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Research ArticleArticle

P-Glycoprotein-Mediated In Vitro Biliary Excretion in Sandwich-Cultured Rat Hepatocytes

Pieter P. Annaert, Ryan Z. Turncliff, Catherine L. Booth, Dhiren R. Thakker and Kim L. R. Brouwer
Drug Metabolism and Disposition October 2001, 29 (10) 1277-1283;

Abstract

Recently, sandwich-cultured (SC) rat hepatocytes have been used as an in vitro model to assess biliary excretion of drugs and xenobiotics. The purpose of the present study was to validate the use of SC rat hepatocytes for the in vitro assessment of P-glycoprotein (P-gp)-mediated biliary drug excretion. The specific and fluorescent P-gp substrate rhodamine 123 (Rh123) and the P-gp substrate digoxin were selected as model compounds. Rh123 and digoxin accumulation and Rh123 efflux under standard and Ca2+-free conditions were quantified in SC rat hepatocytes to determine substrate secretion into canalicular networks in vitro. The major role of P-gp in the biliary excretion of these compounds was confirmed by inhibition experiments with the potent P-gp inhibitor GF120918. Hepatocyte culture conditions, including media type and time in culture, significantly affected Rh123 biliary excretion. P-gp expression, as assessed by Western blot, was increased with culture time. Dexamethasone (an in vivo inducer of P-gp) concentrations ranging from 0.01 to 1 μM in the cell culture medium did not influence P-gp expression or Rh123 biliary excretion. Rh123 and digoxin biliary clearance values, predicted from SC rat hepatocyte data, were consistent with values reported in vivo and in isolated perfused rat liver studies. In conclusion, the results of this study demonstrate the utility of SC rat hepatocytes as an in vitro model to study and predict the biliary excretion of P-gp substrates.

Biliary excretion is an important elimination pathway for many xenobiotics and may influence significantly the concentration-time profile in the systemic circulation. For example, rapid hepatic uptake and efficient biliary excretion of the organic cation vecuronium is responsible for the very short plasma half-life of this muscle relaxant after i.v. administration in cats (Proost et al., 2000). Hence, drug interactions at the biliary excretion level may be responsible for significant changes in drug plasma concentration-time profiles (Angelin et al., 1987; Speeg and Maldonado, 1994; Ellis and Webster, 1999; Kiso et al., 2000). Drugs undergoing significant biliary excretion in addition to urinary excretion may still be used safely in patients with impaired kidney function (e.g., temocapril; Puchler et al., 1997). Therefore, it is crucial to understand the molecular and cellular mechanisms underlying the biliary excretion process. Isolated perfused rat liver (IPRL1) experiments have been used extensively to study hepatic drug disposition and biliary excretion as well as hepatic drug interactions. However, IPRL studies are time- and resource-consuming. In this context, the recently developed in vitro model for biliary drug excretion based on sandwich-cultured (SC) rat hepatocytes appears to be very useful (Liu et al., 1999a,b). Tight junction modulation following incubation with Ca2+-free buffer is used to measure intracellular accumulation of substrates in the absence of canalicular accumulation. In combination with accumulation data obtained during incubations in Ca2+-containing standard buffer, representing both intracellular and canalicular accumulation of the compound, in vitro biliary excretion can be determined. An excellent correlation was established between in vivo intrinsic biliary clearance and in vitro biliary clearance determined in this model for a series of compounds with varying extents of biliary excretion. Recently, SC rat hepatocytes have been validated and used as an in vitro model to study the biliary excretion of organic anions, including taurocholate (Liu et al., 1999a;Chandra et al., 2001).

In the present study, the utility of SC rat hepatocytes was evaluated to study P-gp-mediated biliary drug excretion. The optimization of this biliary excretion model for P-gp substrates could lead to a promising and relevant alternative approach to study hepatic transport. Two isoforms of the “P-gp” family of transport proteins are expressed at the canalicular membrane of the hepatocyte. The code for one transporter is contained in the ABCB4 gene (previously designated as MDR2/MDR3 in humans andmdr2 in rodents) and has been associated with the expression of a phospholipid flippase (Ruetz and Gros, 1994; Oude Elferink et al., 1997). The functional presence of this P-gp isoform at the canalicular membrane of hepatocytes is required for normal bile secretion. The affinity of ABCB4-encoded P-gp seems to be restricted to phospholipids, although transport of certain drugs, including digoxin and vinblastine was demonstrated recently inABCB4-transfected polarized cell monolayers (Smith et al., 2000). The other P-gp isoform(s) encoded by the ABCB1 gene (mdr1a/b in rats and mice and MDR1 in humans) is responsible for the well known multidrug resistance phenotype in tumor cells. The presence of P-gp at epithelial (e.g., intestine) and endothelial (e.g., blood-brain barrier) cell layers throughout the body suggests an important role for this efflux protein in the formation of a biochemical barrier against exogenous substances. The role of ABCB1 P-gp in the biliary excretion of organic cations has been demonstrated in vivo by comparing biliary excretion of P-gp substrates in normal and ABCB1 knockout mice (Watanabe et al., 1992;Speeg and Maldonado, 1994; Smit et al., 1998a,b; Cvetkovic et al., 1999) and recently has been reviewed (Meijer et al., 1997; Kusuhara et al., 1998). Xenobiotics, including drugs and pesticides, that are substrates for P-gp are structurally diverse, relatively lipophilic, and usually have a cationic moiety.

