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
After intake, drugs are usually transformed into new chemical
species that may have either toxic or therapeutic effects. The phar-
macological response of a drug is primarily the result of its effec-
tive interaction with the target which is linked to the concentration
reaching it. Pharmacokinetics (absorption, distribution, metabolism
and excretion) of the compound also plays a key role in its efficacy
as it governs the drug concentration at the site of action [1]. There-
fore, inappropriate pharmacokinetics can result in an inadequate or
variable concentration of the drug at the site of action and in great
variations in the clinical response. Drug metabolism is a major de-
terminant of drug clearance and inter-individual pharmacokinetic
differences, and is an indirect determinant of the clinical efficacy
and toxicity of drugs [2]. The pharmaceutical industry is committed
to marketing safer drugs with fewer side effects, predictable phar-
macokinetic properties and quantifiable drug-drug interactions.
Therefore, it is desirable that poorly behaved compounds are re-
moved early in the discovery phase rather than during the more
costly drug development phases.
Gaining knowledge on the metabolism of a given drug, the
enzymes so far involved, and on their inhibition or induction poten-
tial is a necessary step in the pharmaceutical development of new
compounds. The ultimate goal of these efforts is to ascertain: a)
how the molecule is biotransformed and whether the produced me-
tabolites contribute to the pharmacological action of the drug
(metabolic profile); b) which enzymes are involved in its biotrans-
formation and whether this could be influenced by the concomitant
use of other drugs, c) whether the drug itself influences the expres-
sion or the activity of such enzymes which, in turn, could influence
its metabolism and also that of other drugs and d) the drug toxicity
associated with drug metabolism.
Primary cultured hepatocytes, liver slices and microsomes are
valuable in vitro models for drug metabolism studies [3]. Human
hepatocytes are fully competent metabolic cells. They retain the
*Address correspondence to this author at the Centro de Investigacin,
Hospital La Fe, Avda de Campanar 21, 46009-Valencia, Spain; Tel: +34 96
1973040; Fax: +34 96 1973018; E-mail: gomez_mjo@gva.es
expression of both Phase I and II enzymes for several days in cul-
ture [3-6], and are capable of generating a metabolic profile of a
drug similar to that found in vivo [7], which is when they are con-
sidered the gold standard model for xenobiotic metabolism and
toxicity 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 prepara-
tions obtained from different human liver donors, seriously compli-
cate their use in routine testing.
To overcome these limitations, different cell line models have
been proposed for drug metabolism screening in recent years. Hu-
man 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. Unfortunately, most hepatic
cell lines have not constituted a real alternative to primary cultured
hepatocytes for drug metabolism studies given their very
low/partial expression of cytochrome P-450 (P450). New strategies
are currently being used to up-regulate the expression of drug me-
tabolizing enzymes in these cells. This paper reviews the features of
liver-derived cell lines and their suitability for drug metabolism
studies, as well as the strategies based on the use of genetically
engineered cells expressing individual drug metabolizing enzymes.
HUMAN LIVER-DERIVED CELL LINES
Hepatic cell lines have several advantages when compared with
primary cultured human hepatocytes. Hepatic cell lines grow con-
tinuously, have almost an unlimited life-span and have quite a sta-
ble phenotype. Cell lines are easily available, culture conditions are
simpler than primary hepatocytes, and easily standardized among
laboratories. In addition, some hepatomas retain (in part) a differen-
tiated adult phenotype. These features make hepatic cell lines po-
tentially appropriate for screening purposes. Human hepatoma cell
lines have been widely used as an in vitro model for the study of
hepatocellular functions, as well in toxicity studies. Concerning
drug metabolism, one major drawback is that hepatoma cells ex-
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-
tion to that of the normal adult liver (Table 1). Decreased activity
levels are found particularly for the P450 reactions involved in the
oxidative metabolism of drugs and xenobiotics. The low P450 sys-
tem functionality is likely due to an impaired expression of the
P450 enzymes, as hepatoma cell lines express sufficient levels of
functional NADPH cytochrome P450 reductase (CPR) that would
enable their P450s to act. Specific mRNA levels of major hepatic
P450 enzymes in human hepatoma cell lines and in primary hepato-
cytes are comparatively shown in Table 2. Frequently, the non-
hepatic isoenzymes are expressed, (e.g. CYP1A1, instead of
CYP1A2).The CYP1A1 mRNA decays remarkably quickly (half-
life of 2.4 h), and is one of the most unstable of the P450 mRNAs.
The rapid decay of CYP1A1 mRNA is associated with a rapid loss
in poly(A) tail length, suggesting that deadenylation is the first step
in the decay pathway. The short half-life appears to be conserved
across species, which suggests that this characteristic of the
CYP1A1 mRNA is important for its function [23].
Hepatoma cell lines also express conjugating enzymes, namely
glutathione S-transferase (GST) and UDP-glucuronyltransferase
(UGT) and sulphotranferases (Table 1) [14, 15, 25-28]. For in-
stance, KYN-2 and Mz-Hep-1 cells express one of the two different
UGT isoforms found in hepatocytes [20]. Furthermore, the induci-
bility of UGT1A isoforms in HepG2 cells by
-naphthoflavone has
been reported [29]. Quite frequently however, the isozymes ex-
pressed are not those found in adult liver tissue. For instance, N-
acetyltransferase (NAT) in hepatoma cells has an activity profile
that resembles extra-hepatic tissue NATs rather than liver NATs
(NAT1 predominating over NAT2). Thus, hepatoma cell lines do
not represent a good in vitro model to investigate the human me-
tabolism of arylamines or hydrazines in the liver [30].
