Xenobiotics Inhibit Hepatic Uptake and Biliary Excretion of Taurocholate in Rat Hepatocytes
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《毒物学科学杂志》
Curriculum in Toxicology, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599
ABSTRACT
Reports suggest that troglitazone, and to a lesser extent bosentan, may alter bile acid homeostasis by inhibiting the bile salt export pump. The present studies examined the hypothesis that these xenobiotics may modulate multiple hepatic bile acid transport mechanisms. In suspended rat hepatocytes, troglitazone (10 μM) decreased the initial rate of taurocholate uptake 3-fold; the initial uptake rate of estradiol-17-D-glucuronide, a substrate of the organic anion transporting polypeptides, also was decreased 4-fold. Bosentan (100 μM) decreased the initial uptake rate of taurocholate and estradiol-17-D-glucuronide by 12- and 7-fold, respectively. In sandwich-cultured rat hepatocytes, 10-min accumulation of taurocholate in cells + bile canaliculi (408 ± 57 pmol/mg protein) was decreased significantly by troglitazone (157 ± 17 pmol/mg protein, respectively) only in the presence of Na+, the driving force for the sodium taurocholate cotransporting polypeptide. A similar decrease with 10-fold higher concentrations of bosentan was noted. The biliary excretion index of taurocholate (55 ± 8%) was decreased in the presence of 10 μM troglitazone (27 ± 2%) and 100 μM bosentan (10 ± 6%). In conclusion, xenobiotics may alter hepatic bile acid transport by inhibiting both hepatic uptake and biliary excretion.
Key Words: hepatotoxicity; taurocholate; troglitazone; bosentan; hepatocytes; hepatobiliary transport.
INTRODUCTION
Idiosyncratic hepatotoxicity is defined as toxicity that is observed during drug therapy where no clear mechanism can be determined. In vitro studies have suggested that one mechanism of idiosyncratic hepatotoxicity may be the modulation of hepatobiliary transport mechanisms and subsequent alteration of bile acid homeostasis. For certain drugs, inhibition of canalicular transport may help explain the observed adverse hepatic reactions (Fattinger et al., 2001; Funk et al., 2001a).
The bile salt export pump (Bsep; Abcb11, sister of P-glycoprotein) is the transport protein primarily responsible for biliary excretion of unconjugated bile acids, which are more hepatotoxic than their conjugated metabolites (Byrne et al., 2002). Troglitazone and bosentan inhibited Bsep-mediated taurocholate transport in cell plasma membrane vesicles (Fattinger et al., 2001; Funk et al., 2001b). Although Bsep has been the major focus of transport inhibition as a mechanism of drug-induced hepatotoxicity, other canalicular transport proteins also may be modulated by these xenobiotics. Kostrubsky et al. (2001) reported that the multidrug resistance-associated protein 2 (Mrp2, Abcc2), the transport protein responsible for biliary excretion of conjugated and unconjugated organic anions, including conjugated bile acids, mediated biliary excretion of troglitazone metabolites in rats. Similarly, Fouassier et al. (2002) reported that bosentan may be associated with alterations in canalicular bile formation via modulation of Mrp2. In vivo studies utilizing a radiolabeled tracer dose of taurocholate demonstrated that taurocholate accumulated in liver tissue of rats treated with troglitazone (Funk et al., 2001b). Collectively, these data suggest that inhibition of canalicular transport proteins responsible for biliary excretion of bile acids may be an important pathway leading to intrahepatic cholestasis.
Inhibition of basolateral transport mechanisms responsible for hepatic uptake of bile acids has been addressed only recently. The sodium taurocholate cotransporting polypeptide (Ntcp, Slc10a1) is responsible for the sodium-dependent uptake of bile acids (Trauner and Boyer, 2003). Ntcp is down-regulated in obstructive and hepatocellular cholestasis (Gartung et al., 1996, 1997), alluding to the functional importance of this transport protein in maintaining bile acid homeostasis. The organic anion transporting polypeptide family (Oatp, Slco; formerly Slc21a) is responsible for basolateral uptake of structurally diverse organic anions, cations, and zwitterions, including numerous xenobiotics and endobiotics, such as bile acids, from sinusoidal blood into hepatocytes (Trauner and Boyer, 2003). Hepatic uptake of taurocholate is mediated by both sodium-dependent (Ntcp) and sodium-independent (Oatp) processes; however, the Oatp proteins contribute substantially less than Ntcp to the overall hepatic uptake of bile acids (Kouzuki et al., 2000; Meier et al., 1997). Numerous compounds have been shown to inhibit Oatp isoforms; however; the effect of this inhibition on bile acid homeostasis remains to be fully elucidated (Cattori et al., 2001). Treiber et al. (2004) reported that digoxin, quinidine, and cholecystokinin, inhibitors of the three major rodent hepatic Oatp isoforms, impaired the uptake of bosentan into rat hepatocytes, suggesting that as an Oatp substrate, bosentan may compete with bile acids for Oatp-mediated hepatic uptake. However, given the minor contribution of Oatp to hepatic uptake of bile acids, competitive inhibition of Oatp alone would not be expected to significantly affect the overall bile acid homeostasis. Inhibition of hepatic bile acid uptake may attenuate the elevation of intrahepatic bile acid concentrations that would be expected to occur following impairment of bile acid biliary excretion.
In the present study, freshly isolated suspended rat hepatocytes and sandwich-cultured primary rat hepatocytes were selected to examine the sites of drug-bile acid transport interactions. Suspended hepatocytes are widely used for the study of hepatic uptake because they reliably predict uptake kinetics and mechanisms (Brock and Vore, 1984; Brouwer et al., 1987). Sandwich-cultured hepatocytes currently are the only in vitro cellular model amenable to the study of kinetics and mechanisms of both hepatic uptake and biliary excretion (Zamek-Gliszczynski and Brouwer, 2004). Over time in culture, sandwich-cultured hepatocytes reestablish cell polarity and tight junctional complexes, leading to the formation of sealed bile canaliculi (Liu et al., 1999b). Modulation of the re-formed tight junctions with Ca2+ allows quantification of compound accumulation in cells + bile canaliculi (presence of Ca2+) and cells only (absence of Ca2+), which can be used to calculate biliary clearance and the biliary excretion index (i.e., the fraction of the accumulated substrate that is excreted into bile) (Liu et al., 1999b). Sandwich-cultured rat hepatocytes express and maintain functional Oatp, Ntcp, Bsep, and Mrp2, and reestablish appropriate localization of transport proteins on the basolateral and apical plasma membrane domains, resulting in vectorial transport of taurocholate from extracellular medium into the re-formed bile canaliculi (Hoffmaster et al., 2004; Liu et al., 1999b). Sandwich-cultured hepatocytes have been used successfully to study hepatobiliary transport kinetics and mechanisms, including detailed examination of transport processes responsible for hepatic uptake and subsequent biliary excretion (Annaert et al., 2001; Liu et al., 1998, 1999a; Zamek-Gliszczynski et al., 2003). The present studies examined the effect of troglitazone and bosentan on the hepatic uptake and biliary excretion of taurocholate, using freshly isolated suspended rat hepatocytes and sandwich-cultured primary rat hepatocytes.
