You are on page 1of 11

Current Drug Metabolism, 2008, 9, 1-11 1

Cell Lines: A Tool for In Vitro Drug Metabolism Studies

M.T. Donato1,2,3 A. Lahoz4, J.V Castell1,2,3 and M.J. Gmez-Lechn2,3,*

1
Departamento de Bioqumica y Biologa Molecular, Facultad de Medicina, Universidad de Valencia, Spain; 2CIBERHEPAD, FIS,
Spain. 3Unidad de Hepatologa Experimental. Centro de Investigacin, Hospital La Fe, Valencia, Spain; 4Advancell In Vitro Cell
Technologies, Valencia, Spain

Abstract: Primary cultured hepatocytes are a valuable in vitro model for drug metabolism studies. However, their widespread use is
greatly hindered by the scarcity of suitable human liver samples. Moreover, the well-known in vitro phenotypic instability of hepatocytes,
the irregular availability of fresh human liver for cell harvesting purposes, and the high batch-to-batch functional variability of hepatocyte
preparations obtained from different human liver donors, seriously complicate their use in routine testing. To overcome these limitations,
different cell line models have been proposed for drug metabolism screening. Human liver-derived cell lines would be ideal models for
this purpose given their availability, unlimited life-span, stable phenotype, and the fact that they are easy to handle. However, the human
hepatoma cells currently used (i.e. HepG2, Mz-Hep-1) show negligible levels of drug-metabolizing and do not constitute a real alterna-
tive to primary hepatocytes. Different strategies have been proposed to generate metabolically competent immortalized hepatocytes
(transformation of human hepatocytes with plasmids encoding immortalizing genes, hepatocyte-like cells derived from stem cells, cell
lines generated from transgenic animals, hepatocyte/hepatoma hydrid cells). Moreover, recombinant models heterologously expressing
P450 enzymes in different host cells have been developed and successfully used in drug metabolism testing. In addition, new strategies
have recently been explored to upregulate the expression of drug-metabolizing enzymes in cell lines of a human origin (i.e. transfection
with expression vectors encoding key hepatic transcription factors). Among metabolic-based drug-drug interactions, P450 inhibition
seems to be the most important. A major application of recombinant models expressing a single P450 is the screening of potential en-
zyme inhibitors. Therefore, pharmaceutical companies increasingly make use of cell lines to speed up the selection of new drugs with fa-
vourable pharmacokinetic and metabolic properties.
Keywords: Hepatocytes, hepatoma cells, genetically engineered cells, drug metabolism, P450 induction, P450 inhibition.

INTRODUCTION expression of both Phase I and II enzymes for several days in cul-
After intake, drugs are usually transformed into new chemical ture [3-6], and are capable of generating a metabolic profile of a
species that may have either toxic or therapeutic effects. The phar- drug similar to that found in vivo [7], which is when they are con-
macological response of a drug is primarily the result of its effec- sidered the gold standard model for xenobiotic metabolism and
tive interaction with the target which is linked to the concentration toxicity studies. However, their widespread use is greatly hindered
reaching it. Pharmacokinetics (absorption, distribution, metabolism by the scarcity of suitable human liver samples. Moreover the well-
and excretion) of the compound also plays a key role in its efficacy known in vitro phenotypic instability of hepatocytes, the irregular
as it governs the drug concentration at the site of action [1]. There- availability of fresh human liver for cell harvesting purposes, and
fore, inappropriate pharmacokinetics can result in an inadequate or the high batch-to-batch functional variability of hepatocyte prepara-
variable concentration of the drug at the site of action and in great tions obtained from different human liver donors, seriously compli-
variations in the clinical response. Drug metabolism is a major de- cate their use in routine testing.
terminant of drug clearance and inter-individual pharmacokinetic To overcome these limitations, different cell line models have
differences, and is an indirect determinant of the clinical efficacy been proposed for drug metabolism screening in recent years. Hu-
and toxicity of drugs [2]. The pharmaceutical industry is committed man liver-derived cell lines would be ideal models for this purpose
to marketing safer drugs with fewer side effects, predictable phar- given their availability, unlimited life-span, stable phenotype, and
macokinetic properties and quantifiable drug-drug interactions. the fact that they are easy to handle. Unfortunately, most hepatic
Therefore, it is desirable that poorly behaved compounds are re- cell lines have not constituted a real alternative to primary cultured
moved early in the discovery phase rather than during the more hepatocytes for drug metabolism studies given their very
costly drug development phases. low/partial expression of cytochrome P-450 (P450). New strategies
Gaining knowledge on the metabolism of a given drug, the are currently being used to up-regulate the expression of drug me-
enzymes so far involved, and on their inhibition or induction poten- tabolizing enzymes in these cells. This paper reviews the features of
tial is a necessary step in the pharmaceutical development of new liver-derived cell lines and their suitability for drug metabolism
compounds. The ultimate goal of these efforts is to ascertain: a) studies, as well as the strategies based on the use of genetically
how the molecule is biotransformed and whether the produced me- engineered cells expressing individual drug metabolizing enzymes.
tabolites contribute to the pharmacological action of the drug
HUMAN LIVER-DERIVED CELL LINES
(metabolic profile); b) which enzymes are involved in its biotrans-
formation and whether this could be influenced by the concomitant Hepatic cell lines have several advantages when compared with
use of other drugs, c) whether the drug itself influences the expres- primary cultured human hepatocytes. Hepatic cell lines grow con-
sion or the activity of such enzymes which, in turn, could influence tinuously, have almost an unlimited life-span and have quite a sta-
its metabolism and also that of other drugs and d) the drug toxicity ble phenotype. Cell lines are easily available, culture conditions are
associated with drug metabolism. simpler than primary hepatocytes, and easily standardized among
laboratories. In addition, some hepatomas retain (in part) a differen-
Primary cultured hepatocytes, liver slices and microsomes are
tiated adult phenotype. These features make hepatic cell lines po-
valuable in vitro models for drug metabolism studies [3]. Human
tentially appropriate for screening purposes. Human hepatoma cell
hepatocytes are fully competent metabolic cells. They retain the
lines have been widely used as an in vitro model for the study of
hepatocellular functions, as well in toxicity studies. Concerning
*Address correspondence to this author at the Centro de Investigacin,
Hospital La Fe, Avda de Campanar 21, 46009-Valencia, Spain; Tel: +34 96 drug metabolism, one major drawback is that hepatoma cells ex-
1973040; Fax: +34 96 1973018; E-mail: gomez_mjo@gva.es press biotransformation activities to a very limited extent (i.e. only

1389-2002/08 $55.00+.00 2008 Bentham Science Publishers Ltd.


2 Current Drug Metabolism, 2008, Vol. 9, No. 1 Donato et al.

a few enzymes) and, even if they do so, levels are very low in rela- scription levels of genes [17, 31] or a cocktail of P450 substrate
tion to that of the normal adult liver (Table 1). Decreased activity probes [32] to assess increases in P450 protein activity.
levels are found particularly for the P450 reactions involved in the HepG2 is among the most widely used human hepatoma cell
oxidative metabolism of drugs and xenobiotics. The low P450 sys- line. These cells show many liver-specific functions, express conju-
tem functionality is likely due to an impaired expression of the gating enzymes, but lack a functional expression of almost all the
P450 enzymes, as hepatoma cell lines express sufficient levels of relevant human liver P450s. Consequently, they are a very poor
functional NADPH cytochrome P450 reductase (CPR) that would alternative to primary cultured cells. The activity and protein con-
enable their P450s to act. Specific mRNA levels of major hepatic tent of most P450s is undetectable by conventional methods, and
P450 enzymes in human hepatoma cell lines and in primary hepato- only very sensitive techniques like RT-PCR detect and quantify the
cytes are comparatively shown in Table 2. Frequently, the non- transcription levels of genes. Most of the P450 isoforms (i.e.
hepatic isoenzymes are expressed, (e.g. CYP1A1, instead of CYP2B6, 2C9, 3A4) examined in HepG2 presented values that
CYP1A2).The CYP1A1 mRNA decays remarkably quickly (half- were 2-3 orders of magnitude lower than in hepatocytes [16] (Table
life of 2.4 h), and is one of the most unstable of the P450 mRNAs. 2). A novel differentiated human hepatoma cell line, WGA, derived
The rapid decay of CYP1A1 mRNA is associated with a rapid loss from HepG2 and which expresses CYP2B6 and constitutive andro-
in poly(A) tail length, suggesting that deadenylation is the first step stane receptor (CAR), has been proposed as a powerful system to
in the decay pathway. The short half-life appears to be conserved study the regulation of the CYP2B6 gene expression by phenobar-
across species, which suggests that this characteristic of the bital [33].
CYP1A1 mRNA is important for its function [23].
BC2 is a human hepatocarcinoma-derived cell line possessing
Hepatoma cell lines also express conjugating enzymes, namely the capacity of growing and of undergoing a spontaneous differen-
glutathione S-transferase (GST) and UDP-glucuronyltransferase tiation in vitro after confluence. Once they reach this status, cells
(UGT) and sulphotranferases (Table 1) [14, 15, 25-28]. For in- remain stably differentiated in culture for several weeks, but may
stance, KYN-2 and Mz-Hep-1 cells express one of the two different return to a proliferative status (hence be de-differentiated) upon
UGT isoforms found in hepatocytes [20]. Furthermore, the induci- sub-culturing. Differentiated BC2 cells express the most relevant
bility of UGT1A isoforms in HepG2 cells by -naphthoflavone has P450 isozyme mRNAs (CYP1A1/2, 2A6, 2B6, 2C9, 2E1, and 3A4)
been reported [29]. Quite frequently however, the isozymes ex- (Table 2). Both CYP3A4 activity and conjugating enzymes (GST
pressed are not those found in adult liver tissue. For instance, N- and UGT) are well expressed in these cells. In addition, BC2 cells
acetyltransferase (NAT) in hepatoma cells has an activity profile show the ability to respond to enzyme inducers. A previous study
that resembles extra-hepatic tissue NATs rather than liver NATs showed that BC2 is able to increase P450 activities in response to
(NAT1 predominating over NAT2). Thus, hepatoma cell lines do methylcholantrene, phenobarbital, rifampicin and dexamethasone
not represent a good in vitro model to investigate the human me- [15]. Although P450 activities in BC2 were markedly lower than in
tabolism of arylamines or hydrazines in the liver [30]. human hepatocytes, the effects elicited by some of these model
Few authors have reported the ability of hepatoma cell lines to inducers were, in relative terms, comparable to those observed in
respond to inducers (Table 3). The response of these cells, in terms human cellular models. Further work on BC2 cells showed a het-
of enzyme activity, is very poor except for the non-hepatic isoform erogeneous expression of P-glycoprotein and other relevant markers
CYP1A1, which is easily inducible [14, 20, 26-28]. However, it is [34].
possible to monitor the induction of some P450 isoforms (i.e. A new human hepatoma cell line, named HepaRG, derived
CYP3A4, 2C9) by making use of either reverse transcriptase- from a human hepatocellular carcinoma, also shows features of a
polymerase chain reaction (RT-PCR) techniques to quantify tran- well differentiated hepatocyte [18]. When seeded at a low density,