In the present study, the fluorescent substrate rhodamine 123 (Rh123) was used as a specific probe compound to evaluate the role of P-gp in the biliary excretion process in SC rat hepatocytes. Rh123 is a high-affinity P-gp substrate (apparent Kmof 13.5 μM in membrane vesicles; Shapiro and Ling, 1997). In vivo cumulative biliary excretion of i.v. administered Rh123 was ∼8% of the dose in rats (Sweatman et al., 1990), whereas the intrinsic biliary clearance of intact Rh123 in the isolated perfused rat liver was ∼12 ml/min/kg of body weight (Stapf et al., 1994; Jager et al., 1997). The P-gp substrate digoxin was selected as an additional test compound, since up to 30% of the dose is excreted unchanged in the bile of rats (Hedman and Meijer, 1998) and humans (Hedman et al., 1990).

Experimental Procedures

Animals.

Male Wistar rats (Charles River Laboratories Inc., Raleigh, NC) weighing 220 to 270 g were used for hepatocyte isolation from whole liver. Animals had free access to water and food prior to surgery. All animal procedures were compliant with the guidelines of the Institutional Animal Care and Use Committee (University of North Carolina at Chapel Hill).

Materials.

Collagenase (type 1, class 1) was obtained from Worthington Biochemical Corporation (Freehold, NJ). Dulbecco's modified Eagle's medium (DMEM), Williams' E Medium (WEM), modified Chee's medium (MCM), and insulin were purchased from Invitrogen (Carlsbad, CA). [3H]Digoxin and [3H]taurocholate were obtained from PerkinElmer Life Sciences (Boston, MA). Rh123, taurocholate, dexamethasone, digoxin, bovine serum albumin (BSA), β-glucuronidase (EC 3.2.1.31),p-nitrophenyl-β-d-glucuronide, Triton X-100, 10× DMEM, soybean trypsin inhibitor, and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO). ITS culture supplement (6.25 mg/ml insulin, 6.25 mg/ml transferrin, 6.25 μg/ml selenous acid, 5.35 mg/ml linoleic acid, and 1.25 g/ml BSA) and rat tail collagen (type I) were obtained from Collaborative Biomedical Products (Bedford, MA). GF120918 was kindly provided by Dr. Ron Laethem (Glaxo Wellcome, Research Triangle Park, NC). The reagents for total protein measurement with the bicinchoninic acid method were from Pierce (Rockford, IL). All other chemicals and reagents were of analytical grade and were readily available from commercial sources.

Isolation and In Vitro Culture of Primary Rat Hepatocytes.

Hepatocytes were isolated from male Wistar rats (220–270 g) using a collagenase perfusion, as described previously (Seglen, 1976). Briefly, after the rat was anesthetized (60 mg/kg ketamine + 12 mg/kg xylazine i.p.), the portal vein was cannulated, and the liver was perfused with a Ca+2-free buffer (118.1 mM NaCl, 25 mM NaHCO3, 5.5 mM glucose, 1 mM EGTA, 4.7 mM KCl, and 1.2 mM KH2PO4, pH 7.4) equilibrated with 95% O2/5%CO2 at a rate of 30 ml/min. The inferior vena cava was cannulated to establish a recirculating system (100 ml of Ca+2-free buffer). After 10 min of perfusion, 0.05 to 0.075 g of collagenase and 4 mg of soybean trypsin inhibitor were added to the perfusate reservoir to obtain a final collagenase concentration of ∼200 U/ml; 1 min later, l ml of CaCl2 was added. The liver was perfused for ∼10 min with the collagenase buffer, removed from the rat, and immersed in ice-cold medium (DMEM containing 5% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 4 mg/l insulin, and 1 μM dexamethasone). The capsule surrounding the liver was torn gently to release the hepatocytes. The cells were filtered through a 70-μm mesh filter and then centrifuged (50g) for 2 min at 4°C. The pellet was resuspended in equal parts of medium and isotonic Percoll and centrifuged (70g) for 5 min at 4°C to separate out the nonviable cells. The pellet was resuspended in medium and centrifuged (50g) for 2 min at 4°C. Hepatocytes were counted in a hemocytometer and viability was determined using the trypan blue exclusion method. Viability was always >90% with a yield of 2 to 3 × 108 cells. Cells were resuspended in medium and diluted to a final concentration of 1.0 × 106 cells/ml.

Cell Culture Dish Preparation and Standard Hepatocyte Sandwich-Culture.

