Functional expression and regulation of drug transporters in monolayer- and sandwich-cultured mouse hepatocytes

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Abstract

Primary hepatocyte cultures are now considered as convenient models for in vitro analyzing liver drug transport. However, if primary human and rat hepatocytes have been well-characterized with respect to drug transporter expression and regulation, much less is known for primary mouse hepatocytes. The present study was therefore designed to gain insights about this point. The profile of sinusoidal and canalicular drug transporter mRNA expression in short time (4 h)-cultured mouse hepatocytes was found to be highly correlated with that of freshly isolated hepatocytes; by contrast, those of counterparts cultured for a longer time (until 4 days) either in monolayer configurations on plastic or collagen or in sandwich configuration with matrigel were profoundly altered: uptake drug transporters such as Oct1, Oatps and Oat2 were thus down-regulated, whereas most of efflux transporters such as Mdr1a/b, Mrp3, Mrp4 and Bcrp were induced. Moreover, short time-cultured hepatocytes exhibited the highest levels of sinusoidal influx transporter activities. Transporter-mediated drug secretion into canalicular networks was however only observed in sandwich-cultured hepatocytes. Mouse hepatocytes cultured either in monolayer or sandwich configurations were finally shown to exhibit up-regulation of referent transporters in response to exposure to prototypical activators of the drug sensing receptors pregnane X receptor, aryl hydrocarbon receptor or constitutive androstane receptor. Taken together, these data demonstrate the feasibility of using primary mouse hepatocytes for investigating potential interactions of xenobiotics with hepatic transporter activity or regulation, provided that adequate culture conditions are retained.

Introduction

Hepatic drug transporters belonging to the solute carrier (SLC) or the ATP-binding cassette (ABC) transporter superfamilies are involved in hepato-biliary secretion of drugs and drug–drug interactions (Giacomini et al., 2010). They are located at the sinusoidal or canalicular pole of hepatocytes (Funk, 2008, Hirouchi et al., 2009) (See Fig. 1 for a schematic overview of drug transporter expression by mouse hepatocytes). Putative interactions of some hepatic transporters with new molecular entities developed by pharmaceutical companies have now to be characterized (Giacomini et al., 2010, Zhang et al., 2008). For this purpose, primary hepatocyte cultures have emerged as a valuable and useful tool (Ghibellini et al., 2006, Sahi et al., 2010, Soars et al., 2007).

Cultured hepatocytes can be used either in a monolayer standard configuration, i.e., hepatocytes plated on plastic dishes or collagen-coated dishes, or in a sandwich configuration, i.e., hepatocytes plated on collagen-coated dishes and overlaid with a second layer of collagen or matrigel (LeCluyse, 2001). These two configurations have been successfully retained to study drug transporter activity and regulation (Jigorel et al., 2006, Swift et al., 2010). Rat hepatocytes cultured in conventional monolayer conditions however fail to display bile canalicular structures and show a marked down-regulation of sinusoidal influx drug transporters, associated with a concomitant over-expression of the efflux transporter P-glycoprotein (Abcb1) (Fardel et al., 1993, Jigorel et al., 2005, Luttringer et al., 2002). By contrast, sandwich-cultured rat hepatocytes exhibit functional canalicular networks (LeCluyse et al., 1994, Liu et al., 1999b), even if they also display reduced expression of sinusoidal influx transporters with time in culture when compared to freshly isolated hepatocytes (Borlak and Klutcka, 2004, Kotani et al., 2011, Tchaparian et al., 2011). With regard to human hepatocytes cultured either in monolayer or sandwich conditions, expression of drug transporters appears to be much better preserved with time in culture when compared to rat counterparts (Hoffmaster et al., 2004, Jigorel et al., 2005, Kotani et al., 2011, Li et al., 2009, Schaefer et al., 2012, Takeba et al., 2011).

Unlike primary rat and human hepatocytes, cultured mouse hepatocytes remain poorly characterized with respect to drug transporter expression and activity, even if bile acid transport and multidrug resistance-associated protein (Mrp/Abcc) 4 expression have been recently investigated in sandwich-cultured mouse hepatocytes (Swift and Brouwer, 2010). Primary mouse hepatocytes likely represent an interesting in vitro model for studying liver drug transporters, because they can originate from various and already generated knockout mice in which specific liver transporter has been deleted, thus potentially allowing to address the function and the substrates of the disrupted transporter (Klaassen and Lu, 2008). Primary mouse hepatocytes may also served for identifying signaling ways governing transporter expression, through, for example, the use of hepatocytes from transgenic mice deficient in drug-sensing receptors such as pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR) or constitutive androstane receptor (CAR), known to be involved in transporter regulation (Klaassen and Aleksunes, 2010). Before considering the use of primary hepatocytes from transgenic mice for transporter studies, accurate characterization of basal transporter expression in primary wild-type mouse hepatocytes is however required. The present study was therefore designed to carefully analyze expression, activity and regulation of drug transporters in mouse hepatocytes cultured in monolayer or sandwich configurations; the transporters studied in this work correspond to referent hepatic SLC and ABC transporters (Funk, 2008) and their cellular localization is indicated in Fig. 1.