Table 1. Basal Biotransformation Activities of Human Liver-Derived Cell Lines and Primary Cultured Human Hepatocytes [3, 8-23]

AHHa ECOD EROD MROD BROD PROD COH DXMT pNPH CLZX DOH T6OH CPR mEH GST UGT SULT

Hepatocytes 2.91.0 24.211.3 4.52.0 5.041.87 2.46 1.45 3.31.8 13742 77.647.4 10457 273 31773 25310 23.22.2 18072 301112 3.60.4 0.009

HepG2 1-3 0.50.2 0.900.47 0.35 0.34 0.28 0.26 0.23 0.09 0.05 0.04 <0.01b <1 <0.1b <1 b 0.18 0.16 5.8 b 75 8 34 7 1.5-100 0.2-97

Mz-Hep-1 < 0.10 0.370.26 <0.1 < 0.10 0.0 <0.01 <0.01b 0 <0.1b <1 b 0.15 0.33 7.3 b 134 34 1.60.5

KYN-2 4.1 5.20.2

Chang 7.3 b 118


b
Hep3B 9.9

Fa2N-4 0.20 1.33 2.1

HuFoe-15 135 3.2

BC2 (28- 2.0 0.1 0.28 0.04 0.62 0.05 0.60 0.05 0.16 0.05 0.08 0.03 <0.01 1.4 0.3 0.48 2.54 46.5 5.4 4.80.8 1.3 0.6 0.20 0.03
days)

HepaRG traces 131 11 54 10


differentiated

(a) AHH (arylhydrocarbon hydroxylase), ECOD (7-ethoxycoumarin-O-deethylase), EROD (7-ethoxyresorufin-O-deethylase), MROD (7-methoxyresorufin-O-demethylase),
BROD (7-benzoxyresorufin O-debenzylase), PROD (7-penthoxyresorufin-O-depentylase), COH (coumarin 7-hydroxylation), DXMT (dextromethorphan O-demethylation), pNPH
(p-nitrophenol hydroxylase), CLZX (chlorzoxazone 6-hydroxylation) DOH (diclofenanc 4-hydroxylation) and T-6OH (testosterone 6-hydroxylase) activities are expressed as
pmol/mg protein x min. CPR (NADPH-cytochrome P450 reductase), mEH (microsomal epoxide hydrolase), GST (gluthathione-S-transferase), UGT (UDP-glucuronil transferase),
and SULT (sulfotransferase) are expressed as nmol/mg protein x min. Values are mean standard deviation.
In humans, AHH, EROD and MROD activities are associated to CYP1A1/2, while ECOD is associated to CYP1A1/2, CYP2A6, CYP2B6, CYP2C9 CYP2E1 and CYP3A3/4/5.
BROD activity, and in part PROD, is specific of CYP2B6. COH and DXMT are dependent on CYP2A6 and CYP2D6 respectively. CLZX and pNPH are catalyzed by CYP2E1, and
DOH and T6OH are indicative of CYP2C9 and CYP3A4, respectively.
(b) Unpublished data from our laboratory.
Cell Lines: A Tool for In Vitro Drug Metabolism Studies Current Drug Metabolism, 2008, Vol. 9, No. 1 3

Table 2. Comparative Expression of P450 Transcripts in Primary Human Hepatocytes and Hepatoma cell lines [15-18, 24]

Cell model CYP1A1 CYP1A2 CYP2A6 CYP2B6 CYP2C9 CYP2C19 CYP2D6 CYP2E1 CYP3A4 CYP3A5

a
Hepatocytes 100 100 100 100 100 100 100 100 100 100
HepG2 6.99 0.03 0.25 0.50 0.01 0.05 1.57 0.04 0.03 1.16
Mz-Hep-1 1.37 0.03 0.25 0.09 0.003 0.05 0.011 0.006 0.003 0.03
BC2b 0.01 0.05 0.16 0.02 0.07 <0.01 0.07 0.28 2.49
HepaRG 7.5 4.9 22.1 0.8 0.7 6.8

(a) Results are mean values expressed as a percentage compared to hepatocytes.


(b) Unpublished data from our laboratory.

Table 3. Induction of P450 Activities in Primary Human Hepatocytes and Hepatic Cell Lines [15-18]

Cell model CYP1A1/2 (EROD)a CYP3A4 (T6OH) CYP2C9 (DOH)


control MC control RIF control RIF

Hepatocytes 4.5 2.0 66.1 27 (15)b 253 110 733 202 (2.9) 226 55 276 40 (1.2)
HepG2 0.9 0.5 12.8 4.1 (14) 0.18 0.16 0.76 0.34 (4.2)
Mz-Hep-1 0.4 0.3 2.1 1.2 (5.2) 0.15 0.33 0.21 0.12 (1.4)
BC2 0.4 0.1 3.1 0.7 (7.2) 43 3 52 7 (1.2)
c
Fa2N-4 0.2 5.3 (29) 2.1 17.9 (8.5) 1.33 2.45 (1.8)
HepaRG traces 63 1 54 10 540 68 (9.9) 6.2 1.6d 13.1 2.6 (2.1)

(a) Cells were exposed to 1-2 M 3-methylcholanthrene (MC) or to 10-50 M rifampicin (RIF). Activities (see Table 1 for abbreviations) are expressed as pmol/mg x min. Data are
mean standard deviations.
(b) Data in parentheses correspond to the fold over activity in corresponding controls (untreated cells).
(c) Fa2N-4 cells exposed to -naphtoflavone.
(d) CYP2C9 activity assayed as tolbutamide 4-hydroxylation.

they acquire an elongated undifferentiated morphology, actively model system for induction studies [32] to identify CYP3A4 induc-
divide, and after having reached confluency, form typical hepato- ers in particular [47].
cyte-like colonies surrounded by biliary epithelial-like cells.
Moreover, and contrary to other human hepatoma cell lines, includ- GENETICALLY ENGINEERED CELLS EXPRESSING HU-
ing the HepG2 cells, HepaRG cells not only express various P450s MAN DRUG METABOLIZING ENZYMES
(CYP1A2, 2B6, 2C9, 2E1, 3A4), but also the nuclear receptors Advances in molecular biology have enabled the possibility of
CAR and pregnane X receptor (PXR) at levels comparable to those cloning and heterologously expressing genes of human drug-
found in cultured primary human hepatocytes. They also express metabolizing enzymes. In recent years, recombinant human models
other specific markers of adult hepatocytes, including phase II en- expressing heterologous genes of human P450s, and other genes
zymes and drug transporters, and are able to respond to selective involved in drug metabolism in different host cells, have been de-
inducers of the P450 enzymes [35]. veloped by using expression vectors which encode for human en-
In view of the limited expression of drug-metabolizing enzymes zymes [48]. Genetically engineered cells expressing human drug-
in most hepatoma cell lines, alternative approaches have been ex- metabolizing enzymes have become new potent tools for investigat-
plored to obtain immortalized hepatocytes from a non-hepato- ing drug metabolism, drug-drug interactions and the metabolism-
carcinoma origin (for a review, see [36]). A successful immortaliza- based activation of chemicals.
tion of normal hepatocytes from different species was achieved A variety of expression systems using bacteria, yeast, insect
using different strategies including cell transformation with virus cells, and mammalian cells (-lymphoblastoid cells, COS cells,
genes or oncogenes (i.e. simian virus 40 large T antigen, c-myc, cH- V79 Chinese hamster ovary cells, HepG2 cell line) have been suc-
ras) [37-39], the generation of cell lines from transgenic animals cessfully transfected to transiently or stably express P450 genes.
expressing viral transforming genes, oncogenes or growth factors The catalytic activity of a CYP-transfected cell relies not only on
[40, 41], or hydrid cells obtained by the fusion of hepatocytes and the efficient expression of the transgene, but also on the endoge-
immortalized cell lines [42,43]. Although some hepatic functions nous CPR activity. Hepatoma cells express sufficient CPR to ensure
were maintained following cell transformation, these cells show a sufficient monooxygenase activity, while this can represent a limit-
very low expression of P450 enzymes, which, in general, makes ing step in other cells. As a means to increase the catalytic activity
them unsuitable for drug metabolism studies [44-46]. Recently, the of CYP-transfected cells, the co-expression of the P450 gene, to-
drug-metabolizing capacity of Fa2N-4, a non-tumorigenic immor- gether with CPR, has been successfully achieved [49,50]. Different
talized human hepatic cell line, has been examined. The P450 ex- cell lines have been engineered to individually express either spe-
pression in Fa2N-4 cells is markedly lower than those of primary cific human P450s (CYP1A1, 1A2, 2A6, 2B6, 2B8, 2C6, 2C8, 2C9,
human hepatocytes [17]. However, increases in specific P450 ac- 2C19, 2D6, 2E1 and 3A4) or combinations of the xenobiotic me-
tivities can be observed after exposure to inducers (Table 3). An tabolizing enzymes. Among mammalian cell expression systems,
evaluation of CYP1A2, CYP3A4, CYP2C9, UGT1A, and MDR1 liver-derived cell lines HepG2 [51-54] and human liver epithelial
mRNA levels in Fa2N-4 cells treated with a panel of known induc- cells (THLE), a non-tumorigenic SV40-immortalized human liver
ers showed that Fa2N-4 cells responded in a similar way as primary epithelial cell line which retained most of the phase II enzymes
hepatocytes did [17]. These cells have been proposed as a robust [55,56], have been successfully transfected to express human P450
4 Current Drug Metabolism, 2008, Vol. 9, No. 1 Donato et al.

