Chemico-Biological Interactions 168 (2007) 66–73

The human hepatoma HepaRG cells: A highly differentiated model

for studies of liver metabolism and toxicity of xenobiotics
André Guillouzo a,∗ , Anne Corlu b , Caroline Aninat a , Denise Glaise b ,
Fabrice Morel a , Christiane Guguen-Guillouzo b
a INSERM U620, IFR 140, Université de Rennes1, Rennes, France
b INSERM U522, IFR 140, Université de Rennes1, Rennes, France
Available online 16 December 2006

Abstract
Although they have several important limitations primary human hepatocytes still represent the in vitro gold standard model for
xenobiotic metabolism and toxicity studies. The large use of human liver cell lines either from tumoral origin or obtained by oncogenic
immortalisation is prevented by the loss of various liver-specific functions, especially many cytochrome P450 (CYP)-related enzyme
activities. We review here recent results obtained with a new human hepatoma cell line, named HepaRG, derived from a human
hepatocellular carcinoma. These cells exhibit unique features: when seeded at low density they acquire an elongated undifferentiated
morphology, actively divided and after having reached confluency formed typical hepatocyte-like colonies surrounded by biliary
epithelial-like cells. Moreover contrary to other human hepatoma cell lines including HepG2 cells, HepaRG cells express various
CYPs (CYP1A2, 2B6, 2C9, 2E1, 3A4) and the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor
(PXR) at levels comparable to those found in cultured primary human hepatocytes. They also express various other functions such
phase 2 enzymes, apical and canalicular ABC transporters and basolateral solute carrier transporters, albumin, haptoglobin as well
as aldolase B that is a specific marker of adult hepatocytes. HepaRG cells could represent a surrogate to primary human hepatocytes
for xenobiotic metabolism and toxicity studies and even more, a unique model system for analysing genotoxic compounds.
© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Human hepatoma cells; HepaRG cells; Liver-specific functions; Cytochromes P450; Constitutive androstane receptor; Hepatotoxicity

1. Introduction

During the 1975–1999 period 6 out of 16 drugs with-

Abbreviations: AhR, aryl hydrocarbon receptor; BSEP, bile
salt export pump; CAR, constitutive androstane receptor; CYP,
cytochrome P450; DMSO, dimethylsulfoxide; GST, glutathione
transferase; MRP, multidrug-resistance associated protein; NTCP,
Na+ -taurocholate co-transporting polypeptide; OATP, organic anion
transporting polypeptide; OCT, organic cation transporter; PPAR, per-
oxisome proliferator-activated receptor; PXR, pregnane X receptor;
UGT, UDP glucuronyl transferase
∗ Corresponding author. Tel.: +33 2 23 23 47 91;
fax: +33 2 23 23 47 94.
E-mail address: andre.guillouzo@univ-rennes1.fr (A. Guillouzo).
drawn from the market were for their hepatic toxicity [1].
This may be explained at least in part because human
hepatotoxicity has a poor correlation with correspond-
ing animal toxicity, possibly due to several reasons. In
particular, the rates and routes of xenobiotic metabolism
are frequently different between species and in addition
interindividual variations are observed in humans due to
either genetic, physiopathological and/or environmental
factors.
In vitro liver preparations are frequently used as sur-
rogate models to hepatotoxicity in vivo; they include

