Toxicology in Vitro xxx (2012) xxx–xxx

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Toxicology in Vitro

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Co-cultures of enterocytes and hepatocytes for retinoid transport and metabolism

Carlotta Rossi a,1, Barbara Guantario a,1, Simonetta Ferruzza a, Christiane Guguen-Guillouzo b,Yula Sambuy a,⇑, Maria Laura Scarino a, Diana Bellovino a

a National Research Institute on Food and Nutrition (INRAN), Rome, Italyb INSERM UMR 991, Université de Rennes 1, Faculté de médecine, F-35043 Rennes Cedex, France

a r t i c l e i n f o

Article history:Available online xxxx

Keywords:b-caroteneRetinolCaco-2/TC7CYP26A1HepaRGRetinol Binding Protein 43A cells

0887-2333/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.tiv.2012.04.013

Abbreviations: AP, apical; BC, b-carotene; BCMO1ygenase; BL, basolateral; CM, chylomicrons; FBS, fetalhypoxanthine phosphoribosyltransferase; RA, retinoiprotein 4; ROH, retinol; REs, retinyl esters.⇑ Corresponding author. Address: National Resea

Nutrition (INRAN), via Ardeatina 546, 00178 Rome, Itfax: +39 06 51 494 550.

E-mail address: sambuy@inran.it (Y. Sambuy).1 These authors contributed equally to this work.

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a b s t r a c t

Dietary retinoid bioavailability involves the interplay of the intestine (transport and metabolism) and theliver (secondary metabolism). To reproduce these processes in vitro, differentiated human intestinalCaco-2/TC7 cells were co-cultured with two hepatocyte cell lines. Murine 3A cells and the more highlydifferentiated human HepaRG hepatocytes were both shown to respond to b-carotene (BC) and retinol(ROH) treatment by secreting Retinol Binding Protein 4 (RBP4). In co-culture experiments, Caco-2/TC7were differentiated on filter inserts and transferred for the time of the experiment to culture wells con-taining confluent 3A or differentiated HepaRG cells. Functionality of the co-cultures was assayed using asendpoints the retinol-dependent secretion of RBP4 and the retinoic acid-dependent induction of CYP26A1in hepatocytes. BC and ROH added to intestinal Caco-2/TC7 induced a reduction in intracellular RBP4 lev-els in the underlying hepatocytes and its secretion into the medium. HepaRG hepatocytes were alsoshown to up-regulate the expression of CYP26A1 mRNA in response to retinoid treatment. This in vitromodel represents a useful tool to analyze the absorption and metabolism of retinoids and could be fur-ther developed to investigate other dietary compounds and molecules of pharmacological interest.

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1. Introduction

Vitamin A (retinol, ROH) is an essential fat-soluble nutrient ob-tained from the diet as carotenoids from plant sources, or as retinylesters (REs) from foods of animal origin. Circulating levels of ROHare maintained within a narrow range, despite intake fluctuations,thus preventing risk of hyper- or hypo-dosage. Physiological con-centrations of vitamin A are maintained by the concerted actionof several specific binding proteins, which contribute to vitaminA transport and metabolism (Blaner, 1994; D’Ambrosio et al.,2011). ROH is stored in the liver as lipid droplets, and mobilizedand secreted bound to its specific carrier protein, Retinol BindingProtein 4 (RBP4). RPB4 is abundantly synthesized by hepatocytesand is secreted only in the retinol-bound form (molar ratio of1:1) (Soprano and Blaner, 1994). The ‘‘apo’’ form of this protein isretained in the hepatocyte when ROH levels are low, and its secre-tion is promptly restored upon ROH repletion, as shown in vivo and

ll rights reserved.

, b-carotene 15,150-monoox-bovine serum; HPRT, human

c acid; RBP4, retinol binding

rch Institute on Food andaly. Tel.: +39 06 51 494 497;

Co-cultures of enterocytes and

in vitro (Bellovino et al., 1999, 2003; Kato et al., 1984). The mainrole of RBP4 is the transport of ROH from its storage site to the var-ious vitamin A-dependent tissues. Here ROH is oxidized by specificintracellular enzymes to the active form, retinoic acid (RA), whosepleiotropic effects are transduced by two families of nuclear recep-tors, RARs and RXRs. RA plays a key role in modulating the prolif-eration and differentiation of many cell types, as well as inembryonic development and fetal morphogenesis. IntracellularRA concentration needs to be strictly controlled by the concertedaction of specific binding proteins and enzymes involved in bothRA synthesis (retinal dehydrogenase and retinal oxidases) and deg-radation (CYP26 family) according to cellular requirements (Rossand Zolfaghari, 2004, 2011).