genes. Non-hepatic cell lines AHH-1, a human B lymphoblastoid sponsible for the oxidative metabolism of xenobiotics. As a full
cell line [57], and V79 Chinese hamster cells [58,59] have also been contingent of drug-metabolizing enzymes is not expressed in
genetically engineered for the stable expression of human P450 HepG2 and other hepatoma cell lines, drug metabolic stability or
genes. The P450 expression is confirmed by the determination of metabolite profile studies cannot be performed in these cellular
enzyme activities and by the RT-PCR procedure. Recombinant models, except for compounds metabolized through Phase II conju-
enzymes show catalytic properties comparable to those of human gating reactions or oxidations catalyzed by the CYP1A1 or 1A2
liver microsomes, and the activity levels of the expressed P450 enzymes [68-70]. Hepatoma cells may be more useful for other
subtypes are similar or higher when compared to human hepato- applications in drug discovery such as enzyme induction or the
cytes. Heterologously expressed individual P450s are commercially assessment of the hepatotoxic potential of new drugs. Recently,
available and can be easily used in high-throughput metabolic novel liver-derived cell lines have been proposed as valuable tools
screening assays, in the phenotyping of P450 reactions, and as for the study of the induction of P450 and other drug-metabolizing
bioreactors to generate high amounts of a metabolite [60,61]. The enzymes [18, 47]. Although primary hepatocyte cultures are still
use of these cDNA-expressed P450s to study the enzyme function considered the gold standard in vitro model for induction studies,
has notably contributed to a greater understanding of substrate and promising results obtained after analyzing the utility of highly dif-
inhibitor specificity of individual P450 enzymes. Work to date has ferentiated hepatic cell lines to identify P450 inducers and to pre-
primarily focused on P450s, although cell lines expressing other dict the extent of drug-drug interactions for compounds in devel-
genes involved in xenobiotic metabolism have also been success- opment identified these cellular models as useful alternatives for
fully transfected (i.e. GSTs, UGTs, etc.) [62]. Moreover, cell lines preliminary screening. Similarly, hepatoma cells have been recog-
expressing all P450 enzymes individually have been reported, and nized as a good choice to evaluate non-metabolic dependent toxic-
cell lines which express combinations of the xenobiotic metaboliz- ity [71-73]. However, they do not constitute a real alternative to
ing enzymes have also been developed. The expression of multiple hepatocytes to screen chemicals bioactivated by drug-metabolizing
enzymes is important for a generalized use of engineered cells as enzymes. Cell lines are less sensitive towards bioactivable toxicants
toxicology screening tools [57]. In this line, engineering cellular than primary cells, even for promutagens activated by the
models heterologously co-expressing human CYP1A2 and poly- CYP1A1/2 enzymes (expressed in hepatoma cells but at signifi-
morphic variants of NAT2 in V79 cells [63], or CYP2E1 and cantly lower levels than in hepatocytes) [24].
GSTP1 in hepatoma cells [64], have been developed to evaluate the As indicated above, different alternatives have been explored to
metabolism-dependent toxicity of chemicals. Recently, hepatoma confer metabolic competence to cell lines, and new strategies are
cell lines which incorporate a new transactivator system that allows currently being developed. Nowadays, recombinant systems ex-
a time- and dose-dependent control of the expression of individual pressing a single P450 enzyme are commonly used in drug discov-
gene targets, including CYP2E1 and other drug-metabolizing en- ery and development. These models are particularly useful in the
zymes or transcription factors, have been successfully developed early detection of potential P450 inhibitors and in the identification
[64]. These cellular systems are promising tools to investigate the of P450s responsible for the metabolism of new drugs. Other poten-
precise role and functions of specific proteins and to model varia- tial uses of metabolically competent cell lines include metabolic
tions in human liver and their relevance to drug metabolism and stability screening and the generation of high amounts of drug-
toxicity. derived metabolites (bioreactors).
New strategies are now being currently explored to overcome
the limitations of hepatic cell lines for drug-metabolism studies. IDENTIFICATION OF P450 ENZYMES INVOLVED IN ME-
The low biotransformation activity of liver-derived cell lines is TABOLISM
likely to be the consequence of a very low P450 gene transcription, Reaction phenotyping characterizes the P450 isoforms that
resulting in trace levels of mRNA and protein synthesis [16]. Dif- catalyze a given drug-metabolizing reaction in the human liver.
ferent explanations for this phenomenon have been proposed: an This information is a key issue in drug discovery; it allows to
alteration (possibly a decrease) of specific transcription factors that minimize the role of polymorphic enzymes leading to inter-
control the expression of P450 genes, an increase in the levels of individual variations and to predict potential drug-drug interactions
transcription repressors, an alteration in the isoform pattern or func- that may result upon the co-administration of the new chemical
tions of transcription activators, or a chromatin compactation and with P450 isoform selective inhibitors or inducers. Phenotyping is
alternative mRNA splicing. Promising experimental approaches generally straightforward when a single P450 is involved in a meta-
based on the use of adenoviral expression vectors encoding relevant bolic pathway. However, P450 enzymes exhibit distinct yet over-
liver-specific transcription factors (HNF3, C/EBP) have been lapping substrate specificities, and multiple P450s can contribute to
successfully generated to increase the CYP3A4 and CYP2C expres- the metabolism of a particular compound. Approaches based on the
sions in HepG2 cells [65,66]. Alternatively, the re-expression of use of a battery of recombinant P450 systems, specific chemical or
limiting coactivators could be of critical importance for the antibody inhibitors, and on correlations of probe substrate activities
reactivation of non-functional transcription factors. HNF4 has with metabolic rates of test compounds have been combined and
been shown to be present in HepG2 and Mz-Hep-1 cells at levels as used for P450 phenotyping purposes.
high as in primary human hepatocytes. A low expression of the Genetically engineered cell lines expressing stable human drug-
HNF4 coactivators, PGC1 and SRC1, is shown in these hepa- metabolizing enzymes have been satisfactorily used in the identifi-
toma cell lines, which can seriously impair the expression of cation of those enzymes involved in the metabolism of a drug can-
HNF4 target genes [67]. Transfection of PGC1 and SRC1 coac- didate [56, 74, 75]. Incubating the compound with each separate
tivators caused an important enhancement of the CYP2C9, 1A1, cell line is useful to ascertain which P450 isoform(s) is(are) respon-
and 1A2 expressions in human hepatoma cells, which reveals the sible for metabolite/s formation. Previous studies in our laboratory
key role of coactivators in the transcriptional activation of P450 have demonstrated that the use of a battery of cultured cells ex-
genes by HNF4. pressing P450 enzymes individually is a valuable tool for assessing
USE OF CELL LINES FOR DRUG METABOLISM PUR- whether a given P450 can give rise to a particular drug metabolite
POSES [56]. The formation of diclofenac metabolites was analyzed after a
direct incubation of the drug with intact monolayers of THLE cells
Hepatoma cell lines have been widely used as in vitro models genetically manipulated for the stable expression of the major hu-
for the study of hepatocellular functions as well as hepatotoxicity man P450 enzymes involved in hepatic xenobiotic metabolism (Fig.
studies. However, their application to drug metabolism studies is 1). As incubation with cultured cells can be extended over long
hindered by their marginal expression of major P450 enzymes re-
Cell Lines: A Tool for In Vitro Drug Metabolism Studies Current Drug Metabolism, 2008, Vol. 9, No. 1 5

Fig. (1). Rate of diclofenac metabolism by P450-engineered cell lines. Diclofenac or 5-hidroxy-Diclofenac were incubated with P450-expressing cells. Cell
supernatant was withdrawn at regular time intervals and the content of 4-HO-Diclofenac (A), 4, 5-di-HO-Diclofenac (B), 5-HO-Diclofenac (C) and 3-
HODiclofenac (D) was analyzed by HPLC. Reproduced from [56].

periods (up to 24 hours at least), this in vitro model is particularly pared. By using the relative activity factor (the ratio of the activity
useful for investigating slowly metabolised compounds as well as of individual P450 in the engineered cells and that for the same
metabolism due to minor P450 isoforms [56]. The results revealed probe substrate in liver microsomes or hepatoctyes), an estimation
that as many as seven different P450s were potentially involved in of the degree of participation of each individual P450 in the overall
diclofenac metabolism. The screening approach using engineered metabolism of the drug can be made. A comparison of the results
cells provides an inclusion/exclusion screen of the potential role of obtained with CYP-expressing THLE cells and human hepatocytes
each P450, and the involvement of polymorphic enzymes can easily suggested the relative contribution of the enzymes implicated in the
be identified. Subsequent studies are needed to evaluate the rele- 5-hydroxylation of diclofenac in vivo (CYP2C8> 2C19  2C18 >>
vance of each enzyme in the drug metabolism. 2B6) (Fig. 2) [56].
Indicators for the correct interpretation of kinetic data from
recombinant enzyme models have been discussed [76-80]. A limita-
tion inherent to recombinant models is that some differences in
activity characteristics may exist with regard to P450s in their na-
tive membrane environment. P450 enzyme levels could be far in
excess of their relative amount in human liver and relative concen-
trations of accessory proteins (CPR or cytochrome b5) may differ
with those in human hepatocytes/human liver. Additional expres-
sion system-related factors may have an effect on the coupling effi-
ciency of the CPR and P450 and, hence, influence the results [79].
Moreover, secondary metabolism cannot be identified simply in
recombinant systems expressing a single enzyme, and this may lead
to incorrect predictions. With engineered cells only, neither the
degree of involvement of each P450 enzyme in a particular reaction
in vivo can be estimated, nor the metabolic profile of the drug in
humans be anticipated. However, such limitations have not hin-
dered their utility for P450 reaction phenotyping. Different strate-
gies based on the combined use of in vitro models showing a full
contingent of P450 enzymes (i.e. human liver microsomes, primary Fig. (2). Estimation of the contribution of the different P450s to the
human hepatocytes) and P450 recombinant models have been pro- metabolism of diclofenac in human hepatocytes. The percentage of con-
posed to mathematically reconstruct the relative contribution of tribution of each P450 to the in vivo formation of 5-hydroxy-diclofenac was
each P450 enzyme in the metabolism of a given compound [56, 74, estimated by applying the relative activity factor that relates the levels of
75, 81]. To this end, the formation rates of the different metabolites individual P450s in the cell lines and hepatocytes to the data shown in Fig.
in the engineered cultured cells expressing a single P450, and in (1). Reproduced from [56].
either human liver microsomes or cultured hepatocytes, are com-
6 Current Drug Metabolism, 2008, Vol. 9, No. 1 Donato et al.

