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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Drugs induce cholestasis by diverse and still poorly understood mechanisms in humans. Early hepatic effects of chlorpromazine (CPZ), a neuroleptic drug known for years to induce intrahepatic cholestasis, were investigated using the differentiated human hepatoma HepaRG cells. Generation of reactive oxygen species (ROS) was detected as early as 15 minutes after CPZ treatment and was associated with an altered mitochondrial membrane potential and disruption of the pericanalicular distribution of F-actin. Inhibition of [3H]-taurocholic acid efflux was observed after 30 minutes and was mostly prevented by N-acetyl cysteine (NAC) cotreatment, indicating a major role of oxidative stress in CPZ-induced bile acid (BA) accumulation. Moreover, 24-hour treatment with CPZ decreased messenger RNA (mRNA) expression of the two main canalicular bile transporters, bile salt export pump (BSEP) and multidrug resistance protein 3 (MDR3). Additional CPZ effects included inhibition of Na+-dependent taurocholic cotransporting polypeptide (NTCP) expression and activity, multidrug resistance-associated protein 4 (MRP4) overexpression and CYP8B1 inhibition that are involved in BA uptake, basolateral transport, and BA synthesis, respectively. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Compared to CPZ effects, overloading of HepaRG cells with high concentrations of cholic and chenodeoxycholic acids induced a delayed oxidative stress and, similarly, after 24 hours it down-regulated BSEP and MDR3 in parallel to a decrease of NTCP and CYP8B1 and an increase of MRP4. By contrast, low BA concentrations up-regulated BSEP and MDR3 in the absence of oxidative stress. Conclusion: These data provide evidence that, among other mechanisms, oxidative stress plays a major role as both a primary causal and an aggravating factor in the early CPZ-induced intrahepatic cholestasis in human hepatocytes. (HEPATOLOGY 2013)

Cholestatic liver disorders include a spectrum of hepatobiliary diseases of diverse etiologies that are characterized by impaired hepatocellular secretion of bile, resulting in accumulation of bile acids (BA), bilirubin, and cholesterol. Extrahepatic cholestasis corresponds to biliary obstruction outside the liver that can be due to stones, tumors, biliary atresia, or primary sclerosing cholangitis, whereas intrahepatic cholestasis is caused by drugs, genetic transporter defects, infections, or primary biliary cirrhosis.1 Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury in humans.2 In many cases it results from the hepatobiliary transporter system alteration, in particular the bile salt export pump (BSEP, or ABCB11), which is the most physiologically important canalicular bile transporter.3 However, the mechanisms by which drugs induce cholestasis are diverse and remain poorly understood.4, 5 Indeed, in addition to hepatobiliary transporter changes, other mechanisms, such as altered cell polarity, disruption of cell-to-cell junctions, and cytoskeletal modifications, can participate in cholestasis.6, 7 A role for oxidative stress as a primary causal agent and/or an aggravating factor has been supported in extrahepatic cholestasis induced by bile duct ligation,6, 8, 9 but it remains poorly documented in intrahepatic cholestasis.

Chlorpromazine (CPZ), a neuroleptic drug of the phenothiazine family widely used in the treatment of schizophrenia, has caused several cases of liver injury during its therapeutic use, which mostly include intrahepatic cholestasis10 and phospholipidosis. CPZ has been reported to inhibit bile flow in in vitro perfused rat liver11 and human liver canalicular vesicles.12 However, its initial toxic effects remain largely ignored, likely because current models used for safety assessment in drug development do not accurately predict cholestasis in humans.13 Rat hepatocyte couplets14 and primary rat and human hepatocytes in a sandwich configuration7 have been the most common in vitro cell models used to analyze hepatic transport processes. However, it is now recognized that compounds known to interfere with BSEP function are often not associated with significant liver cell injury in these standard preclinical models, although they have been related to liver damage when administered in humans.15-17 Studies with human liver cells are preferable because species-dependent differences have been reported. For instance, taurocholic acid (TA) elimination through the basolateral membrane was much higher in rat hepatocytes than in their human counterparts.17 The limited availability of fresh cells had led to the use of cryopreserved human hepatocytes for sandwich cultures; however, not all batches are suitable for culturing in a sandwich configuration.7 In the present study we used the differentiated human HepaRG cell line that expresses phases 1 and 2 drug metabolizing enzymes and transporters, and forms functional bile canaliculi,18-20 to analyze features of intrahepatic cholestasis induced by CPZ treatment and to characterize the mechanisms involved in the initiation and progression of the lesions. We demonstrated a role of oxidative stress as both a primary causal and aggravating agent in early intracellular TA accumulation in HepaRG cells treated with CPZ.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chemicals.

CPZ, cholic and chenodeoxycholic acids, salicylic acid (SA), cyclosporine A (CSA), methylthiazoletetrazolium (MTT), N-acetyl-cysteine (NAC), and 6β-hydroxytestosterone were purchased from Sigma (St. Quentin Fallavier, France). Dihydroethidium (DHE), 2′,7′-dichlorodihydrofluorescein (H2-DCFDA), and JC-1 dye were from Invitrogen-Molecular Probe. [3H]-Taurocholic acid was from Perkin Elmer (Boston, MA).

