Stable Expression, Activity, and Inducibility of Cytochromes P450in Differentiated HepaRG Cells□S

Sebastien Antherieu, Christophe Chesne, Ruoya Li, Sandrine Camus, Agustin Lahoz,Laura Picazo, Miia Turpeinen, Ari Tolonen, Jouko Uusitalo, Christiane Guguen-Guillouzo, and

Andre Guillouzo

INSERM U620, Rennes, France (S.A., A.G.); Universite de Rennes 1, Faculte des Sciences Pharmaceutiques et Biologiques,Rennes, France (S.A., A.G., C.G.-G.); Biopredic International, Rennes, France (C.C., R.L., S.C.); Unidad Mixta Fundacion

Hospital La Fe-Advancell, Valencia, Spain (A.L., L.P.); Novamass Ltd., Oulu, Finland (A.T., J.U.); Department of Pharmacologyand Toxicology, University of Oulu, Oulu, Finland (M.T.); and INSERM U522, Hopital Pontchaillou, Rennes, France (C.G.-G.)

Received September 12, 2009; accepted December 17, 2009

ABSTRACT:

HepaRG cells possess the unique property to differentiate in vitroand to express various functions of mature hepatocytes, includingthe major cytochromes P450 (P450s). In the present study, wecarefully analyzed mRNA expression and activity of the majorP450s and their responsiveness to three prototypical inducers,phenobarbital, rifampicin, and omeprazole, in differentiatedHepaRG cell cultures over a 4-week period after low and highseeding. Only minor differences were observed in P450 activitieswhen measured by two cocktails of probe substrates, probablyrelated to the choice and/or concentration of substrates. Similarresults were obtained from the two cell seeding conditions. Ex-pression and activities of several P450s were dimethyl sulfoxide-dependent. However, basal P450 expression and activities aswell as their responsiveness to the prototypical inducers were

well maintained over the 4-week period, and a good correlationwas observed between transcript levels and corresponding ac-tivities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found toaccurately respond to their respective prototypical inducers,i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basalexpression of several phase II enzymes, transporters, and nu-clear receptors, and response to inducers were also well pre-served. More genes were found to be induced in HepaRG cellsthan in primary human hepatocytes, and no marked variationwas noticed between the different passages. Taken together,these data support the conclusion that HepaRG cells representa promising surrogate to primary human hepatocytes for xeno-biotic metabolism and toxicity studies.

Drug-induced hepatotoxicity represents a major clinical problemaccounting for approximately 50% of all cases of acute liver failure,and it is a major cause of attrition in drug development. Estimation ofcytochrome P450 (P450) induction and inhibition and prediction ofdrug-drug interactions are an important consideration for the devel-opment of novel therapeutic agents (Park and Miller, 1996; Pelkonenet al., 2008). Predicting the ability of a drug to modulate P450

expression at an early stage of its discovery and development shouldreduce the risk of failure in the clinic and, more importantly, permitthe identification of alternative noninducing/noninhibiting chemicalstructures. Over the past decades, various in vitro and/or ex vivomodels have been developed to investigate drug metabolism. In vitroliver cell systems represent a good experimental approach to screenpotential hepatotoxic compounds and to investigate mechanisms bywhich chemicals induce liver lesions (Guillouzo, 1998). Primaryhuman hepatocytes are considered to be the most pertinent model forin vitro testing of the induction/inhibition potential of drug candidates(Guillouzo, 1998; Gomez-Lechon et al., 2004; Hewitt et al., 2007).However, their use is limited by scarce availability of donor organs,large interdonor functional variability, and early phenotypic changesin vitro. Most hepatocyte cell lines, mainly originated from tumors,have indefinite proliferative capacity, but they are considered inap-propriate for prediction of hepatotoxicity in preclinical drug develop-

This work was supported by the European Community [Contracts LIINTOP-STREP-037499 and Predict-IV-202222 (to A.G.)]; and the Ministerio Ciencia eInovacion/Instituto de Salud Carlos III for a Miguel Server [Contract CP08/00125](to A.L.).

Article, publication date, and citation information can be found athttp://dmd.aspetjournals.org.

doi:10.1124/dmd.109.030197.□S The online version of this article (available at http://dmd.aspetjournals.org)

contains supplemental material.

ABBREVIATIONS: P450, cytochrome P450; LD, low density; DMSO, dimethyl sulfoxide; HD, high density; PB, phenobarbital; RIF, rifampicin;OME, omeprazole; FCS, fetal calf serum; CDFDA, carboxydichlorofluorescein di-acetate; UAM, Unidad Mixta Fundacion Hospital La Fe-Advancell;NM, Novamass; PCR, polymerase chain reaction; FIH, freshly isolated hepatocytes; HPLC, high-performance liquid chromatography; MRP,multidrug resistance-associated protein; BSEP, bile salt export pump; MDR, multidrug resistance protein; RT-qPCR, reverse transcriptase-quantitative PCR; GSTA1/A2, glutathione transferase A1/A2; UGT1A1, UDP-glucuronosyl transferase 1A1; BCRP, breast cancer resistanceprotein; NTCP, Na�-dependent taurocholic cotransporting polypeptide; AhR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor;PXR, pregnane X receptor.

0090-9556/10/3803-516–525$20.00DRUG METABOLISM AND DISPOSITION Vol. 38, No. 3Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 30197/3568248DMD 38:516–525, 2010 Printed in U.S.A.

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ment due to the low levels, if any, of major P450 enzymes and severaltransporters. An exception is represented by the HepaRG cell linederived from a human liver carcinoma (Gripon et al., 2002). Whenseeded at low density (LD), HepaRG cells transdifferentiate intobipotent hepatic progenitors and actively divide before acquiringtypical morphological and functional characteristics of adult humanhepatocytes in primary culture under appropriate culture conditions(Cerec et al., 2007). Indeed, after 2 weeks at confluence in thepresence of 2% dimethyl sulfoxide (DMSO) and corticosteroids, theyappear as hepatocyte-like colonies surrounded by biliary-like cells.Transdifferentiation of differentiated HepaRG cells can be avoided byseeding at high density (HD) (Aninat et al., 2006; Cerec et al., 2007).

