Elsevier

Toxicology Letters

Volume 224, Issue 1, 3 January 2014, Pages 93-100
Toxicology Letters

Alpha-naphthylisothiocyanate modulates hepatobiliary transporters in sandwich-cultured rat hepatocytes

https://doi.org/10.1016/j.toxlet.2013.09.019Get rights and content

Highlights

  • Elucidation of ANIT-induced cholestasis based on interruption of bile acid transport.

  • Metabonomics study of ANIT-treated sandwich-cultured rat hepatocytes.

  • Discovery of the direct cholestatic effect of ANIT on hepatic parenchymal cells.

Abstract

Alpha-naphthylisothiocyanate (ANIT) induces intra-hepatic cholestasis mixed with hepatocellular injury mainly by bile ductular damage. However, its direct effect on hepatic parenchymal cells (hepatocytes) is unclear. Sandwich-cultured rat hepatocytes (SCRH) were applied to clarify this question. Though cytotoxicity was not observed (0–180 μM) in ANIT-treated SCRH, metabonomics analysis of the hepatocytes revealed a shift in the metabolic pattern and a decrease in cellular cholesterol level, accompanied by an increase in total bile acids after 48 h ANIT (5–45 μM) treatment. To assess the function of major hepatic bile acid transporters, the accumulation and efflux of [D-Pen2,5]-enkephalin (DPDPE), 5 (and 6)-carboxy-2′,7′-dichlorofluorescein (CDF) diacetate promoiety and deuterium-labeled sodium taurocholate (d8-TCA) were measured. ANIT incubation for either 30 min or 48 h led to dose-dependent decreases in the biliary excretion index (BEI) of DPDPE and CDF, as well as the intracellular accumulation of d8-TCA, CDF and DPDPE. The basolateral efflux of d8-TCA was also decreased with its BEI barely changed. mRNA expression of multiple uptake transporters and bile acid synthesizing enzymes was down-regulated after 48 h incubation. In conclusion, ANIT could directly induce retention of bile acids in hepatocytes by inhibiting the function of bile acid transporters, which might contribute to its cholestatic effect.

Introduction

Cholestasis is the manifestation of many pathological conditions and drug-induced liver injuries, such as gallstone, malignancy and estradiol-intoxication (Paumgartner, 2006). Many animal models have been developed to simulate the symptom in clinic. Alpha-naphthylisothiocyanate (ANIT)-induced intra-hepatic cholestasis is one of the most popular rodent models. Biochemical and pathological changes induced by ANIT administration include elevation of total bilirubin in serum, increased total bile acids in serum/liver, etc. (Kossor et al., 1993a, Cui et al., 2009). Morphological and functional changes of bile duct epithelial cells (BECs) and hepatocytes could also be observed (Dahm and Roth, 1991). According to the current understandings, BECs are the primary injury targets of ANIT (Connolly et al., 1988, Jean et al., 1995) and inflammation (Bailie et al., 1995, Dahm et al., 1991, Kobayashi et al., 2010), oxidative stress (Ding et al., 2012, Kongo et al., 1999, Ohta et al., 2007), mitochondrial dysfunction (Rolo et al., 2002) are involved. The disruption of hepatic tight junction (Kan and Coleman, 1986) and bile duct obstruction (Kossor et al., 1993b) lead to the development of cholestasis 24–48 h after ANIT administration.

However, it is unclear whether ANIT could exert cytotoxic, metabolic or cholestatic effects directly on hepatocytes. One report showed that 35-min perfusion of ANIT could decrease the uptake and excretion of bile acids in isolated perfused rat livers (IPRLs), without any interference of tight junctions (Kossor et al., 1993a). This observation suggested the likelihood of ANIT's direct effect on hepatic bile acid disposition. Impaired bile acid transport and/or formation, as well as bile duct damage-induced physical obstruction are the two major mechanisms of drug-induced cholestasis (Yang et al., 2013). Previous studies of ANIT have focused on the latter mechanism while the present study will address the former one. Elucidation of this question will deepen and broaden the understanding of ANIT-induced hepatotoxicity from a novel perspective, and thus facilitate appropriate application of this model compound.