P450 phenotyping can provide valuable information at leading As inhibition is an undesirable feature for a drug candidate,
identification or optimization stages, and may contribute to dis- potent P450 inhibitors should be recognized early in the drug de-
criminate compounds with unfavourable metabolic properties. In velopment process. Considerable progress has recently been made
vitro results can be used to identify new chemical entities, mainly in the development of reliable in vitro screening methods to identify
those metabolized by either polymorphic P450s (i.e. CYP2D6, potent P450 inhibitors. Simple, rapid and reproducible enzyme
2C19, 2C9, or P2A6) or a single P450 enzyme. Such properties are inhibition assays based on measurements of catalytic activities are
associated with large inter-individual variations in drug/metabolite indicative of an enzyme-inhibitor interaction [85, 86]. Human liver
levels in blood/tissue which contribute to variable pharmacological microsomes and cDNA-expressed enzymes are the test systems of
effects or to an increased incidence of drug-drug interactions. From choice for this purpose as they are more readily available than hu-
a throughput screening perspective, phenotyping studies are rec- man hepatocytes. The assays are based on the analysis of potential
ommended when a relatively high contribution (>30%) of P450- reductions in the metabolism of an appropriate P450 probe substrate
dependent oxidations to the compound clearance mechanism has in the presence of various concentrations of the tested compound
been suggested [80]. Although several P450s can be involved in [80, 87]. Probe substrate concentrations at or below the Km value,
hepatic metabolism, phenotyping can be limited to those P450, and and the validation of inhibition experiments by testing known spe-
may be considered to be the most relevant in drug metabolism cific P450 inhibitors (positive controls), are recommended (Fig. 3).
(CYP3A4, 2D6, 2C9, 2C19 and 1A2). Inhibitory effects are usually expressed as a percentage of the con-
trol activity value, and IC50 values are calculated by linear interpo-
INHIBITION STUDIES lation. The use of substrate concentrations close to the KM values of
Multidrug therapy is not uncommon in clinical practice. A si- the reaction allows the application of simple inhibition kinetic rela-
multaneous administration of several drugs may result in pharma- tions for a competitive inhibition to estimate Ki from the IC50 val-
cokinetic drug-drug interactions. Such interactions occur when the ues. These assays enable the inhibitory potency of a series of com-
disposition (i.e. absorption, distribution, metabolism and excretion) pounds to be easily compared (i.e. chemically-related structures or
of a drug is altered by another [82]. Of the pharmacokinetic factors drugs with a similar pharmacological activity) on several P450s.
that control drug action, the rate of metabolic transformation is one Applying recombinant systems to in vitro P450 inhibition as-
of the most important. Hence, interactions that result in changes in says has notably contributed to the advances in high-throughput
the rate of drug metabolism can be of great clinical significance. screening. A common approach used in testing new molecules as
Possible consequences of pharmacokinetic drug-drug interactions P450 inhibitors consists in a heterologously expressed P450 en-
include potential changes in drug levels available to bind to recep- zyme, a non-fluorescent probe (which is metabolized to a fluores-
tors and tissue targets, which can consequently alter their pharma- cent product) and the test compound [88, 89]. Fluorescence-based
cologic efficacy and/or induce cell toxicity. assays are highly sensitive and can be performed in multi-well plate
Few enzymes metabolize hundreds of drugs and other foreign formats which increase sample throughput in comparison with con-
compounds. P450 enzymes play a crucial role in metabolism and ventional analytical methods. These assays are easily adapted to
drugs and, therefore, an alteration of the P450 function is an impor- automated robotic systems and are associated with a drastic reduc-
tant mechanism for metabolic-based interactions [83, 84]. The abil- tion in analysis time and costs. However, most fluorimetric probes
ity of an individual P450 enzyme to metabolize multiple substrates are not selective for a single P450 and their application is limited to
is responsible for a large number of drug-drug interactions associ- models expressing an individual enzyme. Therefore, a major advan-
ated with P450 inhibition. Two compounds or more can compete tage of recombinant P450 systems, in relation to liver microsomes,
with each other as substrates for the same enzyme. Moreover, cer- is the possibility of using non-selective fluorimetric probes for
tain compounds are able to inhibit a particular enzyme that is not high-throughput activity assays, thus avoiding laborious and time-
directly involved in its metabolism. A vast number of molecules consuming methodologies (i.e. radiometry, HPLC separations, LC-
have been identified as competitive, non-competitive or irreversible MS/MS analysis). These assays offer the possibility of easily com-
P450 inhibitors [84]. P450 inhibition by one drug will result in paring the inhibitory potency of a series of compounds (i.e. chemi-
elevated plasma/tissue concentrations of the other drugs, which may cally-related structures or drugs with a similar pharmacological
lead to overdosage and/or toxicity for molecules with a narrow activity) on each P450. On the other hand, interpreting inhibition
therapeutic index [83]. results from recombinant systems requires cautious analysis. Since
several enzymes can be involved in the metabolism of a compound,

Fig. (3). Inhibition of individual P450 activities by model inhibitors. Intact THLE cells expressing individual P450s were incubated with fluorimetric sub-
strates in the presence of increasing concentrations of model chemical inhibitors: furafylline (CYP1A2), methoxalen (CYP2A6), tranylcypromine (CYP2B6
and CYP2C19), quercetin (CYP2C8), sulphaphenazole (CYP2C9), quinidine (CYP2D6), diethyldithiocarbamate (CYP2E1), and ketokonazole (CYP3A4).
Results are expressed as the percentage of control activity (assayed in the absence of inhibitors). Adapted from [89].
Cell Lines: A Tool for In Vitro Drug Metabolism Studies Current Drug Metabolism, 2008, Vol. 9, No. 1 7

the use of recombinant models expressing one single P450 may changes in the P450 protein levels using specific antibodies, al-
lead to an overestimation of the inhibitory effect of a given drug though a greater amount of cells is needed for protein quantifica-
(i.e. compounds actively metabolized into inactive metabolites by tion. Currently, primary human hepatocytes and human liver slices
other enzymes), or may not properly identify certain P450 inhibi- are the most accepted biological models for P450 induction studies
tors (i.e. activation of the drug by other enzymes into an inhibitory [5, 99-101]. The high inter-individual variability observed in re-
metabolite) [89, 90]. sponse to inducers and the scarcity of fresh human tissue for ex-
Although most high-throughput screening inhibition assays perimental purposes constitute serious limitations for the wide-
using heterologously expressed human enzymes are based on the spread use of such in vitro systems. Although hepatocyte cryopre-
use of recombinant P450 proteins isolated in microsomal forms, servation has facilitated their availability for metabolic studies, the
intact transgenic cells stably expressing single P450s can also been quality of cell preparations after thawing should be considerably
used for this purpose [89, 91-93] (Fig. 3). A major limitation in improved to obtain long-term hepatocyte cultures that are appropri-
making conclusive statements from assays in microsomal prepara- ate for induction assessments [102].
tions is that some processes involved in in vivo metabolism are Immortalized liver-derived cell lines were proposed as an ideal
missing in subcellular models. Among these processes, drug trans- alternative because of their unlimited availability and phenotypic
port across membranes, further metabolism by cytosolic enzymes, stability. However, studies in HepG2, the most widely human hepa-
or binding to intracellular proteins can be determinants in the actual toma cell line, showed poor inducibility, except for the extrahepatic
concentration of the substrate and inhibitor available to the P450 CYP1A1 enzyme [69, 89]. More recently, novel human liver-
[83, 94]. Given the presence of membrane barriers, assays per- derived cell lines capable of responding to prototypical P450 induc-
formed in intact cells reflect the in vivo situation more closely and ers have been generated [17, 18] (Table 3). High-throughput assays
are, in some respects, more predictive than subcellular models [83]. using Fa2N4 cells cultured in multi-well plates have been proposed
However, the fact that the chemical inhibitor must be transported to identify CYP3A4, 2C9 and/or 1A2 inducers [32, 47]. By using
through biological membranes could imply longer assay times and reference/model inducers, the relative induction potency of test
certain technical problems [89]. Moreover, only non-cytotoxic con- compounds could be evaluated [47]. These cellular systems consti-
centrations of the compound can be tested when living cells are tute promising tools for the screening of potential inducers early on
used. This should not represent a drawback for reliable and useful in drug discovery. Nonetheless, more studies are needed to confirm
screening assays, and in vitro experiments should be conducted at their utility for predicting in vivo drug-drug interactions.
concentrations similar to the relevant in vivo concentration. Although the phenomenon of P450 induction has long since
been known, only in recent years has the use of molecular biologi-
INDUCTION
cal techniques enabled the identification of mechanisms responsible
Drug-drug interactions may also occur as a consequence of for P450 induction. Most inducible P450 genes can be transcrip-
P450 induction. Hepatic drug-metabolizing P450s, CYP3A4 and tionally activated by a receptor-dependent mechanism, resulting in
CYP1A2, and to a lesser extent, CYP2C9 and CYP2B6, are induc- an up-regulated P450 expression [103]. Induction of CYP1A en-
ible by marketed drugs, which represent the basis of a number of zymes by polycyclic aromatic hydrocarbons was the first mecha-
common drug-drug interactions [95-98]. Metabolic interactions nism identified. Upon binding the inducer to cytosolic aryl hydro-
mediated by enzyme induction are far less frequent than those carbon receptors (AhR), the complex is translocated to the nucleus
caused by inhibition. However, their consequences can be clinically where heterodimerizes with the nuclear factor Arnt, and binds to an
relevant. A drug with inductive properties can accelerate its own enhancer/promotor DNA region of CYP1A [104]. PXR and CAR
metabolism, or that of another co-administered drug, and therapeu- are involved in the regulation and induction of CYP2 and CYP3A
tic inefficacy occurs as a consequence of a faster clearance. Alter- enzymes, as well as other drug-metabolizing enzymes (UGTs, sul-
natively, an exaggerated response or an increased toxicity can be photransferases, glutathione S-transferases) and transporters
take place when induced metabolic pathways are responsible for (MDR1 and MRP2) [105-108]. Once activated, PXR and CAR
converting the drug into a more active metabolite. form heterodimers with the retinoid X receptor and then bind to the
The screening of enzyme inducers is more complex and diffi- target xenobiotic response element located in P450 gene promoters,
cult than the identification of potential inhibitors. Enzyme induction resulting in the transcription of the respective enzyme [109-111].
consists in the increase of new protein synthesis in response to rele- The glucocorticoid receptor and the peroxisome proliferators-
vant stimuli. This process is a result of an increased transcription of activated receptor are other nuclear receptors known to regulate the
specific genes and involves a de novo synthesis of mRNA, protein induction of P450 enzymes [103, 112].
synthesis and a post-translational modification to an active enzyme. AhR is expressed in most immortalized cell lines, including
In drug metabolism research however, compounds capable of in- HepG2 hepatoma, and consequently, CYP1A enzymes are induc-
creasing enzymes by other mechanisms other than transcriptional ible in these cells. Meanwhile, the decreased expression of PXR
activation (i.e. mRNA or protein stabilization) are also considered and CAR, compared to that of primary hepatocytes, is likely the
inducers. reason for the low expression/induction of other P450 enzymes in
Unlike inhibition screens, which are routinely used in early most hepatoma cells. In contrast, the relatively high levels of PXR
drug development, assays that identify potential enzyme inducers seen in other liver-derived cell lines (i.e. HepaRG hepatoma) sup-
are less commonly performed and no general consensus exists as to port the inducibility of CYP3A4 in these cells [18].
how reliable these assays are. Inducers cannot be screened in sub- Increasing knowledge of the nuclear receptors mediating P450
cellular models as they require an intact cellular system that is fully induction has contributed to the development of mechanism-based
capable of expressing genes. Cells are typically treated with the test tests for induction screening. Different cell-based reporter gene
compound for 2-3 days. Then P450 levels are compared with those assays for AhR, PXR and CAR have been used as preliminary as-
of control cells. P450 induction can be easily evaluated by deter- sessments for compounds with an inductive potential [93, 113-117].
mining increases in enzyme activity using the selective probe sub- Human hepatocarcinoma, as well as cell lines with an extrahepatic
strate. Although catalytic activity represents the gold standard or non-human origin, have been manipulated to obtain new tools to
method for quantifying the in vitro induction potential of a com- easily discriminate and rank several molecules as a function of their
pound; other methods have been proposed. Measuring mRNA lev- inductive potential [118-120]. The combined use of HepG2-derived
els by RT-PCR contributes to high throughput screening and is cell lines showing distinct CYP3A4 induction profiles [121], or
particularly helpful when both enzyme inhibition and induction are cell-based bioassays for a simultaneous assessment of P450 enzyme
operative [97]. Alternatively, induction could also be assessed by induction and inhibition [91, 93], could offer additional advantages
8 Current Drug Metabolism, 2008, Vol. 9, No. 1 Donato et al.