Culture dishes were coated with a collagen solution (final concentration, 1.5 mg/ml) prepared by neutralizing a mixture of 4 ml of rat tail type I collagen, 4 ml of deionized water, and 1 ml of 10× DMEM with 1 ml of 0.2 N NaOH solution (final pH ∼7.4). Before plating the hepatocytes, 60-mm Permanox culture dishes (Nalge Nunc International, Rochester, NY) were coated with 0.2 ml of ice-cold neutralized type I collagen solution and placed overnight in a 37°C humidified incubator (Stericult 200; Thermo Forma Scientific, Marietta, OH). Each dish was rinsed with 3.0 ml of warm serum-free DMEM to hydrate the collagen prior to the addition of 3.0 × 106 cells. At 1 to 2 h after plating the cells, unattached cells were removed by replacing the cell plating medium with DMEM containing 5% FBS, 4 mg/l insulin, and 0.1 μM dexamethasone. At 24 h after plating, the medium was aspirated and the cells were overlaid with 200 μl of rat tail collagen type I solution (1.5 mg/ml, pH 7.4) to obtain a “sandwich” configuration. One hour later, FBS-free DMEM (3.0 ml/dish, containing 1% ITS, 50 U/ml penicillin, 50 μg/ml streptomycin, and 0.1 μM dexamethasone) was added onto the cultures. The medium was changed every day with fresh FBS-free DMEM until day 4, when accumulation or efflux experiments were performed.

Accumulation Experiments (Day 4, Unless Stated Otherwise).

For Rh123, digoxin, and taurocholate accumulation studies, cells were rinsed twice with 2.0 ml of warm standard HBSS and incubated in 3.0 ml of the same buffer for 10 min at 37°C. For experiments in which the effect of GF120918 was investigated, cells were preincubated for 60 min with the appropriate concentration of the P-gp modulator (0.5–2.0 μM). Subsequently, cells were incubated in 3.0 ml of 1 μM substrate dissolved either in standard or Ca2+-free HBSS for 1 to 30 min and subsequently rinsed four times with 3.0 ml of ice-cold standard HBSS. For rinsing of [3H]digoxin-treated hepatocytes, 10 μM unlabeled digoxin was added to the rinsing buffer to reduce nonspecific binding. Hepatocytes were lysed with 3.0 ml of 0.5% Triton X-100 solution by placing plates on a rotator for 20 min at room temperature. Cell lysates were analyzed by fluorescence spectroscopy (PerkinElmer LS50B, Norwalk, CT) for Rh123 analysis, and by liquid scintillation spectroscopy in a Packard Tricarb (Meriden, CT) for digoxin and taurocholate. Accumulation was normalized to the protein content of the hepatocytes in each well as measured with the bicinchoninic acid method (Smith et al., 1985) using BSA as standard (0.2–1 mg/ml). All accumulation data were corrected for nonspecific binding (<0.2%) to collagen-coated, hepatocyte-free culture dishes.

Effect of Cell Culture Medium Composition on Rh123 Accumulation in SC Rat Hepatocytes.

Cells were isolated and plated as described above. For experiments examining the effect of cell culture medium composition on Rh123 accumulation and biliary excretion index (BEI), the plating medium was replaced with the appropriate culture medium (based on DMEM, WEM, or MCM) 1 to 2 h after plating. Penicillin (50 U/ml), streptomycin (50 μg/ml), and 0.1 μM dexamethasone were added to all culture media. All culture media were further supplemented either with 1% ITS (DMEM + ITS, MCM + ITS, WEM + ITS) or with 5% FBS and 4 mg/l insulin. When FBS and insulin were used, they were included either for the complete culture period (DMEM + FBS) or only for the initial 24-h culture period and replaced by ITS thereafter (DMEM + FBS24). For the investigation of the effect of dexamethasone concentration in the culture medium on Rh123 BEI, cells were cultured in DMEM + FBS24 with dexamethasone concentrations ranging between 0.01 and 1 μM.

Light Microscopy.

Light microscopy was used to confirm the integrity of the canalicular networks. Light microscopy images were taken with a Nikon TMS light microscope at 200× magnification.

Efflux Experiments (Day 4).

For Rh123 efflux studies, cells were rinsed twice with 2.0 ml of warm standard HBSS and incubated in 3.0 ml of the same buffer for 10 min at 37°C. Cells were then incubated in 3.0 ml of 1 μM Rh123 dissolved in standard HBSS for 30 min and subsequently washed four times with 3.0 ml of ice-cold standard HBSS to remove extracellular Rh123. Hepatocytes then were incubated with either standard or Ca2+-free HBSS for the appropriate incubation time (10–30 min). At the end of the incubation period, a sample was taken from the extracellular medium to determine efflux by measuring Rh123 fluorescence with a LS50B fluorometer. At the end of the efflux experiment, the remainder of the extracellular medium was aspirated and the cells were further processed as described under Accumulation Experiments for determination of protein content. Rh123 efflux was normalized to the protein content of the hepatocytes in each dish.

High-Performance Liquid Chromatography Analysis of Rh123 in Samples from Hepatocyte Accumulation Studies.

Proteins in Triton X-100 samples were first precipitated by adding 1 volume of acetonitrile and centrifuging for 10 min at 10,000 rpm. Fifty microliters of the supernatant was injected onto a 4.6- × 150-mm Zorbax phenyl column (DuPont, Wilmington, DE). Peaks of Rh123 (4.6 min) and rhodamine 110 (Rh110, 1.6 min), a degradation product of Rh123, were detected by a Jasco 920 fluorometer with excitation and emission wavelengths set at 480 and 520 nm, respectively. Since Rh110 concentrations in standards and samples never accounted for >5% of the total area (Rh110 + Rh123), Rh110 concentrations were not calculated. Concentrations of Rh123 in samples were determined by comparing Rh123 peak areas to signals obtained after injection of standards with known concentrations of Rh123 dissolved in a 50:50 mixture of Triton X-100 and acetonitrile.

