Increase in Bile Flow and Biliary Excretion of Glutathione-Derived Sulfhydryls in Rats by Drug-Metabolizing Enzyme Inducers Is Mediated by Multidrug Resistance Protein 2

David R. Johnson,1, Sultan S. M. Habeebu and Curtis D. Klaassen,2

Environmental Health and Occupational Medicine Center, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7417

Received August 16, 2001; accepted October 23, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutathione (GSH) is an important cellular constituent for normal liver homeostasis. Certain drug-metabolizing enzyme inducers (i.e., phenobarbital [PB] and pregnenolone-16{alpha}-carbonitrile [PCN]) increase biliary excretion of GSH-derived sulfhydryls (SH) as well as bile flow, whereas other drug-metabolizing enzyme inducers (i.e., 3-methylcholanthrene [3MC] and benzo(a)pyrene [BaP]), do not. The purpose of the study was to determine whether rat multidrug resistance protein 2 (Mrp2) is the inducible transporter responsible for increasing biliary SH excretion and bile flow. Sprague-Dawley (SD) rats were injected ip daily for 4 days with PB, PCN, 3MC, BaP, or vehicle; Mrp2-null Eisai hyperbilirubinemic (EHBR) rats were injected ip daily for 4 days with PCN or vehicle. Although no drug-metabolizing enzyme inducer altered hepatic GSH in SD rats, PB and PCN significantly increased the rate of biliary SH excretion and bile flow. Neither 3MC nor BaP affected the biliary SH excretion rate or bile flow. In control EHBR rats, despite elevated hepatic GSH, the rate of biliary SH excretion was almost completely eliminated and bile flow was dramatically reduced compared with SD rats. Furthermore, PCN treatment did not affect bile flow or the biliary SH excretion rate in EHBR rats. PB and PCN also increased Mrp2 protein levels, but 3MC and BaP did not. None of the drug-metabolizing enzyme inducers tested significantly increased Mrp2 mRNA levels. PCN increased Mrp2 protein, but not Mrp2 mRNA, in a time-dependent manner. In conclusion, Mrp2 is the inducible efflux transporter responsible for increased biliary SH excretion and bile flow after administration of some drug-metabolizing enzyme inducers.

Key Words: drug-metabolizing enzyme inducers; Mrp2; glutathione; organic anion transport; EHBR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutathione (GSH), an endogenous organic anion, is the most abundant nonprotein thiol in the liver, the major site of GSH biosynthesis in the body (Ballatori and Rebbeor, 1998Go; Stein et al., 1986Go). GSH is an integral constituent in normal liver function. GSH is involved in cellular defense against potentially harmful electrophiles. GSH can be conjugated to electrophilic xenobiotics by glutathione S-transferases (GST), a major Phase II drug-metabolizing enzyme family, to produce less harmful, water-soluble chemicals. In addition, GSH alone can also bind to electrophilic chemicals, thus helping prevent cellular damage due to free radicals and alkylating agents. GSH also provides an important osmotic force that drives the bile acid-independent bile flow (Ballatori and Truong, 1989Go, 1992Go). GSH is the predominant nonprotein thiol in bile (Ballatori and Rebbeor, 1998Go; Stein et al., 1986Go). When eliminated into bile, GSH is catabolized by gamma-glutamyl transpeptidase and dipeptidase, breaking GSH down to its three individual amino acids, cysteine, glutamate, and glycine. This provides three osmolar equivalents in bile, contributing to an increase in bile flow by driving water movement into the bile canaliculus (Ballatori and Rebbeor, 1998Go). Thus, when GSH excretion into bile is increased, bile flow also increases; conversely, when GSH excretion is depressed, bile flow decreases accordingly (Ballatori and Truong, 1989Go, 1992Go).

Different drug-metabolizing enzyme inducers have distinct effects on bile flow as well as the hepatic steady-state of GSH. Phenobarbital (PB) and pregnenolone-16{alpha}-carbonitrile (PCN) have been shown to significantly increase bile flow in rats (Klaassen, 1969Go, 1970Go; Lu et al., 1996Go). These same drug-metabolizing enzyme inducers also increase the biliary excretion of GSH-derived sulfhydryls (SH) in rats (Ballatori and Clarkson, 1983Go; Lu et al., 1996Go; Madhu et al., 1993Go; Sieger et al., 1983). Other drug-metabolizing enzyme inducers, however, have no effect on bile flow and biliary excretion of SH. Neither 3-methylcholanthrene (3MC) nor benzo(a)pyrene (BaP) alters bile flow or biliary excretion of SH (Gregus and Varga, 1985Go; Klaassen, 1969Go, 1970Go). Despite the differential effects on biliary SH excretion, none of the drug-metabolizing enzyme inducers altered hepatic GSH concentrations (Madhu et al., 1993Go), suggesting that the increase in biliary SH after inducer treatment is due to increased transport and not increased GSH availability. The inducible transporter responsible for the increase in bile flow and biliary SH excretion by drug-metabolizing enzyme inducers has not yet been elucidated.

