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Research ArticleArticle

CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

Louise Sivertsson, Monica Ek, Malin Darnell, Irene Edebert, Magnus Ingelman-Sundberg and Etienne P. A. Neve
Drug Metabolism and Disposition June 2010, 38 (6) 995-1002; DOI: https://doi.org/10.1124/dmd.110.032367

Abstract

Drug-induced hepatotoxicity is an important cause for disapproval, limitations of use, or withdrawal of drugs, and there is a high need for reproducible in vitro systems that can predict such toxicity. In this study, we show that confluent growth of the human hepatoma cell line Huh7 up to 5 weeks results in increased gene expression of several cytochromes P450 (P450s), UDP-glucuronosyltransferases, transporters, transcription factors, and several liver-specific genes, as measured by low-density array. The most striking effect was seen for CYP3A4 expression. Western blot analysis revealed increased amounts of CYP3A4 together with increased levels of NADPH-P450 reductase, cytochrome b5, and albumin with prolonged time of confluence. By using the CYP3A4-specific substrates luciferin 6′ benzyl ether, testosterone, and midazolam, we could confirm that the increased CYP3A4 gene expression also was accompanied by a similar increase in catalytic activity, inhibitable by the CYP3A4-selective inhibitor ketoconazole. The CYP3A4 activity in confluent cells was also inducible by rifampicin. Finally, the cell system could support the CYP3A4-dependent hepatotoxic activation of aflatoxin B1, which was effectively inhibited by ketoconazole. Our results show that Huh7 cells grown confluent differentiate into a more metabolically competent cell line, especially with regard to CYP3A4.

Primary human hepatocytes are widely used in drug discovery as an in vitro cell model for drug metabolism and toxicity studies (Soars et al., 2007), but their use has several limitations. Besides the sometimes low availability of fresh liver samples suitable for research purposes and the cost associated with their use, there is also a high batch-to-batch variability of the isolated hepatocytes as a result of interindividual differences between donors. In addition, isolated and two-dimensional cultured hepatocytes dedifferentiate, exhibiting a time-dependent reduction in the expression levels of liver-specific genes such as cytochromes P450 (P450s), an effect already apparent after only a few hours (Donato et al., 2008). As an alternative, cell lines are used with evident advantages with respect to their availability and stable phenotype; however, the majority of the hepatoma cell lines most commonly used today contain very low levels of drug-metabolizing enzymes compared with primary hepatocytes (Donato et al., 2008). Different strategies to up-regulate the expression of drug-metabolizing enzymes have been used with the aim to generate better in vitro systems. The introduction of cDNAs encoding drug-metabolizing enzymes or growing cells in the presence of dimethyl sulfoxide (DMSO) are two different approaches commonly used in attempt to generate more metabolic competent hepatic cell lines (Donato et al., 2008).

The human CYP3A family mainly consists of the isoforms CYP3A4, CYP3A5, CYP3A7, and CYP3A43. These enzymes account for approximately 30% of the total P450 levels in human liver (HL), CYP3A4 being the most important and abundant one (Shimada et al., 1994). CYP3A5 is 83% homologous with CYP3A4 and has similar substrate specificity. However, it is usually expressed at much lower levels in the liver and is only detectable in 20 to 30% of the human population (Guengerich, 1999; Eichelbaum and Burk, 2001). CYP3A7 is 90% homologous with CYP3A4 (Torimoto et al., 2007) and accounts for at least 50% of the total P450 in human fetal liver but is seldom expressed in the adult liver (Eichelbaum and Burk, 2001). CYP3A43 is the most recently discovered isoform (Domanski et al., 2001) and is expressed at approximately 0.1% of CYP3A4 expression levels in the liver, sharing approximately 90% homology (Westlind et al., 2001). CYP3A4 is responsible for most of the CYP3A-mediated drug metabolism and has a wide substrate spectrum. The importance of the enzyme in drug metabolism is highlighted by the fact that it is estimated to contribute to the metabolism of approximately 60% of the marketed drugs (Guengerich, 1999). All the CYP3A genes mentioned are organized in a CYP3A locus located on chromosome 7 and are transcriptionally regulated by xenobiotics, steroid hormones, and other endogenous substrates (Burk and Wojnowski, 2004). The nuclear receptor pregnane X receptor (PXR) is a major modulator of xenobiotic-induced regulation of the CYP3A genes (Goodwin et al., 2002; Burk and Wojnowski, 2004), but the constitutive androstane receptor, the glucocorticoid receptor, and the vitamin D receptor are also highly involved in the induction of CYP3A (Goodwin et al., 2002; Burk and Wojnowski, 2004).

Huh7 is a commercially available, widely distributed human hepatoma cell line, frequently used as an in vitro system to study hepatotoxicity (Kogure et al., 2004), hepatitis C virus infection (Yoo et al., 1995; Seipp et al., 1997), and gene regulation (Gunton et al., 2003), but has not yet been fully characterized with respect to its expression of P450s and other genes involved in drug metabolism. Previous studies have established that confluent hepatoma cells alter their gene expression profile during cultivation, rendering an expression pattern more like hepatocytes (Gómez-Lechón et al., 2001; Butura et al., 2004). In this study, we observe that when grown confluent, Huh7 cells also alter their gene expression and become metabolically competent with respect to CYP3A4, resulting in a phenotype more similar to human hepatocytes.

Materials and Methods

Cell Culture.

The human hepatoma Huh7 cell line (Human Science Research Resources Bank, Tokyo, Japan) was routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. The human hepatoma HepG2 cell line (American Type Culture Collection, Manassas, VA) was grown in minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, nonessential amino acids, 1 mM sodium pyruvate, 100 IU/ml penicillin, and 100 μg/ml streptomycin. The cells were harvested 1, 2, 3, 4, and 5 weeks after reaching confluence, and control cells were harvested immediately after reaching confluence (0 weeks of confluence). In addition, in some experiments cells were grown in the presence of 1% DMSO (Sigma-Aldrich, St. Louis, MO) up to 4 weeks. For induction experiments, the cells were treated for 48 h with 5 μM rifampicin (Sigma-Aldrich). Medium was routinely changed every 3 to 4 days. All the cell culture plates were purchased from Corning Life Sciences (Lowell, MA), culture flasks from Sarstedt (Newton, NC), and culture media and supplements from Invitrogen (Carlsbad, CA).