Differentiated HepaRG cells express various liver functions, in-cluding P450s, phase II enzymes, transporters, and nuclear receptorsat levels comparable with those found in primary hepatocytes and areresponsive to prototypical inducers, suggesting that they could repre-sent a surrogate to the latter in drug metabolism and toxicity studies(Aninat et al., 2006; Le Vee et al., 2006; Guillouzo et al., 2007; Josseet al., 2008; Kanebratt and Andersson, 2008b; Turpeinen et al., 2009).Moreover, some evidence has been provided that LD-seeded HepaRGcells can retain relatively stable expression and activities of P450s forseveral weeks at confluence (Josse et al., 2008; Kanebratt and Anders-son, 2008a). However, long-term maintenance of P450 activities andresponsiveness to inducers has not been fully characterized, especiallyin HD-seeded cultures. The present study was undertaken within theEuropean Community Framework Programme LIINTOP project tocompare transcript and activity levels of the major P450s and theirresponsiveness to the prototypical inducers, phenobarbital (PB), ri-fampicin (RIF), and omeprazole (OME), over a 4-week period indifferentiated HepaRG cells. For this purpose, P450 activities weresimultaneously estimated using two different cocktail substrate set-tings. Such P450-substrate cocktails are increasingly used for thedetermination of basal and induced P450 activities (Zhou et al., 2004).Indeed, the use of a variety of substrate combinations, numbers ofsubstrates, or analytical methods has revealed no significant differ-ences between the results obtained from individual or cocktail anal-yses (Dierks et al., 2001; Kim et al., 2005; Lahoz et al., 2008a). Inaddition, expression of various other genes related to xenobioticmetabolism and transport was also measured for comparison pur-poses. We show that basal mRNA and activity levels of major P450s,expression of several phase II enzymes and transporters, as well asinducibility by RIF, PB, and OME were well maintained in differen-tiated HepaRG cells during the 4-week testing period, whether thecells were seeded at LD or HD.

Materials and Methods

Chemicals. DMSO, RIF, PB, OME, and insulin were purchased fromSigma-Aldrich (St. Quentin Fallavier, France). Williams’ E medium wasobtained from Eurobio Laboratories (Les Ulis, France). Fetal calf serum (FCS)was supplied by Perbio (Brebieres, France). Penicillin streptomycin carboxy-dichlorofluorescein di-acetate (CDFDA) and BODIPY FL vinblastine, a flu-orescent analog of the anticancer drug vinblastine, were from Invitrogen(Cergy Pontoise, France). Hydrocortisone hemisuccinate was obtained fromUpjohn Pharmacia (Guyancourt, France). Substrates and metabolite standardsused for the Unidad Mixta Fundacion Hospital La Fe-Advancell (UAM)cocktail were as follows: 4�-hydroxydiclofenac, 6-hydroxychlorzoxazone, hy-droxybufuralol, midazolam, 1�-hydroxymidazolam, phenacetin, bufuralol, me-phenytoin, hydroxymephenytoin, and acetaminophen (supplied by BD Bio-sciences Discovery Labware, Bedford, MA). Substrates used for the Novamass(NM) cocktail were as follows: bupropion, midazolam, and OME were pur-chased from Sequoia Research Products (Pangbourne, UK); testosterone wasobtained from Fluka (Buchs, Swizerland); and phenacetin was obtainedfrom ICN Biomedicals (Costa Mesa, CA). Metabolite standards

O-desmethyldextromethorphan, 6-hydroxychlorzoxazone, desethylamodia-quine, hydroxytolbutamide, and 6�-hydroxytestosterone were purchased fromBD Biosciences Discovery Labware. All other chemicals were obtained fromSigma-Aldrich (St. Louis, MO).

Cell Cultures. HepaRG cells were cultured at a density of either 2.6 � 104

cells/cm2 (LD) or 0.45 � 106 differentiated cells/cm2 (HD) as describedpreviously (Gripon et al., 2002; Aninat et al., 2006). LD-seeded HepaRG cellswere first incubated in the Williams’ E medium supplemented with 10% FCS,100 units/ml penicillin, 100 �g/ml streptomycin, 5 �g/ml insulin, 2 mMglutamine, and 5 � 10�5 M hydrocortisone hemisuccinate for 2 weeks.Maximal liver-specific activities were attained after two additional weeks inthe same medium added with 2% DMSO (Gripon et al., 2002). HD-seededHepaRG cells were directly incubated in the DMSO-containing medium; theydid not transdifferentiate and retained their morphological and functionalcharacteristics. The medium of both LD and HD cultures was renewed every2 or 3 days.

Human hepatocytes from five adult donors undergoing resection for primaryand secondary tumors were obtained by perfusion of histologically normalliver fragments (Guguen-Guillouzo et al., 1982). In brief, hepatocytes wereseeded at a density of 190,000 cells/cm2 in 24-well plates in Williams’ Emedium supplemented with 10% FCS, 100 units/ml penicillin, 100 �g/mlstreptomycin, 1 �g/ml insulin, 2 mM glutamine, and 1 �g/ml bovine serumalbumin. Cells were allowed to attach for 12 h, at which time medium wasreplaced with the same medium deprived of FCS and supplemented with 10�7

M dexamethasone. The medium was renewed every day.Induction Treatments. For induction studies in HepaRG cells, experimen-

tal conditions were based on the observations that P450 induction by DMSOwas lost 24 h after its depletion, and that no marked changes occurred in P450transcript levels after two additional days (Aninat et al., 2006). HepaRG cells(from passages 14, 17, and 20) were seeded either at LD and cultured for upto 56 days or at HD and cultured for up to 28 days (Fig. 1). Three days beforeinducer addition, the medium was changed to a DMSO-free medium contain-ing 2% FCS, and after that period the cells were exposed to the prototypicalinducers (10 �M RIF, 1 mM PB, or 50 �M OME) or their vehicle, i.e., DMSO(final concentration of 0.5%), for RIF and OME and water for PB, for 48 h(mRNA quantification) or 72 h (measurements of P450 activities). Humanhepatocytes were exposed to the prototypical inducers or their vehicle 1 to 3days after seeding. Treatments also lasted either 48 h (mRNA quantification)or 72 h (measurements of P450 activities).