0009-2797/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2006.12.003
 A. Guillouzo et al. / Chemico-Biological Interactions 168 (2007) 66–73
both cellular (tissue slices, isolated and cultured hep-
atocytes, liver cell lines) and subcellular (microsomes,
recombinant enzymes) systems [2]. However, only the
former are potentially capable of expressing the com-
plete metabolism pathways and mimicking the diverse
mechanisms of toxicity occurring in vivo. Primary
human hepatocytes and immortalized hepatocytes have
become widely used; however, both model systems have
limitations. Indeed, primary human hepatocytes have
scarce and unpredictable availability, limited growth
activity and life-span and undergo early and variable
phenotypic alterations. Moreover, liver-specific func-
tions, particularly CYPs and their responsiveness to
prototypical inducers are not similarly maintained with
time of culture. Various experimental conditions have
been proposed to improve both hepatocyte survival and
functions; they include the use of sophisticated media,
sandwich configuration and co-cultivating. However,
whatever the condition used, liver-specific functions are
usually decreased and are not stable exhibiting variable
and different changes with time in culture. Moreover,
species-differences are observed. Thus, CYP inducibil-
ity is better retained in rat hepatocytes cultured in the
sandwich configuration than in conventional monolayer
but this difference is not observed with human hepato-
cytes [3]. However, primary rat hepatocytes show similar
early gene changes in both culture conditions [4]. Con-
trary to their normal rat counterparts primary human
hepatocytes recover to a certain extent their ability to
transcribe specific genes after around 2–3 days in culture
and this results in increased cytochrome P450-dependent
activities [5]. Adult hepatocytes can be cryopreserved
but the freeze/thaw process leads to further marked
functional alterations; these being minimized by gel
entrapping before cryopreservation [6].
In spite of important limitations, primary hepatocytes
remain the gold standard model for studies on xeno-
biotic metabolism and toxicity. Indeed hepatocyte cell
lines, whether of tumoral origin or obtained by onco-
genic transformation, lack a variable and substantial set
of liver-specific functions and consequently, are unsuit-
able for mimicking in vivo normal parenchymal cells.
The major CYP activities are quite low if detectable.
Even HepG2 cells which have retained various hepatic
functions contain little CYP activity with the exception
of the CYPs expressed in fetal liver, such as CYP1A1 and
CYP3A7 [7,8]. Expression of CYPs has been obtained
by transfection of plasmid constructs expressing CYPs
or liver-specific transcription factors but such trans-
fected cell lines are not suitable to study regulation
of gene expression normally observed in hepatocytes
[9,10]. A new human hepatoma cell line, named Hep-
67
aRG, was recently derived from a liver tumor and shown
to remain capable of expressing most of the liver-specific
functions, including the major CYPs involved in drug
metabolism [11,12]. Its growth and functional charac-
teristics are summarized in the present review.
2. Obtention, growth and morphological features
HepaRG cells were obtained from a liver tumor of
a female patient suffering from hepatocarcinoma [11].
These cells exhibit only limited caryotypic alterations
mainly characterized by a surnumerary and remodeled
chromosome 7 and a translocation t(12;22) with a loss
of the 12p fragment leading to a monosomy 12P [11].
When detached and seeded at low density (2.6 ×
104 cells/cm2 ) confluent well-differentiated HepaRG
cells are able to transdifferentiate [13]; they acquire
an undifferentiated elongated cell morphology able to
actively divide and to reach again confluency within 1
week. At that time two morphologically different cell
types appear: one forms clusters of granular epithelial
cells resembling hepatocytes while the second surround-
ing the former is more flattenned and retains a clear
cytoplasm (Fig. 1). Addition of 2% dimethyl sulfoxide
(DMSO) and 5 × 10−5 M hydrocortisone hemisucci-
nate to the culture medium induces differentiation of
hepatocyte-like cells into more granular cells closely
resembling typical adult primary hepatocytes with one
or two nuclei and bile canaliculus-like structures. Undif-
ferentiated elongated cells express markers of liver
progenitor cells while the two cell types identified at
confluency express markers of hepatocytes and biliary
epithelial cells, respectively. The hepatocyte-like cells
represent around 50–55% of the total cell population
[13]. In this line albumin and haptoglobin mRNAs are
quite low during active proliferation of progenitor cells
and begin to increase when the cells reach confluency and
differentiate. By contrast, high levels of mRNAs encod-
ing aldolase B, a marker of adult hepatocytes, are found
in highly differentiated hepatocyte-like cells and repre-
sent around 20% of the values found in freshly isolated
human hepatocytes. By comparison HepG2 cells do not
contain detectable aldolase B transcripts while albumin
and haptoglobin mRNA levels are much lower than those
measured in HepaRG cells (Fig. 2).
The behavior of HepaRG cells is unique; it has never
been described for another human hepatoma cell line and
resembles the coculture model associating hepatocytes
and undifferentiated biliary cells we have developed 25
years ago that allowed longer and better maintenance
of liver-specific functions in hepatocytes [14]. It must
be borne in mind that hepatocytes and biliary epithelial
68 A. Guillouzo et al. / Chemico-Biological Interactions 168 (2007) 66–73
Fig. 1. Phase-contrast micrographs of HepaRG cells. (A) Proliferating
undifferentiated cells 5 days after seeding at low density; (B) cells cul-
tured for 15 days in the absence of DMSO then 15 additional days in the
presence of DMSO; hepatocyte-like cells with typical bile canaliculi
surrounded by biliary-like cells characterized by a clear cytoplasm.
cells are derived from the same progenitor cell in the
liver. Interestingly, when differentiated hepatocyte-like
cells are seeded at high density (0.45 × 106 cells/cm2 )
they have a limited proliferative activity and retain their
hepatocyte-like features. Consequently they can be used
during the first days following cell seeding [12].
3. Drug metabolizing enzyme activities
The huge differences between HepG2 cells and pri-
mary human hepatocytes were confirmed in our recent
study on HepaRG cells [12] (Fig. 3). No transcripts were
detected for CYPs 2B6, 2C9, 2E1 and 3A4 in HepG2
cells. By contrast HepaRG cells expressed all CYPs mea-
sured, namely CYPs 1A2, 2B6, 2C9, 2E1 and 3A4; the
levels being dependent on the duration of confluency and
for most of them on the presence of DMSO in the culture
medium [12].
Fig. 2. mRNA expression for haptoglobin, albumin and aldolase B.
Transcripts were measured in primary human hepatocyte cultures, con-
fluent HepG2 cells and HepaRG cells after different times of culture
and maintained after day 15 in the absence or presence of 2% DMSO.
The values are expressed as percentages compared to a pool of three
different freshly isolated human hepatocyte populations and arbitrar-
ily set at 100%. They are the mean of two experiments in duplicate.
Adapted from Ref. [12].
In their most differentiated state, HepaRG cells
express CYP mRNAs at levels comparable to those found
in cultured primary human hepatocytes. The most sensi-
tive CYPs to the presence of DMSO are CYPs 2B6, 2E1
and 3A4. The high levels of CYP2C9 in both differenti-
ated HepaRG cells and primary human hepatocytes are
likely due to the addition of hydrocortisone to the cul-
ture medium. Indeed CYP2C9 is known to be inducible
by glucocorticoids [15]. The functionality of CYPs in
HepaRG cells is supported by the demonstration of their
 A. Guillouzo et al. / Chemico-Biological Interactions 168 (2007) 66–73 69