Few authors have reported the ability of hepatoma cell lines to
respond to inducers (Table 3). The response of these cells, in terms
of enzyme activity, is very poor except for the non-hepatic isoform
CYP1A1, which is easily inducible [14, 20, 26-28]. However, it is
possible to monitor the induction of some P450 isoforms (i.e.
CYP3A4, 2C9) by making use of either reverse transcriptase-
polymerase chain reaction (RT-PCR) techniques to quantify tran-
scription levels of genes [17, 31] or a cocktail of P450 substrate
probes [32] to assess increases in P450 protein activity.
HepG2 is among the most widely used human hepatoma cell
line. These cells show many liver-specific functions, express conju-
gating enzymes, but lack a functional expression of almost all the
relevant human liver P450s. Consequently, they are a very poor
alternative to primary cultured cells. The activity and protein con-
tent of most P450s is undetectable by conventional methods, and
only very sensitive techniques like RT-PCR detect and quantify the
transcription levels of genes. Most of the P450 isoforms (i.e.
CYP2B6, 2C9, 3A4) examined in HepG2 presented values that
were 2-3 orders of magnitude lower than in hepatocytes [16] (Table
2). A novel differentiated human hepatoma cell line, WGA, derived
from HepG2 and which expresses CYP2B6 and constitutive andro-
stane receptor (CAR), has been proposed as a powerful system to
study the regulation of the CYP2B6 gene expression by phenobar-
bital [33].
BC2 is a human hepatocarcinoma-derived cell line possessing
the capacity of growing and of undergoing a spontaneous differen-
tiation in vitro after confluence. Once they reach this status, cells
remain stably differentiated in culture for several weeks, but may
return to a proliferative status (hence be de-differentiated) upon
sub-culturing. Differentiated BC2 cells express the most relevant
P450 isozyme mRNAs (CYP1A1/2, 2A6, 2B6, 2C9, 2E1, and 3A4)
(Table 2). Both CYP3A4 activity and conjugating enzymes (GST
and UGT) are well expressed in these cells. In addition, BC2 cells
show the ability to respond to enzyme inducers. A previous study
showed that BC2 is able to increase P450 activities in response to
methylcholantrene, phenobarbital, rifampicin and dexamethasone
[15]. Although P450 activities in BC2 were markedly lower than in
human hepatocytes, the effects elicited by some of these model
inducers were, in relative terms, comparable to those observed in
human cellular models. Further work on BC2 cells showed a het-
erogeneous expression of P-glycoprotein and other relevant markers
[34].
A new human hepatoma cell line, named HepaRG, derived
from a human hepatocellular carcinoma, also shows features of a
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 T6
OH 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-6
OH (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 T6
OH 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 (T6
OH) 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
divide, and after having reached confluency, form typical hepato-
cyte-like colonies surrounded by biliary epithelial-like cells.
Moreover, and contrary to other human hepatoma cell lines, includ-
ing the HepG2 cells, HepaRG cells not only express various P450s
(CYP1A2, 2B6, 2C9, 2E1, 3A4), but also the nuclear receptors
CAR and pregnane X receptor (PXR) at levels comparable to those
found in cultured primary human hepatocytes. They also express
other specific markers of adult hepatocytes, including phase II en-
zymes and drug transporters, and are able to respond to selective
inducers of the P450 enzymes [35].
In view of the limited expression of drug-metabolizing enzymes
in most hepatoma cell lines, alternative approaches have been ex-
plored to obtain immortalized hepatocytes from a non-hepato-
carcinoma origin (for a review, see [36]). A successful immortaliza-
tion of normal hepatocytes from different species was achieved
using different strategies including cell transformation with virus
genes or oncogenes (i.e. simian virus 40 large T antigen, c-myc, cH-
ras) [37-39], the generation of cell lines from transgenic animals
expressing viral transforming genes, oncogenes or growth factors
[40, 41], or hydrid cells obtained by the fusion of hepatocytes and
immortalized cell lines [42,43]. Although some hepatic functions
were maintained following cell transformation, these cells show a
very low expression of P450 enzymes, which, in general, makes
them unsuitable for drug metabolism studies [44-46]. Recently, the
drug-metabolizing capacity of Fa2N-4, a non-tumorigenic immor-
talized human hepatic cell line, has been examined. The P450 ex-
pression in Fa2N-4 cells is markedly lower than those of primary
human hepatocytes [17]. However, increases in specific P450 ac-
tivities can be observed after exposure to inducers (Table 3). An
evaluation of CYP1A2, CYP3A4, CYP2C9, UGT1A, and MDR1
mRNA levels in Fa2N-4 cells treated with a panel of known induc-
ers showed that Fa2N-4 cells responded in a similar way as primary
hepatocytes did [17]. These cells have been proposed as a robust
model system for induction studies [32] to identify CYP3A4 induc-
ers in particular [47].
GENETICALLY ENGINEERED CELLS EXPRESSING HU-
MAN DRUG METABOLIZING ENZYMES
Advances in molecular biology have enabled the possibility of
cloning and heterologously expressing genes of human drug-
metabolizing enzymes. In recent years, recombinant human models
expressing heterologous genes of human P450s, and other genes
involved in drug metabolism in different host cells, have been de-
veloped by using expression vectors which encode for human en-
zymes [48]. Genetically engineered cells expressing human drug-
metabolizing enzymes have become new potent tools for investigat-
ing drug metabolism, drug-drug interactions and the metabolism-
based activation of chemicals.