Isolation of RNA and Reverse Transcriptase-Quantitative PolymeraseChain Reaction Analysis. Total RNA was extracted from 106 HepaRG cellsor 106 human hepatocytes with the SV total RNA isolation system (Promega,Madison, WI), which directly included a DNase treatment step. RNAs werereverse-transcribed into cDNA by using a High-Capacity cDNA Archive kit

FIG. 1. Seeding and culture of HepaRG cells. HepaRG cells were cultured at LD orHD. At LD seeding, HepaRG cells transdifferentiated into bipotent progenitors andactively divided until confluence (15 days); then, they differentiated into bothhepatocyte-like and biliary-like cells (approximately 50% each). Maximal liver-specific activities were attained after 2 weeks in the growth medium supplementedwith 2% DMSO (28 days). When seeded at HD, HepaRG cells were directlyincubated in the DMSO-containing medium; they did not transdifferentiate andretained their morphological and functional characteristics. For induction studies,HepaRG cells were seeded either at LD and cultured for up to 56 days or at HD andcultured for up to 28 days.

517FUNCTIONAL STABILITY OF P450 ENZYMES IN HepaRG CELLS

(Applied Biosystems, Foster City, CA). Real-time quantitative polymerasechain reaction (PCR) was performed by the fluorescent dye SYBR Greenmethodology using the SYBR Green PCR Master Mix (Applied Biosystems)and the ABI Prism 7000 (Applied Biosystems). Table 1 (supplemental data)shows primer pairs for each transcript chosen with Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), except for CYP2A6, CYP2C8,CYP2C19, and CYP2D6, which were provided by Biopredic International(Rennes, France) (Girault et al., 2005). The amplification curves were readwith the ABI Prism 7000 SDS software using the comparative cycle thresholdmethod. The relative quantification of the steady-state mRNA levels wascalculated after normalization of the total amount of cDNA tested using 18SRNA as a reference. Furthermore, a dissociation curve was performed after thePCR analysis to verify the specificity of the amplification. Results wereexpressed as percentage of mRNA levels measured in freshly isolated hepa-tocytes (FIH; arbitrarily set at 100%) for basal expression studies, or aspercentage of mRNA levels measured in the corresponding control samplesexposed to the vehicle (arbitrarily set at 100%) for induction studies.

Evaluation of P450 Activities. Two substrate cocktails were used: theUAM cocktail and the NM cocktail.

The UAM cocktail. Activity assays used direct incubation of cell monolay-ers with a cocktail of eight substrates as described previously (Lahoz et al.,2008b). The substrate mixture stock solutions were prepared in DMSO andthen methodically diluted in the incubation medium to obtain the followingfinal concentrations: 10 �M phenacetin (CYP1A2), 5 �M coumarin(CYP2A6), 10 �M bupropion (CYP2B6), 10 �M diclofenac (CYP2C9), 50�M mephenytoin (CYP2C19), 10 �M bufuralol (CYP2D6), 50 �M chlorzoxa-zone (CYP2E1), and 5 �M midazolam (CYP3A4). The final concentration ofDMSO during incubation was 0.5% (v/v). Incubations with the cocktail ofsubstrates lasted 2 h. Metabolism of all compounds was linear during thisincubation period. At the end, aliquots of media (300 �l) were collected andstored at �80°C until delivery and analysis in UAM laboratories. Sampleswere subsequently extracted twice with ethyl acetate 1:1 (v/v), and dextro-methorphan was used as recovery standard. The organic phase was transferredto a clean tube, evaporated under vacuum, dissolved in 100 �l HEPES (5%acetonitrile), and submitted for analysis. Metabolites formed and released intothe culture medium were quantified by high-performance liquid chromatogra-phy (HPLC) tandem mass spectrometry. This system comprised a MicromassQuattro Micro (Waters, Milford, MA) triple quadrupole mass spectrometer inelectrospray ionization mode, interfaced with an Alliance 2795 HPLC (WatersChromatography). Chromatography was performed at 35°C, and an aliquot (20�l) was injected into a Teknokroma C18 column (100 mm � 2.1 mm, 3-�mparticle size) at a flow rate of 0.4 ml/min. The mobile phase was 0.1% formicacid in acetonitrile (A) and 0.1% formic acid in water (B). The proportion ofacetonitrile was increased linearly from 0 to 90% in 6 min, and then the columnwas allowed to re-equilibrate at the initial conditions for 4 min. The columneluent was directed without splitting to an electrospray ionization interface,operating at 320°C and using nitrogen as cone gas (50 l/h). The tandem massspectrometry experiments were carried out with a triple quadrupole analyzeroperating in multiple reaction monitoring mode. Enzymatic activities wereexpressed as picomole of metabolites formed per hour per milligram of totalprotein.

The NM cocktail. Activity assays were performed by direct incubation ofcell monolayers with a cocktail of 10 substrates as described previously(Turpeinen et al., 2005; Tolonen et al., 2007). The substrate mixture stocksolutions were prepared in DMSO and diluted in incubation media to obtainthe following final concentrations: 5 �M melatonin (CYP1A2), 2 �M couma-rin (CYP2A6), 2 �M bupropion (CYP2B6), 5 �M amodiaquine (CYP2C8), 8�M tolbutamide (CYP2C9), 5 �M OME (CYP2C19), 1 �M dextromethor-phan (CYP2D6), 10 �M chlorzoxazone (CYP2E1), 1 �M midazolam(CYP3A4), and 5 �M testosterone (CYP3A4). The final concentration ofDMSO during incubation was 0.5% (v/v). After a 4-h incubation with thecocktail of substrates, aliquots (300 �l) were collected and stored at �80°Cuntil delivery and analysis. Frozen samples were thawed at room temperatureand centrifuged for 10 min at 13,200g, then 20 �l of the supernatant wasinjected into the liquid chromatography tandem mass spectrometry. A Waters2695 Alliance HPLC system (Waters) was used together with a Waters SunfireRP18 column (2.1 � 100 mm column with 5-�m particle size) and a Luna C18precolumn (2.0 � 4.0 mm) (Phenomenex, Torrance, CA) at 30°C. The HPLC

