Regulation of Rat Multidrug Resistance Protein 2 by Classes of Prototypical Microsomal Enzyme Inducers That Activate Distinct Transcription Pathways

David R. Johnson,1 and Curtis D. Klaassen,2

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

Received November 5, 2001; accepted February 8, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microsomal enzyme inducers are capable of modulating biliary excretion of organic anions and bile flow, but the mechanism for modulation is unknown. Therefore, this study was designed (1) to determine the effects of microsomal enzyme inducers on protein and mRNA expression of rat multidrug resistance protein 2 (Mrp2), a canalicular organic anion transporter; and (2) to determine whether classes of microsomal enzyme inducers affect Mrp2 expression in similar manners, thus implying specific nuclear receptor-activated transcription pathways. Male Sprague-Dawley rats were treated with aryl hydrocarbon (Ah) receptor (AhR) ligands/cytochrome P450 (CYP) 1A inducers, constitutive androstane receptor (CAR) ligands/CYP2B inducers, pregnane-X receptor (PXR) ligands/CYP3A inducers, peroxisomal proliferator-activating receptor-{alpha} (PPAR{alpha}) ligands/CYP4A inducers, antioxidant/electrophile response element (ARE/EpRE) ligands, CYP2E1 inducers, or control vehicle. Mrp2 protein levels were significantly increased by all 3 PXR ligands/CYP3A inducers (pregnenolone-16{alpha}-carbonitrile [PCN], spironolactone [SP], and dexamethasone [DEX]) and by both ARE/EpRE ligands (ethoxyquin [EQ] and oltipraz [OPZ]). In contrast, PPAR{alpha} ligands/CYP4A inducers (clofibric acid [CLOF], di-(2-ethylhexyl)phthalate [DEHP], and perfluorodecanoic acid [PFDA]) tended to decrease Mrp2 protein levels. Mrp2 mRNA expression was not significantly affected by any microsomal enzyme inducer, though ARE/EpRE ligands tended to upregulate Mrp2 mRNA. In summary, this study demonstrates that Mrp2 protein levels are significantly increased by PXR ligands/CYP3A inducers and ARE/EpRE ligands, and appear to be decreased by PPAR{alpha} ligands/CYP4A inducers by posttranscriptional mechanisms. Furthermore, these data suggest that measuring Mrp2 mRNA is not a good indicator for Mrp2 protein expression in vivo.

Key Words: aryl hydrocarbon receptor (AhR); antioxidant/electrophile-response element (ARE/EpRE); branched DNA (bDNA); constitutive androstane receptor (CAR); cytochrome P450 (CYP); multidrug resistance protein 2 (Mrp2); peroxisomal proliferator-activating receptor-{alpha} (PPAR{alpha}); pregnane-X receptor (PXR).


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microsomal enzyme inducers are structurally diverse chemicals that upregulate a wide array of hepatic enzymes (Fig. 1Go). They are most noted for upregulating a superfamily of Phase I drug-metabolizing enzymes called cytochromes P450 (CYP), which are important for the bioactivation and biotransformation of lipophilic chemicals to more water soluble structures. Microsomal enzyme inducers upregulate other drug-metabolizing enzymes as well, such as Phase II drug-metabolizing enzymes UDP-glucuronosyltransferases (UGT) and glutathione S-transferases (GST) that are important for adding cosubstrates to xenobiotics to further enhance the chemicals` water solubility, and thus cellular elimination. Upregulation of CYPs and other drug-metabolizing enzymes by microsomal enzyme inducers generally occurs at the transcription level, resulting in subsequent increases in CYP protein levels and functional activities. Previously, microsomal enzyme inducers have been classified according to which subclass of CYP they upregulate. Recently though, microsomal enzyme inducers of CYPs and other drug-metabolizing enzymes have also been classified as ligands for nuclear receptors and DNA enhancer elements that facilitate the transcriptional activation of these genes. For example, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and other CYP1A inducers bind the aryl hydrocarbon (Ah) receptor (AhR), which, after the intricate addition and removal of cytosolic and nuclear cofactors, binds the xenobiotic response element (XRE) and activates CYP1A transcription (Wilson and Safe, 1998Go). Ethoxyquin (EQ) and other microsomal enzyme inducers that are involved in oxidative stress upregulating of Phase II drug-metabolizing enzymes by binding to the antioxidant/electrophile response element (ARE/EpRE), thus activating transcription of GSTs, UGTs, epoxide hydrolase (EH), and quinone oxidoreductase (QR) (Buetler et al., 1995Go,1996Go; Vargas et al., 1998Go). In contrast to other CYP subfamilies, CYP2E1 inducers such as isoniazid (INH) increase CYP2E1 protein by several different modes of regulation, including mRNA stabilization and decreased protein degradation (Damme et al., 1996Go; Park et al., 1993Go; Tsutsumi et al., 1993Go).