A variety of expression systems using bacteria, yeast, insect
cells, and mammalian cells (-lymphoblastoid cells, COS cells,
V79 Chinese hamster ovary cells, HepG2 cell line) have been suc-
cessfully transfected to transiently or stably express P450 genes.
The catalytic activity of a CYP-transfected cell relies not only on
the efficient expression of the transgene, but also on the endoge-
nous CPR activity. Hepatoma cells express sufficient CPR to ensure
sufficient monooxygenase activity, while this can represent a limit-
ing step in other cells. As a means to increase the catalytic activity
of CYP-transfected cells, the co-expression of the P450 gene, to-
gether with CPR, has been successfully achieved [49,50]. Different
cell lines have been engineered to individually express either spe-
cific human P450s (CYP1A1, 1A2, 2A6, 2B6, 2B8, 2C6, 2C8, 2C9,
2C19, 2D6, 2E1 and 3A4) or combinations of the xenobiotic me-
tabolizing enzymes. Among mammalian cell expression systems,
liver-derived cell lines HepG2 [51-54] and human liver epithelial
cells (THLE), a non-tumorigenic SV40-immortalized human liver
epithelial cell line which retained most of the phase II enzymes
[55,56], have been successfully transfected to express human P450
4 Current Drug Metabolism, 2008, Vol. 9, No. 1
genes. Non-hepatic cell lines AHH-1, a human B lymphoblastoid
cell line [57], and V79 Chinese hamster cells [58,59] have also been
genetically engineered for the stable expression of human P450
genes. The P450 expression is confirmed by the determination of
enzyme activities and by the RT-PCR procedure. Recombinant
enzymes show catalytic properties comparable to those of human
liver microsomes, and the activity levels of the expressed P450
subtypes are similar or higher when compared to human hepato-
cytes. Heterologously expressed individual P450s are commercially
available and can be easily used in high-throughput metabolic
screening assays, in the phenotyping of P450 reactions, and as
bioreactors to generate high amounts of a metabolite [60,61]. The
use of these cDNA-expressed P450s to study the enzyme function
has notably contributed to a greater understanding of substrate and
inhibitor specificity of individual P450 enzymes. Work to date has
primarily focused on P450s, although cell lines expressing other
genes involved in xenobiotic metabolism have also been success-
fully transfected (i.e. GSTs, UGTs, etc.) [62]. Moreover, cell lines
expressing all P450 enzymes individually have been reported, and
cell lines which express combinations of the xenobiotic metaboliz-
ing enzymes have also been developed. The expression of multiple
enzymes is important for a generalized use of engineered cells as
toxicology screening tools [57]. In this line, engineering cellular
models heterologously co-expressing human CYP1A2 and poly-
morphic variants of NAT2 in V79 cells [63], or CYP2E1 and
GSTP1 in hepatoma cells [64], have been developed to evaluate the
metabolism-dependent toxicity of chemicals. Recently, hepatoma
cell lines which incorporate a new transactivator system that allows
a time- and dose-dependent control of the expression of individual
gene targets, including CYP2E1 and other drug-metabolizing en-
zymes or transcription factors, have been successfully developed
[64]. These cellular systems are promising tools to investigate the
precise role and functions of specific proteins and to model varia-
tions in human liver and their relevance to drug metabolism and
toxicity.
New strategies are now being currently explored to overcome
the limitations of hepatic cell lines for drug-metabolism studies.
The low biotransformation activity of liver-derived cell lines is
likely to be the consequence of a very low P450 gene transcription,
resulting in trace levels of mRNA and protein synthesis [16]. Dif-
ferent explanations for this phenomenon have been proposed: an
alteration (possibly a decrease) of specific transcription factors that
control the expression of P450 genes, an increase in the levels of
transcription repressors, an alteration in the isoform pattern or func-
tions of transcription activators, or a chromatin compactation and
alternative mRNA splicing. Promising experimental approaches
based on the use of adenoviral expression vectors encoding relevant
liver-specific transcription factors (HNF3, C/EBP
) have been
successfully generated to increase the CYP3A4 and CYP2C expres-
sions in HepG2 cells [65,66]. Alternatively, the re-expression of
limiting coactivators could be of critical importance for the
reactivation of non-functional transcription factors. HNF4
has
been shown to be present in HepG2 and Mz-Hep-1 cells at levels as
high as in primary human hepatocytes. A low expression of the
HNF4
coactivators, PGC1
and SRC1, is shown in these hepa-
toma cell lines, which can seriously impair the expression of
HNF4
target genes [67]. Transfection of PGC1
and SRC1 coac-
tivators caused an important enhancement of the CYP2C9, 1A1,
and 1A2 expressions in human hepatoma cells, which reveals the
key role of coactivators in the transcriptional activation of P450
genes by HNF4
.