Multidrug resistance protein 2 (Mrp2) is a good candidate for investigation to determine whether it is the transporter responsible for the increase in bile flow and biliary SH excretion after drug-metabolizing enzyme induction. Mrp2 is a canalicular efflux transporter that pumps endogenous and xenobiotic chemicals into bile. Mrp2 also transports reduced and oxidized GSH, as well as GSH-conjugated chemicals (Oude Elferink et al., 1989Go, 1994Go). Therefore, the purpose of this study was to determine whether Mrp2 is inducible by drug-metabolizing enzyme inducers, and whether an increase in Mrp2 protein results in an increase in biliary SH excretion as well as bile flow. To emphasize the role of Mrp2 in biliary SH excretion, Mrp2-null Eisai hyperbilirubinemic (EHBR) rats were used as negative controls. EHBR rats lack a functional Mrp2 protein, resulting in chronic conjugated hyperbilirubinemia, as well as impaired biliary elimination of non-bile acid organic anions (Hosokawa, 1992). It was concluded that Mrp2 was the primary biliary GSH transporter that is increased by select drug-metabolizing enzyme inducers, thus providing insight into potential drug-drug interactions due to changes in the disposition of organic anion efflux.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
PB, 3MC, BaP, sulfosalicylic acid, reduced ß-nicotinamide adenine dinucleotide phosphate, and 5,5`-dithio-bis(nitrobenzoic acid) were purchased from Sigma Chemical Co. (St. Louis, MO). PCN was synthesized from 16-dehydropregnenolone precursor (Steraloids, Newport, RI) according to Sonderfan and Parkinson (1988). All other chemicals and reagents were of the highest grade and purchased from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (St. Louis, MO).

Animals and surgery.
Male Sprague-Dawley (SD) rats (Harlan, Indianapolis, IN) and Mrp2-null Eisai hyperbilirubinemic rats (EHBR; generously provided by Mr. Eiichi Okui of Eisai Ltd. Co., Tokyo, Japan) were housed at 25°C with a 12-h light-dark cycle. Rats received water and food ad libitum, and were allowed to acclimate to their environment for 5 days before the study began. SD rats (250–350 g) were subjected to the following chemical treatments: PB (75 mg/kg), PCN (75 mg/kg), 3MC (20 mg/kg), BaP (20 mg/kg), or control vehicle (saline, corn oil, and propylene glycol); EHBR rats (250–350 g) were subjected to PCN (75 mg/kg) or control vehicle (corn oil). PB was dissolved in saline; PCN, 3MC, and BaP were dissolved in corn oil. All chemicals were injected ip in a final volume of 5 ml/kg. The effect of different control vehicles was determined by using the respective vehicles (saline or corn oil) for each chemical for one set of animals, and by dissolving each chemical in a common vehicle (propylene glycol) for another set of animals. No difference was observed between the control vehicles, so they were combined upon analysis. Rats were injected ip once daily for 4 days. On day 5, rats were anesthetized with pentobarbital (45 mg/kg, ip). An abdominal incision was made and the bile duct was cannulated with PE-10 tubing (Becton Dickinson, Sparks, MD). Rat body temperatures were monitored with a rectal thermometer and maintained at 37°C with a heat lamp. Bile was collected at 10-min intervals in preweighed vials containing 5% sulfosalicylic acid (w/v) dissolved in water. At the completion of the bile collection, livers were removed, snap-frozen in liquid nitrogen, and stored at –80°C. Bile samples were centrifuged to precipitate biliary proteinaceous components. The remaining supernatant was transferred to a new tube and stored at –80°C until analysis.

PCN time course.
Male rats (250–350 g) were injected ip once daily for 4 days with PCN (75 mg/kg) or control vehicle (corn oil). Over the course of the 5-day study, livers were removed at 6, 12, 24, 36, 48, 72, and 96 h after the first PCN injection, snap-frozen in liquid nitrogen, and stored at –80°C until analysis.

Bile flow.
Bile volume from each timed collection was measured gravimetrically, assuming a specific gravity of 1.0, and used to determine the rate of bile flow.

Hepatic GSH concentrations and biliary SH excretion.
Liver was homogenized in 5% sulfosalicylic acid (w/v) dissolved in water. The homogenate was centrifuged at 10,000 x g, and the supernatant was removed and used for SH analysis. Hepatic SH concentrations and the biliary SH excretion were determined according to the GSH enzymatic recycling method of Tietze (1969). SH concentrations were measured kinetically for 6 min at 412 nm.

Membrane-enriched protein preparation.
Liver protein samples were prepared according to the method of Trauner et al. (1997), which was modified. Briefly, 1.0 g liver was minced in 10 ml ice-cold buffer A (0.25 M sucrose, 10 mM Tris-HCl [pH 7.4–7.6], 25 µg/ml leupeptin, 50 µg/ml aprotinin, 40 µg/ml PMSF, 0.5 µg/ml pepstatin, and 50 µg/ml antipain). The minced liver was poured into a Dounce homogenizer (Kontes, Vineland, NJ) and homogenized on ice for 20 strokes. The crude homogenate was then homogenized on ice for 5 strokes with a Teflon homogenizer (Caframo, Wiarton, Ontario, Canada). Homogenate was filtered through two layers of cheesecloth, then centrifuged at 100,000 x g for 1 h at 4°C. The resulting pellet was removed and resuspended in buffer B (0.3 M sucrose, 10 mM HEPES [pH 7.5], and 40 µg/ml PMSF). Protein concentrations were determined with a bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL).

