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Blood Monocyte-Derived Neohepatocytes as in Vitro Test System for Drug Metabolism

Department of Medicine II, Faculty of Medicine at Mannheim, University of Heidelberg, Heidelberg, Germany (S.E., S.D.); Department of Traumatology, Technical University Munich, Munich, Germany (A.K.N.); and Department of General Surgery, University of Medicine Berlin, Campus Virchow, Berlin, Germany (A.L.)

(Received January 16, 2008; Accepted June 12, 2008)

	Abstract

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The gold standard for human drug metabolism studies is primary hepatocytes. However, availability is limited by donor organ scarcity. Therefore, efforts have been made to provide alternatives, e.g., the hepatocyte-like (NeoHep) cell type, which was generated from peripheral blood monocytes. In this study, expression and activity of phase I and phase II drug-metabolizing enzymes were investigated during transdifferentiation of NeoHep cells and compared with primary human hepatocytes. Important drug-metabolizing enzymes are cytochrome P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, and 3A4), microsomal epoxide hydrolase 1, glutathione S-transferase A1 and M1, N-acetyltransferase 1, NAD(P)H menadione oxidoreductase 1, sulfotransferase 1A1, and UDP-glucuronosyltransferase 1A6. Monocytes and programmable cells of monocytic origin expressed only a few of the enzymes investigated. Throughout differentiation, NeoHep cells showed a continuously increasing expression of all drug-metabolizing enzymes investigated, resulting in stable basal activity after approximately 15 days. Fluorescence-based activity assays indicated that NeoHep cells and primary hepatocytes have similar enzyme kinetics, although the basal activities were significantly lower in NeoHep cells. Stimulation with 3-methylcholanthrene and rifampicin markedly increased CYP1A1/2 or CYP3A4 activities, which could be selectively inhibited by nifedipine, verapamil, ketoconazole, and quercetin. Our data reveal similarities in expression, activity, induction, and inhibition of drug-metabolizing enzymes between NeoHep cells and primary human hepatocytes and hence suggest that NeoHep cells are useful as an alternative to human hepatocytes for measuring bioactivation of substances.

Drug metabolism in the liver can be divided into two phases (Williams, 1959). Phase I metabolism adds a functional group (e.g., OH, SH, or NH₂) to the substrate by oxidative, reductive, and hydrolytic pathways. Phase II metabolic enzymes modify the newly introduced functional group to O- and N-glucuronides, sulfate esters, various -carboxyamides, and S-glutathionyl adducts to increase their polarity (Parkinson, 1996), making elimination from the cells more rapid. Thus, hepatocytes mediate detoxification by activation of phase I and II enzymatic pathways.

All members of the cytochrome P450 superfamily, belonging to phase I drug-metabolizing enzymes, can be identified by a highly conserved heme-thiolate functionality, responsible for their catalytic mechanism (Nelson et al., 2004). Amino acid variations in their substrate binding sites confer compound selectivity, regioselectivity, and stereoselectivity of the enzymes (Guengerich and MacDonald, 1990), representing the rate-limiting step of the drug biotransformation processes. Experimental data suggest that most biotransformation of xenobiotics is done by enzymes of the first three families (CYP1, CYP2, and CYP3), whereas other P450s are involved in "housekeeping" metabolism of endogenous molecules (Parkinson, 1996; Pelkonen et al., 1998).

To date, primary human hepatocyte cultures are the most powerful tool for in vitro studies (Hewitt et al., 2007), although they have major limitations owing to donor organ scarcity and rapid cellular changes during culture (Guillouzo et al., 1993), resulting in a strong demand for alternative in vitro systems. The human hepatoma cell line HepG2, which secretes low levels of many plasma proteins characteristic for normal human liver cells (Knowles et al., 1980), is best used to study induction of drug-metabolizing enzymes, as their basal expression is significantly lower than that in primary human hepatocytes (Rodriguez-Antona et al., 2002; Wilkening et al., 2003) and strongly varies during culture time (Wilkening and Bader, 2003).