eluents were aqueous 1% formic acid � 10 mM ammonium acetate, pH 2.4(A) and methanol (B). The gradient elution with 5 to 50 to 80% B was appliedin 0 to 1.0 to 4.0 min, followed by 0.5-min isocratic elution with 80% B andcolumn equilibration, resulting in a total time of 8 min/injection. The eluentflow rate was 0.5 ml/min. The flow was split postcolumn with an AccuratePost-Column Stream Splitter (LC Packings, Amsterdam, The Netherlands)with the mass spectrometry ion source-to-waste ratio of 1:3. Data wereacquired using a Micromass Quattro Micro triple quadrupole mass spectrom-eter (Altrincham, Cheshire, UK), equipped with a Z-spray electrospray ionsource. Multiple reaction monitoring mode using a polarity switching betweenpositive and negative ion mode was applied. The capillary voltage used was4200 V and extraction cone voltage 2 V for all compounds. The dissolutiontemperature and source temperature were 280°C and 150°C, respectively.Nitrogen was used as a drying gas with a flow rate of 700 l/h and as a nebulizergas with a full flow rate. The collision cell argon pressure was set to 3.8 �10�3 mbar. The dwell times for monitoring each reaction were 100 msec, andthe delay between positive and negative polarities during hydroxychlorzoxa-zone detection (2.7–3.8 min) was 300 msec. Enzyme activities were expressedas picomole of metabolites formed per hour per milligram of total protein.

Efflux Transport Assays. Analysis of efflux transport was performed byusing two fluorescent substrates: CDFDA, a substrate of multidrug resistance-associated protein (MRP) 2 and BODIPY FL vinblastine, a substrate of bilesalt export pump (BSEP) and multidrug resistance protein (MDR) 1. Afterwashing with the uptake buffer (136 mM NaCl, 5.3 mM KCl, 1.1 mMKH2PO4, 0.8 mM MgSO4, 1.8 mM CaCl2, 11 mM D-glucose, 10 mM HEPES,pH7.4), HepaRG cells were incubated for 30 min at 37°C with fluorescentsubstrates (3 �M CDFDA or 2 �M vinblastine) then washed with chilledphosphate-buffered saline and observed under fluorescence microscopy.

Statistical Analysis. Each value corresponded to the mean � S.D. of threeindependent experiments. The Kruskal-Wallis nonparametric test was used tocompare mRNA levels and P450 activities between different time points. TheMann-Whitney U test was applied to compare mRNA levels and P450 activ-ities between inducer-treated cultures and corresponding control samples. Datawere considered significantly different when p � 0.05.

Results

Basal mRNA Levels of Phase I and II Metabolizing Enzymes,Membrane Transporters, and Nuclear Receptors. Basal mRNAlevels of 20 genes were analyzed by reverse transcriptase-quantitative PCR (RT-qPCR) in FIH and 1- and 3-day-culturedprimary human hepatocytes and LD- and HD-seeded HepaRG cellsat different times for up to 4 weeks after differentiation. Thesegenes were as follows: 10 P450s (CYP1A1, CYP1A2, CYP2A6,CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, andCYP3A4), two phase II enzymes [glutathione transferase A1/A2(GSTA1/A2) and UDP-glucuronosyl transferase 1A1 (UGT1A1)],five membrane transporters [breast cancer resistance protein(BCRP), BSEP, MDR1, MRP2, and Na�-dependent taurocholiccotransporting polypeptide (NTCP)], and three nuclear receptors [arylhydrocarbon receptor (AhR), constitutive androstane receptor (CAR),and pregnane X receptor (PXR)]. All expression values werecompared with those measured in 1-day human hepatocyte culturesset at 100%. Differential changes were observed in basal geneexpression in primary human hepatocytes (Fig. 2) as a function oftime in culture; they could be classified into five groups charac-terized by either a decrease followed by an increase at day 3(CYP2C8, CYP2C9, CYP3A4, UGT1A1, CAR, NTCP), a contin-uous and strong decrease (CYP1A2, CYPB6, CYP2E1, BSEP), adecrease followed by a relative stability (CYP1A1, CYP2C19,GSTA1/2, BCRP), a continuous increase (MDR1, MRP2), or arelative stability (CYP2A6, CYP2D6, AhR, PXR). Compared with1-day primary hepatocyte cultures, all the genes tested were ex-pressed in DMSO-exposed HepaRG cultures; the levels of tran-scripts were either markedly lower (CYP1A2, CYP2C8, CYP2D6,CYP2E1, BSEP, NTCP), at the 30 to 50% level (CYP1A1,

518 ANTHERIEU ET AL.

CYP2A6, CYP2B6, PXR), or comparable or even higher(CYP2C9, CYP2C19, CYP3A4, GSTA1/2, UGT1A1, MDR1,MRP2, AhR, CAR) (Fig. 3, A and C). However, when HepaRGcells were shifted to a DMSO-free medium for 72 h before mRNAquantification, some major differences were found (Fig. 3, B andD). Transcripts of CYP1A1, CYP2A6, CYP2B6, CYP3A4, andUGT1A1 were decreased, whereas those of CYP1A2, CYP2C8,CYP2C9, CYP2C19, CYP2D6, CYP2E1, GSTA1-A2, AhR, CAR,and transporters were only slightly altered. Moreover, most geneswere much less expressed in both proliferative and poorly differ-entiated HepaRG cells cultured for 5 or 15 days, respectively, inthe absence of DMSO after LD seeding; the only exception wasCYP1A1, which was highly expressed (Fig. 3B). All data showeda long-term, relatively stable gene expression of all tested drug-metabolizing enzymes and transporters in differentiated HepaRG cellsfrom both LD- and HD-seeded cultures maintained at confluence for upto 4 weeks.