Western blot analysis.
Protein samples (50 µg/well) were mixed with sample buffer. Samples were loaded unboiled onto a 7.5% SDS-polyacrylamide gel. Proteins were resolved electrophoretically for 3 h at 130 V (Gibco BRL, Gaithersberg, MD). Protein was transferred to nitrocellulose membranes for 3 h at 20 V (Novex, San Diego, CA). Membranes were blocked for 1–2 h at room temperature with 5% dry milk dissolved in Tris-buffered saline (20 mM Trizma base [pH 7.5], 155 mM NaCl) with 0.1% Tween 20 (TBS/T). Membranes were then incubated overnight at 4°C with a 1:30,000 dilution of anti-Mrp2 peptide antibody EAG15 (a generous gift from Dr. Dietrich Keppler, Heidelberg, Germany) diluted in TBS/T containing 3% bovine serum albumin. Membranes were washed three times with TBS/T, then incubated with donkey antirabbit IgG-horseradish peroxidase-linked secondary antibody (Amersham, Arlington Heights, IL), diluted in TBS/T containing 3% bovine serum albumin for 1 h at room temperature. Membranes were washed again three times with TBS/T. Mrp2 protein was detected using an enhanced chemiluminescence (ECLTM) kit (Amersham).

Immunohistochemistry.
Immunohistochemical localization and analysis of Mrp2 protein was performed on cryosections of the liver as described previously (Rost et al., 1999Go), with minor modifications. Briefly, liver cryosections (4 µm thick) were thaw-mounted on Superfrost Plus microscope slides (Fisher Scientific). The slides were permeabilized in acetone for 10 min at –20°C and air-dried. Sections were blocked for 30 min in 5% goat serum/PBS/0.2% Triton X-100. Mrp2 primary antibody EAG15, generated in our laboratory according to the method of Buchler et al. (1996), was applied at 1:50 dilution for 1 h at room temperature. Fluorescein isothiocyanate (FITC)-labeled secondary antibody to rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to detect Mrp2 and rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR) to detect F-actin were applied simultaneously, each at 1:200 dilution, for 1 h at room temperature. Slides were mounted in Slow-Fade Light antifade solution (Molecular Probes, Eugene, OR) and analyzed under fluorescence microscopy. Images were captured on a Nikon Eclipse TE300 inverted microscope (Nikon Corporation, Tokyo, Japan) equipped with appropriate fluorescence excitation and emission filters, a video camera (SPOT II Cooled Color Digital Camera; Diagnostic Instruments Inc., Sterling Heights, MI), and image analysis software (SPOT II Camera software and Adobe PhotoShop 5.0).

Isolation of total RNA.
Total RNA was isolated using RNAzol B reagent (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's protocol. Each RNA pellet was resuspended in 0.2 ml diethyl pyrocarbonate-treated water. The concentration of total RNA in each sample was quantified spectrophotometrically at 260 nm. The integrity of each RNA sample was analyzed by formaldehyde-agarose (1.2% agarose, 2.1 M formaldehyde in 1X MOPS [3-(N-morpholino)propanesulfanic acid, pH 7.2]) gel electrophoresis.

Mrp2 probe subcloning.
A 267-bp probe was subcloned from the full-length Mrp2 cDNA (GenBank accession number D86086, a generous gift from Dr. Peter Meier, Zurich, Switzerland) (Madon et al., 1997Go). Briefly, competent DH5-{alpha} E. coli cells were transformed with the plasmid containing the full-length Mrp2 cDNA. The plasmid was cut with Pst1 (Promega, Madison, WI) at nucleotides 4514 and 5011. This 497-bp fragment was subcloned into the pBluescript vector, which was further cut with Sac1, to obtain a 267-bp Mrp2 cDNA fragment suitable for use as a probe for RNase protection assays.

Ribonuclease (RNase) protection assay.
For RNase protection assays, the 267-bp Mrp2 cDNA probe was linearized with Hind III (Promega). An alpha-[32P]-labeled antisense cRNA was transcribed from the Mrp2 cDNA with T3 polymerase (Promega). A similar cRNA transcription synthesis was performed with a 400-bp rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA fragment (Ambion, Austin, TX). Signal intensity was determined with a Beckman LS6500 scintillation counter, and probe integrity was determined by electrophoresis on a denaturing urea-6% polyacrylamide gel. RNase protection assays were carried out according to McCarson and Krause (1994). Briefly, 20 µg total RNA and Mrp2 or GAPDH cRNA probes were hybridized overnight at 45°C. The mixture was then digested with RNase T1 (0.4 mg/ml) and RNase A (0.02 mg/ml). The digestion was terminated with proteinase K (Fisher Scientific). The protected probes were isolated by phenol:chloroform:isoamyl alcohol extraction and precipitated with isopropanol. The precipitated samples were then resuspended in loading dye and loaded onto a denaturing urea-6% polyacrylamide gel. RNA was electrophoretically separated, after which the protected probes were detected by autoradiography analysis with a PhosphorImagerSI (Molecular Dynamics, Sunnyvale, CA). Data were analyzed with ImageQuant software (Molecular Dynamics).

Development of specific oligonucleotide probe sets for QuantigeneTM branched DNA (bDNA) analysis of Mrp2 mRNA.
Mrp2-specific oligonucleotide probes were designed in a similar fashion to the methods of Hartley and Klaassen (2000). All oligonucleotide probes were designed with a Tm of approximately 63°C. This feature enables hybridization conditions to be held constant (53°C during each hybridization step and for each oligonucleotide probe set). Every probe developed in ProbeDesignerTM was submitted to the National Center for Biotechnological Information for nucleotide comparison by the basic logarithmic alignment search tool (BLASTn) (Altschul et al., 1997Go) to ensure minimal cross-reactivity with other rat sequences. Probes were synthesized (50 nmol synthesis scale) by Operon Technologies (Palo Alto, CA). The probe set for GAPDH was generously provided by Jeff Donahue (Bayer Corp., Emeryville, CA). All probes (i.e., blocker probes, capture extenders, and label extenders) were diluted in 1.0 ml of 10 mM Tris-HCl, pH 8.0, with 1 mM EDTA, and stored at –20°C. All probes designed for Mrp2 mRNA detection using the QuantigeneTM bDNA assay are listed in Table 1Go.