RNA Isolation and cDNA Synthesis.

Total RNA was isolated using RNeasy Midi Kit, including DNase treatment (QIAGEN, Valencia, CA), according to the manufacturer's protocol. Quantity and purity of the RNA were determined, measuring the 260:280 ratio using a Varian, Inc. (Palo Alto, CA) Cary 400 Bio UV/visible spectrophotometer. cDNA was prepared from 0.5 μg of total RNA using an oligo(dT) primer and the Moloney murine leukemia virus reverse transcriptase enzyme (Invitrogen).

TaqMan Low-Density Array and Real-Time Polymerase Chain Reaction Gene Expression Analysis.

A custom-made TaqMan Low-Density Array (LDA; Applied Biosystems, Foster City, CA) was used to study the expression levels of 48 selected genes in samples obtained from 1- to 4-week confluent Huh7 cells, 4-week confluent HepG2, Huh7, and HepG2 control cells (0-week confluent), and HL (RNA prepared from liver tissue, pool of five donors) as described previously (Ek et al., 2007). Glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase 1, and TATA-box binding protein (TBP) were used as housekeeping genes. The mRNA expression levels of the CYP3A enzymes in confluent Huh7 and HepG2 were also confirmed by ready-to-use TaqMan Gene Expression Assays, according to the manufacturer's instructions (Applied Biosystems). TBP was used as a housekeeping gene. The relative mRNA levels compared with mRNA levels in the 0-week confluent control cells (relative expression value set to 1) were defined by the 2−ΔΔCT method as described by Livak and Schmittgen (2001).

Western Blot Analysis.

Huh7 cells and HepG2 cells cultured confluent up to 5 weeks and Huh7 cells cultured in the presence of 1% DMSO for 4 weeks were used. The cells were harvested, homogenized in 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA and 20% glycerol, sonicated (20 × 1-s pulses), and centrifuged for 10 min at 10,000g. The resulting supernatant was centrifuged at 100,000g for 1 h, and the obtained microsomal pellet was resuspended in 50 mM potassium phosphate buffer, pH 7.4. Protein concentration was determined according to Bradford (1976). Microsomes isolated from a pool of 15 HL tissues, both male and female, were used as a positive control (CellzDirect, Durham, NC). The microsomes were functionally tested by the supplier for selected P450s and Phase II enzyme activities such as CYP1A, CYP3A, and UDP-glucuronosyltransferases (UGTs). Twenty-five micrograms of microsomal fraction were mixed with Laemmli sample buffer, subjected to SDS-polyacrylamide gel electrophoresis using a 10% gel, and then transferred to a Hybond-C extra membrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK). After blocking in Tris-buffered saline (250 mM Tris-HCl, pH 7.4, 200 mM NaCl) containing 0.05% (v/v) Tween 20 and 5% (w/v) fat-free milk powder, the membranes were incubated with rabbit anti-CYP3A4 and anti-CYP3A5 C-terminal peptide antibodies (Edwards et al., 1998), goat antialbumin (Bethyl Laboratories, Montgomery, TX), rabbit anti-cytochrome b5 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), rabbit anti-NADPH-P450 reductase (Abcam plc, Cambridge, UK), or rabbit anti-ERp29 antibodies (Mkrtchian et al., 1998), followed by goat anti-rabbit or rabbit anti-goat conjugated horseradish peroxidase secondary antibodies (Dako Denmark A/S, Glostrup, Denmark). Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

Immunofluorescence Microscopy.

Four-week confluent cells and subconfluent cells (approximately 90% confluent), grown on coverslips, were fixed in 3% paraformaldehyde for 20 min at room temperature. After quenching with 10 mM glycine for 5 min at room temperature, cells were permeabilized with 0.2% Triton-X solution for 20 min and blocked with 5% normal horse serum for 1 h. Cells were stained with rabbit anti-CYP3A4 C-terminal peptide antibody (Edwards et al., 1998) in 0.1% bovine serum albumin for 1 h, followed by Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (Invitrogen) for 1 h. The coverslips were mounted with ProLong Gold antifade reagent containing 4,6-diamidino-2-phenylindole (Invitrogen), and the CYP3A4 expression was examined and photographed using a Zeiss LSM 710 confocal microscopy with Zen 2008 software (Carl Zeiss, Jena, Germany).

Determination of CYP3A4 Enzyme Activity.

CYP3A4 activity in microsomes from 1- to 5-week confluent Huh7 cells, 0-week confluent control cells, and HL (pool of 15 donors) was measured using the luminescent P450-Glo assay (Promega, Madison, WI). Reaction mixture containing 20 μg of microsomes, 200 μM luciferin 6′ benzyl ether, and 200 mM potassium phosphate buffer, pH 7.4, was incubated for 20 min at 37°C. Reaction mixture without microsomes was used as control. The reaction was initiated by the addition of an NADPH-regenerating system, and the reaction mixture was incubated for 90 min at room temperature. Luciferin Detection Reagent (Promega) was added to stop the reaction, and luminescent signal was measured using a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). For CYP3A4 inhibition experiments, the microsomes were preincubated with 5 μM ketoconazole (KCZ) (Sigma-Aldrich) 5 min before activity measurements.

