Prediction of Drug-Induced Liver Injury in HepG2 Cells Cultured with

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Prediction of Drug-Induced Liver Injury in HepG2 Cells Cultured with Human Liver Microsomes Jong Min Choi, Soo Jin Oh, Ji-Yoon Lee, Jang Su Jeon, Chang Seon Ryu, Young-Mi Kim, Kiho Lee, and Sang Kyum Kim Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/tx500504n • Publication Date (Web): 10 Apr 2015 Downloaded from http://pubs.acs.org on April 12, 2015

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Chemical Research in Toxicology

Prediction of Drug-Induced Liver Injury in HepG2 Cells Cultured with Human Liver Microsomes

Jong Min Choi, † Soo Jin Oh, ‡ Ji-Yoon Lee, † Jang Su Jeon, † Chang Seon Ryu, † YoungMi Kim, § Kiho Lee ║,*, Sang Kyum Kim †,*

†

College of Pharmacy, Chungnam National University, Daejeon, 305-764, Republic of Korea

‡

Bio-Evaluation Center, KRIBB, Ochang, Chungbuk, 363-883, Republic of Korea

§

College of Pharmacy, Hanyang University, Ansan, Gyeonggido, 426-791 Republic of Korea

║

*

College of Pharmacy, Korea University, Sejong, 339-700 Republic of Korea

Corresponding author: College of Pharmacy, Korea University, Sejong, 339-700 Republic

of Korea Tel.: +82 44 860 1616. Fax: +82 44 860 1606. E-mail address: [email protected] (K. Lee).

*

Corresponding author: College of Pharmacy, Chungnam National University, 220 Gung-

dong, Yuseong-gu, Daejeon 305-764, Republic of Korea. Tel.: +82 42 821 5930; Fax: +82 42 823 6566. E-mail address: [email protected] (S. K. Kim).

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Table of Contents Graphic

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ABSTRACT Drug-induced liver injury (DILI) via metabolic activation by drug-metabolizing enzymes, especially cytochrome P450 (CYP), is a major cause of drug failure and drug withdrawal. In this study, an in vitro model using HepG2 cells in combination with human liver microsomes was developed for prediction of DILI. The cytotoxicity of cyclophosphamide, a model drug for bioactivation, was augmented in HepG2 cells cultured with microsomes in a manner dependent on exposure time, microsomal protein concentration, and NADPH. Experiments using pan- or isoform-selective CYP inhibitors showed that CYP2B6 and CYP3A4 are responsible for bioactivation of cyclophosphamide. In a metabolite identification study employing LC-ESI-QTrap and LC-ESI-QTOF, cyclophosphamide metabolites including phosphoramide mustard, a toxic metabolite, were detected in HepG2 cells cultured with microsomes, but not without microsomes. The cytotoxic effects of acetaminophen and diclofenac were also potentiated by microsomes. The potentiation of acetaminophen cytotoxicity was dependent on CYP-dependent metabolism, and the augmentation of diclofenac cytotoxicity was not mediated by either CYP- or UDP-glucuronosyltransferasedependent metabolism. The cytotoxic effects of leflunomide, nefazodone, and bakuchiol were attenuated by microsomes. The detoxication of leflunomide by microsomes was attributed to mainly CYP3A4-dependent metabolism. The protective effect of microsomes against nefazodone cytotoxicity was dependent on both CYP-mediated metabolism and nonspecific protein binding. Nonspecific protein binding but not CYP-dependent metabolism played a critical role in the attenuation of bakuchiol cytotoxicity. The present study suggests that HepG2 cells cultured with human liver microsomes can be a reliable model in which to predict DILI via bioactivation by drug metabolizing enzymes.

Keywords: drug-induced toxicity; bioactivation; detoxication; human liver microsomes; cytochrome P450

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INTRODUCTION

Drug-induced liver injury (DILI) is a major cause of failure in drug development and withdrawal approved drugs from the market.1 Thus, studies of DILI in drug discovery and development are recognized as essential means of ensuring drug safety.2 Although the mechanisms of DILI are not always known, metabolic activation has been reported to be one major reason for DILI.3 Various in vitro assays have been used to predict and characterize DILI during the process of drug discovery. Primary cultured human hepatocytes are frequently used as an in

vitro cell model for evaluation of drug metabolism and drug-induced cytotoxicity because they are fully competent metabolic cells and retain the expression of both phase I and II enzymes.4 However, they have several limitations including limited availability, in vitro phenotypic instability, individual variation, difficulty of handling, and high cost for experiments.4,5 To overcome these limitations, various cell lines (e.g., BC2, HepaRG, and confluent Huh7) and genetically engineered cells expressing human drug-metabolizing enzymes have been proposed as alternative models of primary hepatocytes.4,6-11 Although these new cell lines and new strategies have a number of advantages including stable phenotype, unlimited lifespan, availability, and good reproducibility, these in vitro systems also have several disadvantages, including limited expression of drug-metabolizing enzymes, especially cytochrome P450 (CYP), inconsistency with in vivo hepatotoxicity, long culture time, and patents. Unlike these approaches, a method using the rat liver S9 fraction was proposed by Otto et al. (2008).12 This method employs cellular systems in combination with rat S9 fraction retaining metabolic activity to compensate for the low basal metabolic activity of hepatoma cell lines and facilitated simple and rapid high-throughput screening. However, this method

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also has some limitations in terms of species differences and difficulty of analyzing toxic metabolites, because of the complexity of the liver S9 fraction. The problems with method using rat liver S9 fraction mentioned above may be resolved by using human liver microsomes instead of the S9 fraction. Liver microsomes have higher metabolic activity than the liver S9 fraction and it is possible to analyze toxic metabolites without predominant detoxication processes, such as glutathione-dependent metabolism. Thus, application of human liver microsomes can improve prediction of DILI under clinical conditions. The objective of the present study was to develop an in vitro assay using HepG2 cells in combination with human liver microsomes for prediction of DILI. HepG2 is the most widely used human hepatoma cell line that retains liver-specific functions by expressing liverabundant proteins, such as albumin, hepatocyte nuclear factors, and conjugating enzymes.13,14 Although HepG2 cells are not a suitable alternative to primary hepatocytes for estimating DILI because of their low level of CYP expression, microsomes may sufficiently compensate for the low basal activity of CYP isoforms. In this study, cytotoxicity induced by various chemicals, such as cyclophosphamide (CPA), acetaminophen, diclofenac, leflunomide, nefazodone, bakuchiol, coumarin, and tolcapone, was determined in HepG2 cells in the presence of microsomes. To identify CYP isoforms involved in their metabolic activation/inactivation, 1-aminobenzotriazole (ABT), a potent nonspecific CYP inhibitor, and CYP isoform-selective inhibitors were used. To determine the effect of nonspecific protein binding, chemical-induced cytotoxicity was measured in HepG2 cells cultured with microsomes or heat-inactivated microsomes. To verify the model’s reliability, sudoxicam-, meloxicam-, valsartan-, and irbesartan-induced cytotoxicity was determined in HepG2 cells cultured with microsomes. Moreover, metabolite identification of CPA was performed to compare metabolic profiles in HepG2 cells cultured with or without microsomes.

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EXPERIMENTAL PROCEDURES

Reagents.