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TABLE 1 Oligonucleotide Probes Generated for Analysis of Mrp2 mRNA Expression by the bDNA Signal Amplification Assay
 
QuantigeneTM bDNA signal amplification assay.
All reagents for analysis by the bDNA assay (i.e., lysis buffer, capture hybridization buffer, amplifier/label probe buffer, wash A and wash D, and substrate solution) were supplied in the QuantigeneTM bDNA Signal Amplification Kit (Bayer Diagnostics, East Walpole, MA); the components of these reagents have been published previously (Wang et al., 1997Go). Mrp2-specific oligonucleotide probe sets (i.e., blocker probes, capture probes, and label probes) were combined and diluted to 50 fmol/µl in lysis buffer. The GAPDH probe set, used for normalization of mRNA expression between wells, was used at 50, 100, and 200 fmol/µl for capture probes, blocker probes, and label probes, respectively. The bDNA assay was run according to the method of Hartley and Klaassen (2000). Briefly, total RNA (1 µg/µl; 10 µl) was added to each well of a 96-well plate containing capture hybridization buffer and 100 µl of diluted Mrp2 probe set and allowed to hybridize overnight at 53°C in a QuantiplexTM bDNA Heater (Bayer Diagnostics). Subsequently, plates were removed from the heater, cooled to room temperature, and rinsed with wash A. Samples were hybridized with a solution containing the bDNA amplifier molecules (50 µl/well) diluted in amplifier/label probe buffer and incubated for 30 min at 53°C. The plate was again cooled to room temperature, after which the wells were washed three times with wash A. Label probe, diluted in amplifier/label probe buffer, was added to each well (50 µl/well), and hybridized to the bDNA-RNA complex for 15 min at 53°C. The plate was cooled to room temperature, and each well was rinsed two times with wash A, followed by three washes with wash D. Alkaline phosphatase-mediated luminescence was triggered by the addition of a dioxetane substrate solution (50 µl/well). The enzymatic reaction was allowed to proceed for 30 min at 37°C, and luminescence was measured with the QuantiplexTM 320 bDNA Luminometer (Bayer Diagnostics) interfaced with QuantiplexTM Data Management Software Version 5.02 (Bayer Diagnostics) for analysis of luminescence from 96-well plates.

Statistics.
All results were analyzed statistically by one-way ANOVA, followed by Duncan's multiple range post-hoc test. Statistically significant results (p < 0.05) are denoted by an asterisk (*) or a pound sign (#).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatic GSH Content and Biliary SH Excretion Rates
To determine the importance of Mrp2 in GSH homeostasis, liver GSH concentrations and the rates of biliary SH excretion were quantified in control and inducer-treated SD and Mrp2-null EHBR rats (Figs. 1 and 2GoGo, respectively). In SD rats, none of the four inducers tested changed hepatic GSH concentration from control levels (Fig. 1Go, upper panel). However, the biliary excretion rates of SH did differ with the various drug-metabolizing enzyme inducers. PB and PCN significantly increased the biliary SH excretion rate by 98 and 63%, respectively (178 ± 18 nmol/min/kg and 146 ± 24 nmol/min/kg), above control (90 ± 8 nmol/min/kg) (Fig. 1Go, lower panel). 3MC and BaP had no effect on the biliary excretion rate of SH.



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FIG. 1. Hepatic GSH concentrations and SH excretion rates in SD rats after enzyme inducer treatment. SD rats were treated with enzyme inducers or control vehicle as described in Materials and Methods. Twenty-four hours after the last dose, the rates of biliary SH excretion were measured for 10 min. Hepatic GSH concentrations and biliary SH excretion were determined by the GSH enzymatic recycling assay, according to Tietze (1969). GSH concentrations were measured kinetically for 6 min at 412 nm. Upper panel, liver GSH concentrations. Lower panel, biliary SH excretion rates. All data are given as mean ± SE for 17–23 animals in each group. *Statistical difference from control values (p < 0.05).

 


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FIG. 2. Hepatic GSH concentrations and biliary SH excretion rates in EHBR rats after PCN treatment. EHBR rats were treated with PCN or control vehicle as described in Materials and Methods. Twenty-four hours after the last dose, bile was collected in preweighed tubes for 10 min. Hepatic GSH concentrations and biliary SH excretion were determined by the GSH enzymatic recycling assay, according to Tietze (1969). GSH concentrations were measured kinetically for 6 min at 412 nm. Upper panels refer to steady-state liver GSH concentrations, and lower panels refer to biliary SH excretion rates. Upper left panel, comparison of hepatic GSH concentrations between control SD and EHBR rats. Upper right panel, comparison of hepatic GSH concentrations between control and PCN-treated EHBR rats. Lower left panel, comparison of biliary SH excretion rates between control SD and EHBR rats. Lower right panel, comparison of biliary SH excretion rates between control and PCN-treated EHBR rats. SD Con, control SD rat; EHBR Con, control EHBR rats; EHBR PCN, PCN-treated EHBR rats. All data are given as mean ± SE for seven animals in each group. *Statistical difference from control SD rat values (p < 0.05). #Statistical difference from control EHBR rat values (p < 0.05).