Despite the significance of this issue, there have been just a few cell-based investigations on the direct effect of ANIT on hepatic parenchymal cells, and their implications are limited by short-term culture or single acute endpoints, such as lactate dehydrogenase (LDH) and alanine transaminase (ALT) release (Jean et al., 1998, Orsler et al., 1999). In the current study, sandwich-cultured rat primary hepatocytes (SCRH) were applied. When primary hepatocytes are cultured in sandwich configuration, their viability and hepatic functions are maintained over a prolonged culture period (Liu et al., 1999a, Liu et al., 1999b). In addition, formation of canalicular networks and polarized localization of transporters make sandwich-cultured primary hepatocytes an ideal model to study compounds’ cholestatic effects and modulation of transporters.

In the present study, the impact of ANIT on the viability and metabolome of hepatocytes were analyzed. The metabonomics study showed that intracellular cholesterol was decreased and accordingly, total bile acids were increased after ANIT treatment. To explain this novel observation, the function and expression of transporters involved in hepatobiliary disposition of bile acids were evaluated, including uptake transporters sodium/taurocholate cotransporting polypeptide (Ntcp), organic anion transporting polypeptide (Oatp); basolateral efflux transporter multidrug resistance-associated protein 3 (Mrp3); canalicular efflux transporters bile salt export pump (Bsep) and Mrp2. The expression of bile acid synthesizing enzymes, namely cholesterol 7α-hydroxylase (Cyp7a1), sterol 27-hydroxylase (Cyp27a1), and sterol 12α-hydroxylase (Cyp8b1), was also measured. Our results demonstrate that ANIT could modulate the function and expression of several hepatic bile acid transporters, which might contribute to the increase in bile acids within hepatocytes.

Section snippets

Materials

All the reagents used in cell culture were from GIBCO unless otherwise stated. All the chemicals used were from Sigma–Aldrich unless otherwise stated.

Isolation and culture of rat hepatocytes

Hepatocytes were isolated from male Sprague-Dawley rats (Shanghai Lab., Animal Research Center, China) by two-step perfusion and purified by 45% isotonic Percoll (Kotani et al., 2011). The animal use protocol was approved by the Institutional Animal Care and Use Committee of Shanghai Institute of Materia Medica, China. The hepatocytes were seeded

ANIT was not cytotoxic to SCRH

As determined by four cytotoxicity assays which were WST-1 assay, ATP assay, LDH release and ALT release, ANIT was not cytotoxic within 5–180 μM. 180 μM was near the maximum solubility in culture medium containing 0.1% DMSO. VPA, the positive control, decreased ATP level with an IC50 of 14.4 mM and reduced WST-1 product formation with an IC50 of 1.71 mM. The IC50 of VPA was consistent with previous report (Kiang et al., 2010) (Fig. 1).

ANIT altered SCRH metabolome and increased intracellular total bile acids

The GC–MS analysis of the hepatocytes detected 201 peaks (Fig. 2

Discussion

SCRH is widely used to evaluate long-term effect of toxicant treatment and transporter-based or metabolism-mediated hepatotoxicity (Swift and Brouwer, 2010). In the current study, direct effects (i.e. cytotoxic, metabolic, and cholestatic effects) of ANIT on hepatocytes were investigated using SCRH. Results of metabonomics study demonstrated that ANIT treatment shifted metabolite profile of hepatocytes. Interestingly, hepatic cholesterol and total bile acids decreased and increased,

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was supported by National Science Foundation of China (Grant No. 81302836), Hundred Talents Program of the Chinese Academy of Sciences, and Major National Science and Technology Programs (Grant No. 2012ZX09301001-006 and 2012ZX09302003). We thank Kyunghee Yang for her helpful comments and assistance in writing the manuscript, and Xuan Ni for technical assistance.

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    Present address: Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill 27599, NC, USA.

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