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FIG. 1. Chemical structures of prototypical microsomal enzyme inducers. Inducers were classified according to cytochrome P450 (CYP) induction and transcription activation pathways. Ah receptor (AhR) ligands/CYP1A inducers: 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 3, 3`, 4, 4`, 5-pentachlorobiphenyl (PCB 126), indole-3-carbinol (I-3-C), ß-naphthoflavone (BNF); constitutive androstane receptor (CAR) ligands/CYP2B inducers: phenobarbital (PB), 2, 2`, 4, 4`, 5-pentachlorobiphenyl (PCB 99), diallyl sulfide (DAS); pregnane-X-receptor (PXR) ligands/CYP3A inducers: pregnenolone-16{alpha}-carbonitrile (PCN), dexamethasone (DEX), spironolactone (SP); peroxisomal proliferator activating receptor-{alpha} (PPAR{alpha}) ligands/CYP4A inducers: clofibric acid (CLOF), di-(2-ethylhexyl)phthalate (DEHP), perfluorodecanoic acid

 
Transporter proteins are also targets of microsomal enzyme inducers. Several studies using human and rodent models have demonstrated that transporter proteins, such as MDR1-encoding P-glycoprotein (Pgp), organic anion transporting polypeptide 2 (oatp2), and multidrug resistance protein (Mrp) transporter family members, are upregulated by microsomal enzyme inducers (Cherrington, 2002; Gant et al., 1992Go; Geick et al., 2001Go; Guo et al., 2002Go; Johnson et al., 2002; Rausch-Derra, 2001). Alterations in transporter expression can lead to dramatic changes in chemical uptake and elimination from hepatocytes. Multidrug resistance protein 2 (Mrp2; ABCC2) is a 200-kDa transporter protein located on the canalicular membrane of hepatocytes (Buchler et al., 1996Go). Mrp2 is primarily responsible for biliary elimination of nonbile acid organic anions, most of which have been conjugated by Phase II drug-metabolizing enzymes (Oude-Elferink and Jansen, 1994Go). The absence of Mrp2 leads to dramatically impaired biliary excretion of nonbile acid organic anions and hyperbilirubinemia, resulting in Dubin-Johnson syndrome in humans (Javitt et al., 1978Go; Paulusma et al., 1997Go). Previous studies have indicated that Mrp2 expression is induced by various individual microsomal enzyme inducers such as dexamethasone (Courtois et al., 1999aGo), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T; Weilandt et al., 1999), and oltipraz (Courtois et al., 1999bGo). However, it is uncertain whether classes of microsomal enzyme inducers affect Mrp2 expression in similar manners. Furthermore, it is also unknown whether there are any specific mechanisms by which microsomal enzyme inducers affect Mrp2 expression.