 
In EHBR rats, hepatic GSH concentrations (8030 ± 399 nmol/g) were 77% higher than those in SD rats (4530 ± 577 nmol/g) (Fig. 2Go, upper left panel). This result is in agreement with previously published data that hepatic GSH levels are higher in EHBR rats than SD rats (Lu et al., 1996Go). Furthermore, PCN significantly increased hepatic GSH concentrations (9900 ± 369 nmol/g) above control levels by 23% in EHBR rats (Fig. 2Go, upper right panel). The biliary excretion rate of SH in EHBR rats was dramatically different from that observed in SD rats. Despite more available GSH substrate to be transported in the livers of EHBR rats, the rate of biliary SH excretion in control EHBR rats was only 11% (9.68 ± 2 nmol/min/kg) of control SD rats (90.1 ± 8 nmol/min/kg) (Fig. 2Go, lower left panel). The fact that the biliary SH excretion rate in EHBR rats was dramatically reduced from SD rats shows that our results are in agreement with previous reports (Lu et al., 1996Go; Sugawara et al., 1998Go). PCN did not increase the rate of biliary SH excretion in EHBR rats (Fig. 2Go, lower right panel).

Bile Flow
To evaluate the role of Mrp2 in increased bile flow after drug-metabolizing enzyme induction, bile flow was measured in control and inducer-treated SD and Mrp2-null EHBR rats. In SD rats, PB and PCN significantly increased bile flow by 24% (87 ± 4 µl/min/kg) and 22% (86 ± 5 µl/min/kg), respectively, above control (70 ± 3 µl/min/kg) (Fig. 3Go). However, 3MC and BaP did not change bile flow from the control level. Bile flow in control EHBR rats (41 ± 2 µl/min/kg) was 42% less than that observed in control SD rats (70 ± 3 µl/min/kg) (Fig. 4Go, left panel). In contrast to SD rats, PCN did not increase bile flow in EHBR rats (Fig. 4Go, right panel).



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FIG. 3. Bile flow in SD rats after enzyme inducer treatment. SD rats were treated with enzyme inducers or control vehicle as described in Materials and Methods. Twenty-four hours after the last dose, bile was collected in preweighed tubes for 10 min. Bile volume was measured gravimetrically, assuming a specific gravity of 1.0. All data are given as mean ± SE for 17–23 animals in each group. *Statistical difference from control values (p < 0.05).

 


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FIG. 4. Bile flow in Mrp2-null EHBR rats after PCN treatment. EHBR rats were treated with PCN or control vehicle as described in Materials and Methods. Twenty-four hours after the last dose, bile was collected in preweighed tubes for 10 min. Bile volume was measured gravimetrically, assuming a specific gravity of 1.0. Left panel, comparison of bile flow between control SD and EHBR rats. Right panel, comparison of bile flow between control and PCN-treated EHBR rats. SD Con, control SD rat; SD PCN, PCN-treated SD rat; EHBR Con, control EHBR rats; EHBR PCN, PCN-treated EHBR rats. All data are given as mean ± SE for seven animals in each group. *Statistical difference from control values (p < 0.05).

 
Mrp2 Protein Expression
Mrp2 protein expression from control and inducer-treated SD rat liver was quantified by Western blot analysis (Fig. 5Go, upper and lower panels). Protein samples from EHBR rat liver were used as a negative control for Mrp2 protein expression, as indicated by the lack of Mrp2 protein detected in these rats. Mrp2 protein expression was detected at approximately 200 kDa for all samples. Both PB and PCN significantly induced Mrp2 protein expression in SD rat livers by 136 and 191%, respectively (Fig. 5Go, lower panel). Neither 3MC nor BaP treatment affected Mrp2 protein expression. To verify the absence of Mrp2 functional protein in EHBR rats, Western blot detection of Mrp2 protein was also performed in control and PCN-treated EHBR rat livers. As expected, no Mrp2 protein expression was detected in either group (data not shown).



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FIG. 5. Mrp2 protein expression in SD and Mrp2-null EHBR rats after enzyme inducer treatment. SD and EHBR rats were treated with enzyme inducers or control vehicle as described in Materials and Methods. Membrane-enriched protein was isolated and separated by 7.5% SDS-PAGE (50 µg protein/lane). Mrp2 protein was detected by the Mrp2-specific polyclonal antibody EAG15. Upper panel, representative western blot of Mrp2 protein expression in control and drug-metabolizing enzyme-induced SD rats. Mrp2 protein was detected at approximately 200 kDa. Mrp2 protein expression was undetected in Mrp2-null EHBR rat liver samples. Lower panel, graphical representation of Mrp2 protein expression results from control and drug-metabolizing enzyme-induced SD rats. All data are given as mean ± SE for 17–23 animals in each group. *Statistical difference from control values (p < 0.05).