CPA,

acetaminophen,

diclofenac,

nefazodone,

dimethylsulfoxide

(DMSO),

carbamazepine, glucose 6-phosphate (G6P), glucose 6-phosphate dehydrogenase (G6PDH), nicotinamide adenine dinucleotide phosphate (NADP+), 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT), paroxetine (PRX), ketoconazole (KCZ), furafylline (FFL), phenacetin, coumarin, dextromethorphan, bupropion, tolbutamide, chlorzoxazone, testosterone, meloxicam, valsartan, irbesartan, and tolcapone were purchased from SigmaAldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), PenStrep, and trypsin-EDTA were obtained from GIBCO (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). Pooled human liver S9 fraction, pooled human liver microsomes (BD UltraPool HLM 150), and S-mephenytoin were purchased from BD Gentest Co (Woburn, MA, USA). Leflunomide, bakuchiol, sudoxicam, and ABT were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Midazolam was purchased from Bukwang Pharma Co. (Seoul, Republic of Korea). All other reagents were of highest chemical purity and were obtained from commercial sources.

Cell Culture and Cell Viability Assay.

The HepG2 cell line obtained from the Korea Cell Line Bank (Seoul, Republic of Korea) was routinely cultured in high-glucose DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 1 mM sodium pyruvate. Cells were incubated at 37°C in a humidified 5% CO2/95% air atmosphere.

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Cell viability was monitored by measuring MTT activity.15 The medium was aspirated from the cells and 100 µL MTT (0.5 mg/mL) was added to each well. Cell viability was expressed as a percentage of the value for control cells (cells incubated without compound).

HepG2 Cultured with Human Liver Microsomes or Human Liver S9 Fraction.

To sterilize the human liver S9 fraction and human liver microsomes, these fractions were filtered using 0.2-µm syringe filters following 5-fold dilution with serum-free DMEM. Human liver S9 fraction mixture or human liver microsomes mixture (1-mL final volume) consisted of 420 µL of diluted S9 fraction or microsomes (0.75 mg/mL), 20 µL of 100 mM NADP+, 12.7 µL of 1 M G6P, 20 µL of G6PDH (100 U/mL), 50 µL of 80 mM MgCl2, 41 µL of 150 mM KCl, and 436.3 µL of supplemented DMEM. HepG2 cells were cultured in 96well plates (1.5×104 cells/well) for 24 h. And the culture medium in wells was removed and the cells were treated with 50 µL of DMEM containing test compound followed by addition of 50 µL of human liver S9 mixture or human liver microsomes mixture. The final concentration of DMSO as a vehicle in the incubations did not exceed 0.5%. After incubation for 6 or 12 h, medium was changed with 200 µL of serum-free DMEM without the test compound, and cells were incubated for 42 or 36 h, respectively. The delay of incubation time was chosen to amplify the signal in the MTT assay because newly damaged cells may have residual mitochondrial dehydrogenase activity.12

Treatment with CYP Inhibitors and Heat-Inactivation of Human Liver Microsomes.

FFL (6 µM), PRX (10 µM), and KCZ (1 µM) were used as CYP1A2, CYP2B6, and CYP3A4 inhibitors, respectively. The concentrations of CYP inhibitors were determined

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based on our previous study using human liver microsomes.16 Although PRX is known as a CYP2B6 and CYP2D6 inhibitor, PRX was used for CYP2B6 inhibition because the metabolism of CPA or leflunomide is not related with CYP2D6. ABT (2.5 mM) was used as a broad CYP inhibitor.17,18 To inhibit CYP activity in microsomes, CYP inhibitors were added to microsomes before used to treat in HepG2 cells. CYP inhibitor-treated microsomes were incubated for at least 20 min at room temperature and immediately added to each well. None of the CYP inhibitors used in this study resulted in increased cell toxicity at the concentrations used. Heat-inactivated microsomes were prepared by filtering after heating for 30 min at 45°C.

CYP Activities in Heat-Inactivated Human Liver Microsomes and ABT-Treated Human Liver Microsomes.

CYP activities in microsomes treated with CYP inhibitors and in heat-inactivated microsomes were determined according to the method described by Lee and Kim (2013).16 Briefly, the incubation mixtures (final volume, 200 µL) contained 0.2 mg/mL microsomal protein, 0.1 M phosphate buffer (pH 7.4), 1 mM NADPH, and CYP isoform-specific substrate cocktail set (A set: : 50 µM phenacetin for CYP1A2, 10 µM coumarin for CYP2A6, 100 µM S-mephenytoin for CYP2C19, 5 µM dextromethorphan CYP2D6, and 10 µM midazolam for CYP3A4; B set: 50 µM bupropion for CYP2B6, 100 µM tolbutamide for CYP2C9, 50 µM chlorzoxazone for CYP2E1, and 50 µM testosterone for CYP3A4). After a 5 min pre-incubation at 37°C with ABT or after a 30 min heat-inactivation of microsomes at 45°C, the reactions were initiated by addition of 1 mM NADPH. After a 10 min incubation, the samples for each enzyme assay were centrifuged at 3,000g for 20 min at 4°C, and the supernatants of the individual reaction samples and pooled cocktail incubation samples (A

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set : B set, 1:1) were analyzed by LC-MS/MS.

Metabolite Identification of CPA in HepG2 Cells Cultured with or without Human Liver Microsomes.

HepG2 cells were cultured in 96-well plates (1.5×104 cells/well). The cells were treated with 50 µL of DMEM containing 1,000 or 10,000 µM CPA and 50 µL of human liver microsomes mixture for 6 or 12 h. At the end of the incubation period, 50-µL aliquots were collected in 1.5-mL microcentrifuge tubes containing the two volumes of ice-cold acetonitrile-methanol (1:1). The samples were vortex-mixed and centrifuged at 16,000 × g for 15 min. The supernatant was directly injected into a LC-ESI-QTrap or LC-ESI-QTOF system for identification of CPA metabolites. The LC-ESI-QTrap system consisted of a Shimadzu Prominence UFLC system (Shimadzu Instruments Co., Kyoto, Japan) and an Applied Biosystems 3200 QTrap system equipped with a Turbo V IonSpray source (Applied Biosystems, Foster City, CA, USA) operated in positive ion mode. The analysis was performed on an XTerra column (2.1 × 50 mm i.d., 3.5 µm; Waters, Milford, MA, USA) maintained at 30°C and the samples were separated using a mobile phase consisting of solvent A (deionized water containing 0.1% (v/v) formic acid) and solvent B (methanol) under gradient conditions. The TurboIonSpray interface was operated in positive ion mode at 5500 V. The operating conditions were as follows: ion source temperature, 500°C; nebulizing gas flow, 50 L/min; auxiliary gas flow, 40 L/min; curtain gas flow, 20 L/min; and collision gas pressure 3.6 × 10−5 Torr. Nitrogen was used as the curtain gas, collision gas, and nebulizer gas. Enhanced product ion (EPI) mode was used to obtain the product ion spectra for structural identification of the analytes. Multiple reaction monitoring (MRM) mode was used for quantitative analysis. The MRM

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mass transitions used were 199 → 171 for dechloroethylcyclophosphamide, 259 → 140 for iminocyclophosphamide, 277 → 221 for 4-hydroxycyclophosphamide (aldophosphamide), 221 → 163 for phosphoramide mustard, and 237 → 194 for the internal standard carbamazepine. Relative quantitation of the identified metabolites was performed by comparison of their MRM peak area ratios established against the internal standard. Acquisition and analysis of data were performed using the Analyst software (ver. 1.5; Applied Biosystems). To confirm the identified metabolites of CPA, a 5-µL aliquot of supernatant was injected into an accurate mass LC-MS/MS system consisting of an Agilent 1290 Infinity HPLC and Agilent 6530 QTOF mass spectrometer equipped with a dual AJS ESI source (Agilent Technologies Korea, Seoul, Republic of Korea). Chromatographic separations were conducted by gradient elution with a binary mobile phase comprised of deionized water and acetonitrile both containing 0.1 vol% formic acid at a flow rate of 0.5 mL/min on an Agilent Eclipse Plus C18 column (4.6 × 100 mm, 3.5 µm) maintained at 40°C. The QTOF mass spectrometer was operated in positive full scan mode with a mass accuracy < 2 ppm, a mass resolution of 5,000−10,000 (m/z 100−922), a measuring frequency of 10,000 transients/s, and a detection frequency of 2 GHz (200,000 points/transient). Purine (m/z 121.050873 [M+H]+) and standard HP-921 (hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine) (m/z 922.009798 [M+H]+) were infused continuously into the ion source for mass calibration. The data acquisition and post-run analyses were conducted using the software MassHunter Acquisition B.05.00 and MassHunter Qualitative Analysis B.04.00 (Agilent Technologies Korea).