Treatment of Samples with β-Glucuronidase.

Aliquots of samples from accumulation studies with Rh123 in day 4 SC rat hepatocytes were treated with β-glucuronidase (750 U/ml) in sodium acetate (pH 5) or with sodium acetate solution alone. Concentrations of Rh123 and Rh110 in β-glucuronidase-treated and untreated aliquots were measured by high-performance liquid chromatography analysis and compared to determine the presence of rhodamine glucuronides in the samples. Preliminary experiments withp-nitrophenyl-glucuronide had shown that 0.5% Triton X-100 did not affect β-glucuronidase activity.

P-gp Expression by Western Blot.

Cell culture and harvest

Cells were cultured on 60-mm polystyrene dishes (Costar, Cambridge, MA), as described above. Dexamethasone (0.01, 0.1, 1.0 μM, in DMSO <0.1% of final media volume) treatment was initiated 2-h postseeding and continued until cell harvest on days 2 and 4. Dishes (10–20 dishes/treatment group/day) were harvested on days 0, 2, and 4. Day 0 cells were harvested approximately 6 h postseeding with no dexamethasone treatment. Cell culture media were removed from the plate and cells were rinsed with cold HBSS. Cells were lysed with ice-cold hypotonic lysing buffer containing protease inhibitors (10 μg/ml aprotinin, 10 μg/ml leupeptin). After 10 min, cells were scraped from the dishes into a tight fitting Dounce homogenizer and were homogenized (15 strokes). The homogenate was centrifuged at 1000g for 5 min. The supernatant from the first spin was centrifuged at 30,000g for 30 min. The pellet was carefully removed and stored at −80°C until immunoblot analysis.

Immunoblot analysis.

The pellet was resuspended and protein concentration was determined using the Bio-Rad CD Protein Assay kit. Samples (30–40 μg) were separated by SDS-polyacrylamide gel electrophoresis at 150 V. Proteins were electrotransferred (400 mA for 60 min) to nitrocellulose membrane. The blots were blocked with phosphate-buffered saline containing 20% nonfat dry milk and 0.05% Tween 20 and sequentially incubated with the monoclonal anti-MDR antibody, C219 (1:1500; Fujirebio Diagnostics, Inc., Malvern, PA) overnight at 4°C, and goat anti-mouse IgG conjugated with peroxidase (1:10,000; Invitrogen) for 1 h at room temperature. P-gp was detected using enhanced chemiluminescence (Amersham Pharmacia Biotech, Bukinghamshire, England) and transferred to film followed by densitometric analysis (Alpha Imager 2000).

Data Analysis.

The BEI was determined by dividing the difference in substrate accumulation between standard (cellular plus canalicular accumulation) and Ca+2-free (cellular accumulation) buffer by the accumulation in standard buffer (Liu et al., 1999a). Alternatively, in vitro biliary clearance (Clbile; milliliter per minute per milligram of protein) was calculated according to the following equation:Clbile=AccumulationHBSS−AccumulationCa2+−free HBSSConcentrationincubation medium×Incubation time In vitro biliary clearance values were scaled up using 200 mg of protein/g of liver and 40 g of liver/kg of rat body weight to obtain clearance values in milliliters per minute per kilogram (Seglen, 1976).

All results describing Rh123 or digoxin biliary excretion obtained from the literature were expressed as intrinsic biliary clearance (ClBin) values, as described previously (Liu et al., 1999b), by correcting for rat hepatic plasma flow rate (in vivo studies) or reported flow rate of the perfusion buffer (IPRL studies). Biliary clearance values were normalized per kilogram of rat body weight.

Statistics.

Statistically significant differences in BEI were determined by analysis of variance procedures and unpaired Students' ttest with Bonferroni's correction for multiple comparisons. Statistically significant differences in accumulation or efflux were determined by the unpaired Students' t test. In all cases, the criterion for statistical significance was P < 0.05.

Results

Reduced Nonspecific Binding of Rh123 in Permanox Culture Dishes Compared with Polystyrene.

Nonspecific binding (adsorption) of P-gp substrates such as Rh123 to plastic surfaces could potentially lead to misinterpretation of accumulation data. Therefore, adsorption of Rh123 was measured in collagen-coated, hepatocyte-free culture dishes following incubation in 1 μM Rh123 and subsequent rinsing as performed during typical Rh123 accumulation experiments in SC rat hepatocytes (Table1). Irrespective of the type of culture dish used, nonspecific binding of Rh123 was significantly higher in the absence of Ca2+ during the 30-min incubation period. However, Rh123 adsorption was much higher in tissue culture-treated polystyrene culture dishes than in culture dishes consisting of Permanox plastic.

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Table 1

Nonspecific binding of Rh123 to collagen-coated polystyrene and Permanox culture dishes

Time Course of Rh123 Accumulation and Biliary Excretion in SC Rat Hepatocytes.