 
Immunohistochemistry
To evaluate the presence of increased levels of Mrp2 in the canalicular membrane, Mrp2 protein expression and localization in liver were investigated in control and PCN-treated SD and EHBR rats by immunohistochemistry and fluorescence microscopy. The hepatocyte sinusoidal, lateral, and canalicular membranes were visualized with rhodamine-labeled phalloidin to detect the F-actin network (red stain, Figs. 6AGo–D). F-actin immunofluorescence displayed a regular polygonal staining of hepatocytes in liver from all rats. In control SD rats, Mrp2 expression (green stain) was largely confined to the canalicular membrane (Fig. 6AGo). Overlap of Mrp2 and F-actin produced a yellow stain. After 4 days of PCN treatment, an increase in Mrp2 protein expression was clearly demonstrated in SD rats (Fig. 6BGo). Following PCN treatment, Mrp2 remained localized mainly to the canalicular membrane. However, in some areas, the much higher increase in Mrp2 expression resulted in a blotchy and irregular staining pattern, suggestive of an accumulation of Mrp2-containing vesicles in the pericanalicular region. In EHBR rats, Mrp2 protein was not detected in control rats (Fig. 6CGo) and in PCN-treated rats (Fig. 6DGo).



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FIG. 6. Immunohistochemical localization of Mrp2 protein in SD and Mrp2-null EHBR rats after PCN treatment. Double-label immunohistochemical detection of Mrp2 and F-actin in wild type (A and B) and EHBR (C and D) rat livers before (A and C) and after PCN treatment for 4 days (B and D). F-actin was visualized using rhodamine-labeled phalloidin (red stain). Mrp2 was detected using the primary antibody EAG15 or antiserum generated in our laboratory, and a FITC-labeled secondary antibody (green stain). Yellow staining indicates overlap of Mrp2 and F-actin in biliary canaliculi and pericanalicular areas. The absence of green and yellow staining in C and D indicate lack of Mrp2 in livers from EHBR rats, which are natural mutants lacking Mrp2 protein expression.

 
Mrp2 RNA
Mrp2 mRNA expression from both SD and EHBR rats was analyzed by two separate methods, RNase protection assay and QuantigeneTM bDNA assay. GAPDH mRNA was used as the internal control for both assays. RNase protection assay showed Mrp2 mRNA signal detected at 237 bp, the size of the protected Mrp2 probe; the protected GAPDH probe was detected at 316 bp (Fig. 7Go). tRNA was used as a negative control for Mrp2 detection. No nonspecific binding of the Mrp2 probe occurred, as shown by no signal detected with the tRNA sample. PB, PCN, and 3MC tended to increase Mrp2 mRNA expression in SD rats (approximately 60, 90 and 60%, respectively, above control) though none were statistically significant (Fig. 8Go). Mrp2 mRNA expression in EHBR rats was detectable, yet was severely decreased compared with SD rats. PCN did not affect Mrp2 mRNA expression in EHBR rats (Fig. 7Go).



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FIG. 7. Mrp2 mRNA expression in SD and EHBR rats after enzyme inducer treatment. SD and EHBR rats were treated with enzyme inducers or control vehicle as described in Materials and Methods. Twenty-four hours after the last dose, liver was removed, and total RNA was isolated. Mrp2 mRNA expression was analyzed using the Ribonuclease protection assay. The Mrp2 probe produced a protected Mrp2 mRNA band of 237-bp. GAPDH was used as an internal standard for each sample, producing a protected mRNA band of 316-bp.

 


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FIG. 8. Graphical results of RNase protection assay analysis of Mrp2 mRNA expression from control and inducer-treated SD and EHBR rats. Results are expressed as the ratio of Mrp2 signal to GAPDH signal. All data are given as mean ± SE for 7–23 animals in each group.

 
When Mrp2 mRNA was analyzed by the bDNA assay (Fig. 9Go), none of the drug-metabolizing enzyme inducers affected Mrp2 mRNA expression. Mrp2 mRNA expression in control EHBR rats was approximately 30% of control SD rats and was not increased by PCN (data not shown).



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FIG. 9. Graphical results of Mrp2 mRNA expression in SD rats after drug-metabolizing enzyme induction detected by QuantigeneTM bDNA signal amplification. GAPDH mRNA was used as the internal control. All data are given as mean ± SE for 17–23 animals in each group.

 
Time Course of Mrp2 mRNA and Protein Expression after PCN Treatment
Because no increase in Mrp2 mRNA was detected at day 5, yet Mrp2 protein was increased, the time course of Mrp2 mRNA and protein expression during PCN induction was examined to ensure that a peak in mRNA levels was not overlooked. Control samples at 24, 72, and 120 h were not statistically different in both mRNA and protein expression and were thus combined upon analysis. Mrp2 protein expression did increase in a time-dependent manner (Fig. 10Go, upper panel). Mrp2 protein expression reached maximal expression at 72 h, approximately 270% above control. In contrast, Mrp2 mRNA expression remained relatively constant throughout the PCN induction time course (Fig. 10Go, lower panel).