USE OF CELL LINES FOR DRUG METABOLISM PUR-
POSES
Hepatoma cell lines have been widely used as in vitro models
for the study of hepatocellular functions as well as hepatotoxicity
studies. However, their application to drug metabolism studies is
hindered by their marginal expression of major P450 enzymes re-
Donato et al.
sponsible for the oxidative metabolism of xenobiotics. As a full
contingent of drug-metabolizing enzymes is not expressed in
HepG2 and other hepatoma cell lines, drug metabolic stability or
metabolite profile studies cannot be performed in these cellular
models, except for compounds metabolized through Phase II conju-
gating reactions or oxidations catalyzed by the CYP1A1 or 1A2
enzymes [68-70]. Hepatoma cells may be more useful for other
applications in drug discovery such as enzyme induction or the
assessment of the hepatotoxic potential of new drugs. Recently,
novel liver-derived cell lines have been proposed as valuable tools
for the study of the induction of P450 and other drug-metabolizing
enzymes [18, 47]. Although primary hepatocyte cultures are still
considered the gold standard in vitro model for induction studies,
promising results obtained after analyzing the utility of highly dif-
ferentiated hepatic cell lines to identify P450 inducers and to pre-
dict the extent of drug-drug interactions for compounds in devel-
opment identified these cellular models as useful alternatives for
preliminary screening. Similarly, hepatoma cells have been recog-
nized as a good choice to evaluate non-metabolic dependent toxic-
ity [71-73]. However, they do not constitute a real alternative to
hepatocytes to screen chemicals bioactivated by drug-metabolizing
enzymes. Cell lines are less sensitive towards bioactivable toxicants
than primary cells, even for promutagens activated by the
CYP1A1/2 enzymes (expressed in hepatoma cells but at signifi-
cantly lower levels than in hepatocytes) [24].
As indicated above, different alternatives have been explored to
confer metabolic competence to cell lines, and new strategies are
currently being developed. Nowadays, recombinant systems ex-
pressing a single P450 enzyme are commonly used in drug discov-
ery and development. These models are particularly useful in the
early detection of potential P450 inhibitors and in the identification
of P450s responsible for the metabolism of new drugs. Other poten-
tial uses of metabolically competent cell lines include metabolic
stability screening and the generation of high amounts of drug-
derived metabolites (bioreactors).
IDENTIFICATION OF P450 ENZYMES INVOLVED IN ME-
TABOLISM
Reaction phenotyping characterizes the P450 isoforms that
catalyze a given drug-metabolizing reaction in the human liver.
This information is a key issue in drug discovery; it allows to
minimize the role of polymorphic enzymes leading to inter-
individual variations and to predict potential drug-drug interactions
that may result upon the co-administration of the new chemical
with P450 isoform selective inhibitors or inducers. Phenotyping is
generally straightforward when a single P450 is involved in a meta-
bolic pathway. However, P450 enzymes exhibit distinct yet over-
lapping substrate specificities, and multiple P450s can contribute to
the metabolism of a particular compound. Approaches based on the
use of a battery of recombinant P450 systems, specific chemical or
antibody inhibitors, and on correlations of probe substrate activities
with metabolic rates of test compounds have been combined and
used for P450 phenotyping purposes.
Genetically engineered cell lines expressing stable human drug-
metabolizing enzymes have been satisfactorily used in the identifi-
cation of those enzymes involved in the metabolism of a drug can-
didate [56, 74, 75]. Incubating the compound with each separate
cell line is useful to ascertain which P450 isoform(s) is(are) respon-
sible for metabolite/s formation. Previous studies in our laboratory
have demonstrated that the use of a battery of cultured cells ex-
pressing P450 enzymes individually is a valuable tool for assessing
whether a given P450 can give rise to a particular drug metabolite
[56]. The formation of diclofenac metabolites was analyzed after a
direct incubation of the drug with intact monolayers of THLE cells
genetically manipulated for the stable expression of the major hu-
man P450 enzymes involved in hepatic xenobiotic metabolism (Fig.
1). As incubation with cultured cells can be extended over long
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
P450 phenotyping can provide valuable information at leading
identification or optimization stages, and may contribute to dis-
criminate compounds with unfavourable metabolic properties. In
vitro results can be used to identify new chemical entities, mainly
those metabolized by either polymorphic P450s (i.e. CYP2D6,
2C19, 2C9, or P2A6) or a single P450 enzyme. Such properties are
associated with large inter-individual variations in drug/metabolite
levels in blood/tissue which contribute to variable pharmacological
effects or to an increased incidence of drug-drug interactions. From
a throughput screening perspective, phenotyping studies are rec-
ommended when a relatively high contribution (>30%) of P450-
dependent oxidations to the compound clearance mechanism has
been suggested [80]. Although several P450s can be involved in
hepatic metabolism, phenotyping can be limited to those P450, and
may be considered to be the most relevant in drug metabolism
(CYP3A4, 2D6, 2C9, 2C19 and 1A2).
INHIBITION STUDIES
Multidrug therapy is not uncommon in clinical practice. A si-
multaneous administration of several drugs may result in pharma-
cokinetic drug-drug interactions. Such interactions occur when the
disposition (i.e. absorption, distribution, metabolism and excretion)
of a drug is altered by another [82]. Of the pharmacokinetic factors
that control drug action, the rate of metabolic transformation is one
of the most important. Hence, interactions that result in changes in
the rate of drug metabolism can be of great clinical significance.
Possible consequences of pharmacokinetic drug-drug interactions
include potential changes in drug levels available to bind to recep-
tors and tissue targets, which can consequently alter their pharma-
cologic efficacy and/or induce cell toxicity.