Section snippets

Chemicals

Rhodamine 123, probenecid, verapamil, phenobarbital and dexamethasone were purchased from Sigma–Aldrich (Saint-Quentin Fallavier, France), whereas carboxy-2,7-dichlorofluoresceine (CF) diacetate and 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) were provided by Invitrogen/Life Technologies (Villebon sur Yvette, France) and Cambridge Isotope Laboratories (Andover, MA), respectively. [3H(G)] taurocholic acid (sp. act. 1.19 Ci/mmol), [6,7-3H(N)] estrone-3-sulfate (E3S) (sp. act. 57.3 Ci/mmol), [1-14C]

Morphology and canalicular network formation

Primary mouse hepatocytes maintained for 3 or 4 days after seeding either in monolayer on plastic or collagen or in sandwich configuration with collagen and matrigel formed confluent or nearly-confluent cultures (Fig. 2A). Hepatocytes cultured in monolayer exhibited a morphology rather flattener than that of sandwich-cultured counterparts, in agreement with previous data (Richert et al., 2002). Sandwich-cultured hepatocytes, but not monolayer-cultured counterparts, displayed extensive

Discussion

The present study demonstrated that expression of drug transporters in primary mouse hepatocytes is markedly influenced by time in culture, whatever the culture conditions used, i.e., monolayer/plastic, monolayer/collagen or sandwich/matrigel configurations. Indeed, mRNA expression of SLC transporters, especially those of Ntcp, Oatp1b2, Oct1, Oat2 and Mate1, rapidly and remarkably fall during primary culture. This is associated with a concomitant loss of Ntcp protein expression and a reduction

Acknowledgment

The authors thank the animal house platform of the SFR BIOSIT (University of Rennes 1, France) for mouse accommodation.

References (54)

  • L. Richert et al.

    Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes

    Toxicol. In Vitro

    (2002)
  • S.J. Rippin et al.

    Cholestatic expression pattern of sinusoidal and canalicular organic anion transport systems in primary cultured rat hepatocytes

    Hepatology

    (2001)
  • M.G. Soars et al.

    The pivotal role of hepatocytes in drug discovery

    Chem. Biol. Interact.

    (2007)
  • Y. Takeba et al.

    Comparative study of culture conditions for maintaining CYP3A4 and ATP-binding cassette transporters activity in primary cultured human hepatocytes

    J. Pharmacol. Sci.

    (2011)
  • R.Z. Turncliff et al.

    Effect of culture conditions on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured rat hepatocytes

    Biochem. Pharmacol.

    (2006)
  • P.P. Annaert et al.

    P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes

    Drug Metab. Dispos.

    (2001)
  • A.W. Bell et al.

    Phenobarbital regulates nuclear expression of HNF-4alpha in mouse and rat hepatocytes independent of CAR and PXR

    Hepatology

    (2006)
  • J. Borlak et al.

    Expression of basolateral and canalicular transporters in rat liver and cultures of primary hepatocytes

    Xenobiotica

    (2004)
  • P. Chandra et al.

    Optimization of culture conditions for determining hepatobiliary disposition of taurocholate in sandwich-cultured rat hepatocytes

    In Vitro Cell. Dev. Biol. Anim.

    (2001)
  • X. Cheng et al.

    Regulation of mouse organic anion-transporting polypeptides (Oatps) in liver by prototypical microsomal enzyme inducers that activate distinct transcription factor pathways

    Drug Metab. Dispos.

    (2005)
  • G. Elaut et al.

    Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures

    Curr. Drug Metab.

    (2006)
  • O. Fardel et al.

    P-glycoprotein expression in human, mouse, hamster and rat hepatocytes in primary culture

    Carcinogenesis

    (1993)
  • C. Funk

    The role of hepatic transporters in drug elimination

    Expert Opin. Drug Metab. Toxicol.

    (2008)
  • A. Geier et al.

    Hepatocyte nuclear factor-4alpha is a central transactivator of the mouse Ntcp gene

    Am. J. Physiol. Gastrointest. Liver Physiol.

    (2008)
  • G. Ghibellini et al.

    Methods to evaluate biliary excretion of drugs in humans: an updated review

    Mol. Pharm.

    (2006)
  • K.M. Giacomini et al.

    Membrane transporters in drug development

    Nat. Rev. Drug Discov.

    (2010)
  • O. Hankinson

    The aryl hydrocarbon receptor complex

    Ann. Rev. Pharmacol. Toxicol.

    (1995)
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