Cell Cultures.

HepaRG cells were seeded at a density of 2.6 × 104 cells/cm2 in Williams E medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL strep tomycin, 5 mg/mL insulin, 2 mM glutamine, and 50 mM hydrocortisone hemisuccinate.21 After 2 weeks, HepaRG cells were shifted to the same medium supplemented with 2% dimethyl sulfoxide for a further 2 weeks in order to obtain confluent differentiated cultures with maximum functional activities. At this time, these cultures contained hepatocyte-like and progenitors/primitive biliary-like cells.21

Cell Viability.

Cytotoxicity of CPZ and BA was evaluated by the MTT colorimetric assay.18

JC-1 Test.

Mitochondrial membrane potential was analyzed using the JC-1 dye.22

F-actin Distribution.

F-actin was localized using a phalloidin-fluoprobe.22

Measurement of Reactive Oxygen Species (ROS).

Superoxide anions were detected by DHE staining. Cells were incubated with 2 μM DHE and 0.5 μg/mL Hoechst for 30 minutes at 37°C. They were then washed with chilled phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and examined under fluorescence microscopy.

Hydrogen peroxide generation was determined by the H2-DCFDA assay. Cells were incubated for 2 hours at 37°C with 5 μM H2-DCFDA; then they were washed with cold PBS, and scraped in potassium buffer (10 mM, pH 7.4) / methanol (v/v) completed with Triton X-100 (0.1%). Fluorescence intensity of cell extracts was determined by spectrofluorimetry using excitation/emission wavelengths of 498/520 nm.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis.

Total RNA was extracted from 106 HepaRG cells with the SV total RNA isolation system (Promega). RNAs were reverse-transcribed into cDNA and RT-qPCR was performed using a SYBR Green mix. Primer sequences are listed in Supporting Table 1.

Na+-Dependent Taurocholic Cotransporting Polypeptide (NTCP) Activity.

Activity of the NTCP transporter was estimated through determination of sodium-dependent intracellular accumulation of radiolabeled TA.20

Efflux of TA.

Cells were first exposed to [3H]-TA for 30 minutes, then washed with PBS and incubated with or without CPZ at different timepoints (from 0 to 6 hours) in a standard buffer with Ca2+ and Mg2+. After the incubation time, cells were washed with PBS and incubated for 5 minutes with a Ca2+ and Mg2+-free buffer in order to disrupt the canalicular tight junctions.23 Then they were scraped in 0.1 N NaOH; the remaining radiolabeled substrate was measured through scintillation counting to determinate TA efflux. To discriminate between basolateral and canalicular efflux, cells were incubated in parallel in either standard or Ca2+ and Mg2+-free buffer for 30 minutes after TA uptake before measuring the remaining radiolabeled TA. Canalicular efflux was calculated using this equation: Canalicular efflux = Radioactivity in efflux mediumCa2+ and Mg2+ free buffer − Radioactivity in efflux mediumStandard buffer.23

CYP3A4 Activity.

6β-Hydroxylation of testosterone by CYP3A4 was measured as described previously.18

Statistical Analysis.

The Mann-Whitney U test was applied to compare data between treated cells and corresponding control cultures. Data were considered significantly different when P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cytotoxicity and Generation of ROS in CPZ-Treated HepaRG Cells.

The MTT test was first used to evaluate cytotoxic effects of CPZ in HepaRG cells after 6- and 24-hour exposure. Whereas no cytotoxicity was observed after 6 hours (data not shown), CPZ induced dose-dependent toxic effects after a 24-hour treatment, with a median inhibitory concentration (IC50) value equal to 80 μM (Fig. 1A). Based on these data, nontoxic (20 and 35 μM) and subtoxic (50 μM, corresponding to 80% cell viability) concentrations of CPZ were used to investigate early events leading to the toxic response. At these concentrations, CPZ induced formation of intracytoplasmic vesicles. These vesicles appeared as lamellar bodies under electron microscopy and, accordingly, expression of target genes of phospholipidosis was found to be modulated (Supporting data).

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Figure 1. Cytotoxicity and intracellular generation of reactive oxygen species in CPZ-treated HepaRG cells. (A) Cells were incubated for 24 hours with different concentrations of CPZ (0-150 μM). Cytotoxicity was measured by the MTT colorimetric assay. Each point is the mean ± SD of three independent experiments. (B) Cells were treated with 50 μM CPZ for 0 to 24 hours with or without 5 mM NAC. Superoxide anions were detected by DHE staining. Nuclei were stained in blue (Hoechst). (C) Cells were treated with solvent (control) or 50 μM CPZ for 6 or 24 hours and with 50 μM CPZ and NAC for 24 hours. Total RNA was isolated from control and treated cells and mRNA levels of HO-1, MnSOD, and Nrf2 were estimated by RT-qPCR. Data represent the means ± SD of three independent experiments. All results are expressed relative to the levels found in control cells, arbitrarily set at a value of 1. *P < 0.05 compared with control.