In vitro cell culture models proved to be extremely useful to un-ravel the molecular mechanisms of transport, metabolism and tox-icity of nutritionally or pharmacologically relevant molecules.Among their limitations, however, is the need to cultivate singlecell types isolated from other cells that are in constant close phys-iological interplay in vivo. In an attempt to overcome these limita-tions, several interesting co-culture models have recently beenreported, combining intestinal cells with other cells of differentorigin, such as neural, hepatic, pancreatic or monocytic cells. Thesemodels better reproduce the tissue cross talks occurring in vivo(Castell-Auvi et al., 2010a,b; Choi et al., 2004; Lau et al., 2004; Sat-su et al., 2003; Tyrer et al., 2011). Differentiated small intestinal

hepatocytes for retinoid transport and metabolism. Toxicol. in Vitro (2012),

2 C. Rossi et al. / Toxicology in Vitro xxx (2012) xxx–xxx

enterocytes are responsible for transport and first metabolism ofingested molecules. After crossing the intestinal barrier, themetabolites come in contact with local cytotypes such as intra-epi-thelial lymphocytes, monocytes, fibroblasts and enteric neuronalcells, or are transported to the liver and to other organs via the he-patic portal circulation and the lymphatic system. To reproducesuch complex interplay in vitro, we used as intestinal cell model,the human adenocarcinoma Caco-2 cell line that spontaneouslydifferentiates in vitro, expressing several morphological and func-tional characteristics of mature small intestinal enterocytes (Delieand Rubas, 1997; Sambuy et al., 2005). The Caco-2 cell line hasbeen shown to transport and partially metabolize carotenoidsand ROH, and to package them into chylomicrons (CM) (Duringet al., 2005, 2002; During and Harrison, 2007; O’Sullivan et al.,2007). Expression and activity of b-carotene-15,150-monooxygen-ase (BCMO1), one of the enzymes involved in conversion of b-car-otene (BC) to ROH, was detected in the Caco-2/TC7 cell line atlevels higher than in the parental Caco-2 cell line (During et al.,1998a; During et al., 2001). On the hepatic side we used two differ-ent cell models, the murine 3A cells (Guantario et al., 2012) and thehuman HepaRG cells, which are able to undergo a complete hepa-tocyte differentiation program (Gripon et al., 2002). The spontane-ously immortalized 3A cell line, obtained from mouse embryo,expresses several liver key features essential for a functional hepa-tocyte model, including the regulation of RBP4 secretion in re-sponse to vitamin A deficiency (Guantario et al., 2012).Differentiated human HepaRG cells express most of the specific li-ver functions at levels close to those found in primary humanhepatocytes, including detoxifying enzymes and drug transporters(Aninat et al., 2006; Le Vee et al., 2006; Lubberstedt et al., 2011),although retinoid metabolism has never been studied in this cellline. After verifying that the two hepatic cell lines responded toROH and BC treatment by activating RBP4 secretion, they wereco-cultured with intestinal Caco-2/TC7 cells, and their responseto retinoids added to the AP side of intestinal cells was investi-gated. As an additional endpoint of retinoid treatment, expressionof CYP26A1 mRNA was investigated in HepaRG hepatocytes bothalone and in co-culture.

2. Materials and methods

2.1. Cell lines maintenance and differentiation

The human intestinal Caco-2/TC7 cell line (Caro et al., 1995)was kindly provided by Dr. Monique Rousset (Institute Nationalde la Santé et de la Recherche Médicale, INSERM, France). Caco-2/TC7 is a clonal line derived from parental Caco-2 cells at late pas-sage, and has been reported to express higher metabolic and trans-port activities than the line of origin, including the BC metabolizingenzyme BCMO1 (Caro et al., 1995; During et al., 1998b). Cells werecultured according to the Low Density (LD) protocol (Natoli et al.,2011, 2012). Briefly, cells were routinely sub-cultured twice aweek when reaching a density of 5.4 � 103 cells/cm2 correspond-ing to approximately 50% of confluence. The line was sub-culturedat LD at least 10 times before preparing a stock of cells betweenpassages 100 and 105, that was stored in liquid nitrogen. Cellswere thawed-out from frozen stock and experiments were per-formed within four passages from thawing. The Caco-2/TC7 cellline was maintained at 37 �C in a 10% CO2/air atmosphere in Dul-becco’s modified Eagle’s medium containing 25 mM glucose,3.7 g/l NaHCO3, 4 mM L-glutamine, 1% non essential amino acids,100 IU/ml penicillin and 100 lg/ml streptomycin (complete Caco-2 medium) supplemented with 10% heat-inactivated fetal bovineserum (FBS–Hyclone FBS, Fisher Scientific, France). Cells wereseeded for differentiation on polycarbonate filters (Transwell� in-serts, 4.7 cm2 area, 0.4 lm pore diameter; Corning Inc. Lowell,