6β-Hydroxylation of testosterone was also studied as a marker of functional CYP3A4 expression as described previously (Waxman et al., 1988). In brief, microsomes from 4-week confluent Huh7 and HepG2 cells and 0-week confluent control cells were used. The reaction mixture (total volume, 0.5 ml) contained 100 μg of microsomes, 200 μM testosterone, 50 mM potassium phosphate buffer, pH 7.4, and 30 mM magnesium chloride. After 2-min preincubation, the reaction was initiated by the addition of 5 mM NADPH, and after 20-min incubation at 37°C, the reaction was stopped with 2.5 ml of ethyl acetate. Each sample contained 48 nmol of 4-androsterone-3,17-dione as an internal standard. Zero-time incubations served as blanks, and blanks containing 22.3 nmol of 6β-hydroxytestosterone served as external standards. The samples were separated on a reversed-phase Zorbax C18 column (5 μm, 150 × 4.6 mm; Agilent Technologies, Santa Clara, CA) with a binary gradient of mobile phase A and B (60% acetonitrile/40% water, v/v).

A cell-based assay was also used to study the CYP3A4 catalytic activity, where 4-week confluent Huh7 and HepG2 cells and 0-week confluent control cells were treated with three different P450-specific substrates. One milliliter of incubation medium (without phenol red) containing 26 μM phenacetin, 9 μM diclofenac (Sigma-Aldrich), and 3 μM midazolam (Actavis, Hafnarfjordur, Iceland) was added concomitantly to each well and incubated for 3 h at 37°C. The medium was collected and stored at −20°C until analysis. The cells were lysed with 1% Triton X-100 in phosphate-buffered saline, and protein concentration in each well was determined according to Bradford (1976). For induction studies, 1 ml of induction medium containing 5 μM rifampicin was added to the cells and replaced with fresh induction medium after 24 h. After another 24 h, 1 ml of medium containing the P450 substrates was added as described above. The CYP2C9 metabolite 4′-hydroxydiclofenac, the CYP3A4 metabolite 1′-hydroxymidazolam, and the CYP1A2 metabolite paracetamol were analyzed using liquid chromatography/mass spectrometry consisting of an HTS PAL injector (CTC Analytics, Zwingen, Switzerland) combined with an HP 1100 LC binary pump and column oven (Agilent Technologies) as described previously (Kanebratt and Andersson, 2008).

Determination of NADPH-P450 Reductase Enzyme Activity.

NADPH-P450 reductase (POR) activity was assessed by measuring cytochrome c reduction in microsomes obtained from 1- to 5-week confluent Huh7 cells, 0-week confluent control cells, and HL (pool of 15 donors). Microsomal protein (100 μg) was mixed with 0.1 M Tris-HCl, pH 7.8, and 40 μM cytochrome c, and the reaction was started by the addition of 100 μM NADPH. Reduced cytochrome c was determined at 550 nm using Varian, Inc. Cary 400 Bio UV/visible spectrophotometer for 2 min at 25°C. The extinction coefficient (ε) of 21 mM−1cm−1 was used to calculate the specific enzyme activity.

Assessment of CYP3A4-Dependent Cytotoxicity.

Four-week confluent cells and control cells were treated with 0, 5, 10, 20, or 40 μM aflatoxin B1 (Sigma-Aldrich) or tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate; Sigma-Aldrich) for 48 h. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) leakage according to the manufacturer's protocol (Roche Diagnostics, Bromma, Sweden) and expressed relative to 100 μM chlorpromazine-treated cells (100% cytotoxicity). The absorbance at 492 nm was determined with a Labsystems Multiskan MS plate reader (Thermo Fisher Scientific). To confirm the involvement of CYP3A4 in the aflatoxin B1-induced cytotoxicity, cells were incubated with 5 μM KCZ 5 min before the addition of aflatoxin B1 (40 μM). After 24 h, KCZ (5 μM) was added again to sustain the inhibitory effect. After an additional 24 h, the aflatoxin B1-induced cytotoxicity was examined as described above.

Statistical Analysis.

Data were analyzed using one-way analysis of variance (Dunnett's multiple comparison test) or two-way analysis of variance (Bonferroni post-tests), and p value <0.05 was considered to be significant. Data are expressed as mean ± S.E.M. of at least three individual experiments.

Results

Increased Gene Expression in Confluent Huh7 Cells.

We initially used a TaqMan LDA to study the mRNA expression of different P450s, UGTs, transcription factors, and transporters in 0- to 4-week confluent Huh7 cells and 4-week confluent HepG2 cells compared with their expression in HL (Fig. 1). For many of the genes analyzed, the relative gene expression increased with time of confluence, in particular CYP3A4. Increased expression levels were also observed for the Phase II enzyme UGT2B7; transcription factors PXR, constitutive androstane receptor, and retinoid X receptor γ; and the liver-specific genes glucose-6-phosphatase, cholesterol 7-α-hydroxylase, and albumin. Moreover, the increased gene expression was generally more pronounced in Huh7 cells compared with HepG2 cells grown confluent for the same amount of time (Fig. 1). Compared with the expression levels detected in HL, the relative expression levels in the cell lines were generally lower, especially in the HepG2 cells. The relative expression levels of hepatic transcription factors were similar to those in HL for both cell types (Fig. 1). Overall the relative expression levels of most genes analyzed were higher in confluent Huh7 cells than in confluent HepG2 cells (Fig. 1).

Fig. 1.
Fig. 1.

Gene expression analysis of confluent Huh7 and HepG2 cells by TaqMan LDA. Levels of gene expression were measured in 1- to 4-week (1–4w) confluent Huh7 cells, 4-week confluent HepG2 cells, and control (Ctrl) Huh7 and HepG2 cells (0-week confluent), as well as HL samples (mix of five donors). The expression for each gene is normalized against the expression of TBP in each sample, and expression levels in Huh7 and HepG2 cells are shown relative to expression levels observed in HL samples (arbitrarily set at 1). Data are expressed as mean of three independent experiments. N.D., not detected.