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ROS generation was determined by DHE staining in 50 μM CPZ-treated HepaRG cells (Fig. 1B). Superoxide anions were detected in hepatocyte-like cells as early as 15 minutes after CPZ exposure. Superoxide anions formation was totally prevented up to 6 hours and only partially after 24 hours by coincubation with the antioxidant NAC. Moreover, expression of three oxidative stress-related genes was analyzed 6 and 24 hours after addition of 50 μM CPZ (Fig. 1C). The NF-E2-related factor 2 (Nrf2) that regulates antioxidant-responsive element-mediated induction of cytoprotective genes and its target gene heme oxygenase 1 (HO-1) were significantly up-regulated at both timepoints, whereas the expression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD), was enhanced only after a 24-hour CPZ treatment. As expected, fold-induction of the three genes was reduced in the presence of NAC.

Alteration of Mitochondrial Membrane Potential and F-actin Distribution by CPZ Treatment.

ROS production is known to generate mitochondrial injury and to disrupt F-actin distribution. Mitochondrial membrane potential was followed by JC-1 staining. CPZ seemed to alter the inner mitochondrial membrane potential, as the green fluorescence associated with monomer forms of JC-1 was more pronounced in CPZ-treated cells compared to untreated cells in which the red fluorescence associated with JC-1 dimers was predominant (Fig. 2A). The longer the time of treatment the stronger was the effect starting at 30 minutes, being more pronounced at 24 hours. Alteration of the mitochondrial membrane potential by CPZ was prevented by cotreatment with NAC, suggesting a role of ROS. Similar alterations were observed in HepaRG cells treated with 5 mM H2O2 starting at 30 minutes (data not shown).

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Figure 2. Alteration of mitochondrial membrane potential and rearrangement of the F-actin cytoskeleton by CPZ treatment. (A) Cells were treated with 50 μM CPZ for different timepoints with or without 5 mM of NAC. Mitochondrial membrane potential was assessed by JC-1 staining. While red fluorescence indicates a normal mitochondrial potential membrane, green fluorescence reflects an alteration of the latter. (B) Cells were treated with 50 μM CPZ for 30 minutes or 2 hours. F-actin was localized by using phalloidin-fluoprobe. Nuclei were stained in blue (Hoechst). F-actin shows a predominant pericanalicular distribution in untreated cells and a less intense staining around the canalicular region in CPZ-treated cells. Although untreated cells show round shaped canaliculi, CPZ-treated cells exhibit retracted bile canaliculi (×20 magnification [a-c]). Arrows indicate bile canaliculi. Details of one canaliculus of untreated cells (d) and 2-hour CPZ-treated cells (e) are shown. Imaging quantification was done by using vHCS.scan (V6.2.0) cellomics software (Thermo Scientific).

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F-actin cytoskeleton, which is one of the primary targets of oxidative stress, was visualized by phalloidin fluoprobe labeling. Untreated cells showed pericanalicular location of F-actin and large bile canaliculi with rounded shape, whereas 50 μM CPZ-treated cells exhibited a different distribution with lesser pericanalicular F-actin, retraction, and decreased surface area of bile canaliculi (Fig. 2B). Quantification of bile canalicular surface area and intensity of F-actin in the pericanalicular region showed up to 28% and 26% decrease, respectively, after CPZ exposure.

Efflux of TA in CPZ-Treated HepaRG Cells.

To assess the effect of CPZ on TA efflux, cells were incubated with [3H]-TA for 30 minutes and then treated for another 30 minutes with 50 μM CPZ in standard buffer or Ca2+ and Mg2+-free buffer. Radiolabeled TA has been measured in these two different buffers and in the cells to determine canalicular and basolateral efflux as well as intracellular accumulation of TA (7). A 25% increase in TA efflux was noticed in untreated HepaRG cells when canalicular tight junctions were disrupted (Ca2+ and Mg2+-free buffer), indicating that bile canaliculi correspond to a delimited closed compartment in these cells (data not shown). After 30 minutes of treatment with CPZ, a 32% intracellular accumulation of TA was observed; it was associated with 35% decrease in canalicular efflux whereas basolateral efflux remained unchanged (Fig. 3A). These data support the conclusion that intracellular accumulation of TA was caused by a decrease of canalicular efflux rather than a diminution of basolateral efflux.

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Figure 3. Effects of NAC on TA efflux in CPZ-, CSA-, and SA-treated HepaRG cells. (A) Cells were exposed to [3H]-TA for 30 minutes to induce intracellular accumulation of TA and then incubated for another 30 minutes with 50 μM CPZ in parallel with either standard buffer (with Ca2+ and Mg2+) or Ca2+ and Mg2+-free buffer. Radiolabeled TA was measured in these two different buffers as well as in the cells in order to determine canalicular and basolateral efflux and accumulation of TA in cells. Data were expressed relative to the level found in control cells, arbitrarily set at the value of 100%. (B) Cells were exposed to [3H]-TA for 30 minutes to induce an accumulation of TA in cells. TA efflux was then determined at different timepoints in control or 50 μM CPZ-treated cultures, in the absence or presence of 5 mM NAC, by measuring intracellular TA accumulation. Efflux of TA was expressed relative to the levels found in control cells, arbitrarily set at a value of 100%. (C) Cells were exposed to [3H]-TA for 30 minutes and then treated or not for 2 hours with 50 μM CPZ, CSA, or SA in the absence or presence of 5 mM NAC. Bile canaliculi were disrupted by an additional 5 minutes incubation with Ca2+ and Mg2+-free buffer. TA efflux was determined by measuring intracellular TA accumulation. Efflux of TA was expressed relative to the level found in control cells, arbitrarily set at a value of 100%. Data represent the means ± SD of three independent experiments. *P < 0.05 compared with control.