MATERIALS AND METHODS
Chemicals. Troglitazone was purchased from Biomol (Plymouth Meeting, PA). Bosentan was kindly provided by Dr. Alexander Treiber (Actelion Pharmaceuticals Ltd, Switzerland). Taurocholate, estradiol-17-D-glucuronide, and choline were purchased from Sigma (St. Louis, MO). 3H-Estradiol-17-D-glucuronide (45 Ci/mmol) and 3H-taurocholate (2 Ci/mmol) were obtained from Perkin Elmer (Boston, MA), and 14C-inulin (37–111 MBq/g) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Collagenase (type I, class I) was obtained from Worthington Biochemical (Freehold, NJ). Dulbecco's modified Eagle's medium, fetal bovine serum, and insulin were purchased from GIBCO (Grand Island, NY). Rat tail collagen (type I) and ITS (insulin, transferrin, selenium) Premix were obtained from BD Biosciences (Palo Alto, CA). All other chemicals and reagents were of analytical grade and were readily available from commercial sources.
Animals. Male Wistar rats (250–300 g) from Charles River (Raleigh, NC) were used as liver donors. They were housed on a 12-h light and dark cycle with free access to food and water in the temperature and humidity controlled DLAM facility in the School of Pharmacy. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of North Carolina.
Isolation of rat hepatocytes. Rat hepatocytes were isolated with a two-step perfusion method as described by Liu et al. (1998) and modified by Annaert et al. (2001). Briefly, rats were anesthetized with ketamine and xylazine (60 and 12 mg/kg ip, respectively) prior to portal vein cannulation. The liver was perfused in situ with oxygenated Ca2+-free Krebs-Henseleit buffer for 10 min at 37°C, followed by the addition of collagenase type I (0.5 mg/ml) and CaCl2 (0.75 μmol) and perfusion for an additional 10 min. Hepatocytes were released into 100 ml of cold Dulbecco's modified Eagle's medium and were filtered through a sterile nylon mesh (70 microns). Cell suspensions were centrifuged (50 x g, 5 min); the cell pellet was resuspended in medium and an equal volume of 90% isotonic Percoll (pH 7.4) and was centrifuged (100 x g, 6 min); the resulting pellet was resuspended in medium and was centrifuged (50 x g, 2.5 min). The final cell pellet was resuspended in medium or Hank's buffer (1 x 106 hepatocytes/ml). Hepatocyte viability was determined by Trypan Blue exclusion. Only hepatocyte preparations with viability greater than 88% were used for further studies.
Accumulation in suspended hepatocytes. Hepatocytes (1 x 106/ml) were stored (0–2 h) on ice in Hank's buffer modified with 10 mM Tris and 5 mM glucose. Hepatocyte suspensions (4 ml) were incubated in bottom-inverted flasks with either 1 μM taurocholate or 1 μM estradiol-17-D-glucuronide in the absence or presence of 10 μM troglitazone or 100 μM bosentan. Incubation mixtures were sampled every 15 sec by removing an aliquot of incubation mixture and centrifuging the cells through a silicone oil/mineral oil (82:18, v:v) layer into potassium hydroxide (3 M) at the bottom of the microfuge tube. Radioactivity in the supernatant and in the cell pellet was quantified. The adherent fluid volume was determined by incubation of cells with 14C-inulin. Protein and substrate concentrations in the incubation mixture were quantified at the end of the experiment.
Culture of rat hepatocytes. Hepatocyte suspensions (1 x 106 hepatocytes/ml) were prepared in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 0.1 μM dexamethasone, and 4 mg/l insulin. Hepatocyte suspensions were seeded on collagen precoated dishes (0.2 ml, 1.5 mg/ml) at a density of 3 x 106 cells/60-mm dish. Approximately 2–3 h after seeding the cells, the medium was replaced. Twenty-four h after seeding, hepatocyte monolayers were overlaid with a neutralized collagen solution (0.2 ml, 1.5 mg/ml). Collagen-overlaid cultures were incubated for 1.5 h at 37°C in a humidified incubator to allow the collagen to gel before the addition of Dulbecco's modified Eagle's medium containing 1% ITS and 0.1 μM dexamethasone. Thereafter, medium was changed daily. Functional studies were conducted 4 days after seeding.
Accumulation in sandwich-cultured rat hepatocytes. Hepatocytes cultured between two layers of gelled collagen were preincubated with 3 ml of standard Hank's balanced salt solution or Ca2+-free Hank's balanced salt solution containing troglitazone (0.1–10 μM), bosentan (1–100 μM), or vehicle at 37°C for 10 min. After aspiration of the preincubation buffer, taurocholate uptake was initiated by the addition of 3 ml standard Hank's balanced salt solution containing taurocholate (10 μM) and xenobiotic or vehicle. Taurocholate accumulation in the absence of Na+ was measured by replacing Na+ by choline in the incubation buffer. Taurocholate, troglitazone, or bosentan concentrations were selected based on previous literature data (Brock and Vore, 1984; Fattinger et al., 2001; Funk et al., 2001b; Liu et al., 1999b). After incubation of hepatocytes with taurocholate and xenobiotic for 0.5, 1, 2, 5, or 10 min, taurocholate uptake was stopped by rinsing the dishes with ice-cold standard buffer (3 x 3 ml). Cells were lysed with 2 ml of 0.5% Triton X-100. Radioactivity and protein content in the lysate were quantified. Substrate accumulation was corrected for nonspecific binding to the collagen by subtracting taurocholate accumulation in collagen precoated dishes without cells.
Sample analysis. 3H-Taurocholate, 3H-estradiol--D-glucuronide, and 14C-inulin were quantified by liquid scintillation spectroscopy using a Packard Minaxi TriCarb scintillation counter (Meriden, CT). Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL) using bovine serum albumin standards (0.2–2 mg/ml).
Data analysis. Pharmacokinetic model development indicated that the model presented in Figure 1A best described the accumulation data of taurocholate in sandwich-cultured hepatocytes. Differential equations based on the model scheme presented in Figure 1A were resolved simultaneously by nonlinear least-squares regression (WinNonlin 3.2, Pharsight Corporation, Mountain View, CA):
(1)
(2)
where Xcell is the amount of taurocholate taken up by the cells, Xbile is the amount excreted into the bile canaliculi, and Xo is the amount of taurocholate added to the incubation medium (30,000 pmol); k12, k21, and k23 represent first-order rate constants for hepatocyte uptake, basolateral excretion, and biliary excretion of taurocholate, respectively. All data sets were fit simultaneously with the same initial parameter estimates using 1/Y weighting.