The mRNA levels of the CYP3A enzymes in confluent Huh7 and HepG2 cells were also quantified by real-time polymerase chain reaction (PCR) and expressed relative to the levels in 0-week confluent Huh7 cells (Fig. 2A) and HepG2 control cells (Fig. 2B), respectively. In Huh7 cells, a dramatic increase in CYP3A4 mRNA expression was observed with increased time of confluence, reaching maximal levels (approximately 1000-fold) after 4 weeks of confluence (Fig. 2A). The expression levels of the other CYP3A isoforms—CYP3A5, CYP3A7, and CYP3A43—were low and did not change significantly during confluence (Fig. 2A). In contrast to Huh7 cells, HepG2 cells grown confluent for the same amount of time did not display any significant increase in expression in any of the CYP3A genes and remained at levels comparable with those observed in nonconfluent cells (Fig. 2B).

Fig. 2.
Fig. 2.

Relative expression levels of CYP3A quantified by TaqMan real-time PCR. Relative mRNA expression levels in Huh7 cells (A) and HepG2 cells (B) harvested 1, 2, 3, 4, and 5 weeks (w) after reaching confluence as quantified by TaqMan real-time PCR. The expression for each gene is normalized against the expression of TBP and shown as fold change compared with control cells (0-week confluent). Data are expressed as mean ± S.E.M. of three independent experiments. **, p < 0.01 compared with control cells, harvested immediately after reaching confluence.

Increased Hepatic Protein Levels in Confluent Huh7 Cells.

The increased CYP3A4 mRNA expression in confluent Huh7 cells was corroborated by Western blot analysis, where an increase in CYP3A4 protein was observed with increasing time of confluence (Fig. 3A). In control cells, no CYP3A4 protein could be detected, but already after 1 week of confluence, CYP3A4 protein was seen. After 3 weeks of confluence, the levels were comparable with those observed in HL. In addition, when examined by confocal microscopy, confluent Huh7 cells displayed a dramatic increase in CYP3A4-specific staining, which was much less abundant than in control cells (Fig. 3C). Moreover, the staining pattern that was observed for CYP3A4 in confluent cells is typically observed for proteins associated with the endoplasmic reticulum, confirming the correct localization of the enzyme. In contrast to the CYP3A4 protein levels, only low levels of CYP3A5 protein could be detected, and levels did not increase with time of confluence (Fig. 3A). However, we could detect increased expression of the P450 electron donor POR and cytochrome b5 (CYB5A), an important modulator for CYP3A4 activity (Schenkman and Jansson, 2003; Gan et al., 2009). Both proteins gradually increased with increased time of confluence, but CYB5A could not be detected until 2 to 3 weeks of confluence (Fig. 3A). The protein levels of the liver-specific protein albumin also significantly increased during confluence, but like POR and CYB5A it never reached the levels detected in HL (Fig. 3A). As a gel loading control, the protein levels of the endoplasmic reticulum resident protein ERp29 (Mkrtchian et al., 1998) were used and are shown at the bottom of the figure (Fig. 3A). Despite increased mRNA levels (Fig. 1), no significant levels of CYP2A6, CYP2B6, or CYP2C protein could be detected by Western blot analysis (data not shown).

Fig. 3.
Fig. 3.

CYP3A4 and several hepatic proteins show increased expression in confluent Huh7 cells but not in confluent HepG2 cells. A, CYP3A4, CYP3A5, POR, CYB5A, and albumin protein levels in isolated microsomes (mix of three or four separate cultures) from 1- to 5-week confluent Huh7 cells, control Huh7 cells (Ctrl; 0-week confluent), HL (mix of 15 donors), and Huh7 cells treated 4 weeks with (+) or without (−) 1% DMSO. ERp29 was used as a gel loading control. B, CYP3A4 and CYP3A5 protein levels in isolated microsomes (mix of three or four separate cultures) from 1- to 5-week confluent Huh7 and HepG2 cells, control Huh7 and HepG2 cells (Ctrl; 0-week confluent), and HL. C, 4-week confluent (Conf) and control (Ctrl; 0-week confluent) Huh7 cells grown on coverslips were fixed, permeabilized, and stained for CYP3A4 protein (green). Nuclei were visualized by 4,6-diamidino-2-phenylindole staining (blue). Scale bar, 10 μm.

A recent report showed that Huh7 cells grown for several weeks in the presence of DMSO up-regulate the expression of several drug-metabolizing enzymes and liver-specific genes (Choi et al., 2009). In our system, Huh7 cells grown confluent for 4 weeks in the presence of 1% DMSO showed no significant increase in CYP3A4, CYP3A5, POR, or albumin protein levels, compared with cells grown for the same amount of time without the addition of DMSO. Only CYB5A is expressed at higher levels in the presence of DMSO (Fig. 3A, right).

In contrast to Huh7 cells, only very low levels of CYP3A4 and CYP3A5 protein could be detected in confluent HepG2 cells (Fig. 3B), confirming the RNA data (Figs. 1 and 2B). After 1 week of confluence, low amounts of CYP3A5 protein were observed, with levels not changing with time of confluence, whereas CYP3A4 protein expression was barely above detection level (Fig. 3B).

Increased CYP3A4 and POR Enzyme Activity in Confluent Huh7 Cells.

Using a luminescent CYP3A4-specific substrate, we determined CYP3A4 catalytic activity in microsomes isolated from control cells and 1- to 5-week confluent Huh7 cells. We observed a time-dependent increase in catalytic activity that correlated well with CYP3A4 protein levels, after 5 weeks of confluence reaching approximately 30% of the activity measured in HL microsomes (Fig. 4A). Even after 8 weeks of confluence, the cells still possessed CYP3A4 activity, although less than observed after 3 weeks of confluence (data not shown). By preincubating the microsomes with KCZ, this activity was effectively inhibited to levels observed in control cells, providing further evidence for the participation of CYP3A4 catalytic activity in these cells (Fig. 4A). Growing cells confluent for 4 weeks in the presence of 1% DMSO did not have an additional effect on CYP3A4 activity compared with 4-week confluent cells without this treatment (Fig. 4B), indicating that DMSO treatment of confluent cells did not affect the CYP3A4 catalytic activity, further confirming the protein expression data (Fig. 3A). The increase in CYP3A4 activity was accompanied by increased POR activity as measured by cytochrome c reduction. A significant increase in POR activity was already observed after 1 week of confluence and after 3 weeks of activity was approximately 50% of that observed in HL (Fig. 4C).