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Then the effects of CPZ in TA efflux were measured at different timepoints (0-6 hours) in standard buffer (Fig. 3B). The Ca2+ and Mg2+-free buffer was excluded because an incubation exceeding 30 minutes with this buffer caused increased cell death. No efflux inhibition was observed before 30 minutes, whereas maximum inhibition occurred after 2 hours, with a 2-fold TA intracellular accumulation in CPZ-treated HepaRG cells. The CPZ-induced decrease of [3H]-TA efflux was abolished when cells were cotreated with CPZ and NAC; this result showed the involvement of oxidative stress in TA accumulation in CPZ-treated HepaRG cells.

Because intracellular accumulation of TA was measured after treatment with CPZ in a standard buffer and that bile canaliculi were at least partially closed in control HepaRG cells, this accumulation could represent bile canalicular storage in addition to intracellular accumulation. To verify this hypothesis, bile canaliculi were disrupted by 5-minute incubation in Ca2+ and Mg2+-free buffer23 after a 2-hour treatment with CPZ. Intracellular accumulation and a decrease in TA efflux were still demonstrated. This decrease was partially but significantly reversed by cotreating the cells with CPZ and NAC (Fig. 3C).

To confirm the specificity of CPZ-induced cholestasis and its ROS dependency, the effects on TA efflux of 50 μM SA and 50 μM CSA, a noncholestatic drug and a potent inhibitor of BSEP, respectively, were also assessed using the same protocol. Although SA had no effect, CSA induced a strong inhibition of TA efflux that was not prevented by NAC cotreatment (Fig. 3C). These data confirm that CPZ-induced TA efflux decrease, unlike CSA, was ROS-dependent.

Modulation of mRNA Levels of Genes Involved in Transport Systems, BA Synthesis, Metabolizing Enzymes, and Nuclear Receptors by CPZ-Induced Oxidative Stress.

We analyzed by RT-qPCR changes in the expression of 20 potential target genes (Supporting Table 1) after treatment with 20 to 50 μM CPZ. These genes are major nuclear receptors (CAR, FXR, and PXR) or key players in uptake transport (NTCP, OATP-B, OATB-C, OATP8, and OCT1), efflux transport (BCRP, BSEP, multidrug resistance protein 1 [MDR1], MDR3, multidrug resistance-associated protein 2 [MRP2], MRP3, and MRP4), BA synthesis (CYP7A1, CYP8B1, and CYP27A1), and metabolism of exogenous and endogenous substances (CYP3A4 and SULT2A1).

Although no effect was observed after 6-hour treatment whatever the studied concentration (data not shown), CPZ altered expression of several genes after a 24-hour exposure at 50 μM (Table 1). CPZ caused a decrease of mRNA of NTCP, CYP8B1, BSEP, and MDR3, whereas it caused an increase of MRP4 (a basolateral BA transporter) and CYP3A4 mRNA levels. No effects were observed on nuclear receptors transcripts. The lower doses of CPZ (20 and 35 μM) did not affect the measured mRNA levels except for CYP3A4.

Table 1. Effects of CPZ on Expression of mRNAs Encoding Genes Related to Hepatobiliary Transporters, Nuclear Receptors, and Phase I and Phase II Metabolizing Enzymes in HepaRG Cells
24 h CPZ (μM)203550
  1. Cells were incubated with 20, 35, and 50 μM CPZ for 24 hours. mRNAs were analyzed by RT-qPCR. Results are expressed as fold of the value found in control cells arbitrarily set at 1. Data are mean ± SD of three independent experiments. Data with P < 0.05 and ± 50% fold change are indicated in bold.

Influx transporters   
 NTCP1.0 ± 0.10.9 ± 0.20.5 ± 0.1
 OATP-B1.3 ± 0.21.2 ± 0.11.1 ± 0.1
 OATP-C1.1 ± 0.31.2 ± 0.10.9 ± 0.1
 OATP81.1 ± 0.21.3 ± 0.21.2 ± 0.1
 OCT11.4 ± 0.21.2 ± 0.20.9 ± 0.2
Efflux transporters   
 BCRP1.2 ± 0.11.3 ± 0.21.4 ± 0.2
 BSEP1.1 ± 0.21.2 ± 0.30.5 ± 0.2
 MDR11.1 ± 0.31.2 ± 0.11.1 ± 0.1
 MDR30.8 ± 0.20.8 ± 0.20.5 ± 0.2
 MRP21.2 ± 0.11.4 ± 0.21.2 ± 0.2
 MRP30.9 ± 0.20.8 ± 0.10.8 ± 0.2
 MRP41.2 ± 0.21.4 ± 0.21.7 ± 0.2
Phases I and II metabolizing enzymes   
 CYP3A41.5 ± 0.21.7 ± 0.21.9 ± 0.3
 SULT2A11.2 ± 0.11.2 ± 0.11.0 ± 0.1
Bile acid formation   
 CYP7A11.1 ± 0.11.2 ± 0.21.1 ± 0.2
 CYP8B11.0 ± 0.10.7 ± 0.10.4 ± 0.1
 CYP27A11.1 ± 0.11.1 ± 0.10.8 ± 0.1
Nuclear receptors   
 CAR1.1 ± 0.21.0 ± 0.20.8 ± 0.1
 FXR1.2 ± 0.11.2 ± 0.21.4 ± 0.0
 PXR1.1 ± 0.21.2 ± 0.21.3 ± 0.2