Statistical Analysis.

Data were analyzed using an unpaired Student’s t-test for two-group comparison or one-

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way analysis of variance followed by the Newman−Keuls multiple range test. The acceptable level of significance was established at P < 0.05, except where indicated otherwise. Data are presented as the mean ± SD. Reproducibility of the results was confirmed in at least three separate experiments.

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RESULTS

Establishment of an In Vitro Model for Prediction of DILI using CPA.

To establish an in vitro model for prediction of DILI, CPA was used as a positive control for bioactivation. Based on the result of a previous study for evaluation of drug-induced cytotoxicity using rat liver S9 fraction,12 CPA-induced cytotoxicity was determined in HepG2 cells cultured with human liver S9 fraction or human liver microsomes. In the absence of human liver S9 fraction or human liver microsomes, cytotoxicity was not induced by addition of CPA up to 10 mM (Figure 1A). Marginal cytotoxicity was observed in HepG2 cells treated with more than 5 mM CPA in the presence of human liver S9 fraction at a concentration of 1.5 mg/mL but not 0.75 mg/mL. The cytotoxicity of CPA was evident in HepG2 cells cultured with 0.75 mg/mL human liver microsomes in a concentration-dependent manner. The results suggest that utilization of human liver microsomes is more suitable than that of human S9 fraction for evaluating metabolism-dependent cytotoxicity of CPA. To optimize the experimental conditions, CPA-induced cytotoxicity was determined in HepG2 cells cultured with various protein concentrations of human liver microsomes for different exposure times (Figure 1B and C). CPA-induced cytotoxicity was increased in a concentration-dependent manner in HepG2 cells cultured with microsomes for 6 or 12 h (Figure 1B). HepG2 cells incubated with microsomes for 12 h were more sensitive to CPAinduced cytotoxicity than those incubated for 6 h, suggesting that the CPA-induced cytotoxicity in HepG2 cells cultured with microsomes was increased in an exposure timedependent manner. The CPA-induced cytotoxicity was increased in a protein concentrationdependent manner, and the maximal cytotoxicity was observed at 0.75 mg/mL microsomes (Figure 1C). Thus, an exposure time of 12 h and microsomes concentration of 0.75 mg/mL

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were chosen to maximize chemical-induced cytotoxicity. In addition, cytotoxicity by microsomes in the absence of any test compound was examined. The viability of HepG2 cells cultured with 0.75 mg/mL microsomes was not decreased at 12 h, but decreased approximately 80% that of control cells at more than 24 h (data not shown). To determine whether the augmentation of CPA cytotoxicity by microsomes is NADPHdependent, the effects of NADPH-generating components on CPA-induced cytotoxicity were measured (Figure 1D). The absence of NADP+ in the NADPH-generating system completely inhibited the CPA-induced cytotoxicity in HepG2 cells cultured with microsomes. The CPAinduced cytotoxicity was also markedly attenuated by exclusion of G6P or G6PDH. These results showed that augmentation of CPA-induced cytotoxicity by microsomes is NADPHdependent. To confirm the involvement of CYP-dependent metabolism in the augmentation of CPAinduced cytotoxicity by microsomes, the effects of ABT, a broad CYP inhibitor, on CPAinduced cytotoxicity were examined (Figure 1E). The activities of eight CYP isoforms 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4 were decreased to less than 10% by 2.5 mM ABT (data not shown). The cytotoxicity of CPA (5 mM) was markedly inhibited by ABT (Figure 1E), which was consistent with the results observed with exclusion of NADPH-generating components. To investigate the role of each CYP isoform in bioactivation of CPA, CPA-induced cytotoxicity was examined in HepG2 cells treated with CYP isoform selective inhibitors (Figure 1F). In the present study, PRX and KCZ were used as selective inhibitors of CYP2B6 and CYP3A4, respectively. The concentration of each CYP isoform-selective inhibitor used in this study was approximately ten times the IC50 value determined in CYP inhibition study using human liver microsomes.16 The activities of CYP2B6 and CYP3A4 were completely inhibited by PRX and KCZ, respectively, at the concentrations used in this study (data not

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shown). The CPA-induced cytotoxicity in HepG2 cells cultured with microsomes was significantly attenuated by PRX or KCZ, and further attenuated by a mixture of PRX and KCZ. These results suggest roles of CYP2B6 and CYP3A4 in metabolic activation of CPA, consistent with a previous study indicating that CYP2B6 and CYP3A4 are the major isoforms involved in metabolism of CPA.19 The identification of CPA metabolites was performed in HepG2 cells cultured with microsomes using LC-ESI-QTrap and LC-ESI-QTOF (Table 1). Based on previous information on metabolites of CPA,20 the m/z values corresponding to [M+H]+ ions of the four proposed metabolites; i.e., dechloroethylcyclophosphamide, iminocyclophosphamide, 4hydroxycyclophosphamide (aldophosphamide), and phosphoramide mustard, were detected in the full scan mass spectra obtained using the LC-ESI-QTrap system. For the first time in this study, fragmentation patterns of four metabolites were analyzed in EPI scan mode. The four proposed CPA metabolites were analyzed in MRM mode for semi-quantitative comparison between control and microsomes-treated HepG2 cells. These metabolites were observed only in HepG2 cells cultured with microsomes. The MRM peak areas of all proposed CPA metabolites were increased in a CPA concentration-dependent manner. In addition, the peak areas of iminocyclophosphamide and 4-hydroxycyclophosphamide (aldophosphamide) were higher at 6 h than those at 12 h. To obtain more accurate mass values of the proposed CPA metabolites, samples were pooled and analyzed using an LC-ESI-QTOF system. The m/z values ([M+H]+) of dechloroethylcyclophosphamide, iminocyclophosphamide, and phosphoramide mustard measured using the LC-ESI-QTOF system were shown to be accurate with mass error between 1.5 and 3.1 ppm; however, 4-hydroxycyclophosphamide was not detected probably due to low intensity (Table 1). These results suggest that the proposed CPA metabolites may be produced in HepG2 cells cultured with but not in those cultured without microsomes.

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Acetaminophen- and Diclofenac-Induced Cytotoxicity in HepG2 Cells Cultured with Human Liver Microsomes.

