Introduction

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For some drugs and xenobiotics to be biotransformed by the phase I and II drug-metabolizing enzymes, they have to be transported into the liver cell. A family of transporter proteins, known as the organic anion transporting polypeptides (oatp), participates in the transport of drugs and xenobiotics into liver. However, whether the drug-metabolizing enzyme inducers regulate these hepatic uptake transporters has not been studied thoroughly. Whereas activation of drug transporters is beneficial in cases when higher clearance rate is desired, it will be detrimental when effective therapeutic concentrations need to be maintained.

This laboratory has shown that phenobarbital and PCN, which induce the drug-metabolizing enzymes via the CAR and PXR receptor, respectively, enhance the plasma disappearance and biliary excretion of cardiac glycosides (Klaassen and Plaa, 1968; Klaassen, 1970a, 1970b, 1974a). In contrast, 3-methylcholanthrene and 3,4-benzo[a]pyrene, which induce drug-metabolizing enzymes via the Ah receptor, did not increase the biliary excretion of the cardiac glycosides (Klaassen, 1970b, 1974a, 1974b). It was later shown that phenobarbital and PCN, but not 3-methylcholanthrene and 3,4-benzo[a]pyrene, increased the uptake of cardiac glycosides into isolated hepatocytes (Eaton and Klaassen, 1979), indicating a specificity in the ability of drug-metabolizing enzyme inducers to enhance the hepatic uptake of xenobiotics.

Recent advances in cloning transporters during the last decade make the in-depth investigation of the hepatic uptake of xenobiotics possible. Rat oatp2, a recently cloned organic anion transporting polypeptide, is mainly localized to the sinusoidal membranes of hepatocytes (Noe et al., 1997; Abe et al., 1998; Gao et al., 1999; Reichel et al., 1999). Oatp2 has been shown to mediate the uptake of many structurally unrelated compounds, with a very high affinity to cardiac glycosides (Noe et al., 1997; Abe et al., 1998; Kakyo et al., 1999; Reichel et al., 1999). Rats treated with phenobarbital showed increased levels of oatp2 protein, and rats treated with PCN exhibited increased levels of both protein and mRNA (Rausch-Derra et al., 2001).

Marked progress on the mechanisms by which the induction of drug-metabolizing enzymes occurs has been made during the last few years. Several ligand-activated transcription factor pathways involved in the induction of some phase I and II enzymes have been discovered (Waxman, 1999; Fuhr, 2000). For example, the AhR mediates the induction of cytochrome P450 1A1/2 (CYP1A1/2; Safe and Krishnan, 1995), some glutathione S-transferases (Hayes and Pulford, 1995) and some UDP-glucuronosyltransferases (Munzel et al., 1998). The CAR mediates the induction of CYP2B6, 2C9, 3A4, and some UDP-glucuronosyltransferases (Sueyoshi and Negishi, 2001). The PXR mediates the induction of CYP3A (Kliewer et al., 1998; Lehmann et al., 1998; Jones et al., 2000). The PPAR mediates the induction of CYP4A (Corton et al., 2000), and the ARE/EpRE mediates the induction of quinone reductase (Buetler et al., 1995; Talalay et al., 1995).

Therefore, the present study aimed to determine whether a much broader scale of drug-metabolizing enzyme inducers regulates rat oatp2. In the current study, five groups of chemicals that are known to regulate phase I and II drug-metabolizing enzymes via ligand-activated transcription factor pathways were administered to male Sprague-Dawley rats. Their effects on oatp2 protein levels were determined by Western blot analysis. To determine whether alterations in oatp2 protein levels were associated with concomitant alterations in oatp2 mRNA levels, the branched DNA technique (bDNA) was used to determine the levels of oatp2 mRNA (Hartley and Klaassen, 2000; Rausch-Derra et al., 2001). The level of oatp2 mRNA was further determined by Northern blot analysis after treatment of the rats with PXR ligands. Moreover, heterogeneous nuclear RNA (hnRNA) RT-PCR was used to determine whether the alterations in the mRNA levels were due to alterations in transcription of the oatp2 gene by de novo synthesis of hnRNA after treatment of the rats with PXR ligands.