To determine the role of CPZ-induced ROS in modulation of transcripts levels, we studied the effects of a 24-hour NAC cotreatment on expression of NTCP, BSEP, MDR3, MRP4, CYP3A4, and CYP8B1 (Fig. 4A). Only inhibition of BSEP was prevented by a 24-hour coexposure with NAC. Moreover, most expression changes induced by CPZ, i.e., inhibition of NTCP, MDR3, and CYP8B1 were reduced after a 48-hour cotreatment with NAC, whereas CYP3A4 was inhibited by CPZ and induced by a cotreatment with CPZ and NAC.

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Figure 4. Role of ROS generation in expression of mRNAs encoding genes related to hepatobiliary transporters and cytochrome P450 enzymes in CPZ-treated HepaRG cells. (A) Cells were treated with solvent (control) or 50 μM CPZ with or without NAC for 24 or 48 hours (with two additions of NAC at 0 and 24 hours). Total RNA was isolated from control and treated cells and mRNA levels of NTCP, BSEP, MDR3, MRP4, CYP3A4, and CYP38B1 were estimated by RT-qPCR. *P < 0.05 compared with control, #P < 0.05 compared with NAC treatment, ns: no significant. (B,C) Cells were treated with solvent (control) or H202 (0.5-5 mM) for 6 hours (B) or 24 hours (C). Total RNA was isolated from control and treated cells and mRNA levels of NTCP, BSEP, MDR3, MRP4, CYP3A4, CYP38B1, HO-1, and NrF2 were estimated by RT-qPCR. Data represent the means ± SD of three independent experiments. All results are expressed relative to the levels found in control cells, arbitrarily set at a value of 1. *P < 0.05 compared with control.

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To confirm the role of oxidative stress in these transcriptomic deregulations, we further analyzed effects of 6- and 24-hour (Fig. 4B,C) treatments of cells with 0.5-5 mM H2O2 on CPZ-affected genes. A dose-dependent decrease in NTCP, BSEP, MDR3, CYP3A4, and CYP8B1 and an increase in MRP4 and HO-1 were shown after a 24-hour exposure to H2O2. However, after 6-hour H2O2 treatment, only HO-1 was overexpressed starting at 1 mM and CYP8B1 was down-regulated with 5 mM H2O2.

NTCP and CYP3A4 Activities in CPZ-Treated HepaRG Cells.

To assess whether CPZ affected the NTCP activity, cells were treated at different timepoints with 50 μM CPZ in the presence or absence of NAC and then incubated with [3H]-TA for 30 minutes. The NTCP activity was evaluated through measurement of intracellular accumulation of radiolabeled TA. As for the NTCP transcripts, a 45% and 23% inhibition of the corresponding activity was observed in HepaRG cells after 24- and 48-hour treatments, respectively (Fig. 5A). NAC cotreatment did not significantly affect CPZ-inhibited TA uptake up to 24 hours, but partially reduced this inhibition after 48 hours. Noticeably, CPZ did not affect NTCP activity during the first 6 hours of treatment.

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Figure 5. NTCP and CYP3A4 activities in CPZ-treated HepaRG cells. (A) Cells were treated at different timepoints (30 minutes to 48 hours) with solvent (control) or 50 μM CPZ (with or without NAC) and then incubated with [3H]-TA for 30 minutes. NTCP activity was evaluated through measurement of intracellular accumulation of the radiolabeled substrate TA. Data represent the means ± SD of three independent experiments. All results are expressed relative to the levels found in control cells, arbitrarily set at a value of 100%. *P < 0.05 compared with control, #P < 0.05 compared with NAC treatment, ns: no significant. (B) HepaRG cells were treated with solvent (control), 20, 35, and 50 μM CPZ or 50 μM rifampicin (a prototypical inducer of CYP3A4) for 48 hours. Then the cells were incubated with 200 μM testosterone for 2 hours in phenol red-free medium and testosterone 6β-hydroxylation activity was determined by high-performance liquid chromatography (HPLC). Results are expressed as picomoles per minute per mg of protein. Data represent the means ± SD of three independent experiments. *P < 0.05 compared with control.