Fig. 4.
Fig. 4.

Confluent Huh7 cells display increased CYP3A4 and POR catalytic activity. A, CYP3A4 enzyme activity was determined in isolated microsomes obtained from 1- to 5-week confluent and control (Ctrl; 0-week confluent) Huh7 cells using the P450-Glo assay (Promega). KCZ (5 μM) was added to microsomes from 5-week confluent cells 5 min before the activity measurement. The same amount of HL microsomes (HL; mix of 15 donors) was used as positive control. B, confluent Huh7 cells were grown for 4 weeks in the presence (+) or absence (−) of 1% DMSO, and the CYP3A4-dependent catalytic activity was determined in isolated microsomes using the P450-Glo assay. C, POR catalytic activity was determined by monitoring cytochrome c reduction in microsomes isolated from 1- to 5-week confluent Huh7 cells, 0-week confluent control (Ctrl) cells, and HL (mix of 15 donors). Data are expressed as mean ± S.E.M. *, p < 0.05 or ***, p < 0.001 compared with control cells. †, p < 0.05 compared with equally cultured cells without treatment. RLU, relative light units.

We also determined the CYP3A4 activity by monitoring the formation of 6β-hydroxytestosterone in the microsomal fraction isolated from 4-week confluent and control Huh7 and HepG2 cells. Although no metabolism of 6β-hydroxytestosterone could be detected in microsomes from control Huh7 cells, microsomes from 4-week confluent Huh7 cells showed a dramatic increase in the formation of 6β-hydroxytestosterone (Fig. 5A), confirming the previous results that these cells have functional CYP3A4 expression. Microsomes isolated from control and 4-week confluent HepG2 cells did not display any significant formation of 6β-hydroxytestosterone (Fig. 5A), which is in good agreement with the lack of CYP3A protein expression in these cells (Fig. 3B).

Fig. 5.
Fig. 5.

CYP3A4-dependent testosterone 6β-hydroxylation and midazolam 1′-hydroxylation are increased in confluent Huh7 cells but not in confluent HepG2 cells. A, CYP3A4 catalytic activity was determined by monitoring the formation of 6β-hydroxytestosterone in microsomes isolated from control (Ctrl) and 4-week confluent (Conf) Huh7 and HepG2 cells, as measured by high-performance liquid chromatography. B, CYP3A4 catalytic activity was determined by monitoring the formation of 1′-hydroxymidazolam in the medium of control (Ctrl; 0-week confluent) and 4-week confluent (Conf) Huh7 and HepG2 cells, as measured by liquid chromatography/mass spectrometry. For induction studies, 5 μM rifampicin (Rif) was added 48 h before activity measurement. Data are expressed as mean ± S.E.M. of three experiments. ***, p < 0.001 compared with control cells. N.D., not detected.

Finally, using a cell-based assay, the CYP1A2-, CYP2C9-, and CYP3A4-specific substrates phenacetin, diclofenac, and midazolam were used to further evaluate the P450 activity. A significant increase in the CYP3A4-specific metabolite 1′-hydroxymidazolam formation was observed in 4-week confluent Huh7 cells compared with control cells (Fig. 5B). Moreover, the CYP3A4 activity was significantly induced when the cells were treated with the CYP3A4 inducer rifampicin. No significant induction was detected in control cells, but in 4-week confluent cells rifampicin treatment resulted in more than 2-fold induction of CYP3A4 activity compared with vehicle-treated cells (0.1% DMSO; Fig. 5B). Only low levels of 1′-hydroxymidazolam could be detected in HepG2 cells, confirming the testosterone data (Fig. 5A). In addition, we were not able to induce CYP3A4 activity in HepG2 cells using rifampicin (Fig. 5B). No CYP1A2 activity (phenacetin hydroxylation) could be detected, and only low levels of 4′-hydroxydiclofenac (CYP2C9) were observed in the Huh7 and HepG2 cells. Moreover, the CYP1A2 or CYP2C9 activity levels did not change with increased time of confluence in both of the cell lines (data not shown).

CYP3A4-Dependent Cytotoxicity in Confluent Huh7 Cells.

To evaluate whether our confluent, metabolically active Huh7 cell system could be used for studying CYP3A4-mediated cytotoxicity, we treated confluent Huh7 cells with aflatoxin B1, a well known CYP3A4-activated toxin and carcinogen (Kamdem et al., 2006). Treatment of 4-week confluent Huh7 cells with increasing amounts of aflatoxin B1 resulted in a dose-dependent increase in cytotoxicity compared with control cells, as observed by monitoring LDH release (Fig. 6). In confluent cells, a significant effect was already observed with 20 μM aflatoxin B1, but no effect could be detected in control cells, treated with the same amount of toxin (Fig. 6). The fact that the aflatoxin B1-induced toxicity in the confluent cells could effectively be prevented by KCZ pretreatment confirmed that aflatoxin B1 toxicity is CYP3A4-dependent (Fig. 6). Treating 4-week confluent HepG2 cells and 0-week confluent control cells using the same amounts of aflatoxin B1 also resulted in cytotoxicity, although modest, but no significant, differences between confluent and control cells could be observed (data not shown).

Fig. 6.
Fig. 6.