for new molecules screening in the early stages of drug develop- ogy [128]. Stably transfected MCF-7 (human breast carcinoma) and
ment. V79 (hamster lung fibroblasts) cells, that express a range of activi-
Reporter gene assays are amenable for the high throughput ties of human ALDH isozymes, were utilized to demonstrate the
screening of compounds able to activate nuclear receptors, although functional ability of cytosolic ALDH to confer a potent resistance
the relevance of such an activation is questionable. A specific inter- to oxazaphosphorine anticancer drugs, as well as a range of lipid
action of a ligand with a single nuclear receptor fails to account for aldehydes, produced as a result of lipid peroxidation under condi-
the coordinated regulation of the P450 expression/induction by tions of cellular redox imbalance [128]. These studies suggested
multiple receptors and mechanisms. Although these assays are that ALDH-mediated and GSH-dependent cellular resistance
promising tools for the rapid identification of potential inducers, mechanisms may function cooperatively to protect against reactive
cross-talk between different elements can be misleading [122]. aldehydes. Similarly, cell lines stably transfected to overexpress
human superoxide dismutase are very useful in vitro models to
XENOBIOTIC BIOACTIVATION identify the role of reactive oxygen species and mitochondrial anti-
Drug metabolism can result in a bioactivation phenomenon oxidant status in apoptotic cell death [131].
rather than a detoxification process leading to metabolism-based To approach more complex phenomena (i.e. two-stage metabo-
toxicity. Many compounds form metabolites that exhibit a greater lism, bioactivation), cells expressing both activating and detoxify-
toxicity than the parent compounds. The phenomenon cannot be ing enzymes have been constructed (i.e. cell lines simultaneously
assessed by hepatoma cell lines as they express biotransformation expressing a form of human P450 and a Phase II enzyme) and used
activities to a very limited or negligible extent. Compounds known to investigate the bioactivation of carcinogens [62]. V79 Chinese
to exhibit toxicity via metabolism-mediated activation have differ- hamster cells were genetically engineered for the stable co-
ential in vitro cytotoxic effects on primary hepatocytes in compari- expression of human CYP1A2 and NAT2 to generate an in vitro
son with hepatoma cells showing low metabolic competence [123, tool for the study of the metabolism-dependent toxicity of aromatic
124]. In a recent study, the hepatoxicity profile of various chemical amines [63]. Previous studies in rats suggested the activation of
entities was evaluated using two in vitro hepatocyte models: WIF- these compounds by CYP1A2, followed by NAT. These activities
B9 cells (a hybrid cell line obtained by the fusion of rat Fao hepa- result in a de-acetylated compound which is highly reactive and
toma and human fibroblasts) and primary hepatocytes. The cytotox- causes cancer. In contrast, testing 2-aminofluorene in the newly-
icity ranking of compounds, based on the lactate dehydrogenase constructed V79 cell line revealed that the metabolic pathway starts
(LDH) release endpoint, was comparable in both cellular models. with the action of NAT2 in humans, followed by that of CYP1A2,
Few marked differences in the hepatotoxicity potential were ob- resulting in the formation of an acetylated, very stable and non-
served, and is likely related to the respective capacity of the models toxic metabolite [63]. Marked species-dependent differences in the
to express P450s, particularly the CYP3A enzyme [125]. metabolic activation of benzo[a]pyrene were also observed by us-
HepaRG cells could represent a surrogate to primary human ing V79 cells expressing CYP1A1 enzymes cloned from different
hepatocytes for xenobiotic metabolism and toxicity studies since species. The data suggested that human CYP1A1 is more specific
both the analysis of metabolic profiles of several compounds and for the critical 7,8,9,10 position than CYP1A1 from the other spe-
the effects of hepatotoxins suggested certain resemblance of cies tested, which resulted in a higher toxicity in the V79 cells ex-
HepaRG cells to primary hepatocytes [18]. Comparison of cyto- pressing the human CYP1A1 [132]. Ethanol-induced toxicity has
toxic effects of several reference hepatotoxicans in HepaRG and been explored in HepG2 cultures and in recombinant HepG2 cells
HepG2 cells revealed that acetaminophen and aflatoxin B1 (two transfected with alcohol dehydrogenase (ADH) and/or CYP2E1
bioactivable toxicants via the formation of electrophilic metabolites [133]. The results strongly suggest that hepatic ADH is the major
by P450-dependent reactions) were more cytotoxic to HepaRG than factor responsible for ethanol oxidative metabolism, irrespectively
to HepG2 cells [18, 35]. Similarly, the human hepatoma BC2 is of of the CYP2E1 overexpression, and that a diminished ADH activity
potential interest for the evaluation of xenobiotic-mediated cell facilitates the non-oxidative metabolism of ethanol to products that
toxicity under repeated administration conditions given its stability cause apoptosis in HepG2 cells via the intrinsic pathway. These
in culture once cells have reached confluency. Following a repeated cellular models have been suggested to be valuable in vitro tools to
dosage of a toxin, cytotoxicity is generally observed at a lower identify early ethanol-induced molecular targets as well as to ex-
concentration in comparison to a single administration [126]. plore the mechanisms underlying ethanol hepatotoxicity.
Conferring xenobiotic metabolism capability to target cells Overall, engineered cells can provide useful approaches for
would allow their use in the study of phenomena involving xenobi- metabolism-dependent toxicity assessment and for the study of
otic bioactivation and toxicity in comparison with their isogenic molecular and enzymatic mechanisms responsible for the toxicity
parental control cell line [127]. Genetically modified cell lines can and/or protection against reactive toxicants. For the time being
be very useful models for assessing the toxicological effects of the however, a limitation of genetically engineered cells is the reason
modulation of expression on selected Phase I and Phase II enzymes why only one or two enzymes can be satisfactorily transfected into
in comparison with their isogenic parental control cell lines. The cells. Transfecting more than two P450 genes proves difficult, and
role of both activating and detoxifying enzymes on cellular sensitiv- reproducing the in vivo relative abundance of each enzyme is not
ity to several toxic end-points is stressed [128]. feasible.
Several recent studies illustrate the promising use of P450- CONCLUSIONS AND OUTLOOK
expressing cell lines in the toxicity and bioactivation of drugs. Human hepatocytes are still considered the best in vitro model
HepG2 cells expressing CYP2E1 [52, 53] or CYP3A4 [129, 130] to gain a complete picture of how a drug will be metabolized in
were used to evaluate the toxicity of xenobiotics which may be vivo. However, pharmaceutical companies increasingly make use of
activated or metabolized by these P450s in comparison with control cell lines to speed up the selection of new drugs with favourable
cells. For further mimicking of liver metabolism, HepG2 cells have pharmacokinetic and metabolic properties. Moreover, new strate-
also been transformed by the pBudCE-GS-CYP3A4 vector, which gies to overcome the limitations of hepatic cell lines by generating
contains glutamine synthetase and drug-metabolizing CYP3A4 metabolically competent cell lines that mimick the performance of
genes [60]. hepatocytes are currently being developed as tools for drug metabo-
Cells expressing drug metabolizing enzymes other than P450s, lism and toxicity. In fact, cell lines expressing more than one single
such as aldehyde dehydrogenases (ALDH), superoxide dismutase, P450 enzyme (several P450s, Phase II enzymes, etc.), and which
UGTs or GSTs, are evidently of use in pharmacology and toxicol-
Cell Lines: A Tool for In Vitro Drug Metabolism Studies Current Drug Metabolism, 2008, Vol. 9, No. 1 9