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Because CYP3A4 transcripts were augmented after 24-hour CPZ treatment, we also assessed the effects of CPZ on CYP3A4 activity (Fig. 5B). A dose-dependent increase in the formation of 6β-hydroxytestosterone was found after a 48-hour CPZ treatment. NAC had no effect on the induction of CYP3A4 activity after 48 hours of cotreatment, suggesting that CPZ-induced CYP3A4 was ROS-independent (data not shown).

Effects of BA on Cell Viability, ROS Generation, and mRNA Levels of Genes Involved in Transport Systems, BA Synthesis, and Drug-Metabolizing Enzymes.

To compare CPZ-induced cholestasis to cholestasis-like condition caused by BA overload, HepaRG cells were incubated with the two primary BA, cholic and chenodeoxycholic acids, at 25- 500 μM, and cell viability was assessed by the MTT test after 24-hour exposure (Fig. 6A). Whereas no cytotoxicity was observed with up to 200 μM BA, cell viability dropped to 40% with 500 μM cholic and chenodeoxycholic acids. Moreover, ROS generation was assessed by DHE staining and the H2-DCFDA assay at different timepoints, ranging from 30 minutes to 24 hours. Superoxide anions were detected in hepatocyte-like cells after 6 hours of exposure to 500 μM of either BA (Fig. 6B). In parallel, formation of hydrogen peroxides was detected from 6-hour exposure to 500 μM chenodeoxycholic acid and only after 24-hour treatment with 500 μM cholic acid (Fig. 6C). No ROS generation was evidenced with low concentrations up to 200 μM BA whatever the time of treatment (data not shown). Noteworthy, no alteration of the mitochondrial membrane potential was evidenced before 6-hour exposure to 500 μM of either BA (Fig. 6D). In addition, expression of genes modulated by CPZ was analyzed and found to vary depending on BA concentrations after a 24-hour exposure (Table 2A). Thus, genes involved in the canalicular efflux transport system were either strongly (BSEP) or slightly (MDR3) up-regulated, whereas expression of other genes remained unchanged with low concentrations of either BA. By contrast, BSEP and MDR3 were down-regulated with 500 μM BA. Noticeably, genes related to oxidative stress (HO-1 and Nrf2) were overexpressed, whereas NTCP and CYP8B1 were inhibited with 500 μM BA. In addition, MRP4 transcripts were enhanced with 500 μM cholic acid but decreased with 500 μM chenodeoxycholic acid. A decrease in CYP3A4 mRNAs was obtained in cells overloaded with subtoxic or toxic concentrations of BA.

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Figure 6. Cytotoxicity and intracellular generation of ROS in BA-overloaded HepaRG cells. (A) Cells were incubated for 24 hours with different concentrations of cholic and chenodeoxycholic acids (0-500 μM). Cytotoxicity was measured by the MTT colorimetric assay. Each point is the mean ± SD of three independent experiments. (B) Cells were treated with 500 μM cholic and chenodeoxycholic acids at different timepoints ranging from 2 to 24 hours. Superoxide anions were detected by DHE staining. Nuclei were stained in blue (Hoechst). (C) Cells were treated with 500 μM cholic and chenodeoxycholic acids at different timepoints ranging from 30 minutes to 24 hours, or with 5 mM H2O2 for 24 hours. Hydrogen peroxides were quantified by the H2-DCFDA assay. Data represent the means ± SD of three independent experiments. All results are expressed relative to the levels found in control cells, arbitrarily set at a value of 100%. *P < 0.05 compared with control. (D) Cells were treated with 500 μM cholic and chenodeoxycholic acids at different timepoints ranging from 2 to 24 hours. Mitochondrial membrane potential was assessed by JC-1 staining.

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Table 2. Effects of Cholic and Chenodeoxycholic Acids on Expression of mRNAs Encoding Genes Related to Hepatobiliary Transporters, Cytochrome P450 Enzymes, and Oxidative Stress in HepaRG Cells
 Cholic Acid 24 hChenodeoxycholic Acid 24 h
 25 μM50 μM100 μM200 μM500 μM25 μM50 μM100 μM200 μM500 μM
A
Transporters          
 NTCP1.0 ± 0.00.8 ± 0.11.1 ± 0.20.9 ± 0.10.4 ± 0.11.0 ± 0.01.1 ± 0.11.1 ± 0.10.7 ± 0.30.4 ± 0.1
 BSEP3.1 ± 0.53.2 ± 1.54.5 ± 1.21.4 ± 0.20.4 ± 0.38.1 ± 2.213.7 ± 0.421.8 ± 3.63.0 ± 1.00.3 ± 0.2
 MDR31.4 ± 0.01.2 ± 0.41.6 ± 0.31.2 ± 0.20.5 ± 0.01.8 ± 0.02.2 ± 0.52.4 ± 0.21.0 ± 0.40.4 ± 0.1
 MRP40.9 ± 0.00.9 ± 0.21.0 ± 0.21.1 ± 0.32.1 ± 0.41.0 ± 0.01.0 ± 0.21.1 ± 0.11.3 ± 0.30.5 ± 0.2
CYP P450          
 CYP3A40.8 ± 0.10.8 ± 0.30.9 ± 0.10.6 ± 0.10.5 ± 0.10.9 ± 0.10.8 ± 0.00.8 ± 0.00.6 ± 0.30.4 ± 0.2
 CYP8B11.2 ± 0.01.0 ± 0.11.0 ± 0.10.9 ± 0.10.2 ± 0.11.2 ± 0.31.2 ± 0.21.0 ± 0.20.6 ± 0.30.2 ± 0.0
Oxidative stress          
 HO11.0 ± 0.11.0 ± 0.10.9 ± 0.11.3 ± 0.12.8 ± 0.71.0 ± 0.21.0 ± 0.10.9 ± 0.20.8 ± 0.31.7 ± 0.3
 NrF21.0 ± 0.10.9 ± 0.01.0 ± 0.21.2 ± 0.21.7 ± 0.31.0 ± 0.31.0 ± 0.11.0 ± 0.21.4 ± 0.43.5 ± 0.5
 Cholic Acid 6 hChenodeoxycholic Acid 6 h
 100 μM200 μM500 μM100 μM200 μM500 μM
  1. The cells were incubated with different concentrations of cholic and chenodeoxycholic acids for 24 hours (A) or 6 hours (B). mRNAs were analyzed by RT-qPCR. Results are expressed as fold of the value found in control cells arbitrarily set at 1. Data are mean ± SD of three independent experiments. Data with P < 0.05 and ± 50% fold change are indicated in bold.