To validate the in vitro method established through CPA, cytotoxicity was determined in HepG2 cells treated with acetaminophen and diclofenac. Acetaminophen serves as a model compound for DILI study, and the anti-inflammatory drug, diclofenac induces liver damage, although this effect is relatively rare. Metabolic activation plays a critical role in liver injury by these compounds. HepG2 cells were more sensitive to acetaminophen- and diclofenacinduced cytotoxicity in the presence of microsomes (Figure 2A and B). The augmentation of acetaminophen-induced cytotoxicity by microsomes was inhibited by addition of ABT and the absence of the NADPH-generating system (Figure 2C). The increase in diclofenacinduced cytotoxicity by microsomes was not inhibited by either ABT treatment or exclusion of NADPH-generating system (Figure 2D), suggesting that CYPs may not be involved in potentiation of diclofenac-induced cytotoxicity by microsomes. To examine the role of UDPglucuronosyltransferases (UGTs) in diclofenac-induced cytotoxicity, HepG2 cells were cultured with microsomes pretreated with mefenamic acid, a potent inhibitor of UGT2B7 that catalyzes acyl-glucuronide formation from diclofenac. The augmentation of diclofenacinduced toxicity by microsomes was not attenuated by mefenamic acid. Moreover, the formation of diclofenac acyl-glucuronide, a toxic metabolite of diclofenac, was not increased by addition of microsomes (data not shown). Inactivation of microsomes by heating at 95°C but not 45°C inhibited the augmentation of diclofenac-induced toxicity by microsomes (Figure 2E). In addition, the cytotoxic effects of coumarin and tolcapone, metabolismdependent hepatotoxicants,21,22 were examined in HepG2 cells cultured with microsomes. Addition of microsomes augmented the cytotoxicity induced by coumarin and tolcapone

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(data not shown).

Leflunomide-Induced Cytotoxicity in HepG2 Cells Cultured with Human Liver Microsomes.

Leflunomide, an immunosuppressive drug, was reported to be metabolically inactivated.23 Leflunomide-induced cytotoxicity was attenuated in HepG2 cells cultured with microsomes (Figure 3A). The attenuation of leflunomide-induced cytotoxicity by microsomes was partially inhibited by addition of ABT, exclusion of the NADPH-generating system or heat inactivation of microsomes at 45 °C (Figure 3B). Leflunomide-induced cytotoxicity was examined in HepG2 cells treated with CYP isoform-selective inhibitors (Figure 3C). Leflunomide metabolism was mainly mediated by CYP1A, CYP2B, and CYP3A.24 The detoxication of leflunomide by microsomes was attenuated by KCZ and mixture of FFL, PRX, and KCZ but not by FFL or PRX alone. FFL, a selective CYP1A inhibitor, completely inhibited CYP1A2 activity at the concentration of 6 µM used in this study (data not shown). These results suggest that metabolic detoxication of leflunomide is mediated mainly by CYP3A4.

Nefazodone-Induced Cytotoxicity in HepG2 Cells Cultured with Human Liver Microsomes.

The

antidepressant,

nefazodone,

has

been

reported

to

occasionally

induce

hepatotoxicity.1 Nefazodone-induced cytotoxicity was completely inhibited by microsomes (Figure 4A). Based on the strong protein binding property of nefazodone,25 the role of nonspecific protein binding in the attenuation of nefazodone-induced toxicity by microsomes

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was examined in HepG2 cells incubated with heat-inactivated microsomes instead of active microsomes (Figure 4B). The activities of eight CYP isoforms; i.e., 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4, in heat-inactivated microsomes were decreased to less than 10% relative to those of CYP in active microsomes (data not shown). Nefazodone-induced cytotoxicity was completely inhibited by heat-inactivated microsomes. To minimize nonspecific protein binding, HepG2 cells were incubated with 75 µM nefazodone in the presence of 0, 0.094, 0.188, 0.375, or 0.750 mg/mL heat-inactivated microsomes (Figure 4C). The nefazodone-induced cytotoxicity was attenuated by heat-inactivated microsomes in a protein concentration-dependent manner and was evident in HepG2 cells cultured with 0.094 or 0.188 mg/mL heat-inactivated microsomes. To further investigate the mechanism underlying the attenuation of nefazodone-induced cytotoxicity by microsomes, HepG2 cells were incubated for 24 h with 75 µM nefazodone in the presence of 0.094 or 0.188 mg/mL of active or heat-inactivated microsomes (Figure 4D). The nefazodone-induced cytotoxicity was completely inhibited by active microsomes at concentrations of 0.094 or 0.188 mg/mL but was marginally attenuated by the same concentrations of heat-inactivated microsomes or ABT-treated active microsomes, suggesting that the attenuation of nefazodone-induced cytotoxicity by concentrations of microsomes less than 0.188 mg/mL may be mediated by CYPs.

Bakuchiol-Induced Cytotoxicity in HepG2 Cells Cultured with Human Liver Microsomes.

Bakuchiol is a natural compound isolated from plants, such as Psoralea, and was reported to be metabolically inactivated.17 Bakuchiol-induced cytotoxicity was completely inhibited by microsomes (Figure 5A). To investigate the mechanism underlying the

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attenuation of bakuchiol-induced toxicity by microsomes in HepG2 cells, the cytotoxicity was examined in HepG2 cells incubated with heat-inactivated microsomes instead of active microsomes (Figure 5B). Bakuchiol-induced cytotoxicity in HepG2 cells was completely inhibited by heat-inactivated microsomes. In an experiment to examine the protein concentration dependency of the effect, bakuchiol-induced cytotoxicity was evident in HepG2 cells cultured with 0.094 mg/mL heat-inactivated microsomes (data not shown). To further test the role of nonspecific protein binding in the protective effect of microsomes against bakuchiol-induced cytotoxicity, HepG2 cells were incubated for 24 h with 80 µM bakuchiol in the presence of 0.094 mg/mL of active or heat-inactivated microsomes (Figure 5C). The bakuchiol-induced cytotoxicity was attenuated to a similar extent by both active and heat-inactivated microsomes, indicating that the attenuation of bakuchiol-induced cytotoxicity was mediated via a mechanism that was independent of metabolism. Moreover, the attenuation of bakuchiol-induced cytotoxicity by active microsomes was not inhibited by treatment with ABT or exclusion of the NADPH-generating system, suggesting that CYPs may not be involved in attenuation of bakuchiol-induced cytotoxicity by microsomes. To determine the protein binding property of bakuchiol, protein-binding assay was performed using human plasma and human liver microsomes. Bakuchiol at a concentration of 50 µM bound completely to human plasma protein and 0.75 mg/mL human liver microsomes (data not shown).

Sudoxicam-, Meloxicam-, Valsartan-, and Irbesartan-Induced Cytotoxicity in HepG2 Cells Cultured with Human Liver Microsomes.

To verify the model’s reliability, sudoxicam, meloxicam, valsartan, and irbesartan were selected as test compounds and their cytotoxicity was determined in HepG2 cells cultured

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with microsomes (Figure 6). Sudoxicam and meloxicam were reported to have a marked difference in hepatic toxicity, despite their structural similarity.26 The difference in their hepatotoxicity has been attributed to difference in their metabolic process involving thiazole ring scission to a toxic thiourea metabolite.26 In this study, sudoxicam-induced cytotoxicity was augmented in HepG2 cells cultured with microsomes in the presence of NADPHgenerating system, in a concentration-dependent manner (Figure 6A). In contrast, meloxicam-induced cytotoxicity was increased only at 2,000 µM, the highest concentration used in this study (Figure 6B). Valsartan and irbesartan are angiotensin II receptor blockers used to treat hypertension. Valsartan and irbesartan have been linked to rare instances of acute liver injury. Valsartan is excreted largely as unchanged drug via feces in human and is not metabolized by CYP to a major degree,27 while oxidation by CYP2C9 is involved in elimination of irbesartan.28 In the present study, cell viability was decreased to approximately 75% in HepG2 cells treated with 750 µM valsartan and there was no significant difference in cytotoxicity between in the absence and presence of NADPH-generating system (Figure 6C). Irbesartan exhibited cytotoxicity in HepG2 cells cultured with microsomes in a concentration-dependent manner, which was attenuated by addition of NADPH-generating system (Figure 6D).