are able to respond to enzyme induction, are a promising and real [6] Gmez-lechn, M.J.; Castell, J.V. (2000) in The hepatocyte review,
alternative to human hepatocytes. (Berry, M.N. and Edwards, A.M. Eds.), Kluwer Academic Publish-
ers, London, pp.11-17.
Human hepatoma-derived cell lines show very limited drug [7] Ponsoda, X.; Pareja, E.; Gmez-Lechn, M.J.; Fabra, R.; Carrasco,
metabolism activities. This is largely due to a very low expression E.; Trullenque, R.; Castell, J.V. (2001) J. Hepatol., 34(1),19-25.
of P450 genes. The repression of P450 genes in hepatic cells is [8] Grant, M.H.; Duthie, S.J.; Gray, A.G.; Burke, M.D. (1988) Mixed
apparently the consequence of several factors acting simultaneously function oxidase and UDP-glucuronyltransferase activities in the
on the cells genome because of a decreased expression of key acti- human Hep G2 hepatoma cell line. Biochem. Pharmacol., 37(21),
vating liver-enriched transcription factors (e.g. C/EBP, HNF3), a 4111-4116.
lack of co-activators (SRC-1, SRC-2), and increased levels of rep- [9] Glatt, H.; Gemperlein, I.; Setiabudi, F.; Platt, K.L.; Oesch, F.
ressors (COUP TF-I, COUP TF-II) and co-repressors (SMRT). (1990) Expression of xenobiotic-metabolizing enzymes in propa-
Finally, it is also conceivable that DNA methylation/histone deace- gatable cell cultures and induction of micronuclei by 13 com-
tylation and/or a decrease in co-activators with acetyltransferase pounds. Mutagenesis, 5(3), 241-249.
activity play a role by altering chromatin compactation and by si- [10] Montheith, D.K.; Michalopoulos, G.; Strom, S.C. (1990) Conjuga-
tion of chemical carcinogens by primary cultures of human hepato-
lencing the genes. The detection of minimal but consistent amounts
cytes. Xenobiotica, 20(8), 753-763.
of P450 mRNAs in hepatoma cells suggests that P450 genes have [11] Dharmesh, S.M.; Skelton, T.P. Baenziger, J.U. (1993) J. Biol.
not been totally silenced in these cells by mechanisms such as ex- Chem., 268(23), 17096-17102.
tensive methylation or chromatin condensation. A comparative [12] Doostdar, H.; Grant, M.H.; Melvin, W.T.; Wolf, C.R.; Burke, M.D.
analysis of four major liver transcription factors controlling the (1993) The effects of inducing agents on cytochrome P450 and
expression of typical hepatic genes (HNF1, HNF3, HNF4 and UDP-glucuronyltransferase activities in human HEPG2 hepatoma
C/EBP) between HepG2 cells and human hepatocytes showed that cells. Biochem. Pharmacol., 46(4), 629-635.
some are poorly expressed in hepatoma cells [134]. Hence, it is [13] Roe, A.L.; Snawder, J.E.; Benson, R.W., Roberts, D.W.; Casciano,
conceivable that a lack of the P450 gene expression in hepatomas is D.A. (1993) Biochem. Biophys. Res. Commun., 190(1), 15-19.
the consequence of an altered expression of key regulatory tran- [14] Donato, M.T., Bassi, A.; Gmez-Lechn, M.J.; Penco, S.; Herrero,
scription factors. However, relevant results evi-denced that it was E.; Adamo, D.; Castell, J.V; Ferro, M. (1994) In Vitro Cell Dev.
Biol. Anim., 30A, 574-580.
unlikely that restoring a single regulator factor
[15] Gmez-lechn, M.J.; Donato, M.T.; Jover, R.; Rodriguez, C.; Pon-
in hepatoma cells could up-regulate all P450 genes [66, 135]. It soda, X.; Glaise, D.; Castell, J.V.; Guguen-Guillouzo, C. (2001)
seems reasonable to expect that the re-expression of all major P450 Eur. J. Biochem., 268(5), 1448-1459.
isoforms in hepatoma cells will require the concerted action of [16] Rodriguez-Antona, C.; Donato, M.T.; Boobis, A.; Edwards, R.J.;
several liver-enriched transcription factors. Recent reports lend Watts, P.S.; Castell, J.V.; Gomez-Lechon, M.J. (2002) Xenobiotica,
support to the working hypothesis that it is possible to achieve a 32(6), 505-520.
substantial expression of drug metabolism enzymes in hepatoma [17] Mills, J.B.; Rose, K.A.; Sadagopan, N.; Sahi, J.; de Morais, S.M.
cells by the appropriate re-expression of a combination of key (2004) J. Pharmacol. Exp. Ther., 309(1), 303-309.
transcription factor(s) [65] The reactivation of non-functional [18] Aninat, C.; Piton, A.; Glaise, D ; Le Charpentier, T.; Langouet, S.;
transcription factors such as HNF4, which, surprisingly, is present Morel, F.; Guguen-Guillouzo, C.; Guillouzo, A. (2006) Drug Me-
in HepG2 cells at levels as high as in hepatocytes, may be another tab. Dispos., 34(1), 75-83.
strategy [67]. The lack of functionality of endogenously expressed [19] Abid, A.; Sobolovic, N.; Magdalou, J. (1996) Cell. Biol. Toxicol.,
12 (2), 115-123.
HNF4 is the reason why HepG2 cells lack essential co-activators [20] Abid, A.; Sabolovic, N.; Magdalou, J. (1997) Life Sci., 60(22),
for its proper functionality [36, 67]. 1943-1951.
Recent reports have shown that the tailored re-expression of [21] Whitlock, J.P.; Haruko, M.; Gelboin, H.V. (1974) J. Cell Biol.,
these factors in hepatoma cells causes a significant re-expression of 63(1), 136-145.
relevant P450s, thus opening a new experimental strategy to gener- [22] Wiebel, F.J.; Wegenke, M.; Kieger, F. (1996) Toxicol. Lett., 88(1-
ate competent cell lines for human drug metabolism studies [36]. In 3), 335-338.
addition, the generation of differentiated hepatic cells from toti- or [23] Lekas, P.; Tin, K.L.; Lee, C.; Prokipcak, R.D. (2000) Arch. Bio-
chem. Biophys., 384(2), 311-318
multipotent stem cells is a promising, yet still immature technology.
[24] Wilkening, S.; Stahl, F.; Bader, A. (2003) Drug Metab. Dispos,.
The development of hepatic cell lines from hepatic stem cells, 31(8), 1035-1042.
transdifferentiation from either non-hepatic progenitor cells or em- [25] Corcos, L.; Weiss, M.C. (1988) FEBS Lett., 233(1), 37-40.
bryonic stem cells, has been partially achieved by few researchers. [26] Karenlampi, S.O.; Tuomi, K.; Korkalainen, M.; Raunio, H. (1989)
These hepatocyte-like cells showed relevant hepatic features and, Biochem. Pharmacol., 38(9), 1517-1525.
in some cases, a certain degree of xenobiotic metabolism capability. [27] Kobliakov, V.; Kulikova, L.; Samoilov, D.; Lang, M.A. (1993)
Mol. Carcinog., 7(4), 276-280.
ACKNOWLEDGEMENTS [28] Enosawa, S.; Suzuki, S.; Kakefuda, T.; Amemiya, H. (1996) Cell
This work was supported by the ALIVE Foundation, and funds Transplant., 5(5 Suppl 1)S, 39-40.
from the European Commission, grants LSHB-CT-2004-504761, [29] Hanioka, N.; Obika, N.; Nishimura, M.; Jinno, H.; Tanaka-
Kagawa, T.; Saito, K.; Kiryu, K.; Naito, S.; Narimatsu, S. (2006)
LSHB-CT-2004-512051 and LSSB-CT-2005-037499.
Food Chem. Toxicol., 44(8), 1251-1260.
REFERENCES [30] Coroneos, E.; Gordon, J.W.; Kelly, S.L.; Wang, P.D.; Sim, E.
(1991) Biochim. Biophys. Acta, 1073(3), 593-599.
[1] Lin, J.; Sahakian, D.C.; de Morais, S.M.; Xu, J.J.; Polzer, R.J.; [31] Sumida, A.; Fukuen, S.; Yamamoto, I.; Matsuda, H.; Naohara, M.;
Winter, S.M. (2003) Curr. Top. Med. Chem., 3 , 1125-1154. Azuma, J. (2000) Biochem. Biophys. Res. Commun., 267(3), 756-
[2] Spatzenegger, M.; Jaeger, W. (1995) Drug Metab. Rev., 27(3), 397- 760.
417. [32] Youdim, K.A.; Tyman, C.A.; Jones, B.C.; Hyland, R. (2007) Drug
[3] Gmez-Lechn, M.J.; Donato, T.; Ponsoda, X.; Fabra, R.; Trullen- Metab. Dispos., 35(2), 275-282.
que, R.; Castell, J.V. (1997) in In vitro Methods in Pharmaceutical [33] Rencurel, F.; Stenhouse, A.; Hawley, S.A.; Friedberg, T.; Hardie,
Research, (Castell, (J.V. and Gmez-Lechn, M.J. Eds.) Academic D.G; Sutherland, C.; Wolf, C.R. (2005) J. Biol. Chem., 280(6),
Press, New York , pp. 129-154. 4367-4373.
[4] Gmez-lechn, M.J.; Donato, M.T.; Castell, J.V.; Jover, R. (2003) [34] O'connor, J.E.; Martinez, A.; Castell, J.V.; Gmez-Lechn, M.J.
Curr. Drug Metab., 4(4), 292-312. (2005) Cytometry A, 63(1), 48-58.
[5] Gmez-Lechn, M.J.; Donato, M.T.; Castell, J.V.; Jover, R. (2004) [35] Guillouzo, A.; Corlu, A.; Aninat, C.; Glaise, D.; Morel, F.;
Curr. Drug Metab., 5(5), 443-462. Guguen-Guillouzo, C. (2007) Chem. Biol. Interact., 168(1), 66-73.
10 Current Drug Metabolism, 2008, Vol. 9, No. 1 Donato et al.

