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
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ABSTRACT |
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Key Words: drug-metabolizing enzyme inducers; Mrp2; glutathione; organic anion transport; EHBR.
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INTRODUCTION |
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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-carbonitrile (PCN) have been shown to significantly increase bile flow in rats (Klaassen, 1969
, 1970
; Lu et al., 1996
). These same drug-metabolizing enzyme inducers also increase the biliary excretion of GSH-derived sulfhydryls (SH) in rats (Ballatori and Clarkson, 1983
; Lu et al., 1996
; Madhu et al., 1993
; 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, 1985
; Klaassen, 1969
, 1970
). Despite the differential effects on biliary SH excretion, none of the drug-metabolizing enzyme inducers altered hepatic GSH concentrations (Madhu et al., 1993
), 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., 1989, 1994
). 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.
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MATERIALS AND METHODS |
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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 (250350 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 (250350 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 (250350 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 7.47.6], 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 12 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., 1999), 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., 1997). Briefly, competent DH5-
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., 1997) 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 1
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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 (#).
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RESULTS |
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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. 3). 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. 4
, left panel). In contrast to SD rats, PCN did not increase bile flow in EHBR rats (Fig. 4
, right panel).
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DISCUSSION |
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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 5, respectively). The increase in Mrp2 protein corresponded with an increase in Mrp2 localization to the canalicular membrane (Fig. 6B
). 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. 1
, 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. 2
, lower left panel). In addition, treatment of EHBR rats with PCN did not increase the biliary SH excretion rate (Fig. 2
, lower right panel). Similar effects were observed when EHBR rats were treated with PB (Lu et al., 1996
). 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, 1992; Wielandt et al., 1999
). Bile acid-independent bile flow accounts for approximately 40% of total bile flow (Oude Elferink et al., 1989
), most of which is likely due to the biliary excretion of GSH via Mrp2. Bile flow is markedly depressed when Mrp2 is nonfunctional (Fig. 4
, 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. 4
, 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. 59). 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. 10
). 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, 2000
). 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., 1999) and extrahepatic cholestasis (Paulusma et al., 2000
), to chemical treatment such as ethinyl estradiol (Trauner et al., 1997
). 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., 1999
; Fromm et al., 2000
; Hagenbuch et al., 2001
; Payen et al., 2001
; Wielandt et al., 1999
). 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, 1998; Tanaka et al., 1999
). 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, 1999
). 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., 1991; Sathirakul et al., 1993
), 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., 2000]) 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.
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ACKNOWLEDGMENTS |
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This research was supported by NIH grants ES-09716 and ES-03192, as well as NIH training grant ES-07079.
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NOTES |
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2 To whom correspondence should be addressed. Fax: (913) 588-7501. E-mail: cklaasse{at}kumc.edu
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