In recent years, numerous reports have described the generation of hepatocytes or "hepatocyte-like" cells from various types of extrahepatic cells (Hengstler et al., 2005; Ruhnke et al., 2005a,b; Nussler et al., 2006). In this study, we wanted to investigate the usefulness of hepatocyte-like (NeoHep) cells, derived by transdifferentiation of peripheral blood monocytes (PBMCs), as in vitro test system for drug screening purposes. Therefore, we analyzed expression and activity of P450s involved in xenobiotic metabolism with high abundance in the liver, namely CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, and 3A4 (Shimada et al., 1994; Rendic and Di Carlo, 1997; Hewitt et al., 2007). There exist various modes of regulation, defining the relative abundance and activity of each P450 isoform, which include induction (CYP1A1, 1A2, 2A6, 2E1, 2C, and 3A4), inhibition (all P450s), and genetic polymorphism (CYP2A6, 2C9, 2C19, and 2D6) (Rendic and Di Carlo, 1997). These are important to ascertain to minimize potential drug-drug interactions in the development of drug candidates (Lin and Lu, 1998; Hewitt et al., 2007). Therefore, we investigated the effect of model inducers (3-MC and RIF) and inhibitors (NIF, VER, KET, and QUE) on P450 expression and activity.

Most reports only describe the expression of P450s and neglect the expression of phase II enzymes, which are also important for activation and detoxification of many xenobiotics (Cantelli-Forti et al., 1998). To obtain a more complete view about the drug-metabolizing potential of NeoHep cells, expression and activity of phase II enzymes, EPHX1, GST A1 and M1, NAT1, NMO1, SULT1A1, and UGT1A6 were determined in the present work.

	Materials and Methods

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Chemicals. The following chemicals were used: human recombinant fibroblast growth factor 4, human recombinant interleukin 3, and human recombinant macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN); AHMC, 3-(2-N,N-diethyl-N-methylaminoethyl)-7-methoxy-4-methylcoumarin, anti-β-actin antibody, 7-benzyloxy-4-(trifluoromethyl)coumarin, 3-cyano-7-ethoxycoumarin, 3-cyano-7-hydroxycoumarin, CHC, coumarin, dibenzylfluorescein, 7-ethoxy-4-(trifluoromethyl)coumarin, HC, HFC, Histopaque-1077, KET, MCB, 2-mercaptoethanol, 3-MC, 7-methoxy-4-(trifluoromethyl)coumarin, NIF, QUE, resorufin, RIF, SRB, TRIzol, VER, and Williams' E medium (Sigma-Aldrich, München, Germany); fetal bovine serum (European Commission approved, South American origin), L-glutamine, human serum type AB, penicillin/streptomycin, and RPMI 1640 medium (Cambrex, Taufkirchen, Germany); β-glucuronidase/arylsulfatase mix (Roche Diagnostics, Mannheim, Germany); anti-CYP2C8/9/19, anti-CYP2E1, and anti-CYP3A4 antibodies (CHEMICON, Chandlers Ford, UK); anti-CYP1A1/2, anti-CYP2A/B6, and anti-CYP2D6 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology Inc., Danvers, MA); and NuPAGE bis-tris gels (Invitrogen, Karlsruhe, Germany).

Generation of Programmable Cells of Monocytic Origin. PCMOs were generated from peripheral blood of healthy volunteers as described (Ruhnke et al., 2005a,b). Withdrawal of blood was approved by the local ethics committee of the Medical Faculty at Mannheim, University of Heidelberg (Proposal 164/05). In brief, PBMCs were isolated by density gradient centrifugation (Histopaque-1077). The resulting mononuclear cell fraction was allowed to adhere to tissue culture plastic (1.0 x 10⁶ cells/cm²) for 2 h in RPMI 1640 medium (10% human AB serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin). Nonadherent cells were removed by aspiration, and remaining cells were cultured for 6 days in RPMI 1640-based medium (see above) supplemented with 5 ng/ml human recombinant macrophage colony-stimulating factor, 0.4 ng/ml human recombinant interleukin 3, and 0.1 mM 2-mercaptoethanol.

Generation and Culture of NeoHep Cells. For differentiation into NeoHep cells, PCMOs (day 6) were cultured in hepatocyte conditioning medium (RPMI 1640, 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 3 ng/ml human recombinant fibroblast growth factor 4) as reported (Ruhnke et al., 2005a,b). Culture medium was replaced every 3rd day.

Isolation of Primary Human Hepatocytes. Human liver tissue was obtained according to the institutional guidelines from liver resections of tumor patients with primary or secondary liver tumors. Hepatocytes were isolated by a two-step collagenase perfusion technique followed by a Percoll gradient centrifuge for purification, as described previously (Dorko et al., 1994). Hepatocyte viability was consistently greater than 90%, as assessed by the trypan blue exclusion test. Freshly harvested hepatocytes were cultured on rat-tail collagen-coated culture plates in Williams' E medium (10% fetal calf serum, 1 M insulin, 15 mM HEPES, 1.4 M hydrocortisone, 100 U/ml penicillin, and 100 mg/ml streptomycin).