Few enzymes metabolize hundreds of drugs and other foreign
compounds. P450 enzymes play a crucial role in metabolism and
drugs and, therefore, an alteration of the P450 function is an impor-
tant mechanism for metabolic-based interactions [83, 84]. The abil-
ity of an individual P450 enzyme to metabolize multiple substrates
is responsible for a large number of drug-drug interactions associ-
ated with P450 inhibition. Two compounds or more can compete
with each other as substrates for the same enzyme. Moreover, cer-
tain compounds are able to inhibit a particular enzyme that is not
directly involved in its metabolism. A vast number of molecules
have been identified as competitive, non-competitive or irreversible
P450 inhibitors [84]. P450 inhibition by one drug will result in
elevated plasma/tissue concentrations of the other drugs, which may
lead to overdosage and/or toxicity for molecules with a narrow
therapeutic index [83].
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].
Donato et al.
As inhibition is an undesirable feature for a drug candidate,
potent P450 inhibitors should be recognized early in the drug de-
velopment process. Considerable progress has recently been made
in the development of reliable in vitro screening methods to identify
potent P450 inhibitors. Simple, rapid and reproducible enzyme
inhibition assays based on measurements of catalytic activities are
indicative of an enzyme-inhibitor interaction [85, 86]. Human liver
microsomes and cDNA-expressed enzymes are the test systems of
choice for this purpose as they are more readily available than hu-
man hepatocytes. The assays are based on the analysis of potential
reductions in the metabolism of an appropriate P450 probe substrate
in the presence of various concentrations of the tested compound
[80, 87]. Probe substrate concentrations at or below the Km value,
and the validation of inhibition experiments by testing known spe-
cific P450 inhibitors (positive controls), are recommended (Fig. 3).
Inhibitory effects are usually expressed as a percentage of the con-
trol activity value, and IC50 values are calculated by linear interpo-
lation. The use of substrate concentrations close to the KM values of
the reaction allows the application of simple inhibition kinetic rela-
tions for a competitive inhibition to estimate Ki from the IC50 val-
ues. These assays enable the inhibitory potency of a series of com-
pounds to be easily compared (i.e. chemically-related structures or
drugs with a similar pharmacological activity) on several P450s.
Applying recombinant systems to in vitro P450 inhibition as-
says has notably contributed to the advances in high-throughput
screening. A common approach used in testing new molecules as
P450 inhibitors consists in a heterologously expressed P450 en-
zyme, a non-fluorescent probe (which is metabolized to a fluores-
cent product) and the test compound [88, 89]. Fluorescence-based
assays are highly sensitive and can be performed in multi-well plate
formats which increase sample throughput in comparison with con-
ventional analytical methods. These assays are easily adapted to
automated robotic systems and are associated with a drastic reduc-
tion in analysis time and costs. However, most fluorimetric probes
are not selective for a single P450 and their application is limited to
models expressing an individual enzyme. Therefore, a major advan-
tage of recombinant P450 systems, in relation to liver microsomes,
is the possibility of using non-selective fluorimetric probes for
high-throughput activity assays, thus avoiding laborious and time-
consuming methodologies (i.e. radiometry, HPLC separations, LC-
MS/MS analysis). These assays offer the possibility of easily com-
paring the inhibitory potency of a series of compounds (i.e. chemi-
cally-related structures or drugs with a similar pharmacological
activity) on each P450. On the other hand, interpreting inhibition
results from recombinant systems requires cautious analysis. Since
several enzymes can be involved in the metabolism of a compound,
Cell Lines: A Tool for In Vitro Drug Metabolism Studies
the use of recombinant models expressing one single P450 may
lead to an overestimation of the inhibitory effect of a given drug
(i.e. compounds actively metabolized into inactive metabolites by
other enzymes), or may not properly identify certain P450 inhibi-
tors (i.e. activation of the drug by other enzymes into an inhibitory
metabolite) [89, 90].
Although most high-throughput screening inhibition assays
using heterologously expressed human enzymes are based on the
use of recombinant P450 proteins isolated in microsomal forms,
intact transgenic cells stably expressing single P450s can also been
used for this purpose [89, 91-93] (Fig. 3). A major limitation in
making conclusive statements from assays in microsomal prepara-
tions is that some processes involved in in vivo metabolism are
missing in subcellular models. Among these processes, drug trans-
port across membranes, further metabolism by cytosolic enzymes,
or binding to intracellular proteins can be determinants in the actual
concentration of the substrate and inhibitor available to the P450
[83, 94]. Given the presence of membrane barriers, assays per-
formed in intact cells reflect the in vivo situation more closely and
are, in some respects, more predictive than subcellular models [83].
However, the fact that the chemical inhibitor must be transported
through biological membranes could imply longer assay times and
certain technical problems [89]. Moreover, only non-cytotoxic con-
centrations of the compound can be tested when living cells are
used. This should not represent a drawback for reliable and useful
screening assays, and in vitro experiments should be conducted at
concentrations similar to the relevant in vivo concentration.
INDUCTION
Drug-drug interactions may also occur as a consequence of
P450 induction. Hepatic drug-metabolizing P450s, CYP3A4 and
CYP1A2, and to a lesser extent, CYP2C9 and CYP2B6, are induc-
ible by marketed drugs, which represent the basis of a number of
common drug-drug interactions [95-98]. Metabolic interactions
mediated by enzyme induction are far less frequent than those
caused by inhibition. However, their consequences can be clinically
relevant. A drug with inductive properties can accelerate its own
metabolism, or that of another co-administered drug, and therapeu-
tic inefficacy occurs as a consequence of a faster clearance. Alter-
natively, an exaggerated response or an increased toxicity can be
take place when induced metabolic pathways are responsible for
converting the drug into a more active metabolite.