[36] Castell, J.V.; Jover, R.; Martinez-Jimenez, C.P.; Gomez-Lechon, [68] Lancon, A.; Hanet, N.; Jannin, B.; Delmas, D.; Heydel, J.M.; Li-
M.J. (2006) Expert Opin. Drug Metab. Toxicol., 2(2), 183-212. zard, G.; Chagnon, M.C.; Artur, Y.; Latruffe, N. (2007) Drug Me-
[37] Sack, G.H. Jr.; Obie, C. (1981) Exp. Cell Res., 134(2), 425-432. tab. Dispos., 35(5),699-703.
[38] Fischbach, M.; Cao, H.W.; Diez Ibanez, M.; Tsaconas, C.; Alouani, [69] Zhang, Z.Y.; Pelletier, R.D.; Wong, Y.N.; Sugawara, M.; Zhao, N.;
S.; Montandon, F., el Baraka, M.; Padieu, P.; Dreano, M.; Chesse- Littlefield, B.A. (2006) Biochem. Biophys. Res. Commun., 341(2),
beuf-Padieu, M. (1991) Cell Biol. Toxicol., 7(4), 327-345. 399-407.
[39] Osanai, M.; Ogawa, K.; Lee, G.H. (1997) Cancer Res., 57(14), [70] Brandon, E.F.; Meijerman, I.; Klijn, J.S.; den Arend, D.; Sparidans,
2896-2903. R.W.; Lazaro, L.L.; Beijnen, J.H.; Schellens, J.H. (2005) Antican-
[40] Yanai, N.; Suzuki, M.; Obinata, M. (1991) Exp. Cell Res., 197(1), cer Drugs, 16(9), 935-943.
50-56. [71] Pinti, M.; Troiano L.; Nasi, M.; Ferraresi, R.; Dobrucki, J.;
[41] Klocke, R.; Gomez-Lechon, M.J.; Ehrhardt, A.; Mendoza-Figu- Cossarizza, A. (2003) J. Biol. Regul. Homeost. Agents, 17(2),166-
eroa, T.; Donato, M.T.; Lopez-Revilla, R.; Castell, J.V.; Paul, D. 171.
(2002) Biochem. Biophys. Res. Commun., 294(4), 864-871. [72] Bhadauria, S.; Singh, G.; Sinha, N.; Srivastava, S. (2007) Cell Mol.
[42] Widman, L.E.; Golden, J.J.; Chasin, L.A. (1979) J. Cell Physiol., Biol., 53(1), 102-114.
100(3), 391-400. [73] Schoonen, W.G.; de Roos, J.A.; Westerink, W.M.; Debiton, E.
[43] Cassio, D.; Hamon-Benais, C.; Guerin M, Lecoq, O. (1991) J. Cell. (2005) Toxicol. In Vitro, 19(4), 491-503.
Biol., 115(5), 1397-1408. [74] Chang, Y.; Moody, D.E.; McCamce-Katz, E.F. (2006) Drug Me-
[44] Bayad, J.; Bagrel, D.; Sabolovic, N.; Magdalou, J.; Siestm G. tab. Dispos., 34, 440-448.
(1991) Biochem. Pharmacol., 42(7), 1345-1351. [75] Emoto, C.; Murase, K.; Iwasaki, K. (2006) Xenobiotica, 36, 671-
[45] Roberts, E.A.; Letarte, M.; Squire, J.; Yang, S. (1994) Hepatology, 683.
19(6), 1390-1399. [76] Rodrigues, A.D. (1999) Biochem. Pharmacol., 57, 465-480.
[46] Donato, M.T.; Klocke, R.; Castell, J.V.; Stenzel, K.; Paul, D.; [77] Venkatakrishnan, K.; von Moltke, L.L.; Court, M.H.; Harmatz,
Gmez-Lechn, M.J. (2003) Xenobiotica, 33(5), 459-473. J.S.; Crespi, C.L.; Greenblatt, D.J. (2000) Drug Metab. Dispos., 28,
[47] Ripp, S.L.; Mills, J.B.; Fahmi, O.A.; Trevena, K.A.; Liras, J.L.; 1493-1504.
Maurer, T.S.; de Morais, S.M. (2006) Drug Metab. Dispos., 34(10), [78] Venkatakrishnan, K., von Moltke, L.L.; Greenblatt, D.J. (2001a) J.
1742-1748. Pharmacol. Exp. Ther., 297, 326-337.
[48] Tang, W.; Wang, R.W.; Lu, A.Y.; (2005) Curr. Drug Metab., 6, [79] Masimirembwa, C.M.; Thompson, R.; Andersson, T.B. (2001)
503-517. Comb. Chem. High Throughput Screen., 4(3), 245-263.
[49] Pritchard, M.P.; Glancey, M.J.; Blake, J.A.; Gilham, D.E.; [80] Bjornsson, T.D.; Callaghan, J.T.; Einolf, H.J.; Fischer, V.; Gan, L.;
Burchell, B.; Wolf, C.R.; Friedberg, T. (1998) Pharmacogenetics, Grimm, S.; Kao, J.; King, S.P.; Miwa, G.; Ni, L.; Kumar, G.; Mc-
8(1), 33-42. Leod, J.; Obach, R.S.; Roberts, S.; Roe, A.; Shah, A.; Snikeris, F.;
[50] Chen, L.; Buters, J.T.; Hardwick, J.P.; Tamura, S.; Penman, B.W.; Sullivan, J.T.; Tweedie, D.; Vega, J.M.; Walsh, J.; Wrighton, S.A.
Gonzalez, F.J.; Crespi, C.L. (1997) Drug Metab. Dispos., 25(4), (2003) Drug Metab. Dispos., 31(7), 815-832.
399-405. [81] Venkatakrishnan, K.; Von Moltke, L.L.; Greenblatt, D.J. (2001b) J.
[51] Yoshitomi, S.; Ikemoto, K.; Takahashi, J.M.; Miki, H.; Namba, M.; Clin. Pharmacol., 41(11), 1149-1179.
Asahi, S. (2001) Toxicol. In Vitro, 15(3), 245-256. [82] Singh, S.S. (2006) Curr. Drug Metab., 7, 165-182.
[52] Lu, Y.; Cederbaum, A.I. (2006) Toxicol. Sci., 89(2), 515-523. [83] Lin, J.H.; Lu, A.Y.H. (1998) Clin. Pharmacokinet., 35 , 361-390.
[53] Zhuge, J.; Luo, Y.; Yu, Y.N. (2003) World J. Gastroenterol., 9(12), [84] Donato, M.T.; Gmez-Lechn, M.J. (2006) Curr. Enzyme Inhibiti-
2732-2736. on, 2, 281-304.
[54] Zhuge, J.; Yu, Y.N.; Wu, X.D. (2004) World J. Gastroenterol., [85] Moody, G.C.; Griffin, S.J.; Mather, A.N.; McGinnity, D.F.; Riley,
10(2), 234-237. R.J. (1999) Xenobiotica, 29, 53-75.
[55] Mac, K.; Offord, E.; Pfeifer, A. (1997) in In vitro Methods in [86] Atkinson, A.; Kenny, J.R.; Grime, K. (2005) Drug Metab. Dispos.,
Pharmaceutical Research (Castell J.V. and Gmez-Lechn M.J. 33, 1637-1647.
Eds.), Academic Press, New York, pp. 433-456. [87] Baranczewski, P.; Stanczak, A.; Sundberg, K.; Svensson, R.; Wal-
[56] Bort, R.; Mac, K.; Boobis, A.; Gmez-Lechn, M.J.; Pfeifer, A.; lin, A.; Jansson, J.; Garberg, P.; Postlind, H. (2006) Pharmacol.
Castell, J.V. (1999) Biochem. Pharmacol., 58 , 787-796. Rep., 58, 453-472.
[57] Crespi, C.L.; Miller, V.P. (1999) Pharmacol. Ther., 84 ,121-131. [88] Cohen, L.H.; Remley, M.J.; Raunig, D. and Vaz, A.D. (2003) Drug
[58] Schneider, A.; Schmalix, W.A.; Siruguri, V.; de Groene, E.M.; Metab. Dispos., 31(8), 1005-1015.
Horbach, G.J.; Kleingeist, B.; Lang, D.; Bocker, R.; Belloc, C.; [89] Donato, M.T.; Jimenez, N.; Castell, J.V. and Gomez-Lechon, M.J.
Beaune, P.; Greim, H.; Doehmer, J. (1996) Arch. Biochem. Bio- (2004) Drug Metab. Dispos., 32(7), 699-706.
phys., 332(2), 295-304. [90] Yueh, M.F.; Kawahara, M.; Raucy, J. (2005) Toxicol. In Vitro,
[59] Krebsfaenger, N.; Murdter, T.E.; Zanger, U.M.; Eichelbaum, M.F.; 19(2), 275-287.
Doehmer, J. (2003) ALTEX, 20(3), 143-154. [91] Yueh, M.F.; Kawahara, M.; Raucy, J. (2005) (b) Drug Metab.
[60] Omasa, T.; Kim, K.; Hiramatsu, S.; Katakura, Y.; Kishimoto, M.; Dispos., 33(1), 38-48.
Enosawa, S.; Ohtake,H. (2005) Biotechnol. Prog., 21(1), 161-167. [92] Trubetskoy, O.; Marks, B.; Zielinski, T.; Yueh, M.F.; Raucy, J.
[61] Akiyama, I.; Tomiyama, K.; Sakaguchi, M.; Takaishi, M.; Mori, (2005) AAPS J., 7(1), E 6-13.
M.; Hosokawa, M.; Nagamori, S.; Shimizu, N.; Huh, N.H.; Miya- [93] Ito, K.; Iwatsubo, T.; Kanamitsu, S.; Ueda, K.; Suzuki, H.; Sugi-
zaki, M. (2004) Int. J. Mol. Med., 14(4), 663-668. yama, Y. (1998) Pharmacol. Rev., 50, 387-412.
[62] Sawada, M.; Kamataki, T. (1998) Mutat. Res., 411(1), 19-43. [94] Finch, C.K.; Chrisman, C.R.; Baciewicz, A.M.; Self, T.H. (2002)
[63] Scheuenpflug, J.; Krebsfanger, N.; Doehmer, J. (2005) Altern. Lab. Arch. Intern. Med., 162(9), 985-992.
Anim., 33(6), 561-577. [95] Meunier, V.; Bourrie, M.; Julian, B.; Marti, E.; Guillou, F.; Berger,
[64] Goldring, C.E.; Kitteringham, N.R.; Jenkins, R.; Lovatt, C.A.; Y.; Fabre, G. (2000) Xenobiotica, 30(6), 589-607.
Randle, L.E.; Abdullah, A.; Owen, A.; Liu, X.; Butler, P.J.; Willi- [96] Luo, G.; Cunningham, M.; Kim, S.; Burn, T.; Lin, J.; Sinz, M.;
ams, D.P.; Metcalfe, P.; Berens, C.; Hillen, W.; Foster, B.; Simp- Hamilton, G.; Rizzo, C.; Jolley, S.; Gilbert, D.; Downey, A.; Mu-
son, A.; McLellan, L.; Park, B.K. (2006) Am. J. Physiol. Cell Phy- dra, D.; Graham, R.; Carroll, K., Xie, J.; Madan, A.; Parkinson, A.;
siol., 290(1), C 104-115. Christ, D.; Selling, B.; LeCluyse, E.; Gan, L.S. (2002) Drug Metab.
[65] Rodriguez-Antona, C.; Bort, R.; Jover, R.; Tindberg, N.; Ingelman- Dispos., 30(7), 795-804.
Sundberg, M.; Gomez-Lechon, M.J.; Castell, J.V. (2003) Mol. [97] Madan, A.; Graham, R.A.; Carroll, K.M.; Mudra, D.R.; Burton,
Pharmacol., 63(5), 1180-1189. L.A.; Krueger, L.A.; Downey, A.D.; Czerwinski, M.; Forster, J.;
[66] Bort, R.; Gomez-Lechon, M.J.; Castell, J.V.; Jover, R. (2004) Arch. Ribadeneira, M.D.; Gan, L.S.; LeCluyse, E.L.; Zech, K.; Robert-
Biochem. Biophys., 426(1), 63-72. son, P.; Koch, P.; Antonian, L.; Wagner, G.; Yu, L.; Parkinson, A.
[67] Martinez-Jimenez, C.P.; Gomez-Lechon, M.J.; Castell, J.V.; Jover, (2003) Drug Metab. Dispos., 31(4), 421-431.
R. (2006) J. Biol. Chem., 281(40), 29840-29849. [98] LeCluyse, E.L. (2001) Eur. J. Pharm. Sci., 13(4), 343-368.
Cell Lines: A Tool for In Vitro Drug Metabolism Studies Current Drug Metabolism, 2008, Vol. 9, No. 1 11