Treatment of Cells. Before all experiments, the cells were serum-starved overnight in William's E medium (2 mM L-glutamine). Treatment with the different substances was performed in serum-free medium for the indicated times and concentrations. Control conditions included cells maintained for the same period in serum-free medium supplemented with the solvent chemical.

Conventional RT-PCR. Total cellular RNA was isolated using TRIzol reagent according to the manufacturer's protocol. First-strand cDNA was synthesized from 1 µg of total RNA using the QuantiTect reverse transcription kit (QIAGEN, Hilden, Germany). Primer sequences and the corresponding annealing temperatures are summarized in Table 1. Appropriate cDNA dilutions and PCR cycling numbers were determined for each gene to ensure that the PCR did not reach saturation. PCR products resolved by gel electrophoresis in a 1.5% (w/v) agarose gel (in Tris-borate EDTA) were visualized by ethidium bromide.

Western Blot Analysis. Cells were lysed in ice-cold RIPA buffer (50 mM Tris, 250 mM NaCl, 2% Nonidet P-40, 2.5 mM EDTA, 0.1% SDS, 0.5% deoxycholic acid, protease inhibitor, and 1.0% phosphatase inhibitor, pH 7.2) as described (Wiercinska et al., 2006). Protein concentration was measured with the DC Protein Assay Kit (Bio-Rad, Munich, Germany). Total protein lysates were separated by SDS-polyacrylamide gel electrophoresis using NuPAGE bis-tris gels and transferred to nitrocellulose membranes (VWR, Darmstadt, Germany). Immunoblotting proceeded as described (Weng et al., 2007).

Cytochrome P450 Activity Assays. Fluorescence-based P450 assays were performed by incubation of intact cells (in 96-well plates) with selected substrates as reported (Donato et al., 2004). In brief, 100 µl of reaction buffer (1 mM Na₂HPO₄, 137 mM NaCl, 5 mM KCl, 0.5 mM MgCl₂, 2 mM CaCl₂, 10 mM O-(+)-glucose, and 10 mM HEPES, pH 7.4) containing the appropriate amount of fluorogenic substrate were added to each well. After incubation at 37°C, supernatants were transferred to white/black 96-well plates, and cells were fixed for protein quantification by SRB staining. Potential metabolite conjugates formed were hydrolyzed by incubation of supernatants with β-glucuronidase/arylsulfatase (150 Fishman units/ml and 1200 Roy units/ml, respectively) for 2 h at 37.0°C. Samples were diluted (1:4) with the appropriate quenching solution. Formation of fluorescent metabolite was quantified by means of a Fluoroskan Ascent fluorescence microplate reader (Thermo Fisher Scientific, Egelsbach, Germany). Results are given as picomoles of metabolite formed per minute normalized to total protein content. Experimental conditions are summarized in Table 2. Methanol-fixed cells were used for background subtraction.

Phase II Enzyme Activity Assays. Cells, cultured in 96-well plates, were incubated at 37°C with 100 µl of reaction buffer (1 mM Na₂HPO₄, 137 mM NaCl, 5 mM KCl, 0.5 mM MgCl₂, 2 mM CaCl₂, 10 mM O-(+)-glucose, and 10 mM HEPES, pH 7.4), containing the appropriate amount of fluorescent substrate (products from P450 assays). After 15, 30, 45, 60, 90, and 120 min the remaining fluorescent signal in the supernatant (Fluoroskan Ascent fluorescence microplate reader) was determined, and cells were fixed for protein quantification by SRB staining. Results are given as nanomoles of fluorescent substrate reduced per minute normalized to total protein content. Methanolfixed cells were used as negative control (background subtraction).

SRB Staining. SRB staining was performed as reported (Skehan et al., 1990). In brief, cells were covered with ice-cold fixation buffer (95% ethanol and 5% acetic acid) and kept at –20°C for 1 h. Fixed cells were stained with 0.4% SRB (w/v) in 1% acetic acid for 30 min. Unbound dye was removed by washing with 1% acetic acid. Bound SRB was resolved in 10 mM unbuffered Tris solution and optical densities at 565 nm (SRB) and 690 nm (background) were determined. From the optical densities, we calculated the total protein content with the standard curve (y = 1.0305x – 1.6519; R² = 0.9868), obtained by plotting the optical density from SRB staining versus total protein contents measured with the DC Protein Assay Kit.