	Materials and Methods

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Chemicals. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was a gift from Dr. Karl Rozman (University of Kansas Medical Center, KS). Oltipraz was a gift from Dr. R. Lubet (National Cancer Institute, Bethesda, MD). Polychlorinated biphenyl 99 (PCB 99) and polychlorinated biphenyl 126 (PCB 126) were obtained from AccuStandard (New Haven, CT). All other chemicals, unless otherwise indicated, were purchased from Sigma-Aldrich Co. (St. Louis, MO).

Animals and Treatment. Male Sprague-Dawley (SD) rats (Harlan Sprague-Dawley Inc., Indianapolis, IN), weighing approximately 225 g, were used throughout the study except for the rats administered PCB 126 and PCB 99, which were from Sasco Laboratories, Inc. (Wilmington, MA). Five animals as a group were administered one of the following chemicals: AhR ligands: TCDD (3.9 µg/kg, 1 day, i.p. in corn oil), indole-3-carbinol (I-3-C, 56 mg/kg, 4 days, gavage in corn oil), -naphthoflavone (BNF, 100 mg/kg, 4 days, i.p. in corn oil), and PCB 126 (40 µg/kg, 7 days, gavage in corn oil); CAR ligands: phenobarbital (PB, 80 mg/kg, 4 days, i.p. in saline), diallyl sulfide (DAS, 500 mg/kg, 4 days, i.p. in corn oil), and PCB 99 (16 mg/kg, 7 days, gavage in corn oil); PXR ligands: pregnenolone-16-carbonitrile (PCN, 50 mg/kg, 4 days, i.p. in corn oil), spironolactone (SPIRO, 75 mg/kg, 4 days, i.p. in corn oil), and dexamethasone (DEX, 50 mg/kg, 4 days, i.p. in corn oil); PPAR ligands: clofibric acid (CLOF, 200 mg/kg, 4 days, i.p. in saline), diethylhexylphthalate (DEHP, 1200 mg/kg, 4 days, gavage in corn oil), and perflurodecanoic acid (PFDA, 40 mg/kg, 1 day, killed on day 5, i.p. in corn oil); and ARE/EpRE activators: butylated hydroxyanisole (BHA, 75 mg/kg, 4 days, gavage in corn oil), ethoxyquin (EQ, 150 mg/kg, 4 days, gavage in corn oil), and oltipraz (OLTI, 150 mg/kg, 4 days, gavage in corn oil). All injections were at a volume of 5 ml/kg. Livers were removed on day 5, snap-frozen in liquid nitrogen, and stored at 80°C.

Preparation of Membranes. Liver membrane samples were prepared by homogenizing liver samples from each individual animal (0.1-0.5 g) in 10 ml of buffer (0.25 M sucrose, 10 mM Tris-HCl [pH 7.5], 25 µg/ml leupeptin, 50 µg/ml aprotinin and antipain, 0.5 µg/ml pepstatin, and 40 µg/ml phenylmethylsulfonyl fluoride), using a Teflon pestle and a 15-ml glass homogenizing vessel (Wheaton, Millville, NJ). The homogenate was centrifuged at 4°C at 100,000g for 1 h. The pellet was then resuspended in 0.3 mM sucrose and 20 mM HEPES (pH 7.5). The protein concentration of each sample was determined by the bicinchoninic acid procedure using a BCA kit by Pierce (Rockford, IL).