B
Transporters      
 NTCP1.3 ± 0.41.4 ± 0.31.2 ± 0.11.2 ± 0.21.3 ± 0.31.3 ± 0.1
 BSEP1.3 ± 0.21.1 ± 0.20.8 ± 0.16.3 ± 2.43.9 ± 1.30.8 ± 0.2
 MDR31.2 ± 0.30.9 ± 0.10.9 ± 0.21.8 ± 0.41.8 ± 0.30.9 ± 0.2
 MRP41.0 ± 0.10.9 ± 0.10.8 ± 0.01.0 ± 0.01.0 ± 0.21.2 ± 0.2
CYP P450      
 CYP3A41.0 ± 0.10.9 ± 0.01.2 ± 0.11.0 ± 0.11.2 ± 0.21.2 ± 0.4
 CYP8B11.1 ± 0.31.0 ± 0.10.9 ± 0.11.2 ± 0.31.2 ± 0.20.6 ± 0.1
Oxidative stress      
 HO10.7 ± 0.20.7 ± 0.22.2 ± 0.70.9 ± 0.22.0 ± 0.33.4 ± 0.9
 NrF21.0 ± 0.01.0 ± 0.02.2 ± 0.41.2 ± 0.13.0 ± 0.95.1 ± 1.2

In addition, after a 6-hour exposure of HepaRG cells to the two BA, BSEP and MDR3 were overexpressed by 200 μM or lower concentrations, whereas only CYP8B1 was down-regulated by 500 μM chenodeoxycholic acid. Moreover, HO-1 and Nrf2 expression was induced by 200 and 500 μM chenodeoxycholic acid and only by 500 μM cholic acid (Table 2B).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Although the first studies were carried out about 30 years ago, the mechanisms involved in cholestasis caused by CPZ remain poorly understood. In the current work we show that CPZ caused early intracellular TA accumulation by way of induction of an oxidative stress in human HepaRG cells. Later on this cholestatic effect was associated with deregulated expression of several influx and efflux transporters.

We show for the first time that bile canaliculi correspond to a delimited closed compartment in HepaRG cells and that CPZ impairs canalicular secretion rather than basolateral efflux of TA using buffers with or without Ca2+ and Mg2+. TA efflux inhibition occurred after 30 minutes, whereas increased levels of superoxide anions were already observed 15 minutes after CPZ exposure and were associated at 6 hours with up-regulation of Nrf2 and HO-1, two genes related to oxidative stress. These data help establish that following CPZ treatment, ROS generation occurred before efflux inhibition, favoring a role of the oxidative stress in this inhibition rather than a direct effect of CPZ on canalicular efflux. The absence of TA accumulation by cotreatment with NAC confirmed this role of oxidative stress. Moreover, CPZ-induced oxidative stress was associated with an alteration of mitochondrial membrane potential and with an impairment of pericanalicular F-actin distribution. These data are in agreement with several studies demonstrating that oxidative stress might play an important role in the pathogenesis of hepatic injury during cholestasis in both rodents and humans.6, 8, 24 It impaired secretion of bile salts by internalizing BSEP by way of a disarrangement of cytoskeletal F-actin in rat hepatocyte couplets.6, 9, 25

Although CPZ has been shown to inhibit bile flow in the rat11 and human BSEP activity in the transfected SK-E2 cell line,12 studies on isolated plasma membrane vesicles expressing human or rat BSEP failed to show an effect of CPZ on BSEP activity,13, 26 most likely because of absence of ROS generation in these latter. Noteworthy, unlike SA (a noncholestatic drug), CSA (a potent inhibitor of BSEP) also strongly reduced TA efflux; however, NAC cotreatment with CSA, unlike with CPZ, had no effect on this efflux inhibition excluding any ROS involvement in CSA-induced TA accumulation.