Statistics. Results are expressed as mean ± S.E.M. Curve fitting was performed using GraphPad Prism software (GraphPad Software Inc., El Camino Real, CA), which allowed determination of R², kinetics parameters, and EC₅₀ values. Results were analyzed by analysis of variance followed by paired comparison (Bonferroni), as appropriate. P < 0.05 was taken as the minimal level of significance.

View larger version (34K): [in this window] [in a new window]   FIG. 1. NeoHep cells express phase I and II drug-metabolizing enzymes. RT-PCR and Western blot analysis was performed with pooled samples from five individual preparations of monocytes (mono), PCMOs, NeoHep cells from healthy controls, and human hepatocytes (Heps). The cDNA template and total protein contents loaded were adjusted so as not to reach saturation of signal. a, RT-PCR for basal mRNA levels of human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP3A4. *, unexpected PCR product, which is not present in Heps. b, Western blots showing basal protein levels of CYP1A1/2, CYP2A/B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4. c, RT-PCR showing basal mRNA levels of the human phase II enzymes, EPHX1, GST A1, GST M1, NAT1, NMO1, SULT1A1, and UGT1A6. In a through c β-actin was used as loading control. h, human; nc, negative control.

	Results

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View larger version (34K): [in this window] [in a new window]   FIG. 2. NeoHep cells display cytochrome P450 metabolite release over time similar to that of human hepatocytes. NeoHep cells (N = 4; n = 4; ——) and human hepatocytes (N = 4; n = 4; ·····) were incubated for 0 to 120 min with the individual substrates. After hydrolysis of metabolite conjugates with β-glucuronidase/arylsulfatase, the amount of metabolite formed was normalized to total protein content and plotted versus the corresponding incubation times. Curve fitting for kinetics of CYP1A1/2 (a), CYP2A6 (b), CYP2B6 (c), CYP2C8/9 (d), CYP2D6 (e), CYP2E1 (f), and CYP3A4 (g) was done using GraphPad Prism software. Background signal was determined by incubation of methanol-fixed cells and subtracted from each measurement value.

All phase II drug-metabolizing enzymes investigated were expressed (mRNA) in NeoHep cells (Fig. 1c). Similar to P450 isoforms, phase II enzyme expression was generally lower in NeoHep cells (EPHX1 68%, GST A1 87%, GST M1 72%, NAT1 66%, NMO1 66%, SULT1 73%, and UGT1A6 29%) compared with human hepatocytes. Most phase II enzymes except GST A1 and UGT 1A6, which were exclusively expressed in NeoHep cells and primary hepatocytes, were also expressed in monocytes and PCMOs.

Incubation with P450 Isozyme-Selective Substrates Leads to Comparable Metabolite Release over Time in NeoHep Cells and Human Hepatocytes. Enzyme kinetic experiments were performed with substrates that are isozyme-selective for CYP1A1/2, CYP2A6, CYP2B6, CYP2C8/9, CYP2D6, CYP2E1, and CYP3A4 in both NeoHep cells (N = 4; n = 4) and human hepatocytes (N = 4; n = 4) (Fig. 2, a–g). Cells were incubated for 0 to 120 min with the appropriate substrates. Metabolite conjugates formed were hydrolyzed for 2 h at 37.0°C with β-glucuronidase/arylsulfatase. The amount of metabolite formed was normalized to total protein content and plotted versus the incubation time. The correlation coefficients (R²) from curve-fitting are summarized in Table 3. RT-PCR and Western blot analysis indicated lower expression of P450 isoforms in NeoHep cells compared with that in human hepatocytes (Fig. 1, a and b); thus, it was not surprising that the plateau values (Table 3) were lower in NeoHep cells. However, the time points at which the plateau is half-reached (t_{1/2 Plateau}) (Table 3) are comparable between both cell types. Based on these results, substrate incubation times for all further experiments were determined to be between t_{1/2 Plateau} and t_Plateau (Table 2) not to measure product release during steady-state (plateau) conditions.