Western Blot Analysis. Polyacrylamide-gel electrophoresis (running buffer: 25 mM Tris, 192 mM glycine, and 0.1% sodium dodecyl sulfate, pH 8.4) was performed to separate membrane proteins and molecular markers (Bio-Rad, Hercules, CA) on 12% Tris-glycine gels from Novex (San Diego, CA). Ten-well gels were used throughout the study, and 50 µg of protein per sample was loaded into each lane. Two universal internal standards (two samples) were used in each gel to correct for gel to gel differences. Proteins in the gels were transferred to polyvinylidene difluoride membranes (Novex) for 3 h at 20 V (transfer buffer: 12 mM Tris base, 96 mM glycine, and 10% methanol). Membranes were placed in blocking solution [5% nonfat dry milk in Tris-buffered saline/Tween 20 (TBST)] overnight at 4°C. Blots were then incubated with primary antibody (1:1000 in TBST with 2.5% nonfat dry milk) for 3 h at room temperature. After thorough washing, blots were incubated with secondary antibody (1:5000 in TBST) (goat anti-rabbit IgG; Amersham Life Sciences, Arlington Heights, IL) for 1 h at room temperature. Blots were then washed thoroughly, and detection of antibody was through enhanced chemiluminescence detection (Amersham Biosciences, Inc., Piscataway, NJ). Blots were exposed to radiographic film (X-MOAT AR; Eastman Kodak, Rochester, NY) for 30 s to 1 min. The films were developed and examined by densitometric analysis, followed by quantitation with ImageQuant software, version 4.2a (Molecular Dynamics, Sunnyvale, CA).

Total RNA Isolation. Total RNA was isolated using RNAzol B reagent (Tel-Test Inc., Friendswood, TX) according to the instructions by the manufacturer. The integrity, concentration, and quality of the total RNA were accessed by agarose gel electrophoresis, determination of absorption at A₂₆₀, and A₂₆₀ to A₂₈₀ ratio, respectively.

Oatp2 Gene Specific Probe Sets for bDNA. The specific oligonucleotide probe sets (consisting of capture extenders, label extenders, and blockers) were designed utilizing the probe Designer software, version 1.0 (Bayer, Emeryville, CA), to have a melting temperature of 63°C. Every probe was submitted to the National Center for Biotechnological Information for nucleotide comparison by the basic logarithmic alignment tool (BLASTn). All probes were synthesized by Operon Technologies (Palo Alto, CA). The complete list of rat oatp2 oligonucleotide probes has been published (Rausch-Derra et al., 2001).

Branched DNA (bDNA) Technique. Rat oatp2 mRNA levels were assessed using the Quantigene expression kit (bDNA technique; Bayer, Walpole, MA) as described in the manufacturer's protocol and validated in this laboratory (Hartley and Klaassen, 2000). Briefly, the oligonucleotide probes (capture extenders, label extenders, and blockers) were combined and diluted to 50 fmol/ml in lysis buffer (100 mM HEPES buffer, pH 7.6, 0.65 mg/ml proteinase K, 1% lithium lauryl sulfate, 800 mM lithium chloride, 8 mM EDTA, and 0.5% Micro-O-protect). Total RNA (1 µg/µl, 10 µl) was added to each well containing 50 µl of capture hybridization buffer (100 mM HEPES buffer, pH 7.6, 3 mg/ml Boehringer-Mannheim blocking reagent, 1% lithium lauryl sulfate, 8 mM EDTA, and 0.5% Micro-O-protect) and 50 µl of each probe set in lysis buffer. RNA was allowed to hybridize to each probe set overnight at 53°C. Subsequently, the plate was allowed to cool to 46°C and washed with washing buffer [twice with 0.1× SSC (1× SSC contains 0.15 M sodium chloride and 0.015 M sodium citrate), 1% sodium dodecyl sulfate, and 0.5 mg/ml sodium azide]. Samples were hybridized with the bDNA (branched DNA) amplification molecule (100 µl/well at 0.2 fmol/µl bDNA) in the amplifier/label probe buffer (100 mM HEPES buffer, pH 7.6, 1.5 mg/ml Boehringer-Mannheim blocking reagent, 1% lithium lauryl sulfate, 10 µM ZnCl₂, 1 mM MgCl₂, 1% brij 35, and 0.5 Micro-O protect) for 1 h at 46°C. The plate was allowed to cool to room temperature, and the wells were washed with high volume wash buffer (twice). Label probe diluted in amplifier/label probe buffer was added to each well (100 µl/well at 0.4 fmol/µl of alkaline phosphatase), and the alkaline phosphatase-conjugated label probe was allowed to hybridize to the bDNA-RNA complex for 1 h at 46°C. Each plate was cooled to room temperature and washed with high volume wash buffer (twice). The reaction was triggered by the addition of a dioxetane substrate solution [100 µl/well of Lumiphos Plus (Lumigen, Inc., Southfield, MI) containing 0.3% sodium dodecyl sulfate]. The enzymatic reaction was allowed to proceed for 1 h at 37°C, and the luminescence was measured with the Quantiplex 320 Luminomiter equipped to read 96-well plates (Chiron Corp., Emeryville, CA).