Therefore, the purpose of this study was 2-fold: first, to comprehensively analyze the effects of 18 prototypical microsomal enzyme inducers on rat Mrp2 protein expression, and second, to determine whether the effects of microsomal enzyme inducers on Mrp2 protein were due to the upregulation of Mrp2 mRNA expression. Furthermore, the data will provide essential information as to whether classes of microsomal enzyme inducers affect rat Mrp2 expression in similar manners, thus implicating the potential roles of specific ligand-activated transcription factor pathways. Microsomal enzyme inducers were divided according to the following classes of transcription factor ligands/CYP inducers (Fig. 1Go): AhR ligands/CYP1A inducers (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD], indole-3-carbinol [I-3-C], ß-naphthoflavone [BNF], and 3,3`4,4`,5-pentachlorobiphenyl [PCB 126]), constitutive androstane receptor (CAR) ligands/CYP2B inducers (phenobarbital [PB], 2,2`,4,4`,5-pentachlorobiphenyl [PCB 99], and diallyl sulfide [DAS]), pregnane-x receptor (PXR) ligands/CYP3A inducers (pregnenolone-16{alpha}-carbonitrile [PCN], spironolactone [SP], and dexamethasone [DEX]), peroxisomal proliferator-activating receptor-{alpha} (PPAR{alpha}) ligands/CYP4A inducers (clofibric acid [CLOF], di-(2-ethylhexyl)phthalate [DEHP], and perfluorodecanoic acid [PFDA]), antioxidant/electrophile response element (ARE/EpRE) ligands (ethoxyquin [EQ] and oltipraz [OPZ]), and CYP2E1 inducers (isoniazid [INH], acetylsalicylic acid [ASA], and streptozotocin [STZ]). Results from this study indicate that rat Mrp2 protein is significantly increased by PXR and ARE/EpRE ligands, and PPAR{alpha} ligands tend to decrease Mrp2 protein. In contrast, rat Mrp2 mRNA expression is not significantly affected by any microsomal enzyme inducer used in this study, thus suggesting that rat Mrp2 protein is increased by posttranscriptional modifications that are regulated by PXR and ARE/EpRE ligands.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
ß-Naphthoflavone (BNF), indole-3-carbinol (I-3-C), diallyl sulfide (DAS), phenobarbital (PB), spironolactone (SP), dexamethasone (DEX), clofibric acid (CLOF), di-(2-ethylhexyl)phthalate (DEHP), perfluorodecanoic acid (PFDA), ethoxyquin (EQ), oltipraz (OTP), acetylsalicylic acid (ASA), isoniazid (INH), and streptozotocin (STZ) were purchased from Sigma Chemical Co. (St. Louis, MO). Pregnenolone-16{alpha}-carbonitrile (PCN) was synthesized from 16-dehydropregnenolone precursor (Steraloids, Newport, RI) according to Sonderfan and Parkinson (1988). PCB 126 (3,3`,4,4`,5-pentachlorobiphenyl) and PCB 99 (2,2`,4,4`,5-pentachlorobiphenyl) were purchased from Accustandard (New Haven, CT). TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) was generously provided by Dr. Karl Rozman (Kansas City, KS). 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). Chemicals were handled and disposed in accordance to the guidelines set forth by the Chemical Safety Office at the University of Kansas Medical Center.

Treatment of rats with microsomal enzyme inducers.
Male Sprague-Dawley rats (Sasco, Kingston, NY) were housed at 250C with a 12-h light-dark cycle according to AAALAC guidelines. Rats received water and food ad libitum, and were allowed to acclimate to their environment for 5 days before the study began. Rats (~225 g) were subjected to the following microsomal enzyme-inducing chemicals: TCDD (3.9 µg/kg, corn oil, ip, 1 day), I-3-C (56 mg/kg, corn oil, po), BNF (100 mg/kg, corn oil, ip), PCB 126 (40 µg/kg, corn oil, po, 7 days), PB (80 mg/kg, saline, ip), PCB 99 (16 mg/kg, corn oil, po, 7 days), DAS (500 mg/kg, corn oil, ip), PCN (50 mg/kg, corn oil, ip), SP (75 mg/kg, corn oil, ip), DEX (50 mg/kg, corn oil, ip), CLOF (200 mg/kg, saline, pH 7, ip), DEHP (1200 mg/kg, corn oil, po), PFDA (40 mg/kg, corn oil, ip, 1 day), EQ (150 mg/kg, corn oil, po), OPZ (150 mg/kg, corn oil, po), INH (200 mg/kg, saline, ip), ASA (500 mg/kg, corn oil, po), STZ (100 mg/kg, 100 mM sodium citrate, pH 4.5, ip, 1 day), corn oil (ip), corn oil (po), and saline (ip). All animals were treated for 4 days unless otherwise noted, and injections were in a volume of 5 ml/kg. On day 5, livers were removed, snap frozen in liquid nitrogen, and stored at –800C.

Total membrane protein preparation.
Liver protein samples were prepared as previously described (Johnson et al., 2002). Protein concentrations were determined using a bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL).