[99] Edwards, R.J.; Price, R.J.; Watts, P.S.; Renwick, A.B.; Tredger, [117] Wang, K.; Mendy, A.J.; Dai, G.; Luo, H.R.; He, L.; Wan, Y. (2006)
J.M.; Boobis, A.R.; Lake, B.G. (2003) Drug Metab. Dispos., 31(3), J. Toxicol. Sci., 92(1), 51-60.
282-288. [118] El-Sankary, W.; Gibson, G.G.; Ayrton, A.; Plant, N. (2001) Drug
[100] Nomeir, A.A.; Ruegg, C.; Shoemaker, M.; Favreau, L.V.; Pala- Metab. Dispos., 29(11), 1499-1504.
manda, J.R.; Silber, P. and Lin, C.C. (2001) Drug Metab. Dispos., [119] Puzyn, T.; Falandysz, J.; Jones, P.D.; Giesy, J.P. (2007) J. Environ.
29(5), 748 -753. Sci. Health A Tox. Hazard Subst. Environ. Eng., 42(5), 573-590.
[101] Kafert-Kasting, S.; Alexandrova, K.; Barthold, M.; Laube, B.; [120] Sinz, M.; Kim, S.; Zhu, Z.; Chen, T.; Anthony, M.; Dickinson, K.;
Friedrich, G.; Arseniev, L.; Hengstler, J.G. (2006) Toxicology, Rodrigues, A.D. (2006) Curr. Drug Metab., 7(4), 375-388.
220(2-3), 117-125. [121] Noracharttiyapot, W.; Nagai, Y.; Matsubara, T.; Miyata, M.; Shi-
[102] Garcia, M.; Rager, J.; Wang, Q.; Strab, R.; Hidalgo, I.J.; Owen, A.; mada, M.; Nagata, K.; Yamazoe, Y. (2006) Drug Metab. Pharma-
Li, J. (2003) In Vitro Cell Dev. Biol. Anim., 39(7), 283-287. cokinet., 21(2), 99-108.
[103] Waxman, D.J. (1999) Arch. Biochem. Biophys., 369(1), 11-23. [122] Gerbal-Chaloin, S.; Pichard-Garcia, L.; Fabre, J.M.; Sa-Cunha, A.;
[104] Whitlock, J.P. (1999) Ann. Rev. Pharmacol. Toxicol., 39, 103-125. Poellinger, L.; Maurel, P.; Daujat-Chavanieu, M. (2006) Cell. Si-
[105] Kullak-Ublick, G.A.; Becker, M.B. (2003) Drug Metab. Rev., gnal., 18(5), 740-750.
35(4), 305-317. [123] Wang, K.; Shindoh, H.; Inoue, T.; Horii, I. (2002) J. Toxicol. Sci.,
[106] Saini, S.P.; Sonoda, J.; Xu, L.; Toma, D.; Uppal, H.; Mu, Y.; Ren, 27(3), 229-237.
S.; Moore, D.D.; Evans, R.M.; Xie, W. (2004) Mol. Pharmacol., [124] Mingoia, R.T.; Nabb, D.L.; Yang, C.H.; Han, X. (2007) Toxicol. In
65(2), 292-300. Vitro, 21(1), 165-173.
[107] Verreault, M.; Senekeo-Effenberger, K.; Trottier, J.; Bonzo, J.A.; [125] Biagini, C.P.; Boissel, E.; Borde, F.; Bender, V.E.; Bouskila, M.;
Belanger, J.; Kaeding, J.; Staels, B.; Caron, P.; Tukey, R.H.; Bar- Blazy, F.; Nicaise, L.; Mignot, A.; Cassio, D.; Chevalier, S. (2006)
bier, O. (2006) Hepatology, 44(2), 368-378. Toxicol. In Vitro, 20(6), 1051-1059.
[108] Wang, H.; LeCluyse, E.L. (2003) Clin. Pharmacokinet., 42(15), [126] Fabre, N.; Arrivet, E.; Trancard, J.; Bichet, N.; Roome, N.O.;
1331-1357. Prenez, A.; Vericat, J.A. (2003) Cell Biol. Toxicol., 19(2), 71-82.
[109] Goodwin, B.; Hodgson, E., Liddle, C. (1999) Mol. Pharmacol., [127] Langenbach, R.; Smith, P.B.; Crespi. C. (1992) Mutat. Res., 277(3),
56(6), 1329-1339. 251-275.
[110] Sueyoshi, T.; Kawamoto, T.; Zelko, I.; Honkakoski, P.; Negishi, [128] Townsend, A.J.; Kiningham, K.K.; St. Clair, D.; Tephly, T.R.;
M.T. (1999) J. Biol. Chem., 274(10), 6043-6046. Morrow, C.S.; Guengerich, F.P. (1999) Toxicol. Sci., 48(2), 143-
[111] Moore, L.B.; Parks, D.J.; Jones, S.A.; Bledsoe, R.K.; Consler, 150.
T.G.; Stimmel, J.B.; Goodwin, B.; Liddle, C.; Blanchard, S.G.; [129] Holownia, A.; Braszko, J.J. (2004) Biochem. Pharmacol., 67(6),
Willson, T.M.; Collins, J.L.; Kliewer, S.A. (2000) J. Biol. Chem., 1057-1064.
275(20), 15122-15127. [130] Vignati, L.; Turlizzi, E.; Monaci, S.; Grossi, P.; Kanter, R.; Mons-
[112] Gerbal-Chaloin, S.; Daujat, M.; Pascussi, J.M.; Pichard-Garcia, L.; houwer, M. (2005) Toxicolog, 216(2-3), 154-167.
Vilarem, M.J.; Maurel, P. (2002) J. Biol. Chem., 277(1), 209-217. [131] Majima, H.J.; Oberley, T.D.; Furukawa, K.; Mattson, M.P.; Yen,
[113] Honkakoski, P.; Jaaskelainen, I.; Kortelahti, M.; Urtti, A. (2001) H.C.; Szweda, L.I.; St Clair, D.K. (1998) J. Biol. Chem., 273(14),
Pharm. Res., 18(2), 146-150. 8217-8224.
[114] Persson, K.P.; Ekehed, S.; Otter, C.; Lutz, E.S.; McPheat, J.; Ma- [132] Doehmer, J. (2006) Altern. Lab. Anim., 34(6), 561-575.
simirembwa, C.M.; Andersson, T.B. (2006) Pharm. Res., 23(1), 56- [133] Wu, H.; Cai, P.; Clemens, D.L.; Jerrells, T.R.; Ansari, G.A.;
69. Kaphalia, B.S. (2006) Toxicol. Appl. Pharmacol., 216(2), 238-247.
[115] Nallani, S.C.; Goodwin, B.; Buckley, A.R.; Buckley, D.J.; Desai, [134] Jover, R.; Bort, R., Gmez-Lechn, M.J.; Castell, J.V. (1998)
P.B. (2004) Cancer Chemother. Pharmacol., 54(3), 219-229. FEBS Lett., 431(2), 227-230.
[116] Ogg, M.S.; Williams, J.M.; Tarbit, M.; Goldfarb, P.S.; Gray, T.J.; [135] Schrem, H.; Klempnauer, J.; Borlak, J. (2004) Pharmacol. Rev.,
Gibson, G.G. (1999) Xenobiotica, 29(3), 269-279. 56(2), 291-330.

Received: September 05, 2007 Revised: September 28, 2007 Accepted: October 01, 2007