Since the data set was not sufficient to simultaneously estimate both uptake and basolateral excretion (i.e., in the presence of xenobiotics the model often converged at an uptake rate constant similar to control with a greatly increased basolateral excretion rate constant) the basolateral excretion rate constant (k21) was fixed at the average estimate from individual control data sets (0.98 ± 0.24 min–1), while uptake and biliary excretion rate constants were estimated. Physiologically, basolateral excretion from sandwich-cultured rat hepatocytes may be attributed to the up-regulation of Mrp3 (Abcc3), the basolateral efflux transporter for taurocholate (Hirohashi et al., 2000), over time in culture (Zhang et al., 2001). The assumption of a treatment-independent basolateral excretion rate constant was validated by measurement of basolateral excretion of taurocholate from taurocholate preloaded sandwich-cultured hepatocytes with intact bile canaliculi; the basolateral excretion rate of taurocholate was not altered by troglitazone or bosentan (data not shown).
The fraction of the accumulated compound that resides in bile canaliculi was quantified with the biliary excretion index (BEI), calculated using B-CLEARTM technology (Qualyst, Inc., Research Triangle Park, NC) based on the following equation (Liu et al., 1999b):
(3)
The biliary excretion index was calculated using 10-min accumulation data from four different rat liver preparations. A sigmoidal inhibition model was fit to the biliary excretion index–xenobiotic concentration data in order to estimate an apparent IC50 of troglitazone and bosentan for inhibition of taurocholate biliary excretion using the following equation:
(4)
where C represents the xenobiotic concentration, IC50 is the concentration of xenobiotic producing 50% of maximal inhibition, and is the Hill coefficient.
Statistics. Data were analyzed by analysis of variance (ANOVA) with Tukey's or Fisher's post hoc multiple comparison test. All statistical tests were performed with SigmaStat (Chicago, IL). All data are reported as mean ± SEM from three or four different rat liver preparations, each measurement performed in triplicate in each preparation.
RESULTS
Suspended Rat Hepatocytes
In suspended rat hepatocytes, 10 μM troglitazone significantly decreased the accumulation of 1 μM taurocholate by 3-fold (Fig. 2A); taurocholate accumulation in the presence of troglitazone was significantly lower relative to control at all time points. The initial uptake rate of taurocholate was significantly decreased from 0.88 ± 0.17 to 0.28 ± 0.15 pmol/sec/mg protein by troglitazone. Estradiol-17-D-glucuronide was utilized to assess the effect of troglitazone on Oatp-mediated hepatic uptake. Troglitazone (10 μM) decreased the initial uptake rate of 1 μM estradiol-17-D-glucuronide from 0.87 ± 0.23 to 0.20 ± 0.12 pmol/sec/mg protein (p = 0.06; Fig. 2B).
In suspended rat hepatocytes, 100 μM bosentan significantly decreased the initial uptake rate of taurocholate from 0.88 ± 0.17 to 0.07 ± 0.05 pmol/sec/mg protein (Fig. 3A). Bosentan also significantly decreased the initial uptake rate of 1 μM estradiol-17-D-glucuronide from 0.87 ± 0.23 to 0.13 ± 0.04 pmol/sec/mg protein (p < 0.05, Fig. 3B).
Sandwich-Cultured Rat Hepatocytes
To determine whether impaired taurocholate uptake was due to inhibition of Ntcp- or Oatp-mediated transport, accumulation of taurocholate at 10 min in sandwich-cultured rat hepatocytes was assessed in the presence or absence of Na+ (Tables 1 and 2). Taurocholate accumulation in the absence of Na+ was significantly lower than in the presence of Na+ at lower xenobiotic concentrations. Higher xenobiotic concentrations significantly decreased taurocholate accumulation in the presence of Na+ to values observed in the absence of Na+. Xenobiotics appeared to slightly impair Na+-independent accumulation, but these differences failed to reach statistical significance.
Troglitazone (10 μM) significantly decreased taurocholate accumulation at 10 min in cells + bile canaliculi from 408 ± 57 to 157 ± 17 pmol/mg protein (Fig. 4A). Accumulation of taurocholate in cells + bile canaliculi in the presence of 10 μM troglitazone was significantly lower than in control sandwich-cultured rat hepatocytes at all time points. At high concentrations of troglitazone, taurocholate accumulation in cells + bile canaliculi was comparable to the cellular accumulation of taurocholate in control sandwich-cultured rat hepatocytes with disrupted bile canaliculi (Fig. 4B). Cellular 10-min accumulation of taurocholate in sandwich-cultured rat hepatocytes with disrupted bile canaliculi was decreased by 10 μM troglitazone (116 ± 15 vs. 172 ± 27 pmol/mg protein), but the difference was not statistically significant. Troglitazone (5 μM) significantly decreased the biliary excretion index of taurocholate (Fig. 4C).
Bosentan (100 μM) significantly decreased taurocholate accumulation at 10 min in cells + bile canaliculi from control values of 408 ± 57 to 154 ± 28 pmol/mg protein (Fig. 5A). Accumulation of taurocholate in cells + bile canaliculi in the presence of 100 μM bosentan was significantly lower than in control sandwich-cultured rat hepatocytes at all time points except 0.5 min. Taurocholate accumulation in cells + bile canaliculi at higher concentrations of bosentan was comparable to the cellular accumulation of taurocholate in control sandwich-cultured rat hepatocytes with disrupted bile canaliculi (Fig. 5B). Bosentan did not significantly decrease the cellular accumulation of taurocholate in sandwich-cultured rat hepatocytes with disrupted bile canaliculi (Fig. 5B). Bosentan at the two highest concentrations (50 and 100 μM) significantly decreased the biliary excretion index of taurocholate (Fig. 5C).
Pharmacokinetic Modeling of Sandwich-Cultured Rat Hepatocyte Data
Representative fits of Equations (1) and (2) to taurocholate accumulation-time data in sandwich-cultured rat hepatocytes in the presence (cells + bile canaliculi) or absence (cells) of Ca2+ are shown in Figure 1B. Troglitazone (10 μM) significantly decreased the rate constant governing taurocholate uptake (from 0.0079 ± 0.0007 to 0.0047 ± 0.0009 min–1) and the rate constant governing biliary excretion (from 0.29 ± 0.07 to 0.10 ± 0.03 min–1). Likewise, bosentan (100 μM) significantly decreased the rate constant for taurocholate uptake (from 0.0079 ± 0.0007 to 0.0040 ± 0.0007 min–1) and the rate constant for biliary excretion (from 0.29 ± 0.07 to 0.07 ± 0.03 min–1).
A sigmoidal inhibition model was fit to the taurocholate biliary excretion index–xenobiotic concentration data to estimate the IC50 of xenobiotics for biliary excretion of taurocholate. Troglitazone (IC50 = 0.91 ± 0.12 μM) was a much more potent inhibitor of taurocholate biliary excretion than bosentan (IC50 = 31 ± 11 μM).
DISCUSSION
Numerous xenobiotics impair the biliary excretion of taurocholate, including troglitazone and bosentan (Fattinger et al., 2001; Funk et al., 2001a,b). Inhibition of Bsep has been suggested to be a potential mechanism of adverse hepatic reactions associated with troglitazone. The current work expands on the known bile acid transport modulation associated with these xenobiotics by demonstrating that these xenobiotics also may inhibit the hepatic uptake of bile acids in addition to biliary excretion in primary rat hepatocytes.