Northern Blot Analysis of Rat oatp2 mRNA. Total RNA was isolated from livers of the aforementioned SD male rats treated with corn oil, PCN, spironolactone, and dexamethasone (five rats for each treatment group), using RNAzol B reagent following the manufacturer's directions. Poly(A)⁺ mRNA from each tissue was purified from total RNA using poly(A)⁺ pure mRNA isolation kits from Ambion (Austin, TX). Approximately 5 µg of poly(A)⁺-enriched RNA was electrophoretically resolved in 1% formaldehyde-agarose gel, capillary-blotted overnight onto -probe nylon membranes (Bio-Rad), UV cross-linked, and hybridized at 50°C overnight in Expresshyb hybridization buffer (CLONTECH, Palo Alto, CA). Post hybridization washing was done at 55°C. Antisense oligonucleotide probes were 3'-end labeled using terminal transferase (Roche Bioscience, Indianapolis, IN), and the average specific activity was about 6 × 10⁸ cpm/µg DNA. A "cocktail" of oligonucleotide probe sets was used to increase the sensitivity of detection. All probes were chosen from the 3'-untranslated region of oatp-2 mRNA, and their specificity were confirmed by BLAST search. The probe sequences were as follows: probe 1, 5'-GGGTGAAGGCCACTATAGAAGTTTTCCCTGG-3'; probe 2, 5'-GTGTGGGAAACATCCACTTCCCTTGATTGAG-3'; probe 3, 5'-GACAGATGCAGAAGTTTTGCACATGCAGAGG-3'; probe 4, 5'-GAAAACAGGAGATGGCACACTCTGAAGAGTC-3'; probe 5, 5'-CAGTGATGTATGTGAAATGATCAACTGTGTG-3'; probe 6, 5'-CAGCTTTGAATTGTTAGAGAAAAAGAGTCCCAATC-3'.

hnRNA RT-PCR Analysis of oatp2 Gene Transcription. RT-PCR of hnRNA has been used to detect transcription activity as a substitute for the nuclear run-on assay (Jakubowski and Roberts, 1994; Elferink and Reiners, 1996). DNA-free total RNA was isolated by Absolutely RNA RT-PCR Miniprep Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. To detect hnRNA, oatp2 gene-specific primers were designed from intron regions of rat oatp2, flanking exon 7 (forward primer, 5'-ATG AGC TTA GGA CTG AAA GAA GGC-3'; reverse primers, 5'-GGA GCT ACT GGT CAA CCC CAC-3'). The predicted length of the oatp2 amplicon is 415 base pairs. RT-PCR was performed using Access RT-PCT System from Promega (Madison, WI) per the manufacturer's protocol in a Mastercycler (Eppendorf Scientific Inc., Westbury, NY). Primers designed from rat ribosomal protein L19 cDNA were a gift from Dr Karl Rozman and used as an internal standard for comparison of loading differences between tubes; the RT-PCR product of L19 is 196 base pairs long. The samples were then analyzed by electrophoresis on 2% agarose gel in 1× Tris-Boric acid-EDTA buffer.

Statistical Analysis. The data were expressed as mean ± S.E. Statistical significance was determined by one-way analysis of variance, followed by Duncan's posthoc test. The significant level was set at P < 0.05.