Basal Activities of Various P450s. The UAM and NM cocktailswere used for the determination of P450 activities in HepaRG cellsand primary human hepatocytes. In addition to the use of severaldifferent substrates, the major differences between the two assayprocedures were the use of lower substrate concentrations andlonger incubation times in the NM assay. Concentrations of thefour common substrates were 5-fold (bupropion, chlorzoxazone,midazolam) and 2.5-fold (coumarin) higher in the UAM (Fig. 4)than in the NM (Fig. 5) assay. Lower values were frequentlyobtained with the NM cocktail. Compared with the values mea-sured in freshly isolated hepatocyte suspensions, only CYP2A6,CYP2C9, and CYP2E1 activities were markedly decreased after24 h of culture using the UAM probe cocktail (Fig. 4A). Ourresults showed important interdonor variability between humanhepatocyte populations for some activities, especially forCYP2C19 (from 0.98 to 10.04 pmol/h/mg protein; n � 5) andCYP2B6 (from 1.04 to 31.62 pmol/h/mg protein; n � 5).

In DMSO-exposed HepaRG cultures (Fig. 4B), P450 activities

were found to be either lower (CYP1A2, CYP2A6, CYP2C9,CYP2E1) or relatively close (CYP2B6, CYP2C19, CYP2D6,CYP3A4) to those of 1-day primary hepatocyte cultures. In HepaRGcells maintained in DMSO-free medium (Figs. 4C and 5B), most P450activities were lower, especially CYP2B6 and CYP3A4. As expected,P450 activities were all quite low in 5- and 15-day LD-seededcultures, corresponding to proliferating and poorly differentiatedHepaRG cells, respectively.

All activities were relatively well maintained over the 28-dayperiod of confluence. Using the NM cocktail, P450 activity valueswere either comparable with (CYP1A2, CYP2B6, CYP2C19,CYP2D6) or lower than (CYP2C9, CYP3A4) those estimated with theUAM one. Furthermore, low CYP2A6 and CYP2E1 activities werefound with the UAM cocktail, whereas they were undetectable withthe NM cocktail. However, other P450 activities were found to berelatively well maintained by using the NM cocktail, and CYP2C8activity was demonstrated (amodiaquine deethylation).

Functional Activities of Membrane Transporters. To verifywhether canalicular efflux transporters remained functional in hep-atocyte-like HepaRG cells after several weeks at confluence, trans-port of specific fluorescent substrates was analyzed on day 56 afterLD seeding. First, no morphological modification of bile canalic-ular structures was observed when HepaRG cells were cultured inDMSO-free medium for 5 days (Fig. 6A). Moreover, accumulationof CDFDA, a fluorescent substrate of MRP2, was restricted to bilecanaliculi of HepaRG hepatocytes (Fig. 6B). In addition, specificaccumulation of BODIPY FL vinblastine, a fluorescent substrate ofBSEP/MDR1, was also evidenced in canalicular spaces of HepaRGcells. Similar observations were made in HD-seeded HepaRG cellcultures (data not shown).

Induction of P450s by Prototypical Inducers at mRNA and Activ-ity Levels. To further estimate the functional capacity of HepaRG cellsmaintained at confluence for several weeks, their responsiveness tothree prototypical inducers (RIF, PB, and OME) was assessed over a4-week period. At appropriate time points, HepaRG cultures were

FIG. 2. Basal mRNA levels of phase I and IImetabolizing enzymes, membrane transport-ers, and nuclear receptors in human hepato-cytes. Human hepatocytes were used eitherfreshly isolated or 1 to 3 days after seeding.Transcripts of P450s (A), phase II metaboliz-ing enzymes, membrane transporters, and nu-clear receptors (B) were then analyzed usingRT-qPCR assays. Results are the mean �S.D. of three independent experiments andare expressed as percentage compared with1-day cultured human hepatocytes, arbitrarilyset at 100%. The Mann-Whitney U test wasapplied to compare mRNA levels (�, p �0.05; ns, no significant difference).

519FUNCTIONAL STABILITY OF P450 ENZYMES IN HepaRG CELLS

shifted to a DMSO-free medium supplemented with 2% FCS for 72 hand then treated with the prototypical inducers for 48 and 72 h formRNA (Fig. 7) and activity (Fig. 8) measurements, respectively.Comparable results were obtained in LD- and HD-seeded HepaRGcultures, and the levels of P450 induction were relatively consistentfor 4 weeks in differentiated HepaRG cells in both culture conditions(nonsignificant variations, Kruskal-Wallis test).

The main results are summarized in Table 1. As expectedCYP1A2, CYP2B6, and CYP3A4 transcripts and activities werestrongly increased by their prototypical inducers. Thus, CYP1A2mRNA and activity were induced by OME (Figs. 7C and 8C), andCYP2B6 and CYP3A4 mRNA and corresponding activities wereinduced by both PB and RIF (Figs. 7, A and B, and 8, A and B).In addition, various other P450s were modulated by the threeinducers (Supplemental Tables 2, 3, and 4). CYP1A1 and CYP1A2transcripts were augmented in both HepaRG cells and human

hepatocytes by OME. CYP1A1 expression was strongly increasedby OME (450 – 850-fold) and also weakly increased by PB (3– 6-fold) in HepaRG cells. CYP1A2 activity was induced by the threecompounds in HepaRG cells. CYP1A1 activity was not specifi-cally measured by the probe substrates. Expression and activity ofCYP2B6 and CYP3A4 were also induced by the three compounds inboth LD- and HD-seeded HepaRG cells. However, induction of CYP3A4was demonstrated only by 3-hydroxylation of omeprazole (NM) and1-hydroxylation of midazolam (UAM). No induction of 1-hydroxylationof midazolam or 6�-hydroxylation of testosterone was evidenced withthe NM cocktail.

Expression of CYP2A6 was induced by PB in HepaRG cells,whereas it was increased by the three prototypical inducers inhuman hepatocytes. On the other hand, CYP2E1 expression wasdecreased by all three inducers in both human hepatocytes andHepaRG cells. However, CYP2A6 and CYP2E1 activities were not

FIG. 3. Basal mRNA levels of phase I andII metabolizing enzymes, membrane trans-porters, and nuclear receptors in HepaRGcells. HepaRG cells were seeded at LD andcultured for 5 to 56 days or at HD andcultured for 5 to 28 days. Three days beforeexpression analysis, they were shifted toDMSO-free medium (B and D) or main-tained in presence of DMSO (A and C).Transcripts of P450s (A and B), phase IImetabolizing enzymes, membrane trans-porters, and nuclear receptors (C and D)were then analyzed using RT-qPCR as-says. Results are the mean � S.D. ofthree independent experiments (forDMSO-free cultured HepaRG cells) andare expressed as percentages comparedwith 1-day cultured human hepatocytes,arbitrarily set at 100%. The Kruskal-Wallis nonparametric test was applied tocompare mRNA levels between differenttime points in differentiated HepaRGcells (LD-D28 to LD-D56 and HD-D5 toHD-D28; ns, no significant difference).