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MA, USA) and maintained for 2 days in complete medium supple-mented with 10% FBS in both apical (AP) and basolateral (BL) com-partments. On day 3 the medium was changed to complete Caco-2medium in both compartments, supplemented with 10% FBS onlyin the BL compartment and regularly changed three times a weekthereafter (Ferruzza et al., 2012). Differentiated Caco-2/TC7 cellswere used for co-culture experiments after 21 days from seeding.The murine 3A cell line was derived from liver cultures of 14.5 dayspost coitum wild type mice embryos (Guantario et al., 2012). 3Acells were grown in RPMI 1640 supplemented with 10% FBS (FBSEU category; EuroClone, Milan, Italy), 50 ng/ml EGF, 30 ng/ml IGFII (Millipore, Italy), 10 lg/ml insulin, 4 mM L-glutamine and100 IU/ml penicillin and 100 lg/l streptomycin. Cells were platedon collagen I-coated culture dishes at 37 �C in 5% CO2. Human hep-atoma HepaRG cells were maintained and differentiated as previ-ously described (Gripon et al., 2002). Briefly, human HepaRGcells were seeded at a density of 2.6 � 104 cells/cm2 in Williams’E medium supplemented with 10% FBS (EuroClone), 100 IU/mlpenicillin, 100 lg/ml streptomycin, 5 lg/ml insulin, 2 mM gluta-mine, and 50 lM hydrocortisone hemisuccinate. After 2 weeks,during which they reached confluence, HepaRG cells were trans-ferred to the same medium supplemented with 2% dimethylsulfox-ide (HepaRG differentiation medium) for a further 2 weeks, toobtain confluent differentiated cultures containing both hepato-cyte-like and biliary-like cells (in approximately equal amounts).To further enrich the culture, the hepatocyte colonies were selec-tively detached using 0.025% trypsin–2.65 mM EDTA in PBS, leav-ing most of the biliary-like cells behind, and were seeded at highdensity (150,000/cm2) in differentiation medium in 6 wells plates(Corning Inc.) to be used for the experiments (Pernelle et al.,2011). After 5–7 days, experiments were carried out on thesehighly enriched hepatocyte-like cultures. For co-culture experi-ments, Caco-2/TC7 cells differentiated on filter inserts were trans-ferred to culture plates containing confluent 3A cells ordifferentiated HepaRG cells, as shown in Fig. 1. Retinoids wereadded to the AP compartment of Caco-2 cells and the culture plateswere incubated at 37 �C for 24 h. All chemicals were from SigmaAldrich, Italy, unless otherwise stated.

2.2. Carotenoids treatment and micellar preparations

To induce vitamin A deficiency in hepatocytes, both 3A and He-paRG cells were maintained prior to the experiments for 24 h inRPMI 1640 or Williams’ E medium respectively, supplementedwith 10% delipidized FBS (Lonza, Basel, Switzerland). For hepato-cytes treatment, ROH was prepared in 100% ethanol and BC in100% chloroform, the solvent was evaporated and the residue dis-solved in 0.1% Tween 40 in acetone; the solvent was evaporatedagain and the residue re-suspended in the appropriate hepatocytemedium. To reduce photodecomposition and oxidation of the reti-noids, all solutions were prepared in dark tubes.

For treatment of Caco-2/TC7 cells in co-culture, stock solutionsof BC and ROH were prepared in chloroform or ethanol, respec-tively. Appropriate volumes of stock solution were transferred toglass tubes to give the required concentration, solvent was evapo-rated and the residue dissolved in 0.1% Tween 40 in acetone. Thesolvent was evaporated again and the residue dissolved in com-plete Caco-2 medium. Where stated, BC and ROH were deliveredto Caco-2/TC7 cells in mixed micelles prepared as described by(During et al., 1998a) and modified by (Garrett et al., 1999). Com-plex lipid emulsion containing 0.6 mM oleic acid, 0.2 mM lyso-phosphatidylcholine, 0.05 mM cholesterol, 0.2 mM 2-mono-oleylglycerol and 2 mM taurocholic acid was prepared in chloro-form/methanol (3:1 v/v), the solvent evaporated under N2 andthe lipids re-suspended in complete Caco-2 medium containingthe appropriate concentration of ROH or BC.

hepatocytes for retinoid transport and metabolism. Toxicol. in Vitro (2012),

Fig. 1. Schematic representation of the co-culture model. Caco-2/TC7 cells were grown and differentiated on polycarbonate filter membranes for 21 days. Confluent murine3A cells or differentiated human HepaRG hepatocytes were seeded in 6-wells culture plates in their specific differentiation medium. For co-culture experiments, filter insertswith differentiated Caco-2/TC7 cells were transferred to wells containing hepatic cell cultures and treated with retinoids for 24 h.