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DISCUSSION

In the present study, we developed a cell-based system to evaluate the metabolismdependent cytotoxicity and to identify CYP isoforms and metabolites involved in druginduced cytotoxicity for prediction of DILI using HepG2 cells cultured with human liver microsomes. CPA was used as a positive control for developing the assay system. CPA has known as a representative compound for CYP-mediated bioactivation, although CPA is not caused DILI. CPA is an alkylating prodrug that is commonly used in anticancer therapy. CPA is primarily metabolized to 4-hydroxy-CPA by CYP2B6 and CYP3A4.19,29 4-Hydroxy-CPA is activated to the reactive intermediates phosphoramide mustard and acrolein via ring opening. Phosphoramide mustard is primarily responsible for the cytotoxic effects of CPA. In the present study, CPA-induced cytotoxicity was markedly increased by microsomes, which was completely inhibited by exclusion of NADP+ only or the NADPH-generating system. The results obtained in HepG2 cells treated with ABT, a broad CYP inhibitor, clearly demonstrated that augmentation of CPA-induced cytotoxicity by microsomes is mediated by NADPH-dependent CYP reactions. Moreover, experiments using selective CYP inhibitors, including PRX for CYP2B6 and KCZ for CYP3A4, provided information on the contributions of these CYP isoforms to metabolic activation of CPA and the reaction phenotype involved in CPA-induced cytotoxicity. In fact, the inhibitory effect of PRX on CPA-induced cytotoxicity was greater than that of KCZ, suggesting that CYP2B6 may have a greater contribution to CPA-induced cytotoxicity in HepG2 cells cultured with microsomes than CYP3A4. The results were consistent with a previous study on the contribution of each CYP isoform to the metabolism of CPA.19 Moreover, identification of CPA metabolites showed that increased cytotoxicity of CPA by microsomes was mediated by formation of phosphoramide mustard, a cytotoxic metabolite. Thus, the in vitro assay system established in

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this study can be used for characterization of CPA-induced cytotoxicity in HepG2 cells and provides information for prediction of in vivo DILI via bioactivation. Acetaminophen at normal dosages is primarily metabolized through glucuronidation and sulfation, leading to elimination of acetaminophen. In contrast, some CYP isoforms, including CYP1A2, CYP2E1, and CYP3A4, can metabolize acetaminophen to N-acetyl-pbenzo-quinone imine (NAPQI), a toxic alkylating metabolite in cases of acetaminophen overdose.30 As NAPQI can be detoxified by conjugation with GSH, NAPQI-induced liver injury is evident following depletion of GSH. In the present study, the viability of HepG2 cells treated with 40 mM acetaminophen was decreased to less than 75% of controls, suggesting that HepG2 cells are resistant to acetaminophen-induced cytotoxicity through low basal CYP activity. In contrast, acetaminophen-induced cytotoxicity was markedly augmented by addition of microsomes, which was attenuated by treatment with ABT and exclusion of the NADPH-generating system. These results suggest a role for CYP-dependent metabolism in potentiation of acetaminophen-induced cytotoxicity by microsomes. Diclofenac, a nonsteroidal anti-inflammatory drug, was reported to be associated with idiosyncratic hepatotoxicity. Diclofenac is metabolized by CYP2C9, CYP3A4, and UGT2B7,31-33 and metabolism of diclofenac is important in its toxicity assessment due to the formation of reactive metabolites.34 The hepatotoxicity of diclofenac may be attributable to metabolic activation of the drug via the formation of reactive acyl glucuronide and benzoquinone imine intermediates.32 In the present study, diclofenac-induced cytotoxicity was potentiated by the addition of microsomes, which was not inhibited by addition of ABT or the exclusion of the NADPH-generating system. In addition, the potentiation of diclofenac-induced toxicity by microsomes was not attenuated by mefenamic acid, a potent inhibitor of UGT2B7, and the formation of diclofenac acyl-glucuronides was not increased by addition of microsomes. The results suggest that the increase in diclofenac-induced

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cytotoxicity by microsomes may not be due to CYP- or UGT-dependent metabolism.35 However, these results do not rule out the possibility of the involvement of acyl-glucuronide in diclofenac-induced cytotoxicity. On the other hand, inactivation of microsomes by heating at 95°C but not 45°C attenuated the increase in diclofenac-induced toxicity by microsomes, raising the possibility that hepatic microsomal proteins but not CYP may be involved in the potentiation of diclofenac-toxicity by microsomes. Therefore, further studies are required to determine the mechanism underlying the potentiation of diclofenac-induced cytotoxicity by microsomes. Leflunomide is a pyrimidine synthesis inhibitor belonging to the disease-modifying antirheumatic drugs. A recent study showed that leflunomide was detoxified through metabolism by CYP1A, CYP2B/2C, and CYP3A in rat hepatocytes.23 In the present study, leflunomide-induced cytotoxicity was attenuated in HepG2 cells cultured with microsomes, and this effect was inhibited by addition of ABT, exclusion of the NADPH-generating system or heat inactivation of microsomes. The results suggest that detoxication of leflunomide is attributable to CYP-dependent metabolism, although nonspecific protein binding may be involved in the attenuation of leflunomide-induced toxicity by microsomes. Moreover, the experiments using selective CYP inhibitors, including FFL for CYP1A2, PRX for CYP2B6, or KCZ for CYP3A4, provided information on the contribution of CYP3A4 to detoxication of leflunomide. The antidepressant drug, nefazodone, has been shown to induce idiosyncratic hepatotoxicity.1 The incidence of liver injury by nefazodone was estimated to be 28.9 per 100,000 patient years. The common clinical symptoms of nefazodone-induced liver disease include jaundice and elevation of serum ALT, AST, and bilirubin levels.36 The mechanism of nefazodone-induced liver injury is not fully understood. Some of the previous studies have suggested that nefazodone is mainly metabolized by CYP3A4 to form toxic reactive

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metabolites.37-39 In contrast, other studies showed that nefazodone-induced hepatotoxicity is caused by mitochondrial dysfunction and inhibition of bile acid transport by the parent drug itself.40,41 In the present study, addition of active or heat-inactivated microsomes at 0.75 mg/mL attenuated nefazodone-induced cytotoxicity in HepG2 cells, which suggested that the protective effect of microsomes on nefazodone-induced cytotoxicity may be due to nonspecific protein binding. However, the results of experiments using low concentrations (less than 0.188 mg/mL) of active or heat-inactivated microsomes showed that nefazodoneinduced cytotoxicity was markedly attenuated by active microsomes, relative to inactivated microsomes by heating or ABT treatment. Taken together, all these findings suggest that compounds with high protein-binding activity can be tested using low concentrations of microsomes for long incubation times, such as 24 h. In fact, nefazodone shows more than 98% plasma protein binding.25 Although reactive metabolites of nefazodone are generated by CYPs, it should be noted that various nontoxic metabolites of nefazodone are also produced by CYPs.42 Finally, our data support the results of prior studies showing that the parent drug itself is involved in the nefazodone-induced hepatotoxicity.40,41 Bakuchiol has been used in traditional medicine as an antimicrobial agent and for prevention of bone loss.43,44 Irrespective of the microsomes protein concentration, the heatinactivated microsomes similarly attenuated bakuchiol-induced cytotoxicity compared to active microsomes. Moreover, neither addition of ABT nor exclusion of the NADPHgenerating system inhibited the protective effect of microsomes, suggesting that detoxication of bakuchiol by microsomes may be attributed to nonspecific protein binding rather than CYP-dependent metabolism. Sudoxicam and meloxicam are nonsteroidal anti-inflammatory drugs in the enolcarboxamide class. The only structural difference between sudoxicam and meloxicam is the presence of a methyl group on the C5-position of the thiazole ring in meloxicam.26 This