Fig. 3. mRNA expression for CYPs 1A2, 2C9, 2E1 and 3A4. Transcripts were measured in one human hepatocyte (HH) population at different
times of culture (days 1–7), confluent HepG2 cells and HepaRG cells at different times of culture and maintained after day 15 in the absence or
presence of 2% DMSO. The values are expressed as percentages compared to a pool of three different freshly isolated human hepatocyte populations
and arbitrarily set at 100%. They are the mean of two experiments in duplicate. Adapted from Ref. [12].

specific activities using specific substrates and respon-
siveness to inducers. Indeed, specific activity can be
evidenced for all CYPs and is increased after treatment
with specific inducers. However, both transcripts (Fig. 2)
and basal activity [11] of the non-inducible CYP2D6 are
at the limit of detection suggesting that HepaRG cells
are derived from a CYP2D6 poor metabolizer patient.
The extent of augmentation in the presence of a specific
inducer markedly varies from one CYP to another. Thus,
while basal activity of CYP1A1/2 determined by 7-
ethoxyresorufin-O-deethylation is quite low it is strongly
induced by 5 ␮M 3-methylcholanthrene. CYP1A2 activ-
ity can also be measured by phenacetin deethylation [11].
By contrast, CYP3A4 is no more increased by rifampicin
when the cells are cultured in the presence of DMSO
that strongly augments its content compared to the level
found in DMSO-free confluent cells (Fig. 4). The regula-
tion of certain CYPs, such as CYP3A4, in HepaRG cells
might be explained by transcriptional activation of the
nuclear factors PXR and CAR (Fig. 5). Indeed, one of the
reasons for huge differences in CYP expression between
HepaRG and HepG2 cells is likely down-regulation of
these two major nuclear factors in the latter. A high
expression of CAR has never been reported before in
any hepatoma cell line. As found in primary human
and rodent hepatocytes, PXR and CYP3A4 are inhib-
ited in HepaRG cells by lipopolysaccharide treatment
(Descheemaeker et al., unpublished data).
By contrast, transcripts for the aryl hydrocarbon
receptor (AhR) are present in comparable amounts in
primary human hepatocytes, HepG2 cells and HepaRG
cells, in agreement with the strong induction of CYP1A2
by 3-methylcholanthrene in these cells. Contrary to CAR
and PXR, AhR belongs to the bHLH/PAS (basic helix-
loop-helix/Per-Arnt-Sim) family of transcription factors
and stimulates transcription of CYP1A genes and var-
ious other genes; it is expressed in undifferentiated
liver parenchymal cells and various cell types. Perox-
isome proliferator-activated receptors (PPARs) are other
members of the large ligand-receptor superfamily; they
comprise three isotypes. PPAR␣ is mainly expressed in
liver and PPAR␥ in adipocyte tissue while PPAR␤/␦ has
a ubiquitous distribution. PPARs are receptors for vari-
ous endogenous lipophilic molecules and some synthetic
70 A. Guillouzo et al. / Chemico-Biological Interactions 168 (2007) 66–73
Fig. 4. CYP activities in HepaRG cells. The cells were seeded at low
density and after day 15 were maintained in the presence of 2% DMSO.
During treatment with prototypical inducers, i.e. 3-methylcholanthrene
for 24 h or rifampicin for 72 h the cells were either maintained or
not in the presence of DMSO. CYP activities were estimated by
measuring testosterone 6-␤ hydroxylation (CYP3A4), O-dealkylation
of 7-ethoxyresorufin (CYP1A1/2) and tolbutamide 4-hydroxylation
(CYP2C9). Results are expressed as pmol/mg protein/min. The stu-
dent’s test was applied for statistical analyses between cells exposed to
the vehicle only (control) and cells treated with the inducer (** p < 0.01,