To determine the optimal incubation time for determination of in vitro biliary excretion of Rh123, time-dependent Rh123 accumulation was measured. Accumulation of Rh123 in day 4 SC rat hepatocytes was measured in standard buffer (representing intracellular + canalicular amounts of Rh123) and in Ca2+-free buffer (representing intracellular amounts of Rh123). Differences in accumulation measured in standard and Ca2+-free buffer represent biliary excretion as validated previously for taurocholate by Liu et al. (1999a). Figure1 illustrates time-dependent Rh123 accumulation in SC rat hepatocytes incubated in standard or Ca2+-free buffer. The results show that Rh123 undergoes moderate excretion into biliary networks compared with the bile acid taurocholate. Although in vitro biliary excretion of taurocholate is apparent following an incubation time of only 1 min, 30-min incubation times are required to reach intracellular steady-state levels of Rh123 (Ca2+-free condition) and to measure reliably Rh123 biliary excretion. Treatment of samples from Rh123 accumulation studies with β-glucuronidase did not increase concentrations of Rh123 or Rh110, indicating no significant formation of rhodamine glucuronides in SC rat hepatocytes under the current conditions (data not shown). Following a 30-min incubation with 1 μM Rh123, the average BEI (the percentage of accumulated substrate retained in the bile compartment) of Rh123 was 17.0 ± 1.4%, compared with a BEI of 73.5 ± 2.0% for taurocholate following a 10-min incubation. Corresponding average in vitro biliary clearance values for Rh123 and taurocholate were 8.0 ± 1.4 and 96.4 ± 6.3 ml/min/kg, respectively.

Figure 1
Figure 1

Representative time course of Rh123 (diamonds) and taurocholate (squares) accumulation during incubation of day 4 SC rat hepatocytes in standard (solid symbols) or Ca2+-free (open symbols) buffer.

Compounds were added as 1 μM solutions, and data are presented as mean ± S.D. (n = 4). *P< 0.05, **P < 0.01, ***P < 0.001 for accumulation in standard versus Ca2+-free incubation buffer based on unpaired Student's t test. Differences for accumulation of taurocholate were statistically significant at every time point.

Effect of Ca2+ Depletion on Efflux of Rh123 from Preloaded SC Rat Hepatocytes.

The incubation of SC rat hepatocytes in the absence and in the presence of Ca2+ as an approach to measure in vitro biliary excretion was further validated in Rh123 efflux experiments. Figure 2 illustrates time-dependent Rh123 efflux from day 4 SC rat hepatocytes in the presence of standard and Ca2+-free buffer after preloading the hepatocytes with Rh123 for 30 min. Irrespective of time, significantly higher efflux was measured in the presence of Ca2+-free buffer, consistent with additional release of the substrate from canalicular networks when tight junctions were disrupted.

Figure 2
Figure 2

Time course of Rh123 efflux from day 4 SC rat hepatocytes that were preloaded with 1 μM Rh123 for 30 min.

Points represent average (±S.E.M., n = 4) efflux of Rh123 in standard (♦) and Ca2+-free (■) buffer for a representative experiment. **P < 0.01, ***P < 0.001 for efflux in standard versus Ca2+-free incubation buffer based on unpaired Student'st test.

GF120918 Reduces Biliary Excretion of Rh123 and [3H]Digoxin in Day 4 SC Rat Hepatocytes.

To demonstrate that the observed accumulation differences of Rh123 in SC rat hepatocytes (Fig. 1) are related to P-gp-mediated translocation of Rh123 into canalicular networks, the effect of the potent and selective P-gp inhibitor GF120918 on Rh123 biliary excretion was evaluated. Table 2 illustrates that the difference in Rh123 accumulation in the absence and presence of Ca2+, a measure of biliary excretion, was reduced in the presence of 0.5 μM GF120918; this was reflected by a significant decrease in the Rh123 BEI (Fig.3). As illustrated in Table 2, this effect could be attributed completely to increased Rh123 accumulation during the Ca2+-free incubation, consistent with enhanced Rh123 retention in hepatocytes due to inhibition of P-gp function. As expected, 0.5 μM GF120918 did not affect Rh123 accumulation during incubation with standard buffer, since Rh123 accumulation under these conditions represents both intracellular and canalicular presence of Rh123. In the presence of 2 μM GF120918, the difference in accumulation in the absence and presence of Ca2+ was further reduced, resulting in a Rh123 BEI below 5%. The efficiency of GF120918 to block P-gp-mediated canalicular translocation was established further by studying the effect on in vitro biliary excretion of the P-gp substrate digoxin. Table 2 shows that addition of GF120918 to the incubation medium leads to an increased accumulation of digoxin during Ca2+-free incubation, whereas digoxin accumulation in standard buffer is not affected. Thus, the difference in accumulation in the absence and presence of Ca2+ is lower in the presence of 2 μM GF120918, and digoxin BEI is reduced, as illustrated in Fig. 3. The relative decrease in digoxin and Rh123 BEI was comparable at a GF120918 concentration of 2 μM.