520 ANTHERIEU ET AL.

detected with the cocktails in DMSO-free cultured HepaRG cellswhatever the inducer.

RIF and OME decreased CYP2D6 expression in HepaRG cells but notin primary hepatocyte cultures. CYP2D6 activity was decreased by thethree inducers using the NM cocktail (with dextromethorphan as a sub-strate), but this activity was increased by RIF and PB in both LD- andHD-seeded HepaRG cells by using the UAM cocktail (with bufuralol asa substrate). However, these variations of activities were low comparedwith the -fold inductions of other P450s.

Induction of CYP2C (CYP2C8, CYP2C9, and CYP2C19) expres-

sion by PB and more weakly by RIF was observed in HepaRG cells,whereas only RIF induced CYP2C8 and CYP2C19 expression inhuman hepatocytes. CYP2C9 activity was weakly increased by OMEas well as CYP2C8 by RIF and PB in HepaRG cells using the NMcocktail. Conflicting results were found for CYP2C19 activity withthe two cocktails: a strong decrease of activity by OME and anincrease by RIF were observed with the UAM cocktail (mephenytoinas a substrate), whereas this activity was strongly induced by PB andRIF and more weakly by OME with the NM cocktail (5-hydroxylationof omeprazole as the model reaction).

FIG. 4. Comparative basal activity levelsof P450s in primary human hepatocytesand HepaRG cells using the UAM cock-tail of substrates assay. Human hepato-cytes were used either freshly isolated or1-day after seeding (A). HepaRG cellswere seeded at LD and cultured for 5 to56 days or at HD and cultured for 5 to 28days. Three days before P450 activityanalysis, they were shifted to DMSO-freemedium (C) or maintained in presence ofDMSO (B). P450s activities were thenanalyzed using the UAM cocktail of sub-strates assay. Results are the mean � S.D.of three independent experiments and areexpressed as pmol/min/mg protein. TheMann-Whitney U test was applied tocompare P450 activities in FIH and in1-day-cultured human hepatocytes (�,p � 0.05), and the Kruskal-Wallis non-parametric test was applied to compareP450 activities between different timepoints in differentiated HepaRG cells(LD-D28 to LD-D56 and HD-D5 to HD-D28; §, p � 0.05; ns, no significantdifference).

FIG. 5. Basal activity levels of P450s inHepaRG cells using the NM cocktail ofsubstrates assay. HepaRG cells were seededat LD and cultured for 5 to 56 days or at HDand cultured for 5 to 28 days. Three daysbefore P450 activity analysis, they wereshifted to DMSO-free medium (B) or main-tained in presence of DMSO (A). P450activities were then analyzed using the NMcocktail of substrates assay. Results are themean � S.D. of three independent experi-ments and are expressed as pmol/min/mgprotein. The Kruskal-Wallis nonparametrictest was applied to compare P450 activitiesbetween different time points in differenti-ated HepaRG cells (LD-D28 to LD-D56and HD-D5 to HD-D28; §, p � 0.05; ns, nosignificant difference).

521FUNCTIONAL STABILITY OF P450 ENZYMES IN HepaRG CELLS

It is noteworthy that induction rates were much lower in humanhepatocytes than in HepaRG cells (Fig. 7). CYP1A1, CYP2C8,CYP2C9, CYP2C19, and CYP3A4 were found to be induced by PBin HepaRG cells but not in human hepatocytes (SupplementalTable 2).

Induction of Phase II Enzymes, Transporters, and NuclearReceptors Expression. Expression of phase II enzymes, transporters,and nuclear receptors was investigated in both HepaRG cells andprimary human hepatocytes in response to prototypical inducers (Sup-plemental Table 5). Overall effects were relatively well maintained indifferentiated HepaRG cells whether they were seeded at LD or HDover the 4-week period. GSTA1/2 and UGT1A1 expression wasinduced by the three inducers in LD- and HD-seeded HepaRG cellsand only by RIF and OME in primary human hepatocytes. Likewise,expression of efflux transporters MDR1 and MRP2 was increased bythe three inducers in HepaRG cell cultures and only by RIF and OMEin primary hepatocyte cultures. An induction of BCRP was observedafter PB and OME exposure in HepaRG cells and only after OMEtreatment in human hepatocytes. In addition, an inhibition of theuptake-transporter NTCP expression by OME and a decreased expres-sion of the efflux transporter BSEP by RIF were evidenced in bothdifferentiated HepaRG cells and human hepatocytes. Some responsesof nuclear receptors to inducers were also observed. A RIF-dependentdecrease and a PB-dependent induction of CAR expression wereobserved in HepaRG cells.

Discussion

Because human hepatocytes are a precious, limited resource andshow an extensive interdonor variability in response to inducers,alternatives are required for use in drug discovery. Previous studieshave shown that human hepatoma HepaRG cells express most of themajor drug-metabolizing enzymes when they are differentiated invitro. The present results confirm and extend these observations; they

show that expression and activities of the major P450s as well as theirresponsiveness to prototypical inducers were well maintained over a4-week period in differentiated HepaRG cells obtained from eitherLD- or HD-seeded cultures. Only random and limited differenceswere noticed between these two seeding conditions. Contrary to LDseeding that requires 1 month before HepaRG cells can be used asfully differentiated (Cerec et al., 2007), HD seeding prevents trans-differentiation (Aninat et al., 2006; Cerec et al., 2007) and is associ-ated with only transient and limited decrease in certain liver-specificfunctions. Consequently, HD-seeded cultures are of potentially greatvalue for high volume, convenient use of the HepaRG cell line.