C. Rossi et al. / Toxicology in Vitro xxx (2012) xxx–xxx 3

2.3. Trans-epithelial electrical resistance (TEER) measurement

TEER of differentiated Caco-2/TC7 cells was measured in culturemedium at 37 �C using a commercial apparatus (Millicell ERS; Mil-lipore C., Bedford, MA) as previously described (Ferruzza et al.,1995), to monitor the integrity of the cell monolayer before andafter each experiment.

2.4. Western blotting and immunoprecipitation

Cells were harvested in cold radioimmunoprotein assay buffer(RIPA: 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Nadeoxycholate, 1% Triton X-100) supplemented with Protease Inhib-itor Cocktail (Roche, Milan Italy). Culture media were subjected toimmunoprecipitation with anti-RBP4 antibody, followed by incu-bation with 30 ll Protein A-Sepharose (Roche) for 1 h. Cell lysatesor immunoprecipitates were dissolved in sample buffer (50 mMTris–HCl, pH 6.8, 2% SDS, 10% glycerol, 100 mg/ml bromophenolblue, 10 mM b-mercaptoethanol), heated for 5 min, fractionatedby SDS polyacrylamide gel electrophoresis and transferred to nitro-cellulose filters (Whatman Protran, PerkinElmer, Boston, USA).Membranes were incubated with rabbit polyclonal anti-mouseRBP4 (Alexis Biochemicals-ENZO Life Sciences Inc.), anti-humanRBP4 (Dako Italia, Milan, Italy) or mouse monoclonal anti-tubulin(LLC; MP Biomedicals) antibodies. Proteins were detected withhorseradish peroxidase-conjugated secondary antibodies (GEHealth Care, Italy) and enhanced chemiluminescence reagent(ECL-GE Health Care), followed by analysis of chemiluminescencewith the CCD camera detection system Las4000 Image Quant (GEHealth Care).

2.5. Real time qRT–PCR

Total RNA was isolated from HepaRG cells using silica columns(Nucleo Spin RNA II, Macherey–Nagel, Düren, Germany). RNA(500 ng) was retro-transcribed with a M-MLV Kit (Invitrogen, Mi-lan, Italy) and the cDNA amplified with iTaq Fast SYBR

�Green

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Supermix (Biorad, Milan, Italy). Real time qRT–PCR was performedusing Applied Biosystem 7500 Fast thermal cycler. Specific primerswere designed with the Primer3 software: human CYP26A1 for-ward primer 50-TCCAAAATTTCCATGTCCAA-30, reverse primer 50-CATGTTCTCCAGAAAGTGCG-30; human hypoxanthine phosphori-bosyltransferase 1 (HPRT1) forward primer 50-ACCCTTTCCAA ATCCTCAGC-30, reverse primer 50-GTTATGGCGAC CCGCAG-30. Expres-sion data were normalized using Ct values of the internal controlHPRT1. Fold differences were calculated using the DDct method.

3. Results

To investigate retinoid transport and metabolism in the intes-tine and in the liver, a co-culture in vitro model was set up usinghuman intestinal cells (Caco-2/TC7) and hepatocytes (murine 3Aor human HepaRG) separately cultured and differentiated, andsubsequently combined in the same culture vessel for the 24 h ofthe experiment. During this time, the intestinal cells were treatedwith ROH or BC in the AP medium and RBP4 secretion andCYP26A1 mRNA induction were monitored in the hepatocytes asendpoints of retinoid transport and metabolism. The model isgraphically shown in Fig. 1.

RBP4 secretion from hepatocytes is strictly modulated by theamount of vitamin A availability in vivo. Such ligand-dependentregulation is maintained in murine 3A cells, as shown in Fig. 2.Treatment of confluent 3A cells with delipidized serum for 24 h(retinoid deprivation) led to intracellular RBP4 accumulation,resulting from specific inhibition of secretion. Treatment of reti-noid-deprived 3A cells with 5 lM ROH or 15 lM BC for 24 h re-sulted in reduction of intracellular RBP4, as shown by westernblot analysis of cell lysates (Fig. 2A). ROH treatment induced al-most complete depletion of intracellular RBP4, while about 40%of RBP4 was still retained inside the cells following BC treatment.Immunoprecipitation of RBP4 from the culture medium confirmedthe increased secretion following ROH treatment, while in the caseof BC such increase was not so evident (Fig. 2B).