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structural change causes a remarkable difference in their DILI in human, which can be cause by alterations in metabolic profile of the two drugs.26 While sudoxicam’s clinical development was discontinued due to incidences of severe clinical hepatotoxicity, reports of DILI by meloxicam have been extremely rare.26,45 The present study showed that sudoxicaminduced cytotoxicity was more sensitively increased by microsomes compared with meloxicam, in a NADPH-dependent manner. These results suggest that the model established in this study is suitable for evaluating difference in DILI caused by minor structural change. Valsartan, a low hepatotoxic drug is not metabolized by CYP to a major degree.27 Consistently, cytotoxicity by valsartan was not significantly different in HepG2 cells cultured with microsomes between in the absence and presence of NADPH. On the other hand, cytotoxicity by irbesartan, metabolized by CYP, was attenuated in HepG2 cells cultured with microsomes by addition of NADPH. The difference in cytotoxicity may be attributed to involvement of CYP in their metabolism. The tested compound concentrations were high relative to plasma concentrations observed in human treated with usual therapeutic doses. For example, when nefazodone 500 mg tid is administered to healthy humans, the maximal plasma concentrations are less than 7269 µg/L (less than 16 µM).46 Therapeutic doses of nefazodone associated with liver failure are in the range of 200-400 mg/day. Plasma nefazodone reaches the maximum concentrations of 2-4 µM in healthy humans administered with 200-400 mg/day. Patient-specific factors including hepatic drug accumulation over a long period of time may play a critical role in a nefazodone-induced liver injury, although there are no data documenting the plasma-to-liver ratio of nefazodone. In humans, biliary excretion plays a role in the elimination of nefazodone. Thus, inhibition of bile acid transport by nefazodone may result in accumulation of nefazodone in liver.41 The present results suggest that nefazodone may be detoxified by

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CYP-dependent metabolism, although the concentrations of nefazodone used in this study were higher relative to its concentrations in human plasma. Based on the results of the present study, a decision tree for prediction of DILI is shown Figure 7. Although HepG2 was used for prediction of DILI in the present study, usage of the HepG2 cell line is not an absolute requirement for our in vitro method using human liver microsomes; other hepatoma cell lines or immortalized liver cell lines may be also employed in the system using human liver microsomes. In fact, the augmentation of CPA-induced cytotoxicity by human liver microsomes was also observed in Huh7, human hepatocarcinoma cells (data not shown). Experimental animal liver microsomes can be used for prediction of species-difference in DILI, and such studies are currently underway in our laboratory. The addition of ABT and exclusion of the NADPH-generating system are available to determine CYP- and NADPH-dependent metabolism involved in chemical-induced cytotoxicity. Utilization of CYP isoform selective inhibitors can provide information on the contribution of each CYP isoform to the bioactivation and detoxication of compounds by CYPs. Furthermore, utilization of heat-inactivated microsomes in combination with a low concentration of microsomes can minimize the effect of nonspecific protein binding on chemical-induced cytotoxicity. In addition, the assay system provides a simple and rapid high-throughput screening method for determination of cytotoxicity by chemicals for prediction of DILI possibly with higher reproducibility than human hepatocytes showing individual variation. In the case of specific compounds, this method can provide information on the formation of toxic metabolites using mass spectrometry. In conclusion, we have developed an in vitro assay system using HepG2 cells cultured with human liver microsomes to predict DILI via metabolic activation.

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (K. Lee); Tel.: +82 44 860 1616; Fax: +82 44 860 1606. *E-mail: [email protected] (S. K. Kim); Tel.: +82 42 821 5930; Fax: +82 42 823 6566.

Funding This study was supported by a Grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare, & Family Affairs, Republic of Korea (A100096), by Basic Science Research Program (NRF-2013R1A1A2005981) and Basic Research Lab Program (NRF-2014R1A4A1007304) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education.

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ABBREVIATION ABT, 1-aminobenzotriazole; CPA, cyclophosphamide; CYP, cytochrome P450; DILI, druginduced liver injury; FFL, furafylline; KCZ, ketoconazole; MTT, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide; PRX, paroxetine

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Kalgutkar, A. S., Vaz, A. D., Lame, M. E., Henne, K. R., Soglia, J., Zhao, S. X.,

Abramov, Y. A., Lombardo, F., Collin, C., Hendsch, Z. S., and Hop, C. E. (2005) Bioactivation of the nontricyclic antidepressant nefazodone to a reactive quinone-imine species in human liver microsomes and recombinant cytochrome P450 3A4. Drug Metab.

Dispos. 33, 243-253. (39)

Rotzinger, S., and Baker, G. B. (2002) Human CYP3A4 and the metabolism of

nefazodone and hydroxynefazodone by human liver microsomes and heterologously expressed enzymes. Eur. Neuropsychopharmacol. 12, 91-100. 32


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C., Smith, A. R., and Will, Y. (2008) In vitro assessment of mitochondrial dysfunction and cytotoxicity of nefazodone, trazodone, and buspirone. Toxicol. Sci. 103, 335-345. (41)

Kostrubsky, S. E., Strom, S. C., Kalgutkar, A. S., Kulkarni, S., Atherton, J., Mireles,

R., Feng, B., Kubik, R., Hanson, J., Urda, E., and Mutlib, A. E. (2006) Inhibition of hepatobiliary transport as a predictive method for clinical hepatotoxicity of nefazodone.

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von Moltke, L. L., Greenblatt, D. J., Granda, B. W., Grassi, J. M., Schmider, J.,

Harmatz, J. S., and Shader, R. I. (1999) Nefazodone, meta-chlorophenylpiperazine, and their metabolites in vitro: cytochromes mediating transformation, and P450-3A4 inhibitory actions.

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Clin. Pharmacokinet. 33, 260-275.

33



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Page 34 of 46

Table 1. Identification of CPA metabolites in HepG2 cells cultured with human liver microsomes using LC-ESI-QTrap and LC-ESI-QTOF. LC-ESI-QTrap

LC-ESI-QTOF

Peak area (×104) Metabolite (Formula)

Cyclophosphamide (C7H15Cl2N2O2P) Dechloroethylcyclophosphamide (C5H12ClN2O2P) Iminocyclophosphamide (C7H13Cl2N2O2P) Phosphoramide mustard (C4H11Cl2N2O2P) 4-Hydroxycyclophosphamide (Aldophosphamide) (C7H15Cl2N2O3P)

CPA (µM) 500 5000 500 5000 500 5000 500 5000 500 5000

MRM (EPI)

261 > 140 (233,142,140†,106) 199 > 171 (171†,120,78) 259 > 140 (152,140†,138,120,110) 221 > 163 (185,163†,135,106) 277 > 221 (221†,161,142)

+Microsomes

-Microsomes

6h

12 h

6h

12 h

SA

SA

SA

SA

SA

SA

12.2 ± 0.4

13.0 ± 0.3*

119.0 ± 4.1

124.0 ± 7.7

6.9 ± 0.2

5.8 ± 0.2***

SA

SA

ND

ND

ND 15.0 ± 0.3

13.4 ± 0.3***

4.5 ± 0.2

4.8 ± 0.2 ND

23.3 ± 1.0

23.6 ± 1.1

1.8 ± 0.1

0.6 ± 0.0***

9.6 ± 0.6

2.5 ± 0.1***

ND

ND

ND

ND

† Indicates the most intense fragment ion of each metabolite analyzed by LC-ESI-QTrap in a positive EPI mode. *,***Significantly different from 6 h at P < 0.05 or P < 0.001, respectively (Student’s t-test for two-group comparison). Peak areas measured by LC-ESI-QTrap in a positive MRM mode represent the mean ± SD for four separate samples. Peak areas measured by LC-ESI-QTOF in a full scan mode were obtained using pooled samples. MRM: multiple reaction monitoring. EPI: enhanced product ion. ND: not detected. NA: not applicable. SA: saturated.