An extensive analysis of P450 activities was performed usingsubstrate cocktails. This approach requires fewer sample numbersbecause the same culture is incubated with all the substrates. Mostresults obtained with the UAM and NM cocktails used in the presentstudy were found to be comparable. However, some differences wereobserved, which could be due to differences in the experimentalconditions and/or the choice of probe substrates. Indeed, UAM andNM cocktails were composed of 8 and 10 substrates, respectively(Turpeinen et al., 2005; Lahoz et al., 2008b), and only 4 substrateswere in common: bupropion for CYP2B6, coumarin for CYP2A6,chlorzoxazone for CYP2E1, and midazolam for CYP3A4. P450 ac-tivities were higher with the UAM cocktail, and detectable CYP2A6and CYP2E1 activities were demonstrated only in the presence ofDMSO with this cocktail. This effect could be explained by the factthat substrate concentrations in the UAM cocktail are close to orhigher than their Km values, whereas lower substrate concentrationsare used in the NM cocktail that has been primarily designed fordrug-drug interactions studies. No evidence of any kind of interactionor interference between the substrates has been observed in the latter(Turpeinen et al., 2005). Because testosterone is rapidly metabo-lized in HepaRG cells (Turpeinen et al., 2009), it is likely that theabsence of detectable 6ß-OH-testosterone metabolites with the NMcocktail was due to use of an inadequate concentration of testos-terone and the conjugation of all the metabolites formed during theincubation period. Two major differences were observed afterOME exposure: CYP1A2 induction was much higher using theUAM cocktail and CYP2C19 activity was increased using the NMcocktail and decreased using the UAM cocktail. In both cases, sucheffects could be attributed to the choice of different substrates. Inthe case of CYP1A2, phenacetin and melatonin were used in UAMand NM assays, respectively. Melatonin has been proven to be aless selective substrate (FDA-Guide, 2001) than phenacetin forCYP1A2 induction studies. Regarding CYP2C19, UAM and NMcocktails contained mephenytoin and omeprazole as a substrate,respectively. CYP2C19 inhibition has been described to occurwhen OME was used at high concentrations (Ko et al., 1997).Because OME is a substrate for CYP2C19, its potential intracel-lular accumulation during induction studies could inhibit mephe-nytoin metabolism by CYP2C19.

In support of previous observations (Aninat et al., 2006) using thetwo substrate cocktails, we showed that several P450s that werestrongly increased in the presence of DMSO recovered their basallevels after DMSO withdrawal. Activities of CYP3A4 and CYP2B6were the most sensitive to DMSO, and those of CYP2A6 and CYP2E1became undetectable in cells cultured for 3 days in a DMSO-freemedium, indicating that the DMSO-free conditions were not suitablefor their expression. The cause of the loss of CYP2E1 activity wasunclear. Its inhibition by insulin has been reported in a rat hepatomacell line (Moncion et al., 2002). However, medium depletion or lowerconcentrations of insulin did not prevent CYP2E1 activity loss inHepaRG cells (data not shown).

FIG. 6. Light microscopic appearance of HepaRG cells after DMSO withdrawaland activity of efflux transporters (BSEP/MDR1 and MRP2) in differentiatedHepaRG cells. LD-seeded HepaRG cells were cultured for 56 days and then used5 days after DMSO withdrawal (A). The cells were incubated with fluorescentsubstrates (CDFDA, fluorescent substrate of MRP2 and BODIPY FL vinblas-tine, fluorescent substrate of BSEP and MDR1) for 30 min at 37°C and thenobserved under phase-contrast and fluorescence microscopy (B). The picturesare representative of three independent experiments. BC, bile canaliculus.Original magnification, 150�.

522 ANTHERIEU ET AL.

A good correlation was observed between transcripts and corre-sponding activities. However, -fold inductions of mRNA levels weremuch higher, especially for CYP1A2 (800-fold for transcripts versus30-fold for activity with the UAM cocktail), CYP2B6 (50-fold fortranscripts versus 30-fold for activity with the UAM cocktail), andCYP3A4 (80-fold for transcripts versus 10-fold for activity with theUAM cocktail). As previously emphasized (Aninat et al., 2006),maintenance of HepaRG cells in conditions in which basal P450activities were low resulted in strong increased levels after treatmentwith prototypical inducers. -Fold inductions of P450 transcripts andactivities were much higher than in primary hepatocytes, especiallyfor CYP3A4, demonstrating that HepaRG cells were still highlyresponsive when treatments with inducers were started 3 days afterDMSO withdrawal.

Whereas basal expression and activity of P450s and responsivenessto prototypical inducers are variable in human hepatocyte populations(Morel et al., 1990; Madan et al., 2003), they were always found atcomparable levels in differentiated HepaRG cells when comparingdifferent passages. Several genes, including P450s, which have beenfound to be induced in vitro in only a fraction of human hepatocytepopulations (Goyak et al., 2008), were responsive in differentiated

HepaRG cells. Accordingly, important interdonor variation was foundfor basal CYP2B6 and CYP2C19 activities in primary human hepa-tocytes, and expression of CYP1A1, CYP2C8, CYP2C9, CYP2C19,and CYP3A4 was induced by PB in only HepaRG cells (Table 1).More recently, induction of CYP1A1/2 activities in HepaRG cells, butnot in primary human hepatocytes, was also reported after PB treat-ment (Turpeinen et al., 2009). A major difference between HepaRGcells and primary hepatocytes was the continuous expression ofCYP1A1 in the former; this activity could be related to their trans-formed state rather than to the presence of biliary cells. RIF is anotherwell established inducer, and our data (Table 1) reproduced well theprevious findings in primary hepatocytes (Morel et al., 1990; Lahoz etal., 2008b) and HepaRG cells (Aninat et al., 2006; Kanebratt andAndersson, 2008b). Therefore, HepaRG cells appear to be represen-tative of a large fraction of human hepatocyte populations.