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Table 2

Effect of preincubation with GF120918 (60 min) on accumulation of Rh123 and digoxin in SC rat hepatocytes during incubation with HBSS or Ca2+-free HBSS

Figure 3
Figure 3

Effect of preincubation (60 min) with the P-gp modulator GF120918 on the BEI of Rh123 (▪) and digoxin (▨) in SC rat hepatocytes that were maintained in DMEM + FBS24 (see Fig. 4legend) for 4 days.

Bars represent average (±S.E.M.) BEI obtained using hepatocytes from three different livers. *P < 0.05 compared with control based on unpaired Student's t test with Bonferroni's correction for multiple comparisons. The effect of 0.5 μM GF120918 on digoxin BEI was not determined.

Effect of Culture Medium Composition on in Vitro Biliary Excretion of Rh123.

Various cell culture media conditions for SC rat hepatocytes were compared to optimize the system for evaluation of P-gp-mediated biliary excretion. Accumulation of Rh123 (1 μM, 30 min) in SC rat hepatocytes cultured for 4 days using different cell culture media was determined in standard and Ca2+-free buffer. Rh123 accumulation was significantly (P < 0.05) lower in the presence of Ca2+-free buffer compared with standard buffer under all conditions, except for hepatocytes cultured with MCM + ITS (data not shown). Rh123 BEI values, representing Rh123 biliary excretion, were calculated and are reported in Fig. 4. When FBS in DMEM was replaced by ITS in DMEM 24 h after hepatocyte isolation (DMEM + FBS24), a 3-fold higher Rh123 BEI was observed. When hepatocytes were maintained in serum-free medium for the whole culture period (DMEM + ITS), addition of pyruvate to this medium (DMEM2 + ITS) appeared to enhance Rh123 biliary excretion. The Rh123 BEI was low and variable in hepatocytes cultured in MCM, consistent with less extensive in vitro canalicular network development based on light microscopic inspection of the cultures (data not shown).

Figure 4
Figure 4

Effect of cell culture medium composition on in vitro biliary excretion of Rh123 (expressed as BEI) determined after 4 days in culture.

Values represent average BEI (±S.E.M., n = 3–4) obtained with cultures maintained in various medium conditions. *P < 0.05, **P < 0.01 compared with DMEM + FBS based on unpaired Student's ttest with Bonferroni's correction for multiple comparisons. DMEM2, DMEM containing 1.2 mM pyruvate; FBS24, fetal bovine serum used for first 24-h culture period, ITS thereafter; and ITS, which is an FBS replacement for serum-free culture media.

P-gp Function and Expression in SC Rat Hepatocytes Increases with Culture Time, but Is Unaffected by Dexamethasone Concentration.

In addition to the effect of culture medium on Rh123 BEI, the influence of hepatocyte culture time (1–4 days) and dexamethasone (an inducer of P-gp in vivo) on P-gp expression and Rh123 BEI (reflecting P-gp function) were investigated. Western blot analysis indicated that P-gp expression increased with time in culture (0–4 days), consistent with a tendency of Rh123 BEI to increase with culture time (Fig.5, A and C). Changes in the dexamethasone concentration in the culture medium did not appear to affect expression of the P-gp transporter at any time in culture (Fig. 5C). This was in agreement with Rh123 BEI values obtained in hepatocytes that were cultured in the presence of DMEM (using FBS for the first 24 h and ITS thereafter) with varying dexamethasone concentrations (0.01–1 μM; Fig. 5B).

Figure 5
Figure 5

A, effect of culture time on Rh123 BEI in SC rat hepatocytes maintained in DMEM + FBS24 for 4 days.

Data points represent average BEI (±S.E.M.) for two independent experiments with quadruplicate incubations in standard and Ca2+-free medium. B, effect of dexamethasone concentration (0.01, 0.1, 1 μM as indicated at the top of the lanes) during culture of the hepatocytes in DMEM + FBS24 (see Fig. 4 legend) on Rh123 BEI after 4 days. Columns represent average BEI (±S.E.M.) for three independent experiments with quadruplicate incubations in standard and Ca2+-free medium. C, effect of dexamethasone concentration (0.01, 0.1, 1 μM) during culture as well as culture time on P-glycoprotein expression measured by Western blot in SC rat hepatocytes. +, purified canalicular membrane vesicles from rat liver (positive control).

Prediction of in Vivo Biliary Clearance of Rh123 and Digoxin from in Vitro Data.

Using optimized experimental conditions (DMEM + FBS, 0.1 μM dexamethasone, 30-min incubation, day 4), in vitro biliary clearance values for Rh123 and digoxin were determined (Table3). Predicted in vivo biliary clearance values then were calculated according to the relationship between in vitro and in vivo biliary clearance established previously (Liu et al., 1999b). In Table 3, the predicted clearance values for Rh123 and digoxin are compared with intrinsic biliary clearance values that were calculated based on literature data from in vivo and IPRL studies, as described under Experimental Procedures. An excellent agreement was found between the predicted biliary clearance (13.5 ml/min/kg) of digoxin and the value calculated from IPRL data (14.7 ml/min/kg; Hedman and Meijer, 1998). For Rh123, the predicted value (19.9 ml/min/kg) was in excellent agreement with the intrinsic biliary clearance calculated based on in vivo biliary excretion data (18.3 ml/min/kg; Sweatman et al., 1990), but slightly higher than the intrinsic biliary clearance calculated based on the IPRL study (12.3 ml/min/kg; Jager et al., 1997).