Western blot analysis.
Protein samples (30 µg/well) were mixed with sample buffer, loaded onto a 7.5% SDS-polyacrylamide gel, and run electrophoretically on a Western blot apparatus (Gibco BRL, Gaithersburg, MD). Protein samples were not boiled prior to loading. Each gel contained a loading control that served as a basis for internal comparison. Protein was transferred overnight to nitrocellulose membranes (Novex, San Diego, CA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBS/T). The membranes were then incubated overnight at 40C with a 1:4000 dilution of anti-Mrp2 primary antibody EAG15 (generated in our laboratory according to Buchler et al., 1996Go) in 1% nonfat dry milk in TBS/T. Membranes were washed with TBS/T and then incubated at room temperature with donkey, antirabbit IgG-horseradish peroxidase-linked secondary antibody (Amersham, Arlington Heights, IL), diluted in TBS/T containing 1% nonfat dry milk. Mrp2 protein was detected using an enhanced chemiluminescence (ECLTM) kit (Amersham).

Isolation of total RNA.
Total RNA was isolated using RNAzol B reagent (Tel-Test Inc., Friendswood, TX) as per the manufacturer's protocol. Each RNA pellet was resuspended in 0.2 ml DEPC-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) gel electrophoresis.

Quantigene TM branched DNA (bDNA) signal amplification assay.
Probe sets for detection of all CYPs (CYP1A1, CYP2B1/2, CYP3A1/23, CYP4A2/3, & CYP2E1) and QR, as well as Mrp2-specific oligonucleotide probes, have been previously described (Hartley and Klaassen, 2000Go; Johnson et al., 2002). CYP, QOR, and Mrp2-specific oligonucleotide probe sets (i.e., blocker probes, capture probes, and label probes) were combined and diluted to 50 fmol/µl in the lysis buffer supplied in the QuantigeneTM bDNA Signal Amplification Kit (Bayer Diagnostics, East Walpole, MA). The bDNA assay was run as previously described (Johnson et al., 2002). The enzymatic reaction was allowed to proceed for 30 min at 370C, 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 (*).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytochromes P450 and QR mRNA expression after microsomal enzyme induction.
Prior to analyzing the effects of the 18 microsomal enzyme inducers on Mrp2 expression, total RNA was analyzed by bDNA signal amplification assay to determine mRNA expression of the major chemically inducible CYP subfamilies and QR after microsomal enzyme inducer treatment (Fig. 2Go). CYP1A inducers TCDD, BNF, and PCB 126 upregulated CYP1A1 mRNA by 280- to 390-fold. CYP1A inducer I-3-C upregulated CYP1A1 mRNA, but only by approximately 5-fold. All CYP2B1 inducers upregulated CYP2B1 mRNA by approximately 50- to 70-fold. CYP3A1/23 mRNA was upregulated by approximately 12- to 30-fold by CYP3A ligands. CYP4A2/3 mRNA was upregulated by approximately 10-fold by all CYP4A inducers. ARE/EpRE ligands upregulated QR mRNA by 5- to 6-fold, and CYP2E1 inducers upregulated CYP2E1 mRNA by only 3 to 5 fold. These data correspond with the upregulation of CYP mRNAs in response to microsomal enzyme inducer treatment shown by Hartley and Klaassen (2000), further validating the use of bDNA technology to assess mRNA expression.



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FIG. 2. Effects of prototypical microsomal enzyme inducers on mRNA of hallmark drug-metabolizing enzymes. Rats were treated with microsomal enzyme inducers as described in Materials and Methods. Total RNA was isolated, pooled (n = 5), and analyzed by QuantigeneTM branched DNA (bDNA) signal amplification assay. See Figure 1Go for microsomal enzyme-inducer abbreviations.

 
Mrp2 protein expression after microsomal enzyme induction.
Mrp2 protein expression was determined by Western blot analysis using an Mrp2-specific primary antibody, EAG15 (Fig. 3Go). Two CYP1A inducers tended to increase Mrp2 protein levels (TCDD: 83%; I-3-C: 130%), whereas the other 2 CYP1A inducers tended to decrease Mrp2 protein levels (BNF: 35%; PCB 126: 21%). Two CYP2B inducers tended to increase Mrp2 protein (PB: 77%; DAS: 114%), though neither was statistically significant. The effect of PB on Mrp2 expression seen in this study confirms previous reports from other laboratories (Hagenbuch et al., 2001Go; Ogawa et al., 2000Go). All CYP3A inducers (PCN, SP, and DEX) significantly increased Mrp2 protein levels (approximately 340, 230, and 520%, respectively, above control). Interestingly, all CYP4A inducers (CLOF, DEHP, and PFDA) tended to decrease Mrp2 protein levels approximately 60%, though none were statistically significant. Both ARE/EpRE ligands (EQ and OPZ) significantly increased Mrp2 protein levels above control (~200 and 270%, respectively). CYP2E1 inducers (INH, ASA, STZ) tended to increase Mrp2 protein approximately 90% above control, though none were statistically significant.



