Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line

Abstract

Functional expression of both sinusoidal and canalicular hepatic drug transporters was investigated in the highly differentiated human hepatoma HepaRG cell line and also, for comparison, in primary human hepatocytes and in the hepatoma HepG2 cell line. Using RT-qPCR assays, differentiated HepaRG cells were found to display a pattern of transporter expression close to that found in primary human hepatocytes, i.e. they exhibit substantial mRNA levels of the influx transporters OCT1, OATP-B, OATP-C and NTCP, and of the secretion transporters MRP2, MRP3, BSEP and P-glycoprotein. By contrast, expression of influx transporters was not present or very weak in HepG2 cells. Drug transport assays allowed to detect functional activities of OCT1, OATPs/OAT2, NTCP, MRPs and P-glycoprotein in differentiated HepaRG cells as in primary human hepatocytes whereas HepG2 cells only showed notable MRP and P-glycoprotein activities. In addition, expression of canalicular transporters in HepaRG cells was found to be up-regulated by known inducers of transporters such as rifampicin, phenobarbital and chenodeoxycholate acting on P-glycoprotein, MRP2 and BSEP, respectively. HepaRG cells thus exhibit functional expression of both sinusoidal and canalicular drug transporters and have retained regulatory pathways controlling transporter levels. These data, associated with the known high expression of drug metabolizing enzymes in HepaRG cells, highlight the interest of such hepatoma cells for analysing hepatic drug detoxification pathways.

Introduction

Membrane transport proteins, belonging to the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies of transporters, play a major role in biliary elimination of drugs (Kim, 2002). Indeed, sinusoidal transporters mediate the initial step of hepatic elimination, i.e. drug uptake from blood in hepatocytes (van Montfoort et al., 2003), whereas canalicular transporters are involved in the final stage of hepatic elimination, i.e. secretion of drugs or their metabolites from hepatocytes into bile canaliculi (Schinkel and Jonker, 2003). Major hepatic influx transporters correspond to organic cation transporter 1 (OCT1/SLC22A1), handling organic cations such as tetra-ethylammonium (TEA) and the hypoglycemic agent metformin (Jonker and Schinkel, 2004), organic anion transporting polypeptides (OATP-A/SLCO1A2, OATP-B/SLCO2B1, OATP-C/SLCO1B1 and OATP8/SLCO1B3) and organic anion transporter 2 (OAT2/SLC22A7), mediating the transport of organic anions such as estrone-3-sulfate (ES) (Rippin et al., 2001, Kobayashi et al., 2005) and also of amphipathic organic cations like digoxin (Kim, 2003, Hagenbuch and Meier, 2004), and Na⁺-taurocholate co-transporting polypeptide (NTCP/SLC10A1) involved in Na⁺-dependent uptake of biliary acids (Trauner and Boyer, 2003). Major hepatic canalicular transporters are ABC proteins. They include P-glycoprotein (ABCB1), encoded by MDR1 gene and responsible for efflux of hydrophobic organic cations including anticancer drugs such as doxorubicin, HIV antiprotease inhibitors such as saquinavir and dyes like rhodamine 123 (Schinkel and Jonker, 2003). Multidrug resistance protein 2 (MRP2/ABCC2) constitutes another major canalicular transporter, mediating the secretion of organic anions and drug conjugates, including pravastatin and the fluorescent dye carboxy-2′,7′-dichlorofluorescein (CF) (Fardel et al., 2005). The bile salt export pump (BSEP/ABCB11) is involved in canalicular efflux of biliary acids (Arrese and Ananthanarayanan, 2004). The ABC pump MRP3, located at the sinusoidal pole of hepatocytes, is thought to mediate secretion of drug metabolites into the bloodstream for subsequent urinary elimination (Zelcer et al., 2005). Interestingly, expression of several liver drug transporters are thought to be regulated by xenobiotic treatments through nuclear receptor activation as already described for drug metabolizing enzymes. Thus, rifampin, a pregnane X receptor ligand, and phenobarbital, an activator of the constitutive androstane receptor, have been shown to enhance expression of P-glycoprotein and MRP2 (Geick et al., 2001, Courtois et al., 2002), respectively, whereas BSEP levels can be up-regulated by bile salts via the farnesoid X receptor (Plass et al., 2002). Xenobiotics may also inhibit activity of transporters, through competitive or non-competitive mechanisms (Leonard et al., 2002), which may lead to drug–drug interactions and/or drug adverse effects such as cholestasis or hyperbilirubinemia (Campbell et al., 2004, Shitara et al., 2005).

Primary human hepatocytes have been recently demonstrated to constitute an adequate and useful tool for analysing activity and regulation of drug transporters. Indeed, they exhibit substantial activity of both uptake and efflux transporters (Jigorel et al., 2005, Payen et al., 2000) and retain regulatory ways of transporter expression (Payen et al., 2002). Unfortunately, their unpredictable and scarce availability limits their use, especially for analyses during pre-clinical development of pharmaceutical compounds. Human immortalized hepatic cell lines have been proposed as an alternative to primary hepatocytes in toxicological studies (Knasmuller et al., 2004, Xu et al., 2004). In this context, the newly characterized hepatoma HepaRG cell line may be important to consider since it has been shown to express specific liver functions, including drug detoxifying enzymes, at relatively high levels, in contrast to other hepatoma cell lines such as HepG2 (Gripon et al., 2002, Aninat et al., 2006). Due to the increasing importance of transporters in pharmacokinetic, drug–drug interactions and drug adverse effects, the present study was therefore designed to investigate functional expression of both sinusoidal and canalicular drug transporters in HepaRG cells. Our results indicated that activity and regulation of major hepatic drug transporters can be detected in this cell line, underlining its potential interest as an alternative to primary human hepatocytes for toxicological studies.