NeoHep Cells and Human Hepatocytes Display Similar Phase II Drug Metabolism. Because P450 activities were measured in intact cells, metabolites formed by P450 isoforms are continuously processed by various phase II drug-metabolizing enzymes. Phase II activities in human hepatocytes (N = 4; n = 4) and NeoHep cells (N = 4; n = 4) are presented as reduction (conjugation) of metabolites AHMC, CHC, HC, HFC, fluorescein, and resorufin (Fig. 3, a–f). Furthermore, GST activity was measured by conjugation of MCB (Fig. 3g).

View larger version (32K): [in this window] [in a new window]   FIG. 3. Reduction (conjugation) of metabolites as a measure of active phase II drug metabolism. NeoHep cells (N = 4; n = 4; ——) and human hepatocytes (N = 4; n = 4; ·····) were incubated for 0 to 120 min with a defined concentration of the metabolites formed during P450 enzyme assays. Reduction (conjugation) of AHMC (excitation/emission = 390/460 nm) (a), CHC (excitation/emission = 390/460 nm) (b), HC (excitation/emission = 355/460 nm) (c), HFC (excitation/emission = 390/520 nm) (d), fluorescein (excitation/emission = 485/538 nm) (e), and resorufin (excitation/emission = 530/590 nm) (f) was determined by the decrease in fluorescent signal. The amount of substrate degraded was normalized to total protein content and plotted versus the corresponding incubation times. Background signal was determined by incubation of methanol-fixed cells and subtracted from each measurement value. Curve-fitting was done using GraphPad Prism software. rel., relative.

The amount of metabolite degraded was normalized to total protein content and plotted versus the corresponding incubation times. Correlation coefficients (R²) and half-life times (t_1/2) obtained from curve-fitting were comparable between NeoHep cells and human hepatocytes (Table 4). RT-PCR results indicated that expression of phase II drug-metabolizing enzymes is lower in NeoHep cells compared with that in freshly isolated human hepatocytes (Fig. 1c). This is supported by enhanced degradation of metabolites in human hepatocytes given by lower plateau phase values for CHC, HC, and HFC, whereas those for fluorescein degradation were comparable between both cell types.

Possible metabolite conjugates formed were hydrolyzed by incubating supernatants with β-glucuronidase/arylsulfatase (150 Fishman units/ml and 1200 Roy units/ml, respectively) as described previously (Donato et al., 2004). Kinetics for CHC, HC, HFC, and fluorescein "back-reaction" were measured, and an incubation time of 2 h at 37°C was sufficient to hydrolyze all metabolite conjugates formed (data not shown).

Basal P450 Activities Increase during Differentiation of NeoHep Cells. After having defined reaction conditions for CYP1A1/2, CYP2A6, CYP2B6, CYP2C8/9, CYP2D6, CYP2E1, and CYP3A4, basal P450 activities were measured during transdifferentiation of monocytes to NeoHep cells and compared with those in freshly isolated human hepatocytes (Fig. 4, a–g). Monocytes (N = 11; n = 4) and PCMOs (N = 11; n = 4) showed no significant activities for the P450 isoforms investigated. P450 activities of NeoHep cells (N = 12; n = 4; measured every 5th day in culture) increased during the first 15 days and then reached a stable level. Compared with those in human hepatocytes (N = 10; n = 4), maximal P450 activities of NeoHep cells were significantly lower, which is in line with results from RT-PCR and Western blot analysis (Fig. 1, a and b).

View larger version (27K): [in this window] [in a new window]   FIG. 4. Basal cytochrome P450 activities during transdifferentiation of NeoHep cells. Basal P450 enzyme activities in monocytes (mono) (N = 11; n = 4), PCMOs (N = 11; n = 4), and NeoHep cells (N = 12; n = 4) at days 5, 10, 15, and 20 of differentiation and human hepatocytes (Hep) (N = 10; n = 4). Cytochrome P450 isoforms measured were CYP1A1/2 (a), CYP2A6 (b), CYP2B6 (c), CYP2C8/9 (d), CYP2D6 (e), CYP2E1 (f), and CYP3A4 (g). Activity was given as picomoles of metabolite per minute per milligram of protein. *, p < 0.05; **, p < 0.01; ***, p < 0.001 of values being significantly greater than 0.