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FIG. 3. Effects of microsomal enzyme inducers on Mrp2 protein expression. Rats were treated with microsomal enzyme inducers as described in Materials and Methods. Homogenate protein was isolated and separated by 7.5% SDS-PAGE (30 µg protein/lane). (A) Representative Western blots of Mrp2 protein expression after microsomal enzyme-inducer treatment. Mrp2 protein was detected by the polyclonal antibody EAG15 at approximately 200 kDa. (B) Graphical results of Mrp2 protein expression after microsomal enzyme induction. See Figure 1Go for microsomal enzyme-inducer abbreviations.

 
Mrp2 mRNA expression after microsomal enzyme induction.
Mrp2 mRNA expression after treatment with the various microsomal enzyme inducers was determined by QuantigeneTM bDNA signal amplification assay (Fig. 4Go). None of the microsomal enzyme inducers significantly upregulated Mrp2 mRNA. PCN, EQ, OPZ, and STZ tended to upregulate Mrp2 mRNA (56%, 42%, 75%, and 31%, respectively), though none were statistically significant. In contrast, BNF, PB, DEX, and PFDA tended to downregulate Mrp2 mRNA (48, 33, 43, and 43%, respectively), though these, too, were not statistically significant.



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FIG. 4. Effects of microsomal enzyme inducers on Mrp2 mRNA expression. Rats were treated with microsomal enzyme inducers as described in Materials and Methods. Total RNA was isolated and analyzed by QuantigeneTM branched DNA (bDNA) signal amplification assay. See Figure 1Go for microsomal enzyme inducer abbreviations. Dashed line indicates Mrp2 mRNA level of control samples. All data are given as mean ± SE for 5 animals in each group. *Statistical significance from control values (p < 0.05).

(PFDA);antioxidant/electrophile response element (ARE/EpRE) ligands: ethoxyquin (EQ), oltipraz (OTP); and CYP2E1 inducers: isoniazid (INH), acetylsalicylic acid (ASA), and streptozotocin (STZ).

Dashed line indicates the Mrp2 protein level of control samples. All data are given as mean ± SE for 5 animals in each group. *Statistical significance from control values (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microsomal enzyme inducers have been well characterized in terms of their effects on Phases I and II drug-metabolizing enzymes. However, microsomal enzyme inducers have not been well characterized in regard to their effects on "Phase III" transporters that pump xenobiotics and biotransformed chemicals out of cells. The purpose of this study, therefore, was to generate an extensive profile of Mrp2 protein inducibility by microsomal enzyme inducers. In addition, this study examined whether Mrp2 protein modulation by microsomal enzyme inducers was associated with corresponding changes in Mrp2 mRNA levels. Furthermore, this study examined whether classes of microsomal enzyme inducers affect Mrp2 expression in a similar manner, suggesting a common molecular mechanism (i.e., nuclear receptors and DNA enhancer elements) by which Mrp2 expression may be regulated.

All 3 CYP3A inducers significantly increased Mrp2 protein expression; PCN, SP, and DEX increased Mrp2 protein expression approximately 340, 230, and 520%, respectively, above control (Fig. 3Go). The inductive effects of PXR ligands on Mrp2 protein expression correspond with what is known about their inductive effects on biliary excretion of organic anions, such as GSH, DBSP, GS-BSP, and bilirubin (Faed et al., 1984Go; Klaassen, 1970Go; Madhu et al., 1993Go; Morisoli et al., 1982Go; Pellegrino et al., 1982Go; Zsigmond and Solymoss, 1972Go). In contrast to Mrp2 protein, none of the PXR ligands/CYP3A inducers significantly upregulated Mrp2 mRNA expression (Fig. 4Go). PXR ligands/CYP3A inducers produce a unique phenomenon in that all 3 inducers significantly increase Mrp2 protein expression without significantly affecting Mrp2 mRNA expression. Furthermore, PCN increases Mrp2 protein, but not Mrp2 mRNA, in a time-dependent manner, so up regulation of Mrp2 mRNA at earlier time points was not overlooked (Johnson et al., 2002). Thus, the CYP3A inducers used in this experiment—all of which were rat PXR ligands (Jones et al., 2000Go)—appear to increase Mrp2 protein expression by a PXR-mediated posttranscriptional mechanism.