34


Observed m/z ([M+H]+)

Mass error (ppm)

261.0322

-0.5

199.0391

259.0160

221.0002

ND

Peak area (×104) +Microsomes

6h

12 h

6h

12 h

1796.0

1685.0

1787.4

1820.7

3157.0

2142.0

2935.3

2995.4

70.0

71.1 ND

ND

734.5

757.4

6.2

1.4 ND

ND

67.9

15.2

14.0

15.0 ND

ND

84.0

92.0

ND

ND

ND

ND

3.1

1.5

2.9

NA

-Microsomes

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Figure Legends

Figure 1. Establishment of an in vitro model for prediction of DILI using CPA. (A) HepG2 cells were treated with CPA and NADPH-generating system in the presence of human liver S9 fraction or human liver microsomes. After incubation for 6 h, medium was changed to serum-free DMEM without CPA, and cells were incubated for 42 h. (B) HepG2 cells were treated with CPA and NADPH-generating system in the presence of 0.75 mg/mL microsomes. After incubation for 6 or 12 h, medium was changed to serum-free DMEM without CPA and microsomes, and cells were incubated for 42 or 36 h, respectively. (C) HepG2 cells were treated with CPA and NADPH-generating system in the presence of 0, 0.125, 0.25, 0.50, or 0.75 mg/mL microsomes. (D) HepG2 cells were treated with CPA and 0.75 mg/mL microsomes in the presence or absence of each NADPH-generating component. (E) HepG2 cells were treated with 5 mM CPA and 0.75 mg/mL microsomes in the presence of ABT (2.5 mM), a broad CYP inhibitor, or in the absence of NADPH-generating system. (F) HepG2 cells were treated with 5 mM CPA, NADPH-generating system and 0.75 mg/mL microsomes in the presence of paroxetine (PRX, 10 µM) or ketoconazole (KCZ, 1 µM). “Mix” in the box indicates a combination of PRX and KCZ. After incubation for 12 h, medium was changed to serum-free DMEM without CPA and microsomes, and cells were incubated for 36 h. Cell viability was determined by the MTT assay. Each value represents the mean ± SD for four separate samples. Comparisons among the groups treated with the same concentration of CPA were performed by one-way ANOVA followed by the Newman−Keuls multiple range test. Values with different letters are significantly different (P < 0.05).

Figure 2. Evaluation of acetaminophen- and diclofenac-induced cytotoxicity in HepG2 cells 35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cultured with human liver microsomes. HepG2 cells were treated with acetaminophen (A) or diclofenac (B) in the presence of NADPH-generating system and 0.75 mg/mL microsomes. Addition of ABT (2.5 mM) or exclusion of NADPH-generating system was performed in HepG2 cells treated with acetaminophen (C) or diclofenac (D) in the presence of 0.75 mg/mL microsomes. (E) HepG2 cells were treated with diclofenac in the presence of 0.75 mg/mL active microsomes or inactivated microsomes by heating at 45°C for 30 min or at 95°C for 5 min. After incubation for 12 h, medium was changed to serum-free DMEM without the test compounds and microsomes, and cells were incubated for 36 h. Cell viability was determined by the MTT assay. Each value represents the mean ± SD for four separate samples. ***Significantly different between two groups treated with the same concentration of test compound (P < 0.001, Student’s t-test). Values with different letters are significantly different (P < 0.05, one-way ANOVA followed by Newman−Keuls multiple range test).

Figure 3. Evaluation of leflunomide-induced cytotoxicity in HepG2 cells cultured with human liver microsomes. (A) HepG2 cells were treated with leflunomide in the presence of NADPH-generating system and microsomes. (B) Addition of ABT (2.5 mM) or exclusion of NADPH-generating system was performed in HepG2 cells treated with leflunomide in the presence of 0.75 mg/mL microsomes. In additional experiments, HepG2 cells treated with leflunomide in the presence of 0.75 mg/mL inactivated microsomes by heating at 45°C for 30 min. (C) HepG2 cells were treated with 250 µM leflunomide, NADPH-generating system, and 0.75 mg/mL microsomes in the presence of furafylline (FFL, 6 µM), paroxetine (PRX, 10

µM), or ketoconazole (KCZ, 1 µM). “Mix” in the box indicates a combination of FFL, PRX, and KCZ. After incubation for 12 h, medium was changed to serum-free DMEM without leflunomide and microsomes, and cells were incubated for 36 h. Cell viability was 36


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determined by the MTT assay. Each value represents the mean ± SD for four separate samples. ***Significantly different between two groups treated with the same concentration of test compound (P < 0.001, Student’s t-test). Values with different letters are significantly different (P < 0.05, one-way ANOVA followed by Newman−Keuls multiple range test).

Figure 4. Evaluation of nefazodone-induced cytotoxicity in HepG2 cells cultured with human liver microsomes. (A) HepG2 cells were treated with nefazodone, NADPH-generating system, and 0.75 mg/mL microsomes. (B) HepG2 cells were treated with nefazodone, NADPH-generating system, and heat-inactivated microsomes (0.75 mg/mL). (C) HepG2 cells were treated with 75 µM nefazodone in the presence of 0, 0.094, 0.188, 0.375 or 0.750 mg/mL of heat-inactivated microsomes. After incubation for 12 h, medium was changed to serum-free DMEM without nefazodone and microsomes, and cells were incubated for 36 h. (D) HepG2 cells treated with 75 µM nefazodone were cultured with 0.094 or 0.188 mg/mL active microsomes or inactivated microsomes by heating (at 45°C for 30 min) for 24 h. In additional experiments, HepG2 cells were treated with 75 µM nefazodone in the presence of 0.094 or 0.188 mg/mL active microsomes pretreated by 2.5 mM ABT for 24 h. Medium was changed to serum-free DMEM without nefazodone and microsomes, and cells were incubated for 24 h. Cell viability was determined by the MTT assay. Each value represents the mean ± SD for four separate samples. *,***Significantly different between two groups treated with the same concentration of test compound (P < 0.05 or P < 0.001, respectively, Student’s ttest). Values with different letters are significantly different (P < 0.05, one-way ANOVA followed by Newman−Keuls multiple range test).