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FIG. 10. Time course of Mrp2 protein and mRNA expression in SD rats after PCN induction. SD rats were injected ip once daily for 4 days with 75 mg/kg PCN or control vehicle (corn oil). Livers were removed at 6, 12, 24, 36, 48, 72, and 96 h after the first PCN injection. Upper panel, Mrp2 protein expression was determined by Western blot analysis. Membrane-enriched protein (50 µg protein/lane) from control and PCN-treated livers was separated by 7.5% SDS-PAGE. Mrp2 was detected by the polyclonal EAG15 primary antibody. Lower panel, Mrp2 mRNA expression was detected by bDNA signal amplification assay. GAPDH mRNA was used as the internal control. Dashed line, control SD rats; solid line, PCN-treated SD rats. All data are given as mean ± SE for 4 animals in each group. *Statistical difference from control values (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have provided strong evidence that Mrp2 transports GSH into bile. GSH transport in yeast secretory vesicles is mediated by YCF1, an ATP-dependent, low-affinity ortholog of Mrp (Km ~19 mM) (Rebbeor et al., 1998Go). In addition, Paulusma et al. (1999) determined that rats heterozygous to the Mrp2 mutation (TR/tr) excreted GSH into bile at 37% below control (TR/TR), and that homozygous recessive (tr/tr) rats excreted less than 1% of control GSH levels. They also showed Mrp2 to be a low-affinity ATP-driven transporter of GSH efflux, with a similar Km value of ~20 mM, thus indicating that Mrp2 transports GSH into bile in vivo. Select drug-metabolizing enzyme inducers increase the efficiency of SH elimination into bile (Klaassen, 1970Go; Madhu et al., 1993Go; Zsigmond and Solymoss, 1972Go). The canalicular transporter responsible for the increase in biliary SH excretion after drug-metabolizing enzyme induction has not yet been identified. Thus, Mrp2 is a good candidate protein for explaining the phenomenon of increased biliary excretion of SH after microsomal enzyme induction. Therefore, the purpose of this study was to determine whether Mrp2 is inducible by four drug-metabolizing enzyme inducers, and whether an increase in Mrp2 protein results in an increase in bile flow and rate of biliary SH excretion. It was hypothesized that drug-metabolizing enzyme inducers that increase biliary SH excretion and bile flow (i.e., PB and PCN) do so by increasing Mrp2 expression and function, whereas drug-metabolizing enzyme inducers that do not affect biliary SH excretion and bile flow (i.e., 3MC and BaP) do not affect Mrp2 expression and function. The approaches used in this study provide evidence that Mrp2 is the inducible transporter responsible for increasing the rate of biliary SH excretion after drug-metabolizing enzyme induction.

As shown in this study, PB and PCN treatment significantly increased both the rate of biliary SH excretion and Mrp2 protein levels (Figs. 1 and 5GoGo, respectively). The increase in Mrp2 protein corresponded with an increase in Mrp2 localization to the canalicular membrane (Fig. 6BGo). In contrast, 3MC and BaP treatment did not increase the rate of biliary SH excretion nor did they increase Mrp2 protein expression. The differences in biliary SH excretion cannot be accounted for by alterations in hepatic GSH concentrations, because none of the four inducers examined changed hepatic GSH concentrations above control levels (Fig. 1Go, upper panel). Therefore, PB- and PCN-induced increases in biliary SH excretion are due to increased efflux via Mrp2. Further support for this conclusion comes from the fact that in EHBR rats that lack functional Mrp2 protein, biliary SH excretion was only 10% the rate of SD rats (Fig. 2Go, lower left panel). In addition, treatment of EHBR rats with PCN did not increase the biliary SH excretion rate (Fig. 2Go, lower right panel). Similar effects were observed when EHBR rats were treated with PB (Lu et al., 1996Go). These experiments provide definitive evidence that select enzyme inducers increase the rate of biliary SH excretion by increasing Mrp2 protein expression and function.

This study also adds to the established evidence that the Mrp2-mediated transport of GSH is the primary osmotic factor of bile-acid independent bile flow (Ballatori and Truong, 1992Go; Wielandt et al., 1999Go). Bile acid-independent bile flow accounts for approximately 40% of total bile flow (Oude Elferink et al., 1989Go), most of which is likely due to the biliary excretion of GSH via Mrp2. Bile flow is markedly depressed when Mrp2 is nonfunctional (Fig. 4Go, left panel). Because GSH-driven bile acid-independent bile flow was virtually absent in EHBR rats, bile acid-dependent bile flow was most likely the predominant driving force for bile flow in EHBR rats, as suggested by Kurisu et al. (1991). However, the bile acid-dependent bile flow did not appear to be inducible by PCN, as indicated by no change in bile flow when EHBR rats were treated with PCN (Fig. 4Go, right panel). The effects of PB and PCN on bile flow, therefore, appear to be specific for the bile acid-independent bile flow mechanism, that is, specific for Mrp2.

The mechanism by which PB and PCN increase Mrp2 does not appear to be due to increased transcription. Despite increasing Mrp2 protein after PCN treatment for 4 days, Mrp2 mRNA was not significantly increased (Figs. 5–9GoGoGoGoGo). This is contrary to how PB and PCN induce other proteins, by activating gene transcription via the constitutive androstane receptor (CAR) and the pregnene-X-receptor (PXR), respectively. In addition, Mrp2 protein expression increased in a time-dependent manner, reaching significantly higher expression at day 3 and 4, yet Mrp2 mRNA expression did not show a similar increase, but rather remained relatively constant over time (Fig. 10Go). These results were surprising, because increased transcription seems to be the major mechanism by which drug-metabolizing enzyme inducers increase cytochromes P450, so it was suspected that PB and PCN would also increase Mrp2 protein by increasing Mrp2 mRNA expression. To verify the validity of these results, Mrp2 mRNA expression was quantified by two different experimental methods, RNase protection assay and bDNA assay, a sensitive method for detecting RNA (Hartley and Klaassen, 2000Go). Both assays demonstrated similar results, that the drug-metabolizing enzyme inducers used in this study do not upregulate Mrp2 mRNA. Therefore, PB and PCN must affect Mrp2 protein expression through posttranscriptional mechanisms, such as decreased turnover of the Mrp2 protein.