hepatocytes for retinoid transport and metabolism. Toxicol. in Vitro (2012),

Fig. 2. BC and ROH treatment induce secretion of RBP4 in 3A cells. Confluentmurine hepatocytes (3A) were pre-incubated in delipidized serum for 24 h toinduce retinoid depletion and incubated with ROH (5 lM) or BC (15 lM) for further24 h. RBP4 was detected by western blot analysis in whole cell lysates (A) or inmedium immunoprecipitated with specific RBP4 antibody (B), subjected to 4–20%and 13% SDS–PAGE respectively. Histogram in A shows the ratio of intracellularRBP4 to tubulin levels expressed as a percentage of retinoid-deprived control cells.Data are the means ± SD of a representative experiment performed in triplicate. Arepresentative blot of immunoprecipitated RBP4 from culture medium is shown inB.

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In the co-culture system BC (25 and 15 lM) and ROH (10 and5 lM) were added to the AP medium of differentiated Caco-2/TC7 with or without mixed lipid micelles for 24 h. To monitorthe integrity of Caco-2/TC7 cell monolayer during co-culture andretinoid treatment, TEER was measured before and after the exper-iment. As shown in Fig. 3A, TEER values of Caco-2/TC7 cells werenot affected by the co-culture conditions, nor by the differenttreatments.

RBP4 secretion from hepatocytes was used as an endpoint ofretinoid transport and metabolism across intestinal cell monolayerand hepatocytes. Treatment of Caco-2/TC7 cells with ROH and BCfrom the AP side induced, at all tested concentrations, RBP4 deple-tion of intracellular RBP4 levels of 3A cells in the BL compartment(Fig. 3B). Addition of mixed lipid micelles to favor delivery of reti-noids to Caco-2/TC7 cells, did not significantly affect the responseof the underlying hepatocytes (Fig. 3B). For this reason, in the fol-lowing co-culture experiments, BC and ROH were prepared inTween 40, without addition of lipid micelles, as described inSection 2.

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To test the co-culture system with more highly differentiatedhepatocytes of human origin, the experiments were performedusing human HepaRG hepatocytes beneath the Caco-2/TC7 cellmonolayer. To verify first if HepaRG differentiated hepatocytesexhibited regulated RBP4 secretion, they were incubated with dif-ferent concentrations of ROH (2.5, 5, 10 lM) or BC (1.8, 3.7, 7.5, 15,30 lM) for 24 h. RBP4 secretion appeared to be stimulated by ROHat all tested concentrations, as shown by a decrease of intracellular(Fig. 4A) and an increase of secreted RBP4 (Fig. 4B). Treatment withBC (between 1.8 and 15 lM) also resulted in reduced intracellularRBP4 protein levels (Fig. 4C), as confirmed by the amounts of RBP4immunoprecipitated from the culture medium (Fig. 4D). Treatmentwith 30 lM BC produced very variable and non-significant reduc-tion in intracellular RBP4 (Fig. 4C).

Having established that HepaRG hepatocytes retained vitaminA-dependent regulation of RBP4 release, they were co-culturedwith intestinal cells. Differentiated Caco-2/TC7 cells were grownin co-culture with differentiated HepaRG hepatocytes and incu-bated with different concentrations of BC (15, 30, 60 lM) andROH (5, 10 lM) for 24 h. Caco-2/HepaRG co-culture conditionsand retinoid treatment did not affect the permeability of the cellmonolayer, as shown by TEER measurement, as previously ob-served with 3A cells (Fig. 5A). Treatment of Caco-2/TC7 cells withROH and BC produced, in co-cultured HepaRG cells, similar reduc-tion (about 50%) in intracellular RBP4 levels at all tested concentra-tions (Fig. 5B) and variable increase in secreted RBP4immunoprecipitated from the culture medium (Fig. 5C).

As an additional endpoint to test the intestinal-hepatocyte co-culture model, we investigated the expression of CP26A1 mRNA,which is strongly induced by retinoic acid (RA) in hepatocytes(Ross and Zolfaghari, 2011). The expression of CYP26A1 mRNA byqRT–PCR in HepaRG hepatocytes incubated with different concen-trations of RA, ROH and BC for 6 h is shown in Fig. 6. Since HepaRGcells exhibited undetectable CYP26A1 basal levels (without addi-tion of vitamin A), induction of CYP26A1 expression following ret-inoid treatment was calculated with respect to the lower ROHconcentration employed (1 lM). Treatment with 0.1 and 1 lM RAstrongly induced CYP26A1 mRNA expression by 9- and 13-folds,respectively. Treatment with 3 lM ROH induced the expressionof CYP26A1 by 3-folds, while the increase after 5 lM BC treatmentwas only 2-folds. In co-culture, treatment of Caco-2/TC7 cells with5 and 10 lM ROH for 8 h resulted in low induction of CYP26A1expression in the underlying HepaRG cells, while BC (15, 30 and60 lM) treatment led to undetectable levels in the hepatocytes(data not shown).