*** p < 0.001).
compounds; the three PPARs are expressed in well-
differentiated HepaRG cells [12,16]. It has been recently
shown that CYP4A11, the main fatty acid hydroxylase of
the human liver, considered to be mediated via PPAR␣,
is repressed by retinoic acid in these cells [16].
Phase II enzymes play an important role in detox-
ification and activation of many xenobiotics. mRNAs
Fig. 5. mRNA expression for the CAR nuclear receptor. Transcripts
were measured in one human hepatocyte population at different times
of culture, subconfluent HepG2 cells and HepaRG cells at different
times of culture and maintained after day 15 in the absence or presence
of 2% DMSO. The values are expressed as percentages compared to a
pool of three different freshly isolated human hepatocyte populations
and arbitrarily set at 100%. They are the mean of two experiments in
duplicate. Adapted from Ref. [12].
levels for glutathione transferase (GST) A1/A2, GSTA4,
GSTM1 and UDP-glucuronosyl transferase (UGT)1A1
determined by RT-qPCR are also much higher than
in HepG2 cells, suggesting that, like CYPs, phase II
enzymes are well-maintained in differentiated HepaRG
cells [12]. The values of GSTA1/2 and UGT1A1 in these
latter are equal to or two-fold higher than those measured
in freshly human hepatocytes while GSTM1 represent
around 25%. However, GSTs markedly decrease in
human hepatocytes with time in culture (Table 1).
Hepatocytes possess various transporter systems
expressed at their two polar surface domains [17]. Apical
and canalicular ATP-binding cassette (ABC) trans-
porters such as multidrug-resistance associated protein
(MRP)2, MRP3, multidrug-resistance P-glycoprotein
(MDR1) and bile salt export pump (BSEP) are
responsible for xenobiotic clearance as well as for
secretion of bile salts and other bile constituents.
Basolateral solute carrier transporters such as organic
cation transporter (OCT)1, organic anion transporting
polypeptide (OATP)-B, OATP-C and Na+ -taurocholate
co-transporting polypeptide (NTCP) are involved in the
uptake of endogenous and foreign compounds from the
blood. Transcripts for these transporters have been anal-
ysed in both undifferentiated and differentiated HepaRG
cells using the RT-qPCR method by Le Vee et al. [18].
Transcripts for all the sinusoidal and canalicular trans-
porters tested are expressed. The three ABC transporters,
MDR1, MRP2 and MRP3 which are not liver-specific,
are well expressed in both undifferentiated and differen-
tiated HepaRG cells as well as in HepG2 cells at levels
 A. Guillouzo et al. / Chemico-Biological Interactions 168 (2007) 66–73 71

Table 1
mRNA expression for several phase II enzymes, transporters and thioredoxin
Genes Human hepatocytes HepG2 HepaRG References

FI 1d 3d P D

GSTA 1/2 100 5 5 0 <10 110 [12]

GSTM1 100 20 50 0 <10 25 [12]
100 0 0 5–30 [18]
BSEP
100 190 [19]
100 0 0 10 [18]
OATP-C
100 40 [19]
100 0 0 10 [18]
NTCP
100 80 [19]
100 0 0 50 [18]
OCT-1
100 60 [19]
100 150 150 240 [18]
MDR1
100 250 [19]
100 110 50 50 [18]
MRP2
100 190 [19]
100 80 150 190 [18]
MRP3
100 320 [19]
Thioredoxin 100 950 180 110 1250 304 [12]

Transcripts were measured in primary human hepatocytes, confluent HepG2 cells and undifferentiated proliferating (P) and differentiated (D)
HepaRG cells. Differentiated HepaRG cells were maintained confluent for 15 days in the presence of 2% DMS0. The values are expressed as
percentages compared to either a pool of three different freshly isolated human hepatocyte populations (FI) (phase II enzymes, thioredoxin) or 1-day
human hepatocyte cultures (transporters); 0: undetectable or below 5%. Adapted from Refs. [12,18,19].