Phase II enzymes and plasma transporters are also targets of mi-crosomal enzyme inducers. However only limited studies still exist onthe regulation of UGTs in human hepatocytes (Soars et al., 2004;Nishimura et al., 2008). We show that in addition to the major P450s,several phase II enzymes and transporters were modulated by PB,RIF, and/or OME. The three inducers increased both UGT1A1 and

FIG. 7. Effects of RIF, PB, and OME on expression ofCYP3A4 (A), CYP2B6 (B), and CYP1A2 (C), respectively,in HepaRG cells and human hepatocytes. Human hepato-cytes were used 1 to 4 days after seeding. HepaRG cellswere seeded at LD and cultured for 15 to 56 days or at HDand cultured for 5 to 28 days. Three days before induceraddition, they were cultured in DMSO-free and 2% FCSmedium and then exposed to three prototypical inducers (10�M RIF, 1 mM PB, or 50 �M OME) or their vehicles for48 h in DMSO-free and 2% FCS medium. Transcripts wereanalyzed using RT-qPCR assays. Results are the mean �S.D. of three independent experiments and are expressed aspercentage of mRNA levels measured in the vehicle (arbi-trarily set at 100%). The Kruskal-Wallis nonparametric testwas used to compare mRNA levels between different timepoints in HH and differentiated HepaRG cells (LD-D28 toLD-D56 and HD-D5 to HD-D28; ns, no significant differ-ence), and the Mann-Whitney U test was applied to com-pare mRNA levels between treated cultures with inducersand corresponding control samples (�, p � 0.05).

523FUNCTIONAL STABILITY OF P450 ENZYMES IN HepaRG CELLS

GSTA1/A2 transcripts in differentiated HepaRG cells; this result is inagreement with previous observations in primary hepatocytes. It hasbeen reported that PB is an inducer of GSTA1/2 (Morel et al., 1993)and that OME and RIF are inducers of UGT1A1 (Nishimura et al.,

2008). Note that the -fold inductions of these two genes with the threeinducers were much lower than those observed with P450s, rangingmostly between 1.5- and 5-fold, with the exception of UGT1A1induction by PB and OME that reached 7 to 10-fold (Table 1). Thelow mean increase of UGT1A1 (1.5-fold) and the absence of modu-lation of GSTA1/A2 in 3-day hepatocytes exposed to PB in thepresent study can be explained by interdonor variability in response tothis compound (Morel et al., 1993; Soars et al., 2004).

Various transporters were also analyzed at the transcriptional leveland found to be modulated in HepaRG cells (Table 1). MDR1 andMRP2 were increased by the three inducers, and BCRP was increasedby OME and PB. NTCP was inhibited mainly by OME, and BSEPwas inhibited by RIF and slightly by OME. Our results fully agreewith previous reports showing induction of MDR1 and MRP2 by PBin HepaRG cells (Le Vee et al., 2006; Lambert et al., 2009). Ourresults replicate data previously obtained with human hepatocytes,demonstrating consistent and wide interdonor variability in responseto identical inducers (Jigorel et al., 2006).

In summary, although HepaRG cells are originated from only onedonor and are the product of in vivo transformation, they exhibit adrug metabolism capacity, including responsiveness to chemical mod-ulators, which largely reflects that observed with the majority ofhuman hepatocyte populations in primary culture and, in addition,offer several unique advantages including the following: 1) the dataare reproducible during several passages; 2) the functional activitiesare well maintained for several weeks at confluence; and 3) the levelsof activities can be modulated by selecting appropriate culture con-ditions, especially the composition of the culture medium. Therefore,

FIG. 8. Effects of RIF, PB, and OME on activity of CYP3A4 (A), CYP2B6 (B), and CYP1A2 (C), respectively, in HepaRG cells. HepaRG cells were seeded at LD andcultured for 15 to 56 days or at HD and cultured for 5 to 28 days. Three days before inducer addition, they were cultured in DMSO-free and 2% FCS medium and thenexposed to three prototypical inducers (10 �M RIF, 1 mM PB, or 50 �M OME) or their vehicle for 72 h in DMSO-free and 2% FCS medium. P450 activities were thenanalyzed using the two cocktails of substrate assays (UAM and NM). Results are the mean � S.D. of three independent experiments and are expressed as percentage ofactivity levels measured in the vehicle (arbitrarily set at 100%). The Kruskal-Wallis nonparametric test was used to compare P450 activities between different time points(ns, no significant difference), and the Mann-Whitney U test was applied to compare P450 activities between treated cultures with inducers and corresponding controlsamples (�, p � 0.05).

TABLE 1

-Fold induction of phase I and phase II enzymes, transporters, and nuclearreceptors by RIF, PB, and OME in differentiated HepaRG cells

RIF PB OME

Transcripts1.5 � -Fold �10 CYP2B6 CYP1A1 CYP2C8

CYP2C8 CYP2A6 CYP2C19CYP2C19 CYP2C8

CYP2C9CYP2C19

GSTA1-A2 GSTA1-A2 GSTA1-A2UGTA1 UGTA1 UGTA1BCRP BCRP MDR1MDR1 MDR1 MRP2MRP2 MRP2

CAR10 � -Fold �50 CYP2B6

CYP3A4-Fold �50 CYP3A4 CYP2B6 CYP1A1

CYP3A4 CYP1A2Activities

1.5 � -Fold �10 CYP1A2 CYP1A2 CYP2B6CYP2B6 CYP2C8 CYP2C9CYP2C8 CYP2C9 CYP3A4CYP2C19 CYP2C19CYP2D6 CYP2D6

-Fold �10 CYP3A4 CYP2B6 CYP1A2CYP3A4

524 ANTHERIEU ET AL.

we conclude that HepaRG cells not only represent a promising sur-rogate to primary human hepatocytes for investigating induction ofxenobiotic-metabolizing enzymes and transporters and drug-drug in-teractions, but they also represent a unique metabolically competentcell model for in vitro chronic toxicity studies.

Acknowledgments. We thank Dr. David Steen for critical readingof the manuscript.

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Address correspondence to: Andre Guillouzo, INSERM U991, Faculte desSciences Pharmaceutiques et Biologiques, Universite de Rennes 1, 35043Rennes Cedex, France. E-mail: andre.guillouzo@univ-rennes1.fr

525FUNCTIONAL STABILITY OF P450 ENZYMES IN HepaRG CELLS