4. Discussion

Dietary retinoids are absorbed by mucosal enterocytes, metab-olized and packaged into CM, released to lymph and transported tothe liver and to other tissues within lipoproteins (Harrison, 2005).Once taken up by the liver cells, ROH may be esterified and storedas REs, mostly in stellate cells, bioactivated in a two step process toform RA, or bound to its specific carrier RBP4 and secreted intoplasma as holo-RBP (Ross and Zolfaghari, 2004). This secretory stepis strictly regulated by ligand availability: intracellular RBP4 con-centration in hepatocytes was in fact shown to be exclusively mod-ulated by its rate of secretion, and not by changing rate oftranscription and/or translation, both in vivo and in vitro (Bellovinoet al., 1999; Perozzi et al., 1991; Soprano and Blaner, 1994). ROH-dependent RBP4 secretion was therefore chosen as an endpoint totest the performance of the intestine–liver co-culture system heredescribed.

We show that 3A hepatocyte-like cells activate secretion ofRBP4 not only in response to ROH, as previously reported (Guan-tario et al., 2012), but also after treatment with BC, indicating that

hepatocytes for retinoid transport and metabolism. Toxicol. in Vitro (2012),

Fig. 3. Retinoids supplied to Caco-2/TC7 cells modulate intracellular RBP4 levels in co-cultured 3A cells. Caco-2/TC7 cells were differentiated on polycarbonate filters for21 days and placed in co-culture for 24 h with confluent murine 3A cells that had been previously depleted of retinoids. ROH (5 and 10 lM) or BC (15 and 25 lM) weredelivered to Caco-2/TC7 AP medium with or without mixed lipid micelles. Transepithelial electrical resistance (TEER) was measured before and after 24 h co-culture (A). RBP4was detected in whole cell lysates subjected to 4–20% SDS–PAGE followed by western blotting (B). Histogram in B shows the ratio of intracellular RBP4 to tubulin levelsexpressed as a percentage of cells in co-culture with untreated Caco-2/TC7 cells. Data are the means ± SD of a representative experiment performed in triplicate.

C. Rossi et al. / Toxicology in Vitro xxx (2012) xxx–xxx 5

they express enzyme activities able to convert BC to ROH. How-ever, murine 3A cells, initially employed to test the co-culturemodel with intestinal Caco-2 cells due to their known efficientROH-dependent RBP4 secretion, are not the best available heapto-cyte cell line. The use of human well differentiated HepaRG hepa-tocytes, allowed us to develop a homologous co-culture modelwith human intestinal Caco-2/TC7 cells. ROH and BC supplied inthe culture medium of 3A and HepaRG cells were shown to reduceintracellular RBP4 levels, although with different efficiency be-tween the two molecular species and in the two cell lines. The re-sponse to direct ROH supplementation was found to be stronger in3A cells than in HepaRG hepatocytes, an effect that could be attrib-uted to intrinsic differences between the two models, but may alsoreflect physiological differences related to the species of origin(Tourniaire et al., 2009). BC treatment of HepaRG hepatocytes pro-

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duced a significant reduction in intracellular RBP4 levels in a rangeof concentrations between 1.8 and 15 lM, indicating that BCMO1is active in these cells. This key enzyme in retinoid metabolismwas shown to be expressed, although at levels significantly lowerthan in vivo, in some cell lines such as intestinal Caco-2/TC7 cells(During et al., 1998b), 3T3 adipocytes (Lobo et al., 2010), and stel-late cells (Shmarakov et al., 2010), but its activity has to date neverbeen described in hepatic cell lines, including HepG2 cells (Duringet al., 1998b). BCMO1 expression will be further investigated in 3Aand HepaRG cell to better characterize their response tocarotenoids.