Early alteration of efflux activity was not associated with a decrease in NTCP activity. Indeed, inhibition of NTCP activity was noticeably observed only after 24-hour treatment by CPZ, suggesting that this delayed effect was not triggered by early ROS generation. This conclusion is strongly supported by the failure of NAC to prevent this inhibition. In parallel, CPZ also altered expression of various genes related to hepatobiliary secretion after a 24-hour treatment of HepaRG cells. First, the nearly 50% decrease in BSEP transcript levels could actively contribute to accumulation of BA and therefore to CPZ-induced cholestasis. Second, expression of MDR3, another canalicular efflux transporter, was also decreased. MDR3 is known as an adenosine triphosphate (ATP)-dependent phospholipid flippase, translocating phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane.27 CPZ-induced MDR3 inhibition could prevent phospholipids translocation and formation of biliary micelles with BA and could represent another potential mechanism for drug-induced intrahepatic cholestasis. However, little is known about inhibition of MDR3 by cholestatic drugs, with the exception of a recent study showing the involvement of MDR3 in itraconazole-induced cholestasis.28

If alterations of some transporters appeared to contribute to cholestasis, changes of several others rather represent compensatory mechanisms, which provide alternative excretory routes for accumulated BA in cholestasis.29 As such, expression of the basolateral BA uptake transporter NTCP was down-regulated, whereas that of the alternative basolateral BA export transporter MRP4 was enhanced after 24-hour CPZ exposure of HepaRG cells. NTCP down-regulation and MRP4 up-regulation likely represented a protective response against CPZ-induced cholestasis. Indeed, several studies have reported a reduction of NTCP expression in human and rodent liver cholestasis30-32 as well as an inhibition of CYP8B1 that is involved in the synthesis of cholic acid.29 Accordingly, CPZ showed a decrease of CYP8B1 expression in our study. Overexpression of CYP3A4 usually represents an additional adaptive mechanism facilitating elimination of BA.29 CYP3A4 induction was observed independently of the oxidative stress after 24-hour treatment with low CPZ concentrations, contrary to other compensatory mechanisms. However, CYP3A4 expression was down-regulated by high concentrations of BA, H2O2, and 48-hour CPZ exposure. Such CYP3A4 inhibition could be related to a toxic effect and/or an inflammatory response. A ROS-dependent hepatic inflammatory response has indeed been proposed to explain at least part of the transcriptional alterations occurring in cholestasis.6 Accordingly, CPZ induced expression of the proinflammatory cytokines interleukin (IL)-1β, IL-6, and IL-8 in HepaRG cells (data not shown). IL-1β has been previously found to impair expression of membrane transporters, especially BSEP, in HepaRG cells.33 Undoubtedly, liver cell models have certain limitations for investigating drug-induced idiosyncratic hepatotoxicity, especially due to the absence of immune and other liver cells. Therefore, coculturing HepaRG cells with immune or inflammatory cells should still improve their suitability for investigating idiosyncratic hepatotoxicity of certain drugs.

To compare CPZ-induced cholestasis to cholestasis-like condition caused by BA overload, HepaRG cells were overloaded with two BA, cholic and chenodeoxycholic acids, for 24 hours. This BA overload resulted in the induction of two concentration-dependent responses. BSEP and MDR3 expression was enhanced after treatment with low concentrations of the two BA that comprise 90% of bile salts in humans34 and are known to induce these two transporters by way of activation of FXR.35, 36 In contrast, subtoxic concentrations of BA induced an oxidative stress associated with a decrease of BSEP and MDR3 expression and compensatory mechanisms similar to those observed after 50 μM CPZ exposure. As these mechanisms occurred only when HepaRG cells were overloaded with toxic concentrations of BA or treated with 50 μM CPZ, we suppose that CPZ enhanced accumulation of BA in hepatic cells. Similar gene expression changes were obtained in HepaRG cells treated with H2O2 for 24 hours. Because the oxidative stress was generated only after a 6-hour exposure to high concentrations of BA, it might be concluded that early ROS generation and mitochondrial dysfunction induced by CPZ-treatment were a direct drug effect and not due to BA intracellular accumulation. Likely, BA-induced ROS acted more as an aggravating factor.

In summary, the present work provides the first in vitro study of the mechanisms involved in CPZ-induced intrahepatic cholestasis in human liver, using HepaRG cells. CPZ was found to impair bile acid secretion by multiple and complex mechanisms. First, CPZ induced-ROS generation resulted in a decrease of TA efflux. Second, CPZ-induced cholestasis was associated with an inhibition of BSEP and MDR3 expression. Third, changes in some transporters gene expression induced by CPZ treatment could be considered as an alternative response to escape cholestasis. Altogether, these data provide new insight into the mechanisms of CPZ-induced cholestasis in human hepatocytes, emphasizing both the causal and aggravating role of oxidative stress in drug-induced intrahepatic cholestasis. Moreover, this work suggests that HepaRG cells represent a suitable cell model for a better understanding of the mechanisms regulating transport systems in human cholestatic disorders.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank R. Le Guevel from the ImPACcell platform (Biosit) for imaging analysis.

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  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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HEP_26160_sm_SuppTab1.doc36KSupporting Information Table 1.

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