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Table 3

Comparison of biliary clearance of Rh123 and digoxin (1 μM) in SC rat hepatocytes (in vitro Clbile) with literature values of biliary clearance measured in vivo in rats and/or in IPRL

Discussion

In the present study, the SC rat hepatocyte model was optimized and validated for assessment of biliary excretion of P-gp substrates. Rh123 was selected as a test compound because of its high affinity for P-gp (Km = 13.5 μM; Shapiro and Ling, 1997), and its fluorescent properties enable rapid analysis with high sensitivity. Digoxin, a P-gp substrate that is excreted unchanged (up to 30% of i.v. dose) in human bile (Caldwell and Cline, 1976; Angelin et al., 1987; Hedman et al., 1990) also was evaluated. The extent of Rh123 biliary excretion was reported to be relatively low in rats, approximating 8% of a 5-mg/kg i.v. bolus dose over a 6-h time period (Sweatman et al., 1990). Therefore, successful evaluation of Rh123 biliary excretion in SC rat hepatocytes allows us to extend the utility of this model to compounds exhibiting low-to-moderate biliary excretion. Issues such as nonspecific binding and high intracellular sequestration, typically observed with P-gp substrates (e.g., daunorubicin; Hayes et al., 1999), required optimization of the model system, which initially had been established to study the biliary excretion of the highly excreted bile salt taurocholate (Liu et al., 1999a; Chandra et al., 2001). Extended incubation times of at least 30 min in SC rat hepatocytes were required to reliably measure differences in Rh123 accumulation during standard and Ca2+-free incubations (Fig. 1). Also, Rh123 adsorption to cell culture plates appeared to be a potentially confounding factor in the interpretation of the accumulation data. Table 1 illustrates that nonspecific Rh123 binding to Permanox culture dishes was negligible, whereas significant adsorption of the dye occurred in culture dishes consisting of polystyrene. The absolute difference in Rh123 adsorption to polystyrene dishes incubated in the presence or in the absence of Ca2+ was significant compared with accumulation values measured during Rh123 accumulation studies. Based on the results of these experiments, Permanox culture dishes were used in subsequent accumulation and efflux experiments.

The results from Rh123 efflux experiments shown in Fig. 2 are consistent with the accumulation data. Irrespective of the efflux time (10–30 min), Rh123 efflux was higher when Ca2+-free incubations were performed compared with incubations in standard buffer. The difference in efflux between the two incubation buffers (∼30 pmol/mg of protein) represents the release of Rh123 from canalicular compartments and is in accordance with differences observed in accumulation experiments over the same incubation time (30 min). The Rh123 efflux experiments confirm the validity of tight junction modulation with Ca2+-free buffer as an approach to release canalicular contents. The efflux data also rule out the possibility that the differences observed during accumulation experiments were due to the effects of Ca2+ on the hepatic uptake of Rh123. The results from these efflux studies, unlike data from the accumulation studies, are complicated by potentially extensive intracellular Rh123 sequestration. Furthermore, efflux data cannot be used readily to determine in vitro biliary clearance of substrates.

The primary role of P-gp as a canalicular translocation mechanism for Rh123 and digoxin across the canalicular membrane of SC rat hepatocytes was demonstrated by evaluating the effect of the potent P-gp inhibitor GF120918 on Rh123 and digoxin disposition (Table 2; Fig. 3). Following a 60-min preincubation with 0.5 μM GF120918, an ∼50% reduction in the Rh123 accumulation difference (and thus BEI) was observed compared with control conditions. Increasing the GF120918 concentration to 2 μM further reduced the Rh123 BEI (Fig. 3) below 5%. The concentration range within which GF120918 exhibits its effect on Rh123 transport is in agreement with recent data from Gigliozzi et al. (2000), reporting inhibition of luminal Rh123 secretion by GF120918 in rat cholangiocytes. When hepatocytes were pretreated with 2 μM GF120918 for 60 min, digoxin BEI was reduced to the same extent (by ∼75%) as Rh123 BEI. Preliminary experiments had shown that Rh123 BEI was reduced by only 50% following a 10-min pretreatment with 10 μM GF120918, whereas the length (10–60 min) of GF120918 pretreatment did not appear to affect digoxin disposition. It is not clear why a longer preincubation with GF120918 is required to completely inhibit Rh123 compared with digoxin transport.

As illustrated in Fig. 4, composition of the cell culture medium has a significant influence on biliary excretion of Rh123 in SC hepatocytes. For hepatocytes cultured in DMEM, the use of FBS throughout the 4-day culture period resulted in a significantly lower, albeit reproducible, Rh123 BEI. The use of serum-free media, either for the complete culture time or for the 3 days following the first 24 h after plating the cells, resulted in Rh123 BEI values that were consistently 2- to 3-fold higher than with hepatocytes cultured in FBS-containing DMEM. An increased stability of P-gp in hamster and human MDR cell lines cultured under reduced-serum medium conditions has been reported previously (Muller et al., 1995). A slight increase in Rh123 BEI also was observed when hepatocytes were maintained in serum-free DMEM containing pyruvate. This observation probably is related to enhanced P-gp function due to increased ATP levels following culture in pyruvate-supplemented medium (Tomita et al., 1995). These results illustrate the importance of maintaining consistency in hepatocyte culture conditions, whereas the data also suggest that the model is flexible enough to study the effects of modulation of P-gp expression on P-gp-mediated biliary excretion.