There appears to be a discrepancy between the effects of CYP3A4 inducers/PXR ligands on Mrp2 expression in vitro and in vivo. Previous studies have demonstrated that both human and rodent CYP3A4 inducers upregulate human MRP2 and rodent Mrp2 mRNA expression, respectively, through PXR (also called SXR in humans) (Courtois et al., 1999aGo; Dussault et al., 2001Go; Kast et al., 2001Go). Yet the regulation of Mrp2 by CYP3A4 inducers/PXR ligands appears to be more complex in vivo. In rhesus monkeys, rifampicin, a human PXR ligand, upregulates MRP2 mRNA, but has almost no effect on MRP2 protein (Kauffmann et al., 1998Go). Similarly, this study demonstrated that PXR ligands induce Mrp2 protein, but not Mrp2 mRNA. The differences between in vitro and in vivo induction of Mrp2 expression may be due to the delivery of drug to the target organ. Cell culture allows measurement of effects of chemicals at specific concentrations on target cells, yet in vivo studies also must take into account the complex disposition of chemicals in the body in addition to the effects of chemicals on the target cells. Therefore, target cells may not be exposed to the complete dosage of chemical due to systemic uptake and clearance of chemicals from the body. Thus, CYP3A4 inducers/PXR ligands may directly upregulate rat Mrp2 mRNA expression in cell culture, but in whole animals, rat Mrp2 protein is induced not by the PXR-mediated activation of the Mrp2 gene, but by a PXR-mediated posttranscriptional mechanism.

ARE/EpRE ligands also significantly induced Mrp2 expression. Both EQ and OPZ significantly increased Mrp2 protein approximately 200% and 270%, respectively, above control (Fig. 3Go). This data complements studies in which quercetin and t-butylhydroquinone (tBHQ), two additional ARE/EpRE ligands, induce Mrp2 protein expression in Caco-2 cells (Bock et al., 2000Go). Both EQ and OPZ tended to also upregulate Mrp2 mRNA (approximately 40 and 80% above control, respectively), though neither were statistically significant (Fig. 4Go). Therefore, ARE/EpRE ligands increase Mrp2 protein expression, but not Mrp2 mRNA expression, and that the increase in Mrp2 protein by EQ and OPZ may be largely due to an ARE/EpRE-mediated mechanism independent of the Mrp2 gene. This is supported by the fact that no ARE/EpRE consensus site has yet been found in the rat Mrp2 gene promoter (Kauffmann and Schrenk, 1998Go). An apparent discrepancy exists between the data from this experiment and a study by Courtois et al. (1999b) in which OPZ did not increase Mrp2 protein when administered to rats in the diet (0.075% (w/w)). The differences may be attributed to the smaller dosage of OPZ ingested by the rats when administered in feed versus a higher dosage when administered by gavage. Thus, higher concentrations of OPZ may be needed to increase Mrp2 protein in vivo, as seen in this study.

Another surprising finding was the relative inability of PB and other CYP2B inducers/CAR ligands to significantly affect Mrp2 expression. PB increases biliary excretion of numerous organic anions that are substrates of Mrp2 (Faed et al., 1984Go; Klaassen, 1970Go; Madhu et al., 1993Go; Morisoli et al., 1982Go; Pellegrino et al., 1982Go; Zsigmond and Solymoss, 1972Go) and a previous report demonstrates that PB also significantly increases Mrp2 protein (Johnson et al., 2002). Yet, this study and others have shown that PB has only mild inductive effects on Mrp2 expression (Hagenbuch et al., 2001Go; Ogawa et al., 2000Go), most likely due to the CAR-mediated activation of the ER8 response element in the Mrp2 gene promoter (Kast et al., 2001Go). Perhaps this modest increase in Mrp2 protein expression by CYP2B inducers/CAR ligands is enough to have significant effects on Mrp2 function. An alternative postulation is that PB and other CYP2B inducers/CAR ligands primarily induce other canalicular transporters that have the same organic anion substrates as Mrp2 (Jansen et al., 1987 Jansen et al., 1993; Johnson and Klaassen, 2002Go; Kurisu et al., 1991; Sathirakul et al., 1993), thus explaining the increase in organic anion elimination into bile. In contrast, PB can also decrease biliary excretion of organic anions (Gregus et al., 1990Go; Studenberg and Brouwer, 1992Go; Watkins and Klaassen, 1982Go). Recently, Cherrington et al. (2002) demonstrated that PB and other CAR ligands upregulate mRNA of multidrug resistance protein 3 (Mrp3), a sinusoidal transporter protein that is a member of the MRP subfamily of transporters. Because Mrp2 and Mrp3 share similar substrates, it is possible that the increased Mrp3 results in higher sinusoidal efflux and lower biliary efflux of organic anions.