The concentrations of troglitazone and bosentan employed in this study are within the range of concentrations used in preceding in vitro studies, and are below the lowest observable effect level for toxicity in sandwich-cultured rat hepatocytes (Kemp and Brouwer, 2004). While troglitazone concentrations used in this and other in vitro studies are at or slightly above pharmacologically-relevant concentrations (0.7 μM), the inhibitory effects of bosentan were observed at concentrations 20- to 50-fold higher than total plasma concentrations (CMAX 2 μM) reported in man. Actual unbound plasma concentrations of bosentan are much lower than 2 μM due to extensive protein binding of this drug (Channick et al., 2001; Parke-Davis, 1999). At clinically relevant concentrations of bosentan, no changes in hepatic bile acid disposition were noted.
Suspended hepatocytes were employed to examine the effect of troglitazone and bosentan on the initial uptake rate of taurocholate. Both xenobiotics significantly reduced the cellular accumulation and the initial uptake rate of taurocholate, indicating that these compounds impair taurocholate uptake, which is mediated by both Ntcp and Oatp.
To determine whether these xenobiotics modulate Oatp, uptake of estradiol-17-D-glucuronide was studied. Bosentan decreased the initial uptake rate of estradiol-17-D-glucuronide. Treiber et al. (2004) demonstrated that hepatic uptake of bosentan is mediated by all three hepatic Oatp isoforms. Thus, as demonstrated here, bosentan can compete for uptake with other Oatp substrates. Impaired hepatic uptake of estradiol-17-D-glucuronide in the presence of troglitazone expands on the finding of Nozawa et al. (2004), who demonstrated that the sulfate metabolite of troglitazone inhibits human OATP-C. While competitive inhibition of Oatp-mediated bile acid uptake by these xenobiotics is an explanation consistent with previous literature reports (Nozawa et al., 2004; Treiber et al., 2004), other mechanisms such as alterations in membrane bioenergetics, driving force gradients, and transporter trafficking cannot be ruled out. These data indicate that xenobiotics known to inhibit the biliary excretion of bile acids also may impair hepatic uptake of bile acids, thus attenuating the elevation of intrahepatic bile acid concentrations.
Taurocholate accumulation also was studied in sandwich-cultured rat hepatocytes in the absence or presence of Na+. In the absence of Na+, which is the driving force for Ntcp, Oatp function was maintained, while Ntcp-mediated uptake was negligible. Even though Ntcp is responsible for the majority of taurocholate taken up in sandwich-cultured rat hepatocytes, the amount of taurocholate taken up by Oatp can be quantified (Liu et al., 1998). Both troglitazone and bosentan slightly decreased taurocholate accumulation in the absence of Na+. In contrast, taurocholate accumulation in the presence of Na+ was significantly decreased in a concentration-dependent manner by these xenobiotics. At higher concentrations, troglitazone and bosentan decreased taurocholate accumulation in the presence of Na+ to values observed in the absence of Na+, suggesting nearly complete inhibition of Ntcp. These data suggest that troglitazone and bosentan impair taurocholate uptake primarily by inhibition of Ntcp, which may be a hepato-protective mechanism secondary to the inhibition of Bsep.
In sandwich-cultured rat hepatocytes, decreased taurocholate accumulation in cells + bile canaliculi, combined with a decrease in the biliary excretion index of taurocholate in the presence of troglitazone or bosentan, suggested that both hepatic uptake and biliary excretion were inhibited. In contrast, in sandwich-cultured rat hepatocytes with disrupted bile canaliculi, inhibition of uptake appeared to be offset by the inhibition of biliary excretion such that the extent of change in cellular accumulation of taurocholate was not as great as that in cells + bile canaliculi. Accumulation of taurocholate in sandwich-cultured rat hepatocytes in the presence of xenobiotics demonstrated that inhibition of both taurocholate uptake and biliary excretion helps maintain intrahepatic bile acid homeostasis.
Pharmacokinetic modeling was consistent with the hypothesized inhibition of both hepatic uptake and biliary excretion of taurocholate by troglitazone and bosentan in sandwich-cultured hepatocytes. Both xenobiotics decreased the rate constant for taurocholate biliary excretion, consistent with a decrease in the taurocholate biliary excretion index. In addition, troglitazone and bosentan decreased the rate constant for hepatic uptake of taurocholate.
The apparent IC50 value for the troglitazone-associated decrease in the taurocholate biliary excretion index was 0.91 ± 0.12 μM, which is comparable to the IC50 (3.9 ± 0.6 μM) and Ki (1.3 μM) values estimated in canalicular liver plasma membrane vesicles for the ATP-dependent component of taurocholate uptake (Funk et al., 2001b). The IC50 values determined in sandwich-cultured rat hepatocytes were estimated relative to the extracellular concentration of troglitazone. The intracellular concentration of troglitazone at the site of biliary excretion is likely to be higher than in the extracellular medium, but since only the extracellular concentration is known, the IC50 value may represent an underestimate. In contrast to intact hepatocytes, the incubation medium in vesicles is in direct contact with the canalicular membrane, which may explain the 4-fold higher IC50 estimate in membrane vesicles compared to sandwich-cultured rat hepatocytes.
Bosentan (IC50 = 31 ± 11 μM) was a much less potent inhibitor of taurocholate biliary excretion. Previously, it was reported that 50 μM bosentan inhibited ATP-dependent taurocholate transport by 82% and 55% in canalicular liver plasma membrane vesicles and Bsep-expressing Sf9 cell plasma membrane vesicles, respectively (Fattinger et al., 2001). A Ki value (12 μM) for bosentan inhibition of the ATP-dependent uptake of taurocholate was calculated previously from data obtained in Bsep-expressing Sf9 cell plasma membrane vesicles (Fattinger et al., 2001). The IC50 value estimated for the decrease in taurocholate biliary excretion index in the presence of bosentan in sandwich-cultured rat hepatocytes is in good agreement with previous kinetic studies of this process, considering that the IC50 was estimated with extracellular xenobiotic concentrations.
In conclusion, troglitazone and bosentan modulate transport processes at both the basolateral and canalicular plasma membranes of hepatocytes. In agreement with previous work, biliary excretion of taurocholate was impaired by both xenobiotics. Both xenobiotics impaired hepatic uptake of taurocholate, primarily due to inhibition of Ntcp. Inhibition of hepatic uptake of bile acids may be a hepato-protective mechanism and may help maintain intrahepatic bile acid homeostasis.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Alexander Treiber (Actelion Pharmaceuticals Ltd, Switzerland) for providing bosentan, and for his insightful comments. In addition, the authors are grateful to Dr. Mary Vore (University of Kentucky) for her help with preparation of this manuscript.
This work was supported by grants R21 CA106101, R01 GM41935, and a Toxicology Training Grant 5-T32-ESO7126 from the National Institutes of Health. Daniel C. Kemp was supported by minority grant GM531674 from the National Institutes of Health. Maciej J. Zamek-Gliszczynski was supported by an Eli Lilly and Company Foundation Predoctoral Fellowship Award in Pharmacokinetics and Drug Disposition. This work was submitted to the Graduate School of the University of North Carolina in partial fulfillment of requirements for the Doctor of Philosophy degree in Toxicology.