even higher than in 1-day human hepatocytes. By con-
trast, BSEP, the predominant efflux bile salt system, and
the influx transporters are expressed only in differenti-
ated HepaRG cells and are absent in HepG2 cells. The
levels of influx transporters vary between 10% and 50%
of those found in 1-day human hepatocytes, depending
on the transporter. Interestingly, with the exception of
BSEP and MRP2 which are both involved in the secre-
tion of cholephilic compounds, transporters which were
overexpressed and repressed in HepaRG cells similarly
increased and decreased in human hepatocytes with time
in culture when compared to freshly isolated human hep-
atocytes [18,19]. Regulation and functional activity of
some transporters (P-glycoprotein, MRP2 and BSEP)
have been demonstrated [18].
The thiol-dependent antioxidant protein, thioredoxin
was also analysed and found to be expressed in all cul-
tures, being however strongly increased in 1-day human
hepatocytes and proliferating HepaRG cells.
4. Chemical cytotoxicity
Various chemicals are hepatotoxic, requiring or not
previous metabolism. Due to the lack of some major
CYPs HepG2 cells are not sensitive to certain hepato-
toxic drugs and genotoxic compounds [20,21]. Thus,
compounds such as amiodarone and chlorpromazine that
do not require metabolism to induce toxic effects exhibit
similar cytotoxicity in both HepG2 and HepaRG cells.
By contrast acetaminophen and aflatoxin B1 of which
toxicity is mediated by toxic metabolites are much more
cytotoxic in differentiated HepaRG cells. Thus aflatoxin
B1 , that is cytotoxic via a 8,9-epoxide metabolite formed
by CYP1A2 and 3A4 has a IC50 at around 5 ␮M after a
24 h exposure in agreement with previous findings with
primary human hepatocytes [22]. By contrast, it is not
cytotoxic at all at a concentration of 100 ␮M in HepG2
cells (Fig. 6). Even around 50% of HepG2 cells are still
alive after a 3-day treatment with this mycotoxin.
Other xenobiotics such metals can also be hepa-
totoxic. Iron overload is well known to cause liver
damage. As demonstrated by Troadec et al. [23], like
human hepatocytes HepaRG cells show intracellular iron
accumulation only when they exhibit hepatocyte-like
features after exposure to iron citrate. No iron storage
is observed in proliferating and biliary-like HepaRG
cells. Using a transcriptomic approach through a liver-
dedicated cDNA microarray, iron loading is associated
with both a reduction in cell motility as shown by
a decrease in moesin and RAC1, a small GTP bind-
72 A. Guillouzo et al. / Chemico-Biological Interactions 168 (2007) 66–73
Fig. 6. Comparative cytotoxicity of aflatoxin B1 to subconfluent
HepG2 and differentiated HepaRG cells after a three days treatment.
Cell viability was estimated using a standard MTT test. The values were
normalized to untreated cells and expressed as means ± S.D. (n = 3
cultures).
ing protein, transcripts and appearance of differentiated
functions including xenobiotic (e.g. CYP2E1 and 3A4)
and lipid metabolism (e.g. fatty acid binding protein
1) and response to stress (e.g. superoxide dismutase)
[23]. This observation represents another example of
potential interest of HepaRG cells for hepatic toxicity
studies.
5. Conclusions
All the data obtained in our laboratory and confirmed
by others demonstrate that HepaRG cells represent a
unique human hepatoma cell line, capable of express-
ing both phases I and II drug metabolizing enzymes as
well as membrane transporters normally found in the
liver. Consequently they represent a promising alterna-
tive to primary human hepatocytes. Even more they offer
several advantages over the latter: (i) both undifferenti-
ated and differentiated cells from the same cell line can
be used and compared; (ii) their enzyme activities can
be modulated allowing to more closely mimic quantita-
tive interindividual variations in xenobiotic metabolizing
enzymes usually found when investigating different
human hepatocyte populations; (iii) functional activities
remain relatively stable for 1–2 more weeks after 3–4
weeks at confluency, making the cells a unique model
for chronic toxicity studies; (iv) genotoxic compounds
can be analysed; indeed the cells can be exposed to the
compounds when differentiated, then placed in condi-
tions to proliferate, allowing the use of mutagenic tests
such as the Comet and micronucleus assays. In addition it
may be expected that carcinogens will induce phenotypic
changes after several passages leading to the appearance
of more malignant cell clones, making HepaRG cells
suitable for detecting potential carcinogens for humans;
(v) HepaRG cells can be easily cryopreserved without
marked cell loss.
Acknowledgements
Personal studies were supported by INSERM and the
Association pour la Recherche sur le Cancer (ARC).
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