In addition to the response to ROH and BC in terms of regulatedRBP4 secretion, HepaRG have also been shown to induce CYP26A1mRNA expression after treatment with RA. CYP26A1 is a memberof the cytochrome P450 (CYP) superfamily involved in the homeo-

hepatocytes for retinoid transport and metabolism. Toxicol. in Vitro (2012),

Fig. 4. In differentiated HepaRG cells intracellular RBP4 level is regulated by BC andROH treatment. Differentiated HepaRG hepatocytes were pre-incubated in delip-idized serum for 24 h to induce retinoid depletion and incubated with ROH (10, 5and 2.5 lM) (A and B) or BC (30, 15, 7.5, 3.7, 1.8 lM) (C and D). RBP4 was detectedby western blot analysis in whole cell lysates (A and C) or in medium immuno-precipitated with specific RBP4 antibody (B and D), subjected to 4–20% and 13%SDS–PAGE respectively. Histogram in A shows the ratio of intracellular RBP4 totubulin levels expressed as a percentage of retinoid-deprived control cells. Data arethe means ± SD of a representative experiment performed in triplicate. Represen-tative blots of immunoprecipitated RBP4 from culture medium are shown in B andD.

6 C. Rossi et al. / Toxicology in Vitro xxx (2012) xxx–xxx

stasis of intracellular RA levels (Ross and Zolfaghari, 2011). Thisoxidative enzyme is abundantly expressed in the liver and is highlyregulated by vitamin A; liver CYP26A1 mRNA is present at low lev-els in vitamin A-adequate state, whereas in conditions of vitaminA-deficiency, mRNA amounts fall to nearly undetectable levels(Ross and Zolfaghari, 2011). Cell lines of different origin expressingCYP26A1 can be divided in two groups depending on CYP26A1mRNA basal levels and their response to RA treatment: hepatomaHepG2 and breast cancer MCF-7 cells display low basal levels ofCYP26A1 but exhibit a strong response to ROH or RA, while others,

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such as human embryonic kidney HEK293T and non-small celllung carcinomas SK-LC6 lines, express CYP26A1 mRNA at higherbasal levels but exhibit weaker or no regulation by RA (reportedin Ross and Zolfaghari, 2011). HepaRG displayed very low basallevels and strong responsiveness to RA, placing this cell line inthe first group. CYP26A1 activation in HepaRG was also promotedby treatment with ROH and to a lesser extent by BC, indicatingintracellular metabolism to RA, the principal inducer of this gene(Ross and Zolfaghari, 2011).

Human intestinal Caco-2/TC7 cells undergo spontaneous differ-entiation into a continuous monolayer of cells coupled by func-tional tight junctions, that prevent the non-specific leakage ofmolecules between the AP and BL compartments. TEER measureis a rapid and efficient method to monitor the integrity of the cellmonolayer, which is essential during transport experiments acrossCaco-2 cell monolayers (Press and Di Grandi, 2008). TEER values ofCaco-2/TC7 in co-culture with either 3A or HepaRG cells resultedunaffected, even after treatment with different concentrations ofretinoids. Thus, these cells can be maintained in co-culture for24 h without perturbations of Caco-2/TC7 barrier function thatwould lead to non-specific passage of retinoids across the cellmonolayer. Both ROH and BC treatment of Caco-2/TC7 in co-cul-ture produced a decrease of intracellular RBP4 levels, both in 3Aand in HepaRG cells. These results indicate that ROH, directly orafter esterification to REs, was transported across Caco-2 cellsand delivered to the hepatocytes as ROH to induce RBP4 secretion.BC treatment of Caco-2 cells produced results similar to those ob-tained with ROH in terms of reduction of intracellualr RBP4 in co-cultured hepatocytes, but at higher concentrations. This indicatesthat BC was transported across Caco-2 cells and delivered to theunderlying hepatocytes within CM or metabolized to ROH. The ex-tent of BC metabolism by Caco-2 cells will require further investi-gation. However, the response measured in both hepatic cell linesindicate functional interaction between intestinal and liver cells inthis co-culture model.

It has previously been reported that transport and metabolismof retinoids in Caco-2 cells is facilitated by delivering them inmixed lipid micelles (During et al., 2002; Yonekura and Nagao,2007); we did not however, observe any improvement in RBP4secretion when retinoids were added to the intestinal cells inmixed lipid micelles, compared to delivery with a polysorbateemulsifier (Tween 40). It is possible that further optimization ofthe micellar preparation and/or lipid composition may improvethe performance of the co-culture system by facilitating retinoiddelivery and metabolism, and promoting CM formation and secre-tion from Caco-2 cells. The choice of the intestinal cells may also beimportant in this co-culture model given the heterogeneity ofCaco-2 cell lines of different origin (Sambuy et al., 2005). ParentalCaco-2 cells were in fact reported to be more efficient than Caco-2/TC7 in terms of CM assembly and BC transport (During et al.,2002); on the other hand, Caco-2/TC7 were shown to express high-er levels of BCMO1 than the parental cell line (During et al., 1998b).In the present work, the choice of Caco-2/TC7 cells was made to fa-vor BC conversion to ROH by BCMO1 activity.