NOTES
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ABSTRACT
Reports suggest that troglitazone, and to a lesser extent bosentan, may alter bile acid homeostasis by inhibiting the bile salt export pump. The present studies examined the hypothesis that these xenobiotics may modulate multiple hepatic bile acid transport mechanisms. In suspended rat hepatocytes, troglitazone (10 μM) decreased the initial rate of taurocholate uptake 3-fold; the initial uptake rate of estradiol-17-D-glucuronide, a substrate of the organic anion transporting polypeptides, also was decreased 4-fold. Bosentan (100 μM) decreased the initial uptake rate of taurocholate and estradiol-17-D-glucuronide by 12- and 7-fold, respectively. In sandwich-cultured rat hepatocytes, 10-min accumulation of taurocholate in cells + bile canaliculi (408 ± 57 pmol/mg protein) was decreased significantly by troglitazone (157 ± 17 pmol/mg protein, respectively) only in the presence of Na+, the driving force for the sodium taurocholate cotransporting polypeptide. A similar decrease with 10-fold higher concentrations of bosentan was noted. The biliary excretion index of taurocholate (55 ± 8%) was decreased in the presence of 10 μM troglitazone (27 ± 2%) and 100 μM bosentan (10 ± 6%). In conclusion, xenobiotics may alter hepatic bile acid transport by inhibiting both hepatic uptake and biliary excretion.
Key Words: hepatotoxicity; taurocholate; troglitazone; bosentan; hepatocytes; hepatobiliary transport.
INTRODUCTION
Idiosyncratic hepatotoxicity is defined as toxicity that is observed during drug therapy where no clear mechanism can be determined. In vitro studies have suggested that one mechanism of idiosyncratic hepatotoxicity may be the modulation of hepatobiliary transport mechanisms and subsequent alteration of bile acid homeostasis. For certain drugs, inhibition of canalicular transport may help explain the observed adverse hepatic reactions (Fattinger et al., 2001; Funk et al., 2001a).
The bile salt export pump (Bsep; Abcb11, sister of P-glycoprotein) is the transport protein primarily responsible for biliary excretion of unconjugated bile acids, which are more hepatotoxic than their conjugated metabolites (Byrne et al., 2002). Troglitazone and bosentan inhibited Bsep-mediated taurocholate transport in cell plasma membrane vesicles (Fattinger et al., 2001; Funk et al., 2001b). Although Bsep has been the major focus of transport inhibition as a mechanism of drug-induced hepatotoxicity, other canalicular transport proteins also may be modulated by these xenobiotics. Kostrubsky et al. (2001) reported that the multidrug resistance-associated protein 2 (Mrp2, Abcc2), the transport protein responsible for biliary excretion of conjugated and unconjugated organic anions, including conjugated bile acids, mediated biliary excretion of troglitazone metabolites in rats. Similarly, Fouassier et al. (2002) reported that bosentan may be associated with alterations in canalicular bile formation via modulation of Mrp2. In vivo studies utilizing a radiolabeled tracer dose of taurocholate demonstrated that taurocholate accumulated in liver tissue of rats treated with troglitazone (Funk et al., 2001b). Collectively, these data suggest that inhibition of canalicular transport proteins responsible for biliary excretion of bile acids may be an important pathway leading to intrahepatic cholestasis.
Inhibition of basolateral transport mechanisms responsible for hepatic uptake of bile acids has been addressed only recently. The sodium taurocholate cotransporting polypeptide (Ntcp, Slc10a1) is responsible for the sodium-dependent uptake of bile acids (Trauner and Boyer, 2003). Ntcp is down-regulated in obstructive and hepatocellular cholestasis (Gartung et al., 1996, 1997), alluding to the functional importance of this transport protein in maintaining bile acid homeostasis. The organic anion transporting polypeptide family (Oatp, Slco; formerly Slc21a) is responsible for basolateral uptake of structurally diverse organic anions, cations, and zwitterions, including numerous xenobiotics and endobiotics, such as bile acids, from sinusoidal blood into hepatocytes (Trauner and Boyer, 2003). Hepatic uptake of taurocholate is mediated by both sodium-dependent (Ntcp) and sodium-independent (Oatp) processes; however, the Oatp proteins contribute substantially less than Ntcp to the overall hepatic uptake of bile acids (Kouzuki et al., 2000; Meier et al., 1997). Numerous compounds have been shown to inhibit Oatp isoforms; however; the effect of this inhibition on bile acid homeostasis remains to be fully elucidated (Cattori et al., 2001). Treiber et al. (2004) reported that digoxin, quinidine, and cholecystokinin, inhibitors of the three major rodent hepatic Oatp isoforms, impaired the uptake of bosentan into rat hepatocytes, suggesting that as an Oatp substrate, bosentan may compete with bile acids for Oatp-mediated hepatic uptake. However, given the minor contribution of Oatp to hepatic uptake of bile acids, competitive inhibition of Oatp alone would not be expected to significantly affect the overall bile acid homeostasis. Inhibition of hepatic bile acid uptake may attenuate the elevation of intrahepatic bile acid concentrations that would be expected to occur following impairment of bile acid biliary excretion.
In the present study, freshly isolated suspended rat hepatocytes and sandwich-cultured primary rat hepatocytes were selected to examine the sites of drug-bile acid transport interactions. Suspended hepatocytes are widely used for the study of hepatic uptake because they reliably predict uptake kinetics and mechanisms (Brock and Vore, 1984; Brouwer et al., 1987). Sandwich-cultured hepatocytes currently are the only in vitro cellular model amenable to the study of kinetics and mechanisms of both hepatic uptake and biliary excretion (Zamek-Gliszczynski and Brouwer, 2004). Over time in culture, sandwich-cultured hepatocytes reestablish cell polarity and tight junctional complexes, leading to the formation of sealed bile canaliculi (Liu et al., 1999b). Modulation of the re-formed tight junctions with Ca2+ allows quantification of compound accumulation in cells + bile canaliculi (presence of Ca2+) and cells only (absence of Ca2+), which can be used to calculate biliary clearance and the biliary excretion index (i.e., the fraction of the accumulated substrate that is excreted into bile) (Liu et al., 1999b). Sandwich-cultured rat hepatocytes express and maintain functional Oatp, Ntcp, Bsep, and Mrp2, and reestablish appropriate localization of transport proteins on the basolateral and apical plasma membrane domains, resulting in vectorial transport of taurocholate from extracellular medium into the re-formed bile canaliculi (Hoffmaster et al., 2004; Liu et al., 1999b). Sandwich-cultured hepatocytes have been used successfully to study hepatobiliary transport kinetics and mechanisms, including detailed examination of transport processes responsible for hepatic uptake and subsequent biliary excretion (Annaert et al., 2001; Liu et al., 1998, 1999a; Zamek-Gliszczynski et al., 2003). The present studies examined the effect of troglitazone and bosentan on the hepatic uptake and biliary excretion of taurocholate, using freshly isolated suspended rat hepatocytes and sandwich-cultured primary rat hepatocytes.