This conclusion appears at odds with the current dogma of transcription upregulation being the main mechanism by which drug-metabolizing enzyme inducers increase cytochromes P450. Yet, several studies demonstrate that Mrp2 protein expression does not always correlate with Mrp2 mRNA expression in rat. For example, Mottino et al. (2000) demonstrated that rat Mrp2 protein expression did not correspond with Mrp2 mRNA expression along the intestinal tract. Their findings are supported by Gotoh et al. studies (2000) in which intestinal excretion of dinitrophenol-glutathione (GS-DNP), a Mrp2 substrate, corresponds best with Mrp2 protein expression along the intestine, and not Mrp2 mRNA expression. In addition, this difference between rat Mrp2 protein and mRNA expression have been noted in other studies ranging from pathological events, such as liver regeneration (Gerloff et al., 1999Go) and extrahepatic cholestasis (Paulusma et al., 2000Go), to chemical treatment such as ethinyl estradiol (Trauner et al., 1997Go). Therefore, it is likely that the induction of rat Mrp2 protein expression by PB and PCN occurs in a nontraditional manner, i.e., not by transcriptional upregulation, but by posttranscriptional mechanisms. However, the fact remains that some studies have shown that Mrp2 mRNA and protein are upregulated by chemical inducers (Demeule et al., 1999Go; Fromm et al., 2000Go; Hagenbuch et al., 2001Go; Payen et al., 2001Go; Wielandt et al., 1999Go). Possible explanations for the differences in results may be due to interspecies variation, as well as divergence in response due to primary cultured hepatocytes versus whole-animal models.

Promoter analysis of rat and human Mrp2 genes supports the conclusion that PB and PCN do not regulate Mrp2 at the transcription level. Response elements for CAR and PXR nuclear receptors have not been identified in the human or rat Mrp2 gene 5`-flanking regions (Kauffmann and Schrenk, 1998Go; Tanaka et al., 1999Go). However, Courtois et al. (1999) reported finding a putative PXR response element arranged as an ER8 motif in the promoter region of the rat Mrp2 gene. This putative ER8 PXR response element is contrary to typical PXR response elements, consisting of either a DR3 motif or an ER6 motif (Waxman, 1999Go). Neither a DR3 nor an ER6 motif is present in the promoter region of the rat Mrp2 gene. Nonetheless, there is still a possibility that these response elements are further upstream of the reported promoter sequences. Kauffmann and Schrenk (1998) speculate that PB upregulates Mrp2 mRNA in rat hepatoma H4IIE cells in a region upstream of the sequenced rat Mrp2 promoter. There is also the possibility that hepatocyte culture conditions affect normal transcription of Mrp2, thus altering the normal physiological expression patterns of the Mrp2 gene. Clearly, further experiments need to be performed to elucidate how Mrp2 protein expression is regulated by PB and PCN.

In conclusion, this study shows that Mrp2 is the transporter responsible for increasing the rate of biliary SH transport and bile flow after induction with select drug-metabolizing enzyme inducers. PB and PCN increase Mrp2 protein in hepatocytes, where it localizes in the canalicular membrane, thus increasing biliary SH excretion rates and bile flow. In contrast, induction with PCN does not alter the biliary SH excretion rate or bile flow in Mrp2-null EHBR rats. Because Mrp2 protein expression is unaffected by 3MC or BaP, the rate of biliary SH excretion and bile flow are unaffected. Therefore, the results of this study support the hypothesis that that PB and PCN increase Mrp2 protein expression, which results in increased biliary SH excretion rates as well as bile flow, and that 3MC and BaP do not affect Mrp2 protein levels, thus producing no effect on biliary SH excretion rates or bile flow. It is important to point out that this study did not attempt to characterize the effects of these enzyme inducers on other efflux transporters, and with mounting evidence that other organic anion efflux transporters exist (Kurisu et al., 1991Go; Sathirakul et al., 1993Go), it is possible that these other efflux transporters were also affected by the enzyme inducers used in this study, albeit to a lesser degree than Mrp2.

This study demonstrates that drug-drug interactions can occur with a hepatic transporter protein, thus altering the disposition of endogenous and xenobiotic chemicals. These chemical-chemical interactions may lead to and explain existing alterations in systemic pharmacokinetics and biliary excretion of xenobiotics. This can be either beneficial or detrimental for organisms. For example, the enhanced excretion of GSH-conjugated xenobiotics (e.g., GS-arsenite [Kala et al., 2000Go]) by Mrp2 protein may be beneficial because it would decrease chemical half-life in the body, thus diminishing toxicity. In contrast, enhanced biliary excretion of divalent anionic antibiotics (e.g., ceftriaxone) by Mrp2 may cause a decrease in the therapeutic concentration of the antibiotic in the body.


    ACKNOWLEDGMENTS
 
We would like to thank the following people for their assistance: Dr. Kenneth McCarson and Michelle Winter for their assistance in cDNA probe designing, cDNA subcloning, and instruction in performing RNase protection assays; Dr. Dylan Hartley for the use of EAG15 primary antibody generated in our laboratory, for designing the Mrp2 bDNA assay probes, and for his instruction in performing the bDNA assay; and Dr. Nichole Vansell for performing the bile duct and blood vessel cannulations.

This research was supported by NIH grants ES-09716 and ES-03192, as well as NIH training grant ES-07079.


    NOTES
 
1 Present address: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. Back

2 To whom correspondence should be addressed. Fax: (913) 588-7501. E-mail: cklaasse{at}kumc.edu Back


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