Expression of CYP26A1 mRNA in co-cultured HepaRG was alsoinvestigated after treatment of Caco-2/TC7 cells with ROH andBC. However, given the low levels of induction obtained by directtreatment of HepaRG cells with ROH and BC, activation of CYP26A1expression could not be detected under co-culture conditions, sug-gesting that the concentrations of BC and ROH reaching the hepa-tocytes from intestinal cells were within the physiological range,not requiring CYP26A induction.

Human intestinal Caco-2 cells are still the best available modelfor studies of bioavailability of drugs and toxic compounds, andtheir permeability has been extensively characterized with respectto human enterocytes in vivo and to human pharmacokinetic mod-

hepatocytes for retinoid transport and metabolism. Toxicol. in Vitro (2012),

Fig. 5. Retinoids supplied to Caco-2/TC7 cells modulate intracellular RBP4 in co-cultured HepaRG hepatocytes. Caco-2/TC7 cells were differentiated on polycarbonate filtersfor 21 days and placed in co-culture with HepaRG hepatocytes that had been previously depleted of retinoids. Caco-2/TC7 cells in co-culture were treated from the AP sidewith ROH (10 and 5 lM) or BC (60, 30 and 15 lM) for 24 h. Transepithelial electrical resistance (TEER) was measured before and after 24 h co-culture (A). RBP4 was detectedin whole cell lysates subjected to 4–20% SDS–PAGE followed by western blotting (B) or in medium immunoprecipitated with specific RBP4 antibody, subjected to 13% SDS–PAGE and detected by western blotting (C). Histogram in B shows the ratio of intracellular RBP4 to tubulin levels expressed as a percentage of cells in co-culture withuntreated Caco-2/TC7 cells. Data are the means ± SD of a representative experiment performed in triplicate. A representative blot of immunoprecipitated RBP4 from theculture medium is shown in C.

C. Rossi et al. / Toxicology in Vitro xxx (2012) xxx–xxx 7

els (Press and Di Grandi, 2008). For in vitro toxicity studies, com-bining intestinal Caco-2 cells with well differentiated and meta-bolic competent hepatocytes in co-culture may represent aninteresting approach to evaluate the importance of intestinal bio-availability in the assessment of hepatotoxicity. Other examplesof co-cultures of Caco-2 cells with hepatoma HepG2 cells have re-cently been reported (Castell-Auvi et al., 2010a; Choi et al., 2004;

Please cite this article in press as: Rossi, C., et al. Co-cultures of enterocytes andhttp://dx.doi.org/10.1016/j.tiv.2012.04.013

Lau et al., 2004). However, the use of a poorly differentiated hepa-tic cell line such as HepG2 limits the metabolic potential of themodel. The use of a well differentiated human hepatocyte modelsuch as HepaRG cells provides added-value to the co-culture sys-tem due to the high levels of expression of metabolic and transportactivities, resembling freshly isolated human hepatocytes (Gugu-en-Guillouzo and Guillouzo, 2010). Limitations of the model in-

hepatocytes for retinoid transport and metabolism. Toxicol. in Vitro (2012),

Fig. 6. CYP26A1 expression in HepaRG hepatocytes is regulated by retinoids.Differentiated HepaRG hepatocytes were incubated with ROH (1 and 3 lM), RA (0.1and 1 lM), or BC (5 lM) for 6 h. Results of qRT–PCR for CYP26A1 mRNA are shown.Since control cells did not express CYP26A1, fold changes were calculated withrespect to cells treated with 1 lM ROH. Data are means ± SD of a representativeexperiment performed in triplicate.

8 C. Rossi et al. / Toxicology in Vitro xxx (2012) xxx–xxx

clude the reduced metabolic activity of Caco-2 cells compared toenterocytes in vivo and the absence of bloodborne factors (i.e.transport proteins) that may participate in tissue cross-talks.

In conclusion, we have shown that an homologous human co-culture system of intestinal and hepatic cells can be used for atleast 24 h, allowing detection of endpoints relevant to transportand metabolism of retinoids. The model could be applied to thestudy of other molecules of nutritional, toxicological or pharmaco-logical interest, provided kinetic studies are performed addressingtransport and metabolism in the two cell types of the moleculesunder investigation.

Conflict of interest statement

The authors have declared that there is no conflict of interest.

Acknowledgements

Work supported by EU FP6 Project ‘‘LIINTOP’’ Contract numberSTREP-037499 and by grant ‘‘NUME’’ (DM3888/7303/08) from theItalian Ministry of Agriculture, Food & Forestry (MiPAAF).

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