MATERIALS AND METHODS
Chemicals. Troglitazone was purchased from Biomol (Plymouth Meeting, PA). Bosentan was kindly provided by Dr. Alexander Treiber (Actelion Pharmaceuticals Ltd, Switzerland). Taurocholate, estradiol-17-D-glucuronide, and choline were purchased from Sigma (St. Louis, MO). 3H-Estradiol-17-D-glucuronide (45 Ci/mmol) and 3H-taurocholate (2 Ci/mmol) were obtained from Perkin Elmer (Boston, MA), and 14C-inulin (37–111 MBq/g) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Collagenase (type I, class I) was obtained from Worthington Biochemical (Freehold, NJ). Dulbecco's modified Eagle's medium, fetal bovine serum, and insulin were purchased from GIBCO (Grand Island, NY). Rat tail collagen (type I) and ITS (insulin, transferrin, selenium) Premix were obtained from BD Biosciences (Palo Alto, CA). All other chemicals and reagents were of analytical grade and were readily available from commercial sources.
Animals. Male Wistar rats (250–300 g) from Charles River (Raleigh, NC) were used as liver donors. They were housed on a 12-h light and dark cycle with free access to food and water in the temperature and humidity controlled DLAM facility in the School of Pharmacy. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of North Carolina.
Isolation of rat hepatocytes. Rat hepatocytes were isolated with a two-step perfusion method as described by Liu et al. (1998) and modified by Annaert et al. (2001). Briefly, rats were anesthetized with ketamine and xylazine (60 and 12 mg/kg ip, respectively) prior to portal vein cannulation. The liver was perfused in situ with oxygenated Ca2+-free Krebs-Henseleit buffer for 10 min at 37°C, followed by the addition of collagenase type I (0.5 mg/ml) and CaCl2 (0.75 μmol) and perfusion for an additional 10 min. Hepatocytes were released into 100 ml of cold Dulbecco's modified Eagle's medium and were filtered through a sterile nylon mesh (70 microns). Cell suspensions were centrifuged (50 x g, 5 min); the cell pellet was resuspended in medium and an equal volume of 90% isotonic Percoll (pH 7.4) and was centrifuged (100 x g, 6 min); the resulting pellet was resuspended in medium and was centrifuged (50 x g, 2.5 min). The final cell pellet was resuspended in medium or Hank's buffer (1 x 106 hepatocytes/ml). Hepatocyte viability was determined by Trypan Blue exclusion. Only hepatocyte preparations with viability greater than 88% were used for further studies.
Accumulation in suspended hepatocytes. Hepatocytes (1 x 106/ml) were stored (0–2 h) on ice in Hank's buffer modified with 10 mM Tris and 5 mM glucose. Hepatocyte suspensions (4 ml) were incubated in bottom-inverted flasks with either 1 μM taurocholate or 1 μM estradiol-17-D-glucuronide in the absence or presence of 10 μM troglitazone or 100 μM bosentan. Incubation mixtures were sampled every 15 sec by removing an aliquot of incubation mixture and centrifuging the cells through a silicone oil/mineral oil (82:18, v:v) layer into potassium hydroxide (3 M) at the bottom of the microfuge tube. Radioactivity in the supernatant and in the cell pellet was quantified. The adherent fluid volume was determined by incubation of cells with 14C-inulin. Protein and substrate concentrations in the incubation mixture were quantified at the end of the experiment.
Culture of rat hepatocytes. Hepatocyte suspensions (1 x 106 hepatocytes/ml) were prepared in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 0.1 μM dexamethasone, and 4 mg/l insulin. Hepatocyte suspensions were seeded on collagen precoated dishes (0.2 ml, 1.5 mg/ml) at a density of 3 x 106 cells/60-mm dish. Approximately 2–3 h after seeding the cells, the medium was replaced. Twenty-four h after seeding, hepatocyte monolayers were overlaid with a neutralized collagen solution (0.2 ml, 1.5 mg/ml). Collagen-overlaid cultures were incubated for 1.5 h at 37°C in a humidified incubator to allow the collagen to gel before the addition of Dulbecco's modified Eagle's medium containing 1% ITS and 0.1 μM dexamethasone. Thereafter, medium was changed daily. Functional studies were conducted 4 days after seeding.
Accumulation in sandwich-cultured rat hepatocytes. Hepatocytes cultured between two layers of gelled collagen were preincubated with 3 ml of standard Hank's balanced salt solution or Ca2+-free Hank's balanced salt solution containing troglitazone (0.1–10 μM), bosentan (1–100 μM), or vehicle at 37°C for 10 min. After aspiration of the preincubation buffer, taurocholate uptake was initiated by the addition of 3 ml standard Hank's balanced salt solution containing taurocholate (10 μM) and xenobiotic or vehicle. Taurocholate accumulation in the absence of Na+ was measured by replacing Na+ by choline in the incubation buffer. Taurocholate, troglitazone, or bosentan concentrations were selected based on previous literature data (Brock and Vore, 1984; Fattinger et al., 2001; Funk et al., 2001b; Liu et al., 1999b). After incubation of hepatocytes with taurocholate and xenobiotic for 0.5, 1, 2, 5, or 10 min, taurocholate uptake was stopped by rinsing the dishes with ice-cold standard buffer (3 x 3 ml). Cells were lysed with 2 ml of 0.5% Triton X-100. Radioactivity and protein content in the lysate were quantified. Substrate accumulation was corrected for nonspecific binding to the collagen by subtracting taurocholate accumulation in collagen precoated dishes without cells.
Sample analysis. 3H-Taurocholate, 3H-estradiol--D-glucuronide, and 14C-inulin were quantified by liquid scintillation spectroscopy using a Packard Minaxi TriCarb scintillation counter (Meriden, CT). Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL) using bovine serum albumin standards (0.2–2 mg/ml).
Data analysis. Pharmacokinetic model development indicated that the model presented in Figure 1A best described the accumulation data of taurocholate in sandwich-cultured hepatocytes. Differential equations based on the model scheme presented in Figure 1A were resolved simultaneously by nonlinear least-squares regression (WinNonlin 3.2, Pharsight Corporation, Mountain View, CA):
(1)
(2)
where Xcell is the amount of taurocholate taken up by the cells, Xbile is the amount excreted into the bile canaliculi, and Xo is the amount of taurocholate added to the incubation medium (30,000 pmol); k12, k21, and k23 represent first-order rate constants for hepatocyte uptake, basolateral excretion, and biliary excretion of taurocholate, respectively. All data sets were fit simultaneously with the same initial parameter estimates using 1/Y weighting.
Since the data set was not sufficient to simultaneously estimate both uptake and basolateral excretion (i.e., in the presence of xenobiotics the model often converged at an uptake rate constant similar to control with a greatly increased basolateral excretion rate constant) the basolateral excretion rate constant (k21) was fixed at the average estimate from individual control data sets (0.98 ± 0.24 min–1), while uptake and biliary excretion rate constants were estimated. Physiologically, basolateral excretion from sandwich-cultured rat hepatocytes may be attributed to the up-regulation of Mrp3 (Abcc3), the basolateral efflux transporter for taurocholate (Hirohashi et al., 2000), over time in culture (Zhang et al., 2001). The assumption of a treatment-independent basolateral excretion rate constant was validated by measurement of basolateral excretion of taurocholate from taurocholate preloaded sandwich-cultured hepatocytes with intact bile canaliculi; the basolateral excretion rate of taurocholate was not altered by troglitazone or bosentan (data not shown).