The screening of enzyme inducers is more complex and diffi-
cult than the identification of potential inhibitors. Enzyme induction
consists in the increase of new protein synthesis in response to rele-
vant stimuli. This process is a result of an increased transcription of
specific genes and involves a de novo synthesis of mRNA, protein
synthesis and a post-translational modification to an active enzyme.
In drug metabolism research however, compounds capable of in-
creasing enzymes by other mechanisms other than transcriptional
activation (i.e. mRNA or protein stabilization) are also considered
inducers.
Unlike inhibition screens, which are routinely used in early
drug development, assays that identify potential enzyme inducers
are less commonly performed and no general consensus exists as to
how reliable these assays are. Inducers cannot be screened in sub-
cellular models as they require an intact cellular system that is fully
capable of expressing genes. Cells are typically treated with the test
compound for 2-3 days. Then P450 levels are compared with those
of control cells. P450 induction can be easily evaluated by deter-
mining increases in enzyme activity using the selective probe sub-
strate. Although catalytic activity represents the gold standard
method for quantifying the in vitro induction potential of a com-
pound; other methods have been proposed. Measuring mRNA lev-
els by RT-PCR contributes to high throughput screening and is
particularly helpful when both enzyme inhibition and induction are
operative [97]. Alternatively, induction could also be assessed by
Current Drug Metabolism, 2008, Vol. 9, No. 1 7
changes in the P450 protein levels using specific antibodies, al-
though a greater amount of cells is needed for protein quantifica-
tion. Currently, primary human hepatocytes and human liver slices
are the most accepted biological models for P450 induction studies
[5, 99-101]. The high inter-individual variability observed in re-
sponse to inducers and the scarcity of fresh human tissue for ex-
perimental purposes constitute serious limitations for the wide-
spread use of such in vitro systems. Although hepatocyte cryopre-
servation has facilitated their availability for metabolic studies, the
quality of cell preparations after thawing should be considerably
improved to obtain long-term hepatocyte cultures that are appropri-
ate for induction assessments [102].
Immortalized liver-derived cell lines were proposed as an ideal
alternative because of their unlimited availability and phenotypic
stability. However, studies in HepG2, the most widely human hepa-
toma cell line, showed poor inducibility, except for the extrahepatic
CYP1A1 enzyme [69, 89]. More recently, novel human liver-
derived cell lines capable of responding to prototypical P450 induc-
ers have been generated [17, 18] (Table 3). High-throughput assays
using Fa2N4 cells cultured in multi-well plates have been proposed
to identify CYP3A4, 2C9 and/or 1A2 inducers [32, 47]. By using
reference/model inducers, the relative induction potency of test
compounds could be evaluated [47]. These cellular systems consti-
tute promising tools for the screening of potential inducers early on
in drug discovery. Nonetheless, more studies are needed to confirm
their utility for predicting in vivo drug-drug interactions.
Although the phenomenon of P450 induction has long since
been known, only in recent years has the use of molecular biologi-
cal techniques enabled the identification of mechanisms responsible
for P450 induction. Most inducible P450 genes can be transcrip-
tionally activated by a receptor-dependent mechanism, resulting in
an up-regulated P450 expression [103]. Induction of CYP1A en-
zymes by polycyclic aromatic hydrocarbons was the first mecha-
nism identified. Upon binding the inducer to cytosolic aryl hydro-
carbon receptors (AhR), the complex is translocated to the nucleus
where heterodimerizes with the nuclear factor Arnt, and binds to an
enhancer/promotor DNA region of CYP1A [104]. PXR and CAR
are involved in the regulation and induction of CYP2 and CYP3A
enzymes, as well as other drug-metabolizing enzymes (UGTs, sul-
photransferases, glutathione S-transferases) and transporters
(MDR1 and MRP2) [105-108]. Once activated, PXR and CAR
form heterodimers with the retinoid X receptor and then bind to the
target xenobiotic response element located in P450 gene promoters,
resulting in the transcription of the respective enzyme [109-111].
The glucocorticoid receptor and the peroxisome proliferators-
activated receptor are other nuclear receptors known to regulate the
induction of P450 enzymes [103, 112].
AhR is expressed in most immortalized cell lines, including
HepG2 hepatoma, and consequently, CYP1A enzymes are induc-
ible in these cells. Meanwhile, the decreased expression of PXR
and CAR, compared to that of primary hepatocytes, is likely the
reason for the low expression/induction of other P450 enzymes in
most hepatoma cells. In contrast, the relatively high levels of PXR
seen in other liver-derived cell lines (i.e. HepaRG hepatoma) sup-
port the inducibility of CYP3A4 in these cells [18].
Increasing knowledge of the nuclear receptors mediating P450
induction has contributed to the development of mechanism-based
tests for induction screening. Different cell-based reporter gene
assays for AhR, PXR and CAR have been used as preliminary as-
sessments for compounds with an inductive potential [93, 113-117].
Human hepatocarcinoma, as well as cell lines with an extrahepatic
or non-human origin, have been manipulated to obtain new tools to
easily discriminate and rank several molecules as a function of their
inductive potential [118-120]. The combined use of HepG2-derived
cell lines showing distinct CYP3A4 induction profiles [121], or
cell-based bioassays for a simultaneous assessment of P450 enzyme
induction and inhibition [91, 93], could offer additional advantages
8 Current Drug Metabolism, 2008, Vol. 9, No. 1
for new molecules screening in the early stages of drug develop-
ment.