Cytotoxicity is increased in confluent Huh7 cells when treated with the CYP3A4-dependent hepatotoxin aflatoxin B1. Four-week confluent cells (filled bars) and control cells (0-week confluent; open bars) were treated for 48 h with increasing concentrations of aflatoxin B1 as indicated. Confluent cells were also treated with (+) or without (−) 5 μM KCZ 5 min before the addition of 40 μM aflatoxin B1. Cytotoxicity is measured as LDH release into the medium and expressed relative to 100 μM chlorpromazine-treated cells (100% cytotoxicity). Data are expressed as mean ± S.E.M. from three or four independent experiments. ***, p < 0.001 compared with equally treated control cells (0-week confluent).

As a control, a non–CYP3A4-dependent toxin, tacrine, was used (Spaldin et al., 1994; Lagadic-Gossmann et al., 1998). Four-week confluent Huh7 cells and control cells (0-week confluent) treated with increasing amounts of tacrine did not display any significant increase in cytotoxicity as measured by monitoring LDH leakage (data not shown).

Discussion

One of the major limitations with the use of cell lines in drug discovery is the altered hepatocyte-like functions and metabolic capabilities. In the present study we show that growing the human hepatoma cell line Huh7 confluent up to 5 weeks gradually increases the expression of several drug-metabolizing genes, in particular CYP3A4 (Figs. 1 and 2). Moreover, the elevated mRNA expression levels correlated well with the increase in CYP3A4 protein expression (Fig. 3) and CYP3A4-dependent catalytic activity, which could further be induced by treatment with rifampicin (Figs. 4 and 5). In addition, CYP3A4 activity in the confluent cells was confirmed by increased aflatoxin B1 cytotoxicity (Fig. 6). The fact that the aflatoxin B1-induced toxicity was abolished by KCZ corroborated not only that aflatoxin B1 toxicity is CYP3A4-dependent but also that our system could be used to study CYP3A4 activity. In addition, when treating the Huh7 cells with tacrine, a known hepatotoxic substance that has been suggested to require metabolic activation by CYP1A2 and not by CYP3A4 (Spaldin et al., 1994), no significant difference in toxicity could be observed between confluent and nonconfluent cells (data not shown). Contrary to CYP3A4 only very low amounts of CYP3A5 protein could be detected (Fig. 3A). Despite increased mRNA levels that were observed for CYP2A6, CYP2B6, and CYP2C in confluent Huh7 cells, no significant protein expression could be detected. This might suggest that these P450s are regulated at the post-transcriptional level. The mechanism behind this specific increase of CYP3A4 activity in Huh7 cells during confluence warrants further mechanistic studies.

In the liver, as in most tissues, proliferation is associated with reduced expression of normal differentiated functions. In this study we describe that when Huh7 cells are grown confluent, proliferation is inhibited, resulting in a dense layer of cells with more hepatocyte-like characteristics as illustrated by higher expression levels of drug-metabolizing enzymes, transcription factors, and liver-specific genes such as albumin and glucose-6-phosphatase. We previously observed a similar phenomenon when another human hepatoma cell line (namely, B16A2 cells) was grown confluent (Butura et al., 2004). This phenotypical change that occurs during confluence is not a phenomenon common for all the hepatoma cell lines, as illustrated by our results for HepG2 cells. Proliferation of HepG2 cells, when grown confluent, was not inhibited to the same extent as was observed for Huh7 cells. When studying confluent HepG2 cells, we did not see any significant increase in P450 mRNA expression (Figs. 1 and 2B), CYP3A protein expression (Fig. 3B), or CYP3A catalytic activity (Fig. 5) as was observed in confluent Huh7 cells. Moreover, no significant difference in toxic response to aflatoxin B1 could be seen between confluent and nonconfluent control HepG2 cells (data not shown), further confirming the lack of catalytically active CYP3A4 enzyme.

The expression of most P450s is under strict control of various transcription factors that are regulated during differentiation (Burk and Wojnowski, 2004). PXR is considered to be the most important modulator of xenobiotic-induced regulation of the CYP3A genes (Burk and Wojnowski, 2004), together with hepatic nuclear factor (HNF)-4α (Li and Chiang, 2006). In confluent Huh7 cells, the mRNA levels were increased for both PXR and HNF4α already 1 week after reaching confluence. Overall, the LDA results showed that the relative expression levels of many of the hepatic transcription factors in confluent Huh7 were similar to those observed in HL (Fig. 1). In HepG2, where CYP3A4 protein and activity levels were much lower and induced to a lesser extent, the PXR mRNA levels had not changed with time of confluence, and the level of HNF4α decreased (Fig. 1, right). Treatment of confluent Huh7 cells with rifampicin, a well known inducer of CYP3A4 through its interaction with PXR, showed a significant increase in CYP3A4 activity in 4-week confluent cells (Fig. 5B). Together these results indicate that the observed increase in CYP3A4 mRNA, protein, and activity levels are, at least partly, mediated through transcriptional activation via the PXR receptor. However, at present we cannot rule out other mechanisms, such as epigenetic regulation. This was illustrated recently by Li et al. (2009), who showed an important role of histone modifications in the ontogenic expression of CYP3A genes in mice. The increase in CYP3A4 catalytic activity observed in confluent Huh7 cells can be explained by the concomitant and time-dependent increase of CYP3A4 and its electron donor POR. It is believed that under certain conditions, POR can be the rate-limiting factor for P450 activity, because the POR protein is expressed at much lower stoichiometric amounts than the P450s in the liver (Schmucker et al., 1990). Like POR, CYB5A, another well known modulator of CYP3A4 activity (Gan et al., 2009), also gradually increased with time of confluence, although it could not be detected until 3 weeks of confluence.