The fraction of the accumulated compound that resides in bile canaliculi was quantified with the biliary excretion index (BEI), calculated using B-CLEARTM technology (Qualyst, Inc., Research Triangle Park, NC) based on the following equation (Liu et al., 1999b):
(3)
The biliary excretion index was calculated using 10-min accumulation data from four different rat liver preparations. A sigmoidal inhibition model was fit to the biliary excretion index–xenobiotic concentration data in order to estimate an apparent IC50 of troglitazone and bosentan for inhibition of taurocholate biliary excretion using the following equation:
(4)
where C represents the xenobiotic concentration, IC50 is the concentration of xenobiotic producing 50% of maximal inhibition, and is the Hill coefficient.
Statistics. Data were analyzed by analysis of variance (ANOVA) with Tukey's or Fisher's post hoc multiple comparison test. All statistical tests were performed with SigmaStat (Chicago, IL). All data are reported as mean ± SEM from three or four different rat liver preparations, each measurement performed in triplicate in each preparation.
RESULTS
Suspended Rat Hepatocytes
In suspended rat hepatocytes, 10 μM troglitazone significantly decreased the accumulation of 1 μM taurocholate by 3-fold (Fig. 2A); taurocholate accumulation in the presence of troglitazone was significantly lower relative to control at all time points. The initial uptake rate of taurocholate was significantly decreased from 0.88 ± 0.17 to 0.28 ± 0.15 pmol/sec/mg protein by troglitazone. Estradiol-17-D-glucuronide was utilized to assess the effect of troglitazone on Oatp-mediated hepatic uptake. Troglitazone (10 μM) decreased the initial uptake rate of 1 μM estradiol-17-D-glucuronide from 0.87 ± 0.23 to 0.20 ± 0.12 pmol/sec/mg protein (p = 0.06; Fig. 2B).
In suspended rat hepatocytes, 100 μM bosentan significantly decreased the initial uptake rate of taurocholate from 0.88 ± 0.17 to 0.07 ± 0.05 pmol/sec/mg protein (Fig. 3A). Bosentan also significantly decreased the initial uptake rate of 1 μM estradiol-17-D-glucuronide from 0.87 ± 0.23 to 0.13 ± 0.04 pmol/sec/mg protein (p < 0.05, Fig. 3B).
Sandwich-Cultured Rat Hepatocytes
To determine whether impaired taurocholate uptake was due to inhibition of Ntcp- or Oatp-mediated transport, accumulation of taurocholate at 10 min in sandwich-cultured rat hepatocytes was assessed in the presence or absence of Na+ (Tables 1 and 2). Taurocholate accumulation in the absence of Na+ was significantly lower than in the presence of Na+ at lower xenobiotic concentrations. Higher xenobiotic concentrations significantly decreased taurocholate accumulation in the presence of Na+ to values observed in the absence of Na+. Xenobiotics appeared to slightly impair Na+-independent accumulation, but these differences failed to reach statistical significance.
Troglitazone (10 μM) significantly decreased taurocholate accumulation at 10 min in cells + bile canaliculi from 408 ± 57 to 157 ± 17 pmol/mg protein (Fig. 4A). Accumulation of taurocholate in cells + bile canaliculi in the presence of 10 μM troglitazone was significantly lower than in control sandwich-cultured rat hepatocytes at all time points. At high concentrations of troglitazone, taurocholate accumulation in cells + bile canaliculi was comparable to the cellular accumulation of taurocholate in control sandwich-cultured rat hepatocytes with disrupted bile canaliculi (Fig. 4B). Cellular 10-min accumulation of taurocholate in sandwich-cultured rat hepatocytes with disrupted bile canaliculi was decreased by 10 μM troglitazone (116 ± 15 vs. 172 ± 27 pmol/mg protein), but the difference was not statistically significant. Troglitazone (5 μM) significantly decreased the biliary excretion index of taurocholate (Fig. 4C).
Bosentan (100 μM) significantly decreased taurocholate accumulation at 10 min in cells + bile canaliculi from control values of 408 ± 57 to 154 ± 28 pmol/mg protein (Fig. 5A). Accumulation of taurocholate in cells + bile canaliculi in the presence of 100 μM bosentan was significantly lower than in control sandwich-cultured rat hepatocytes at all time points except 0.5 min. Taurocholate accumulation in cells + bile canaliculi at higher concentrations of bosentan was comparable to the cellular accumulation of taurocholate in control sandwich-cultured rat hepatocytes with disrupted bile canaliculi (Fig. 5B). Bosentan did not significantly decrease the cellular accumulation of taurocholate in sandwich-cultured rat hepatocytes with disrupted bile canaliculi (Fig. 5B). Bosentan at the two highest concentrations (50 and 100 μM) significantly decreased the biliary excretion index of taurocholate (Fig. 5C).
Pharmacokinetic Modeling of Sandwich-Cultured Rat Hepatocyte Data
Representative fits of Equations (1) and (2) to taurocholate accumulation-time data in sandwich-cultured rat hepatocytes in the presence (cells + bile canaliculi) or absence (cells) of Ca2+ are shown in Figure 1B. Troglitazone (10 μM) significantly decreased the rate constant governing taurocholate uptake (from 0.0079 ± 0.0007 to 0.0047 ± 0.0009 min–1) and the rate constant governing biliary excretion (from 0.29 ± 0.07 to 0.10 ± 0.03 min–1). Likewise, bosentan (100 μM) significantly decreased the rate constant for taurocholate uptake (from 0.0079 ± 0.0007 to 0.0040 ± 0.0007 min–1) and the rate constant for biliary excretion (from 0.29 ± 0.07 to 0.07 ± 0.03 min–1).
A sigmoidal inhibition model was fit to the taurocholate biliary excretion index–xenobiotic concentration data to estimate the IC50 of xenobiotics for biliary excretion of taurocholate. Troglitazone (IC50 = 0.91 ± 0.12 μM) was a much more potent inhibitor of taurocholate biliary excretion than bosentan (IC50 = 31 ± 11 μM).
DISCUSSION
Numerous xenobiotics impair the biliary excretion of taurocholate, including troglitazone and bosentan (Fattinger et al., 2001; Funk et al., 2001a,b). Inhibition of Bsep has been suggested to be a potential mechanism of adverse hepatic reactions associated with troglitazone. The current work expands on the known bile acid transport modulation associated with these xenobiotics by demonstrating that these xenobiotics also may inhibit the hepatic uptake of bile acids in addition to biliary excretion in primary rat hepatocytes.