View larger version (56K): [in this window] [in a new window]   FIG. 5. Induction of CYP1A1/2 and CYP3A4 in NeoHep cells and human hepatocytes with rifampicin and 3-methylcholanthrene, NeoHep cells (N = 12; n = 3) () and human hepatocytes (N = 4; n = 4) () were incubated for 72 h with 25 µM 3-MC or 25 µM RIF. DMSO (0.1%) was used as a solvent control. CYP1A1/2 activity (a) and CYP3A4 activity (b) were determined and fold induction by 3-MC and RIF was calculated. c, protein lysates were taken to confirm increased expression of CYP1A1/2 and CYP3A4 by Western blotting in both cell types. Total protein lysate was loaded (30 µg/lane). β-Actin was used as a loading control. **, p < 0.01; ***, p < 0.001.

Effect of Model Inhibitors on CYP1A1/2 and CYP3A4 Activity Is Comparable in NeoHep Cells and Human Hepatocytes. NIF, VER, KET, and QUE are model inhibitors for various P450 isoforms. Because basal levels of all P450 isoforms were significantly lower in NeoHep cells than in freshly isolated human hepatocytes, the effects (EC₅₀) of these P450 inhibitors on induced CYP1A1/2 and CYP3A4 activities were investigated in both cell types. Human hepatocytes (N = 2; n = 2) and NeoHep cells (N = 4; n = 2) were treated with 25 µM 3-MC or RIF. DMSO was used as a solvent control. After 72 h the stimulation medium was removed, and the cells were preincubated for 15 min with different concentrations of nifedipine, verapamil, ketoconazole, or quercetin, before CYP1A1/2 and CYP3A4 activities were measured in the presence of the inhibitors. The corresponding correlation coefficients (R²) and EC₅₀ values are summarized in Table 5.

	Discussion

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In the present study, NeoHep cells were generated from PBMCs donated by healthy volunteers to evaluate their potential as an alternative in vitro test system for drug screening. Secretion of urea and glucose and production of alanine aminotransferase as well as aspartate aminotransferase confirmed earlier observations (Ruhnke et al., 2005a), ensuring the quality of our NeoHep cells. We compared expression and activity of phase I drug-metabolizing enzymes of NeoHep cells and primary human hepatocytes, which comprise more than 90% of drug oxidation in humans, namely CYP1A2 (4–6%), CYP2A6 (2%), CYP2B6 (25%), CYP2C9 (10–11%), CYP2D6 (25%), CYP2E1 (2–5%), and CYP3A4 (52%) (Shimada et al., 1994). In contrast to PCMOs, which only express CYP2E1, expression of all P450 isoforms was increased during differentiation of NeoHep cells but remained much lower compared with that in isolated human hepatocytes. Several reports show a gradual decrease in P450 expression over time in primary human hepatocytes (LeCluyse, 2001; Rodriguez-Antona et al., 2002; Gomez-Lechon et al., 2004), limiting their use for long-term studies. To overcome this problem hepatocytes have been cultured between two layers of collagen (Kern et al., 1997; Tuschl and Mueller, 2006) or in the presence of appropriate cocktails of inducers (Pichard-Garcia et al., 2002).

Therefore, for in vitro screening of new substances or to predict their possible toxicology, there are many hepatocyte-like cell lines in use. However, most of these cell lines have major limitations, e.g., reduced expression of xenosensors, leading to differences in drug metabolism (Hewitt et al., 2007), as well as incomplete and very low expression levels of drug-metabolizing enzymes (Wilkening et al., 2003, Donato et al., 2008). HepG2 and Hep1c1c7 cells, for example, display strongly down-regulated expression of the pregnane X receptor and the constitutive androstane receptor (Pascussi et al., 2001), which are strongly involved in the regulation of CYP3A4. Increased CYP3A4 expression in NeoHep cells by RIF indicates a sufficient expression level of both receptors in this cell type (Gerbal-Chaloin et al., 2006). Similar to HepG2 and Hepa1c1c7 cells, the arylhydrocarbon receptor, strongly involved in the regulation of CYP1A isoenzymes, is expected to be expressed in NeoHep cells, because CYP1A1 and CYP1A2 were inducible with 3-MC (Pascussi et al., 2001). A detailed analysis indicated that P450 expression levels in HepG2 cells varied greatly during culture, leading to strongly variable passage-dependent results (Wilkening and Bader, 2003). In contrast, P450 activity levels of NeoHep cells remained constant for several days.