Reporter gene assays are amenable for the high throughput
screening of compounds able to activate nuclear receptors, although
the relevance of such an activation is questionable. A specific inter-
action of a ligand with a single nuclear receptor fails to account for
the coordinated regulation of the P450 expression/induction by
multiple receptors and mechanisms. Although these assays are
promising tools for the rapid identification of potential inducers,
cross-talk between different elements can be misleading [122].
XENOBIOTIC BIOACTIVATION
Drug metabolism can result in a bioactivation phenomenon
rather than a detoxification process leading to metabolism-based
toxicity. Many compounds form metabolites that exhibit a greater
toxicity than the parent compounds. The phenomenon cannot be
assessed by hepatoma cell lines as they express biotransformation
activities to a very limited or negligible extent. Compounds known
to exhibit toxicity via metabolism-mediated activation have differ-
ential in vitro cytotoxic effects on primary hepatocytes in compari-
son with hepatoma cells showing low metabolic competence [123,
124]. In a recent study, the hepatoxicity profile of various chemical
entities was evaluated using two in vitro hepatocyte models: WIF-
B9 cells (a hybrid cell line obtained by the fusion of rat Fao hepa-
toma and human fibroblasts) and primary hepatocytes. The cytotox-
icity ranking of compounds, based on the lactate dehydrogenase
(LDH) release endpoint, was comparable in both cellular models.
Few marked differences in the hepatotoxicity potential were ob-
served, and is likely related to the respective capacity of the models
to express P450s, particularly the CYP3A enzyme [125].
HepaRG cells could represent a surrogate to primary human
hepatocytes for xenobiotic metabolism and toxicity studies since
both the analysis of metabolic profiles of several compounds and
the effects of hepatotoxins suggested certain resemblance of
HepaRG cells to primary hepatocytes [18]. Comparison of cyto-
toxic effects of several reference hepatotoxicans in HepaRG and
HepG2 cells revealed that acetaminophen and aflatoxin B1 (two
bioactivable toxicants via the formation of electrophilic metabolites
by P450-dependent reactions) were more cytotoxic to HepaRG than
to HepG2 cells [18, 35]. Similarly, the human hepatoma BC2 is of
potential interest for the evaluation of xenobiotic-mediated cell
toxicity under repeated administration conditions given its stability
in culture once cells have reached confluency. Following a repeated
dosage of a toxin, cytotoxicity is generally observed at a lower
concentration in comparison to a single administration [126].
Conferring xenobiotic metabolism capability to target cells
would allow their use in the study of phenomena involving xenobi-
otic bioactivation and toxicity in comparison with their isogenic
parental control cell line [127]. Genetically modified cell lines can
be very useful models for assessing the toxicological effects of the
modulation of expression on selected Phase I and Phase II enzymes
in comparison with their isogenic parental control cell lines. The
role of both activating and detoxifying enzymes on cellular sensitiv-
ity to several toxic end-points is stressed [128].
Several recent studies illustrate the promising use of P450-
expressing cell lines in the toxicity and bioactivation of drugs.
HepG2 cells expressing CYP2E1 [52, 53] or CYP3A4 [129, 130]
were used to evaluate the toxicity of xenobiotics which may be
activated or metabolized by these P450s in comparison with control
cells. For further mimicking of liver metabolism, HepG2 cells have
also been transformed by the pBudCE-GS-CYP3A4 vector, which
contains glutamine synthetase and drug-metabolizing CYP3A4
genes [60].
Cells expressing drug metabolizing enzymes other than P450s,
such as aldehyde dehydrogenases (ALDH), superoxide dismutase,
UGTs or GSTs, are evidently of use in pharmacology and toxicol-
Donato et al.
ogy [128]. Stably transfected MCF-7 (human breast carcinoma) and
V79 (hamster lung fibroblasts) cells, that express a range of activi-
ties of human ALDH isozymes, were utilized to demonstrate the
functional ability of cytosolic ALDH to confer a potent resistance
to oxazaphosphorine anticancer drugs, as well as a range of lipid
aldehydes, produced as a result of lipid peroxidation under condi-
tions of cellular redox imbalance [128]. These studies suggested
that ALDH-mediated and GSH-dependent cellular resistance
mechanisms may function cooperatively to protect against reactive
aldehydes. Similarly, cell lines stably transfected to overexpress
human superoxide dismutase are very useful in vitro models to
identify the role of reactive oxygen species and mitochondrial anti-
oxidant status in apoptotic cell death [131].