DMSO is commonly used in drug metabolism and toxicity studies for dissolving hydrophobic compounds. In addition, it is used extensively to alter cell proliferation (Sainz and Chisari, 2006) and to promote differentiation in cell lines (Aninat et al., 2006), although the mechanism by which DMSO induces differentiation of certain cell types still remains obscure. Yuan et al. (2002) have shown that different solvents such as DMSO can affect P450 reactions, thereby providing an inaccurate assessment of drug interaction potential. Moreover, it has been shown that DMSO itself can cause cytotoxicity by impairing the mitochondria (Vignati et al., 2005). In a recently published study, DMSO was shown to increase the expression of several drug-metabolizing enzymes and other liver-specific genes in Huh7 cells (Choi et al., 2009). Our results show that growing Huh7 cells for 4 weeks in the presence of 1% DMSO does not further increase CYP3A4 protein levels or catalytic activity compared with cells grown for the same time without DMSO (Fig. 3, A and B). Moreover, we show that growing Huh7 cells confluent in the presence of DMSO does not result in any increase in CYP3A5, POR, or albumin protein levels (Fig. 3A), suggesting that growing cells confluent for a few weeks without the addition of DMSO is sufficient to induce CYP3A4. In fact, only CYB5A protein levels were significantly increased after DMSO treatment. Taken together, confluent Huh7 cells are an efficient, CYP3A4-expressing in vitro cell system that does not require the addition of any organic solvents or other inducers. In our opinion, this makes it a suitable model system for drug metabolism and toxicity studies.

In conclusion, in the present study we show that when Huh7 cells are grown confluent they display a change in phenotype resulting in a CYP3A4-competent cell line stable up to 8 weeks. This event cannot be induced in HepG2 cells when grown under the same conditions. Moreover, this change in the confluent Huh7 cells occurs without the addition of organic solvents such as DMSO or any other inducers. We propose that confluent Huh7 cells can be used as an in vitro system for the prediction of CYP3A4-dependent metabolism, as well as a model system for studying CYP3A4 regulation.

Acknowledgments.

We thank Åsa Nordling for the high-performance liquid chromatography analysis. We also thank Dr. Robert J. Edwards for the supply of CYP3A4 and CYP3A5 antibodies and Dr. Souren Mkrtchian for the ERp29 antibodies.

Footnotes

  • This work was supported by The Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning; AstraZeneca; and The Swedish Research Council.

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

    doi:10.1124/dmd.110.032367.

  • ABBREVIATIONS:

    P450
    cytochrome P450
    DMSO
    dimethyl sulfoxide
    HL
    human liver
    PXR
    pregnane X receptor
    LDA
    low-density array
    TBP
    TATA-box binding protein
    UGT
    UDP-glucuronosyltransferase
    KCZ
    ketoconazole
    POR
    NADPH-cytochrome P450 reductase
    LDH
    lactate dehydrogenase
    PCR
    polymerase chain reaction
    CYB5A
    cytochrome b5 type A
    HNF
    hepatic nuclear factor.

  • Received January 22, 2010.
  • Accepted March 16, 2010.