Figure 5. Evaluation of bakuchiol-induced cytotoxicity in HepG2 cells cultured with human 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

liver microsomes. (A) HepG2 cells were treated with bakuchiol, NADPH-generating system, and 0.75 mg/mL microsomes. (B) HepG2 cells were treated with bakuchiol, NADPHgenerating system, and 0.75 mg/mL heat-inactivated microsomes. After incubation for 12 h, medium was changed to serum-free DMEM without bakuchiol and microsomes, and cells were incubated for 36 h. (C) HepG2 cells treated with 80 µM bakuchiol were cultured with 0.094 mg/mL active microsomes or inactivated microsomes by heating (at 45°C for 30 min) for 24 h. In additional experiments, HepG2 cells were treated with 80 µM bakuchiol in the presence of 0.094 mg/mL active microsomes pretreated with 2.5 mM ABT for 24 h. Medium was changed to serum-free DMEM without bakuchiol and microsomes, and cells were incubated for 24 h. Cell viability was determined by the MTT assay. Each value represents the mean ± SD for four separate samples. ***Significantly different between two groups treated with the same concentration of test compound (P < 0.001, Student’s t-test). Values with different letters are significantly different (P < 0.05, one-way ANOVA followed by Newman−Keuls multiple range test).

Figure 6. Evaluation of sudoxicam-, meloxicam-, valsartan-, and irbesartan-induced cytotoxicity in HepG2 cells cultured with human liver microsomes. HepG2 cells cultured with microsomes were treated with sudoxicam (A), meloxicam (B), valsartan (C), or irbesartan (D) in the presence or absence of NADPH-generating system. Cell viability was determined by the MTT assay. Each value represents the mean ± SD for four separate samples. *,***Significantly different between two groups treated with the same concentration of test compound (P < 0.05, P < 0.01 or P < 0.001, respectively, Student’s t-test).

Figure 7. Decision tree for prediction of metabolism-dependent hepatotoxicity. 38


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


39



Figure 1

A

120 a

a

a

a

a

a

aa

aab

aa

b

100

B

a

b

a

140 120

Cell viability (% of control)


80 c

60

40

a

aaa

b

aa a

100

a

a

a

a

b

a

b

a

c 80 b

60

c 40

c 20

bb

20

0

0 0.1

0.5

1.0

5.0

10.0

0.1

0.5

Cyclophosphamide (mM)

1.0

140 120

a


aa

a aa a

100

a

a

c

c

c

60 d

40

a a

a

aa aa

b

b 80

140 120

a b

b

a

D

a

aaa

d

100

a

a

b

a

a a a

a aa

a

b

c

80

d

60 c 40 20

0

0 0.1

0.5

1.0

5.0

10.0

0.1

0.5

5.0

10.0

Without Microsomes

Without Microsomes Microsomes (0.125 mg/mL) Microsomes (0.25 mg/mL) Microsomes (0.50 mg/mL) Microsomes (0.75 mg/mL)

Microsomes without NADP+ Microsomes without G6P Microsomes without G6PDH Microsomes with NADPH generating system

120

F

a a

a

1.0



120

a 100


100

aa

e e

20

E

10.0

Without Microsomes (6 h) Without Microsomes (12 h) Microsomes (0.75 mg/mL, 6 h) Microsomes (0.75 mg/mL, 12h)


C

5.0


CPA only S9 (0.75 mg/mL) S9 (1.5 mg/mL) Microsomes (0.75 mg/mL)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 46

80

60

40

b

80

e c

60

40

d

b

20

20

0

0

Cyclophosphamide (5 mM)

Cyclophosphamide (5 mM)

Without Microsomes Microsomes Microsomes + ABT (2.5 mM) Microsomes without NADPH generating system

Without Microsomes Microsomes Microsomes + PRX (10 µM) Microsomes + KCZ (1 µM) Microsomes + Mix

40


Page 41 of 46

Figure 2

A

120

B

100

80



100

80

60

40

***

60

***

40

20

20

*** 0 5

10

20

30

40

50

100

Acetaminophen (mM)

200

400

100

D a

c



1000

100

a 80

c

60

b 40

20

b 60

b

b

40

20

0

0

Acetaminophen (40 mM)

Diclofenac (600 µM)



100

80

a c

60

40

800

Without Microsomes Microsomes

80

E

600

Diclofenac (µ µM)


C

***

0 2


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b

b

20

0

Diclofenac (600 µM) Without Microsomes Microsomes Heat-inactivated Microsomes (45 °C) Heat-inactivated Microsomes (95 °C)

41



Figure 3

A

120


100

*** 80

***

60

40

*** 20

0 50

100

150

250

Leflunomide (µ µM) Without Microsomes Microsomes

B

100

b


80

c

c c

60

a 40

20

0

Leflunomide (250 µM) Without Microsomes Microsomes Microsomes + ABT (2.5 mM) Microsomes without NADPH generating system Heat-inactivated Microsomes

C

100

80


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

b b b c

c

60

a a 40

20

0

Leflunomide (250 µM) Without Microsomes Microsomes Microsomes + FFL (6 µM) Microsomes + PRX (10 µM) Microsomes + KCZ (1 µM) Microsomes + Mix

42


Page 42 of 46

Page 43 of 46

Figure 4

A

120

***

*** ***

***

***

*** ***

B

100

120

b

*

b ***

100

b

***



*** 80

60

40

20

80

60

40

a

20

0

0 10

25

50

75

10

25

Nefazodone (µ µM)

C

140

e

50

75

Nefazodone (µ µM)


Without Microsomes Heat-inactivated Microsomes

D

120

120

b b 100

c 100



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


d 80

c

60

b 40

a

80

a

c a

a

a

60

40

20

20 0

0 0.094

Nefazodone (75 µM)

0.188

Microsomes (mg/mL)

Without Microsomes Heat-inactivated Microsomes (0.094 mg/mL) Heat-inactivated Microsomes (0.188 mg/mL) Heat-inactivated Microsomes (0.375 mg/mL) Heat-inactivated Microsomes (0.750 mg/mL)

Without Microsomes Active Microsomes Heat-inactivated Microsomes Active Microsomes + ABT (2.5 mM)

43



Figure 5

A

120

***

***

***


100

80

60

40

20

0 5

10

20

40

60

80

Bakuchiol (µ µM) Without Microsomes Microsomes

B

120


***

***

100

80

60

40

20

0 10

40

80

Bakuchiol (µ µM) Without Microsomes Heat-inactivated Microsomes

C

100

80


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

b

b

b

60

40

20

a 0

Bakuchiol (80 µM) Without Microsomes Active Microsomes Heat-inactivated Microsomes Active Microsomes + ABT (2.5 mM)

44


Page 44 of 46

Page 45 of 46

Figure 6

120

A 120 A 100

B B

120

120 100

Cell viability Cell viability (% of control) (% of control)

*

* *

80 60

***

***

60 40

***

40 20

***

viability CellCell viability (%control) of control) (% of

*

100 80

20

100 80 80 60 60 40 40 20

***

20 0

0 10

50

100

250

500

1000

2000

1000

2000

10

0

50

100

250

500

1000

2000 ***

0 10

50

Sudoxicam (µ µM) 100 250 500

Sudoxicam (µ µM)

120

C 120 C 100

10

D D

10080 8060 6040

4020

50

Meloxicam µM) 100 250 (µ 500

1000

2000

Meloxicam (µ µM)

120

120 100

Cell viability Cell viability of control) (% of(% control)

Cell viability Cell viability (% of control) (% of control)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100 80 80 60

**

60 40

**

20 40

***

*** 20 0

0 20 125

250

500

750

125

Valsartan (µ µM)

0 125

250

500

750

250

500

750

Irbesartan (µ µM)

0 125

250

500

Irbesartan (µ µM)

Valsartan (µ µM)

750

HLM without NADPH generating system HLM Microsomes without NADPH generating system Microsomes

45



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Figure 7

46


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Prediction of Drug-Induced Liver Injury in HepG2 Cells Cultured with

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