Similar to hepatoma cell lines, the basal P450 activities in NeoHep cells were also very low, possibly limiting their use for inhibition experiments. Nevertheless, we were able to show (competitive) inhibition of CYP1A1/2 and CYP3A4 activity induced by 3-MC and RIF, using NIF, VER, KET, and QUE. The resulting EC₅₀ values in NeoHep cells were lower than those in human hepatocytes. This might be explained by relatively low P450 baseline levels in NeoHep cells or nonspecific binding of substrate to cell membranes and albumin.

Phase II drug-metabolizing enzymes were only slightly reduced in NeoHep cells compared with primary human hepatocytes (Ruhnke et al., 2005a). Although P450 isoform activities are reduced in the presence of human growth factor, phase II enzymes UGT and GST are not regulated by this growth factor. In contrast to P450 isoforms, almost all phase II drug-metabolizing enzymes, except GST A1 and UGT 1A6, were already expressed in monocytes and PCMOs, and their expression level did not increase significantly during differentiation to NeoHep cells. Thus, it was not surprising that degradation kinetics of AHMC, CHC, HC, HFC, fluorescein, and resorufin were comparable between NeoHep cells and primary hepatocytes, with higher plateau values for NeoHep cells. Similar results were observed for GST activity, represented by MCB conjugation. These results are supported by an earlier observation (Ruhnke et al., 2005b) showing that UGT activity, measured by 4-methylumbelliferone conjugation, is comparable between both NeoHep cells and primary human hepatocytes.

Phenotypic as well as genotypic differences in the expression of drug-metabolizing enzymes are the main causes for high interindividual metabolic variations (Hewitt et al., 2007), not taking into consideration nongenetic factors such as smoking, age, diet, hormonal status, environmental chemicals, and disease state (Shah, 2005; Singh, 2006). There are many polymorphisms reported, which may lead to complete inactivation, decreased activity, or altered substrate specificity of P450s (Ingelman-Sundberg, 2005). In line with this, expression and activity of drug-metabolizing enzymes varied strongly among hepatocytes from different donors (Rodriguez-Antona et al., 2001). Similar donor-dependent variations were observed for NeoHep cells. Donor variations in the response to different inducers further complicate predictions of therapeutic doses, which can lead to severe side effects. In case of the anticoagulant warfarin, certain variants of CYP2C9 and vitamin K epoxide reductase complex subunit 1 require lower doses of warfarin than average to obtain the same therapeutic effect and are more likely to cause bleeding complications at standard doses (Jones, 2007). This finding underlines the need for "personalized medicine," which comprises screening of different therapeutic approaches in vitro before applying a treatment to the patient (Singh, 2006). Although direct comparison is still missing, NeoHep cells display individual variability in P450 expression and activity similar to that seen in human hepatocytes. Because NeoHep cells can be generated from blood samples, they represent a promising tool for personalized therapeutic screenings in the future.

	Acknowledgments

 
We appreciate excellent technical assistance from A. Müller.

	Footnotes

 
This work was supported by the Dietmar Hopp Foundation, the Federal Ministry of Research (BMBF 0313081B/HepatoSys), and Fresenius Biotech GmbH.

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

doi:10.1124/dmd.108.020453.

ABBREVIATIONS: P450, cytochrome P450; PBMC, peripheral blood monocyte; 3-MC, 3-methylcholanthrene; RIF, rifampicin; NIF, nifedipine; VER, verapamil; KET, ketoconazole; QUE, quercetin dehydrate; EPHX1, microsomal epoxide hydrolase 1; GST, glutathione S-transferase; NAT1, N-acetyltransferase 1; NMO, NAD(P)H menadione oxidoreductase 1; SULT1, sulfotransferase 1A1; UGT1A6, UDP-glucuronosyltransferase 1A6; AHMC, 3-(2-(N,N-diethylamino)ethyl)-7-hydroxy-4-methylcoumarin; CHC, 3-cyano-7-hydroxycoumarin; SRB, sulforhodamine B; HC, 7-hydroxycoumarin; HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; MCB, monochlorobimane; PCMO, programmable cell of monocytic origin; RT, reverse transcriptase; PCR, polymerase chain reaction; N, number of individual experiments/donors; n, number of replicates.

¹ Current affiliation: Department of Traumatology, Technical University Munich, Munich, Germany.

Address correspondence to: Dr. Steven Dooley, Department of Medicine II, Gastroenterology and Hepatology, University Hospital Mannheim, Theodor-Kutzer Ufer 1-3, 68167 Mannheim, Germany. E-mail: steven.dooley{at}med.ma.uni-heidelberg.de

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