To approach more complex phenomena (i.e. two-stage metabo-
lism, bioactivation), cells expressing both activating and detoxify-
ing enzymes have been constructed (i.e. cell lines simultaneously
expressing a form of human P450 and a Phase II enzyme) and used
to investigate the bioactivation of carcinogens [62]. V79 Chinese
hamster cells were genetically engineered for the stable co-
expression of human CYP1A2 and NAT2 to generate an in vitro
tool for the study of the metabolism-dependent toxicity of aromatic
amines [63]. Previous studies in rats suggested the activation of
these compounds by CYP1A2, followed by NAT. These activities
result in a de-acetylated compound which is highly reactive and
causes cancer. In contrast, testing 2-aminofluorene in the newly-
constructed V79 cell line revealed that the metabolic pathway starts
with the action of NAT2 in humans, followed by that of CYP1A2,
resulting in the formation of an acetylated, very stable and non-
toxic metabolite [63]. Marked species-dependent differences in the
metabolic activation of benzo[a]pyrene were also observed by us-
ing V79 cells expressing CYP1A1 enzymes cloned from different
species. The data suggested that human CYP1A1 is more specific
for the critical 7,8,9,10 position than CYP1A1 from the other spe-
cies tested, which resulted in a higher toxicity in the V79 cells ex-
pressing the human CYP1A1 [132]. Ethanol-induced toxicity has
been explored in HepG2 cultures and in recombinant HepG2 cells
transfected with alcohol dehydrogenase (ADH) and/or CYP2E1
[133]. The results strongly suggest that hepatic ADH is the major
factor responsible for ethanol oxidative metabolism, irrespectively
of the CYP2E1 overexpression, and that a diminished ADH activity
facilitates the non-oxidative metabolism of ethanol to products that
cause apoptosis in HepG2 cells via the intrinsic pathway. These
cellular models have been suggested to be valuable in vitro tools to
identify early ethanol-induced molecular targets as well as to ex-
plore the mechanisms underlying ethanol hepatotoxicity.
Overall, engineered cells can provide useful approaches for
metabolism-dependent toxicity assessment and for the study of
molecular and enzymatic mechanisms responsible for the toxicity
and/or protection against reactive toxicants. For the time being
however, a limitation of genetically engineered cells is the reason
why only one or two enzymes can be satisfactorily transfected into
cells. Transfecting more than two P450 genes proves difficult, and
reproducing the in vivo relative abundance of each enzyme is not
feasible.
CONCLUSIONS AND OUTLOOK
Human hepatocytes are still considered the best in vitro model
to gain a complete picture of how a drug will be metabolized in
vivo. However, pharmaceutical companies increasingly make use of
cell lines to speed up the selection of new drugs with favourable
pharmacokinetic and metabolic properties. Moreover, new strate-
gies to overcome the limitations of hepatic cell lines by generating
metabolically competent cell lines that mimick the performance of
hepatocytes are currently being developed as tools for drug metabo-
lism and toxicity. In fact, cell lines expressing more than one single
P450 enzyme (several P450s, Phase II enzymes, etc.), and which
Cell Lines: A Tool for In Vitro Drug Metabolism Studies
are able to respond to enzyme induction, are a promising and real
alternative to human hepatocytes.
Human hepatoma-derived cell lines show very limited drug
metabolism activities. This is largely due to a very low expression
of P450 genes. The repression of P450 genes in hepatic cells is
apparently the consequence of several factors acting simultaneously
on the cells genome because of a decreased expression of key acti-
vating liver-enriched transcription factors (e.g. C/EBP
, HNF3), a
lack of co-activators (SRC-1, SRC-2), and increased levels of rep-
ressors (COUP TF-I, COUP TF-II) and co-repressors (SMRT).
Finally, it is also conceivable that DNA methylation/histone deace-
tylation and/or a decrease in co-activators with acetyltransferase
activity play a role by altering chromatin compactation and by si-
lencing the genes. The detection of minimal but consistent amounts
of P450 mRNAs in hepatoma cells suggests that P450 genes have
not been totally silenced in these cells by mechanisms such as ex-
tensive methylation or chromatin condensation. A comparative
analysis of four major liver transcription factors controlling the
expression of typical hepatic genes (HNF1, HNF3, HNF4 and
C/EBP
) between HepG2 cells and human hepatocytes showed that
some are poorly expressed in hepatoma cells [134]. Hence, it is
conceivable that a lack of the P450 gene expression in hepatomas is
the consequence of an altered expression of key regulatory tran-
scription factors. However, relevant results evi-denced that it was
unlikely that restoring a single regulator factor
in hepatoma cells could up-regulate all P450 genes [66, 135]. It
seems reasonable to expect that the re-expression of all major P450
isoforms in hepatoma cells will require the concerted action of
several liver-enriched transcription factors. Recent reports lend
support to the working hypothesis that it is possible to achieve a
substantial expression of drug metabolism enzymes in hepatoma
cells by the appropriate re-expression of a combination of key
transcription factor(s) [65] The reactivation of non-functional
transcription factors such as HNF4
, which, surprisingly, is present
in HepG2 cells at levels as high as in hepatocytes, may be another
strategy [67]. The lack of functionality of endogenously expressed
HNF4
is the reason why HepG2 cells lack essential co-activators
for its proper functionality [36, 67].
Recent reports have shown that the tailored re-expression of
these factors in hepatoma cells causes a significant re-expression of
relevant P450s, thus opening a new experimental strategy to gener-
ate competent cell lines for human drug metabolism studies [36]. In
addition, the generation of differentiated hepatic cells from toti- or
multipotent stem cells is a promising, yet still immature technology.
The development of hepatic cell lines from hepatic stem cells,
transdifferentiation from either non-hepatic progenitor cells or em-
bryonic stem cells, has been partially achieved by few researchers.
These hepatocyte-like cells showed relevant hepatic features and,
in some cases, a certain degree of xenobiotic metabolism capability.
ACKNOWLEDGEMENTS
This work was supported by the ALIVE Foundation, and funds
from the European Commission, grants LSHB-CT-2004-504761,
LSHB-CT-2004-512051 and LSSB-CT-2005-037499.
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Received: September 05, 2007 Revised: September 28, 2007 Accepted: October 01, 2007