The concentrations of troglitazone and bosentan employed in this study are within the range of concentrations used in preceding in vitro studies, and are below the lowest observable effect level for toxicity in sandwich-cultured rat hepatocytes (Kemp and Brouwer, 2004). While troglitazone concentrations used in this and other in vitro studies are at or slightly above pharmacologically-relevant concentrations (0.7 μM), the inhibitory effects of bosentan were observed at concentrations 20- to 50-fold higher than total plasma concentrations (CMAX 2 μM) reported in man. Actual unbound plasma concentrations of bosentan are much lower than 2 μM due to extensive protein binding of this drug (Channick et al., 2001; Parke-Davis, 1999). At clinically relevant concentrations of bosentan, no changes in hepatic bile acid disposition were noted.
Suspended hepatocytes were employed to examine the effect of troglitazone and bosentan on the initial uptake rate of taurocholate. Both xenobiotics significantly reduced the cellular accumulation and the initial uptake rate of taurocholate, indicating that these compounds impair taurocholate uptake, which is mediated by both Ntcp and Oatp.
To determine whether these xenobiotics modulate Oatp, uptake of estradiol-17-D-glucuronide was studied. Bosentan decreased the initial uptake rate of estradiol-17-D-glucuronide. Treiber et al. (2004) demonstrated that hepatic uptake of bosentan is mediated by all three hepatic Oatp isoforms. Thus, as demonstrated here, bosentan can compete for uptake with other Oatp substrates. Impaired hepatic uptake of estradiol-17-D-glucuronide in the presence of troglitazone expands on the finding of Nozawa et al. (2004), who demonstrated that the sulfate metabolite of troglitazone inhibits human OATP-C. While competitive inhibition of Oatp-mediated bile acid uptake by these xenobiotics is an explanation consistent with previous literature reports (Nozawa et al., 2004; Treiber et al., 2004), other mechanisms such as alterations in membrane bioenergetics, driving force gradients, and transporter trafficking cannot be ruled out. These data indicate that xenobiotics known to inhibit the biliary excretion of bile acids also may impair hepatic uptake of bile acids, thus attenuating the elevation of intrahepatic bile acid concentrations.
Taurocholate accumulation also was studied in sandwich-cultured rat hepatocytes in the absence or presence of Na+. In the absence of Na+, which is the driving force for Ntcp, Oatp function was maintained, while Ntcp-mediated uptake was negligible. Even though Ntcp is responsible for the majority of taurocholate taken up in sandwich-cultured rat hepatocytes, the amount of taurocholate taken up by Oatp can be quantified (Liu et al., 1998). Both troglitazone and bosentan slightly decreased taurocholate accumulation in the absence of Na+. In contrast, taurocholate accumulation in the presence of Na+ was significantly decreased in a concentration-dependent manner by these xenobiotics. At higher concentrations, troglitazone and bosentan decreased taurocholate accumulation in the presence of Na+ to values observed in the absence of Na+, suggesting nearly complete inhibition of Ntcp. These data suggest that troglitazone and bosentan impair taurocholate uptake primarily by inhibition of Ntcp, which may be a hepato-protective mechanism secondary to the inhibition of Bsep.
In sandwich-cultured rat hepatocytes, decreased taurocholate accumulation in cells + bile canaliculi, combined with a decrease in the biliary excretion index of taurocholate in the presence of troglitazone or bosentan, suggested that both hepatic uptake and biliary excretion were inhibited. In contrast, in sandwich-cultured rat hepatocytes with disrupted bile canaliculi, inhibition of uptake appeared to be offset by the inhibition of biliary excretion such that the extent of change in cellular accumulation of taurocholate was not as great as that in cells + bile canaliculi. Accumulation of taurocholate in sandwich-cultured rat hepatocytes in the presence of xenobiotics demonstrated that inhibition of both taurocholate uptake and biliary excretion helps maintain intrahepatic bile acid homeostasis.
Pharmacokinetic modeling was consistent with the hypothesized inhibition of both hepatic uptake and biliary excretion of taurocholate by troglitazone and bosentan in sandwich-cultured hepatocytes. Both xenobiotics decreased the rate constant for taurocholate biliary excretion, consistent with a decrease in the taurocholate biliary excretion index. In addition, troglitazone and bosentan decreased the rate constant for hepatic uptake of taurocholate.
The apparent IC50 value for the troglitazone-associated decrease in the taurocholate biliary excretion index was 0.91 ± 0.12 μM, which is comparable to the IC50 (3.9 ± 0.6 μM) and Ki (1.3 μM) values estimated in canalicular liver plasma membrane vesicles for the ATP-dependent component of taurocholate uptake (Funk et al., 2001b). The IC50 values determined in sandwich-cultured rat hepatocytes were estimated relative to the extracellular concentration of troglitazone. The intracellular concentration of troglitazone at the site of biliary excretion is likely to be higher than in the extracellular medium, but since only the extracellular concentration is known, the IC50 value may represent an underestimate. In contrast to intact hepatocytes, the incubation medium in vesicles is in direct contact with the canalicular membrane, which may explain the 4-fold higher IC50 estimate in membrane vesicles compared to sandwich-cultured rat hepatocytes.
Bosentan (IC50 = 31 ± 11 μM) was a much less potent inhibitor of taurocholate biliary excretion. Previously, it was reported that 50 μM bosentan inhibited ATP-dependent taurocholate transport by 82% and 55% in canalicular liver plasma membrane vesicles and Bsep-expressing Sf9 cell plasma membrane vesicles, respectively (Fattinger et al., 2001). A Ki value (12 μM) for bosentan inhibition of the ATP-dependent uptake of taurocholate was calculated previously from data obtained in Bsep-expressing Sf9 cell plasma membrane vesicles (Fattinger et al., 2001). The IC50 value estimated for the decrease in taurocholate biliary excretion index in the presence of bosentan in sandwich-cultured rat hepatocytes is in good agreement with previous kinetic studies of this process, considering that the IC50 was estimated with extracellular xenobiotic concentrations.
In conclusion, troglitazone and bosentan modulate transport processes at both the basolateral and canalicular plasma membranes of hepatocytes. In agreement with previous work, biliary excretion of taurocholate was impaired by both xenobiotics. Both xenobiotics impaired hepatic uptake of taurocholate, primarily due to inhibition of Ntcp. Inhibition of hepatic uptake of bile acids may be a hepato-protective mechanism and may help maintain intrahepatic bile acid homeostasis.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Alexander Treiber (Actelion Pharmaceuticals Ltd, Switzerland) for providing bosentan, and for his insightful comments. In addition, the authors are grateful to Dr. Mary Vore (University of Kentucky) for her help with preparation of this manuscript.
This work was supported by grants R21 CA106101, R01 GM41935, and a Toxicology Training Grant 5-T32-ESO7126 from the National Institutes of Health. Daniel C. Kemp was supported by minority grant GM531674 from the National Institutes of Health. Maciej J. Zamek-Gliszczynski was supported by an Eli Lilly and Company Foundation Predoctoral Fellowship Award in Pharmacokinetics and Drug Disposition. This work was submitted to the Graduate School of the University of North Carolina in partial fulfillment of requirements for the Doctor of Philosophy degree in Toxicology.
NOTES
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