Dexamethasone (25–100 nM) is used widely as a cell culture medium additive for the maintenance of primary hepatocytes in culture (LeCluyse et al., 1996). However, results from several in vitro studies have demonstrated a significant reduction in P-gp expression and function when primary rat hepatocytes were cultured in the presence of this glucocorticoid (Fardel et al., 1993; Chieli et al., 1994; Schuetz et al., 1995). On the other hand, treatment of animals with dexamethasone was shown recently to increase P-gp expression in various tissues, including the liver (Salphati and Benet, 1998; Demeule et al., 1999). In the present study, varying dexamethasone concentrations between 0.01 and 1 μM during culture of SC rat hepatocytes did not affect significantly Rh123 BEI (Fig. 5B). Consistent with this finding, P-gp expression in SC rat hepatocytes was not dependent on dexamethasone concentration within the same range. Figure 5C also illustrates that P-gp expression increased continuously throughout the 4-day culture period. This finding is in agreement with previous reports demonstrating increased P-gp expression with culture time in hepatocytes (Chieli et al., 1994; Lee et al., 1995). Accordingly, a trend toward increasing Rh123 BEI with culture time also was noted (Fig. 5A).

The ability of the in vitro SC hepatocyte model to predict biliary clearance values for Rh123 and digoxin was evaluated. As illustrated in Table 3, the predicted in vivo biliary clearance values were generally in good agreement with the intrinsic biliary clearance values calculated from literature data. The difference in biliary clearance of Rh123 between the IPRL study (Jager et al., 1997) and the in vivo study (Sweatman et al., 1990) may be attributed to the fact that the data were obtained in different model systems (IPRL versus in vivo) at different Rh123 concentrations (∼0.3 and ∼0.5 μM, respectively). Differences between the predicted and measured Rh123 biliary clearance values may be related to differences in Rh123 metabolism. Formation and biliary excretion of Rh123 metabolites (mainly Rh123 glucuronide) had been reported during IPRL and in vivo experiments with Rh123 in rats (Sweatman et al., 1990; Jager et al., 1997). In contrast, Rh123 glucuronide was not observed in SC rat hepatocytes, as demonstrated by unchanged Rh110 and Rh123 concentrations in samples following treatment with β-glucuronidase compared with mock-incubated samples. The lack of Rh123 metabolism in SC rat hepatocytes may lead to enhanced biliary excretion of intact Rh123 in the in vitro SC hepatocyte model. Interestingly, this hypothesis is consistent with results from the IPRL study (Jager et al., 1997) demonstrating that the intrinsic biliary clearance of intact Rh123 increased from 12.3 to 21.3 ml/min/kg in the presence of genistein, a putative inhibitor of glucuronosyltransferases (responsible for formation of Rh123 glucuronide). Low in vitro expression of glucuronosyltransferases in SC rat hepatocytes may be responsible for the absence of Rh123 glucuronide. On the other hand, the relatively short duration of the in vitro experiments (30 min) compared with the in vivo studies (bile sampling between 1 and 6 h) also may contribute to this observation.

In conclusion, results from these studies illustrate the utility of SC rat hepatocytes to assess P-gp-mediated biliary drug excretion in vitro. Additional studies with other P-gp substrates are required to confirm the ability of the model to predict accurately biliary clearance of P-gp substrates. The absence of rhodamine glucuronide in this in vitro system requires further studies to determine the potential utility of the model to assess simultaneously hepatic drug transport and metabolism. On the other hand, the ability to study hepatic transport without interference of metabolism could be a distinct advantage of this model in its current state.

Acknowledgments

We thank Dr. E. LeCluyse for advice on rat hepatocyte cultures.

Footnotes

  • This study was supported by National Institutes of Health Grant GM41935.

  • Abbreviations used are::
    IPRL
    isolated perfused rat liver
    SC
    sandwich-cultured
    P-gp
    P-glycoprotein
    Rh123
    rhodamine 123
    DMEM
    Dulbecco's modified Eagle's medium
    WEM
    Williams' E medium
    MCM
    modified Chee's medium
    BSA
    bovine serum albumin
    FBS
    fetal bovine serum
    ITS
    insulin-transferrin-selenium supplement
    HBSS
    Hanks' balanced salt solution
    BEI
    biliary excretion index
    MDR
    multidrug resistance
    • Received January 2, 2001.
    • Accepted June 19, 2001.

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P-Glycoprotein-Mediated In Vitro Biliary Excretion in Sandwich-Cultured Rat Hepatocytes

Pieter P. Annaert, Ryan Z. Turncliff, Catherine L. Booth, Dhiren R. Thakker and Kim L. R. Brouwer
Drug Metabolism and Disposition October 1, 2001, 29 (10) 1277-1283;
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