Other classes of enzyme inducers used in this experiment did not significantly affect Mrp2 protein expression, and, more importantly, the inducer within these other classes did not exhibit related effects on Mrp2 protein expression. A notable exception was the class of CYP4A inducers/PPAR{alpha} ligands. Though not statistically significant, all 3 CYP4A inducers/PPAR{alpha} ligands tended to decrease Mrp2 protein approximately 60% below control (Fig. 3Go). This was supported by the fact that CLOF decreases biliary excretion of GS-BSP, an Mrp2 substrate (James and Ahokas, 1992Go). In addition to Mrp2 protein, none of these other inducer classes significantly affected Mrp2 mRNA expression, nor did the inducers within the other classes exhibit related effects on Mrp2 mRNA expression. Therefore, the data in this study suggest that the AhR, CAR, and PPAR{alpha} are not molecular mechanisms by which microsomal enzyme inducers affect Mrp2 expression in vivo. This conclusion is supported by the findings that the xenobiotic response element (XRE), the CAR response element (PBREM), and the PPAR response element (PPRE) are either absent or nonfunctional in the 5`-flanking region of the rat Mrp2 gene (Denson et al., 2000Go; Kauffmann and Schrenk, 1998Go).

In summary, PXR ligands/CYP3A inducers (PCN, SP, and DEX) and ARE/EpRE ligands (EQ and OPZ) significantly increase Mrp2 protein. CYP4A inducers (CLOF, DEHP, and PFDA), however, tend to decrease Mrp2 protein, though not significantly. In contrast to Mrp2 protein, none of the microsomal enzyme inducers affect Mrp2 mRNA, though ARE/EpRE ligands (EQ and OPZ) tended to upregulate Mrp2 mRNA. Thus, PXR, PPAR{alpha}, and ARE/EpRE ligands appear to regulate Mrp2 protein expression by posttranscriptional mechanisms. This conclusion appears at odds with the current dogma of transcriptional upregulation being the main mechanism by which drug-metabolizing enzyme inducers increase CYPs. Yet, several studies demonstrate that Mrp2 protein expression does not always correlate with Mrp2 mRNA expression in rat. For example, rat Mrp2 protein expression does not correspond with Mrp2 mRNA expression along the intestinal tract (Mottino et al., 2000Go), but, more importantly, intestinal excretion of dinitrophenol-glutathione (GS-DNP), an Mrp2 substrate, corresponds best with Mrp2 protein expression, and not Mrp2 mRNA expression, along the intestinal tract (Gotoh et al., 2000Go). In addition, differences 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 regulation of rat Mrp2 protein expression in vivo occurs in a nontraditional manner, i.e., not by transcriptional upregulation but by posttranscriptional mechanisms. The data from this study also indicate that the measurement of rat Mrp2 mRNA after microsomal enzyme induction is not an adequate indication of Mrp2 protein expression and function. Therefore, additional protein screening technologies should be employed in order to adequately assess the effects of chemicals on Mrp2 protein expression and function.


    ACKNOWLEDGMENTS
 
This work was supported by NIH grants ES-09716 and ES-03192, as well as NIH training grant ES-07079. The authors would like to thank Susan Buist, Ning Li, Dr. Grace Guo, Dr. Nichole Vansell, Dr. Dylan Hartley, and Dr. Nathan Cherrington for their excellent technical expertise. We would like to further thank Dr. Nathan Cherrington for his assistance in performing the bDNA signal amplification assay.


    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. Email: cklaasse{at}kumc.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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