug-induced liver disorders: implications for drug development and regulation

Drug Saf.

Why drugs fail – A study on side effects in new chemical entities

Curr. Pharm. Des

The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions

Clin. Pharmacol. Ther.

Inhibition of hepatobiliary transport as a predictive method for clinical hepatotoxicity of nefazodone

Toxicol. Sci.

Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development

Toxicol. Sci.

Pregnane X receptor activation protects rat hepatocytes against deoxycholic acid-induced apoptosis

Liver Int.