References

  1. ↵
    1. Aninat C,
    2. Piton A,
    3. Glaise D,
    4. Le Charpentier T,
    5. Langouët S,
    6. Morel F,
    7. Guguen-Guillouzo C,
    8. Guillouzo A
    (2006) Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab Dispos 34:75–83.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Bradford MM
    (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Burk O,
    2. Wojnowski L
    (2004) Cytochrome P450 3A and their regulation. Naunyn Schmiedebergs Arch Pharmacol 369:105–124.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Butura A,
    2. Johansson I,
    3. Nilsson K,
    4. Wärngård L,
    5. Ingelman-Sundberg M,
    6. Schuppe-Koistinen I
    (2004) Differentiation of human hepatoma cells during confluence as revealed by gene expression profiling. Biochem Pharmacol 67:1249–1258.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Choi S,
    2. Sainz B Jr,
    3. Corcoran P,
    4. Uprichard S,
    5. Jeong H
    (2009) Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells. Xenobiotica 39:205–217.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Domanski TL,
    2. Finta C,
    3. Halpert JR,
    4. Zaphiropoulos PG
    (2001) cDNA cloning and initial characterization of CYP3A43, a novel human cytochrome P450. Mol Pharmacol 59:386–392.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Donato MT,
    2. Lahoz A,
    3. Castell JV,
    4. Gómez-Lechón MJ
    (2008) Cell lines: a tool for in vitro drug metabolism studies. Curr Drug Metab 9:1–11.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Edwards RJ,
    2. Adams DA,
    3. Watts PS,
    4. Davies DS,
    5. Boobis AR
    (1998) Development of a comprehensive panel of antibodies against the major xenobiotic metabolising forms of cytochrome P450 in humans. Biochem Pharmacol 56:377–387.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Eichelbaum M,
    2. Burk O
    (2001) CYP3A genetics in drug metabolism. Nat Med 7:285–287.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Ek M,
    2. Söderdahl T,
    3. Küppers-Munther B,
    4. Edsbagge J,
    5. Andersson TB,
    6. Björquist P,
    7. Cotgreave I,
    8. Jernström B,
    9. Ingelman-Sundberg M,
    10. Johansson I
    (2007) Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells. Biochem Pharmacol 74:496–503.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Gan L,
    2. von Moltke LL,
    3. Trepanier LA,
    4. Harmatz JS,
    5. Greenblatt DJ,
    6. Court MH
    (2009) Role of NADPH-cytochrome P450 reductase and cytochrome-b5/NADH-b5 reductase in variability of CYP3A activity in human liver microsomes. Drug Metab Dispos 37:90–96.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Gómez-Lechón MJ,
    2. Donato T,
    3. Jover R,
    4. Rodriguez C,
    5. Ponsoda X,
    6. Glaise D,
    7. Castell JV,
    8. Guguen-Guillouzo C
    (2001) Expression and induction of a large set of drug-metabolizing enzymes by the highly differentiated human hepatoma cell line BC2. Eur J Biochem 268:1448–1459.
    OpenUrlPubMed
  13. ↵
    1. Goodwin B,
    2. Redinbo MR,
    3. Kliewer SA
    (2002) Regulation of CYP3A gene transcription by the pregnane X receptor. Annu Rev Pharmacol Toxicol 42:1–23.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Guengerich FP
    (1999) Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol 39:1–17.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Gunton JE,
    2. Delhanty PJ,
    3. Takahashi S,
    4. Baxter RC
    (2003) Metformin rapidly increases insulin receptor activation in human liver and signals preferentially through insulin-receptor substrate-2. J Clin Endocrinol Metab 88:1323–1332.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Kamdem LK,
    2. Meineke I,
    3. Gödtel-Armbrust U,
    4. Brockmöller J,
    5. Wojnowski L
    (2006) Dominant contribution of P450 3A4 to the hepatic carcinogenic activation of aflatoxin B1. Chem Res Toxicol 19:577–586.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Kanebratt KP,
    2. Andersson TB
    (2008) Evaluation of HepaRG cells as an in vitro model for human drug metabolism studies. Drug Metab Dispos 36:1444–1452.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Kogure T,
    2. Ueno Y,
    3. Iwasaki T,
    4. Shimosegawa T
    (2004) The efficacy of the combination therapy of 5-fluorouracil, cisplatin and leucovorin for hepatocellular carcinoma and its predictable factors. Cancer Chemother Pharmacol 53:296–304.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Lagadic-Gossmann D,
    2. Rissel M,
    3. Le Bot MA,
    4. Guillouzo A
    (1998) Toxic effects of tacrine on primary hepatocytes and liver epithelial cells in culture. Cell Biol Toxicol 14:361–373.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Li T,
    2. Chiang JY
    (2006) Rifampicin induction of CYP3A4 requires pregnane X receptor cross talk with hepatocyte nuclear factor 4alpha and coactivators, and suppression of small heterodimer partner gene expression. Drug Metab Dispos 34:756–764.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Li Y,
    2. Cui Y,
    3. Hart SN,
    4. Klaassen CD,
    5. Zhong XB
    (2009) Dynamic patterns of histone methylation are associated with ontogenic expression of the Cyp3a genes during mouse liver maturation. Mol Pharmacol 75:1171–1179.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Livak KJ,
    2. Schmittgen TD
    (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Mkrtchian S,
    2. Fang C,
    3. Hellman U,
    4. Ingelman-Sundberg M
    (1998) A stress-inducible rat liver endoplasmic reticulum protein, ERp29. Eur J Biochem 251:304–313.
    OpenUrlPubMed
  24. ↵
    1. Sainz B Jr,
    2. Chisari FV
    (2006) Production of infectious hepatitis C virus by well-differentiated, growth-arrested human hepatoma-derived cells. J Virol 80:10253–10257.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Schenkman JB,
    2. Jansson I
    (2003) The many roles of cytochrome b5. Pharmacol Ther 97:139–152.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Schmucker DL,
    2. Woodhouse KW,
    3. Wang RK,
    4. Wynne H,
    5. James OF,
    6. McManus M,
    7. Kremers P
    (1990) Effects of age and gender on in vitro properties of human liver microsomal monooxygenases. Clin Pharmacol Ther 48:365–374.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Seipp S,
    2. Mueller HM,
    3. Pfaff E,
    4. Stremmel W,
    5. Theilmann L,
    6. Goeser T
    (1997) Establishment of persistent hepatitis C virus infection and replication in vitro. J Gen Virol 78:2467–2476.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Shimada T,
    2. Yamazaki H,
    3. Mimura M,
    4. Inui Y,
    5. Guengerich FP
    (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–423.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Soars MG,
    2. McGinnity DF,
    3. Grime K,
    4. Riley RJ
    (2007) The pivotal role of hepatocytes in drug discovery. Chem Biol Interact 168:2–15.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Spaldin V,
    2. Madden S,
    3. Pool WF,
    4. Woolf TF,
    5. Park BK
    (1994) The effect of enzyme inhibition on the metabolism and activation of tacrine by human liver microsomes. Br J Clin Pharmacol 38:15–22.
    OpenUrlPubMed
  31. ↵
    1. Torimoto N,
    2. Ishii I,
    3. Toyama K,
    4. Hata M,
    5. Tanaka K,
    6. Shimomura H,
    7. Nakamura H,
    8. Ariyoshi N,
    9. Ohmori S,
    10. Kitada M
    (2007) Helices F-G are important for the substrate specificities of CYP3A7. Drug Metab Dispos 35:484–492.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Vignati L,
    2. Turlizzi E,
    3. Monaci S,
    4. Grossi P,
    5. Kanter R,
    6. Monshouwer M
    (2005) An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics. Toxicology 216:154–167.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Waxman DJ,
    2. Attisano C,
    3. Guengerich FP,
    4. Lapenson DP
    (1988) Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme. Arch Biochem Biophys 263:424–436.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Westlind A,
    2. Malmebo S,
    3. Johansson I,
    4. Otter C,
    5. Andersson TB,
    6. Ingelman-Sundberg M,
    7. Oscarson M
    (2001) Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43. Biochem Biophys Res Commun 281:1349–1355.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Yoo BJ,
    2. Selby MJ,
    3. Choe J,
    4. Suh BS,
    5. Choi SH,
    6. Joh JS,
    7. Nuovo GJ,
    8. Lee HS,
    9. Houghton M,
    10. Han JH
    (1995) Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV. J Virol 69:32–38.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Yuan R,
    2. Madani S,
    3. Wei XX,
    4. Reynolds K,
    5. Huang SM
    (2002) Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos 30:1311–1319.
    OpenUrlAbstract/FREE Full Text
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CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

Louise Sivertsson, Monica Ek, Malin Darnell, Irene Edebert, Magnus Ingelman-Sundberg and Etienne P. A. Neve
Drug Metabolism and Disposition June 1, 2010, 38 (6) 995-1002; DOI: https://doi.org/10.1124/dmd.110.032367
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