	Results

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Male SD rats were dosed with five groups of prototypical drug-metabolizing enzyme inducers. The dosing paradigm was designed according to what is commonly cited in the literature. Furthermore, the mRNA levels of CYP1A1, CYP2B1/2, CYP3A1, CYP4A2/3, and quinone reductase were determined by the bDNA technique after treatment with these microsomal enzyme inducers to confirm the induction (data not shown). For example, the mRNA levels of CYP1A1 were increased by the treatment of AhR ligands (TCDD, 388-fold; I-3-C, 4.7-fold; BNF, 294-fold; and PCB 126, 279-fold). The mRNA levels of CYP2B1/2 were increased by the CAR ligand PB (53-fold) and phenobarbital-like compounds DAS (65-fold) and PCB 99 (55-fold). The mRNA levels of CYP3A1 were increased by the PXR ligands (PCN, 20-fold; SPIRO, 12-fold; and DEX, 33-fold). The mRNA levels of CYP4A2/3 were increased by the PPAR ligands (CLOF, 6.8-fold; DEHP, 10.3-fold; and PFDA, 10.8-fold). The mRNA levels of quinone reductase were increased by the ARE/EpRE activators (BHA, 2.1-fold; EQ, 4.6-fold; and OLTI, 6.3-fold).

Effects of the Prototypical Drug-Metabolizing Enzyme Inducers on the Protein Levels of Rat Hepatic oatp2. To determine whether oatp2 is regulated by the prototypical drug-metabolizing enzyme inducers that induce phase I and II enzymes through ligand-activated transcription factor pathways, oatp2 protein levels were determined by Western blotting using the anti-oatp2 antiserum developed in this laboratory (Rausch-Derra et al., 2001). Shown in Fig. 1A is a representative Western blot of liver membrane fractions probed with anti-oatp2 antibody after treatment with the aforementioned drug-metabolizing enzyme inducers. The data are also quantitatively expressed as the optical density after scanning each Western blot from each rat (n = 5) (Fig. 1B). All four AhR ligands (TCDD, I-3-C, BNF, and PCB 126) reduced oatp2 protein levels by 56, 56, 60, and 72%, respectively. All three CAR ligands (PB, DAS, and PCB 99) increased oatp2 protein levels moderately, by 80, 100, and 130%, respectively. All three PXR ligands (PCN, SPIRO, and DEX) dramatically increased oatp2 protein levels by 360, 270, and 230%, respectively. The PPAR ligands (CLOF, DEHP, and PFDA) and the ARE/EpRE activators (BHA, EQ, and OLTI) had minimal effects on oatp2 protein levels, except for the PPAR ligand, DEHP, which increased oatp2 protein levels significantly (120%).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Effects of the prototypical drug-metabolizing enzyme inducers on rat oatp2 protein expression. A, a representative Western blot of oatp2 proteins after AhR, CAR, PXR, PPAR, and ARE/EpRE ligand or activator treatment. Oatp2 protein was detected by the polyclonal antibody designed against a unique peptide region of oatp2 protein. B, the quantitative data obtained from five rats in each group. All data were expressed as mean ± S.E. for five animals in each group, except for controls, which were combined from the three individual controls after it was determined that they were not statistically different. *, statistical significance from control values (p < 0.05).

Effects of the Prototypical Drug-Metabolizing Enzyme Inducers on Rat Hepatic oatp2 mRNA Levels. In general, the major mechanism by which chemicals increase drug-metabolizing enzyme mRNA levels is via increase of gene transcription. Although all AhR ligands (TCDD, I-3-C, BNF, and PCB 126) decreased oatp2 protein levels, their effects on oatp2 mRNA levels were not uniform; only PCB 126 decreased oatp2 mRNA levels significantly, TCDD and BNF had no effect, and I-3-C significantly increased oatp2 mRNA levels (Fig. 2). The CAR ligands (PB, DAS, and PCB 99) all increased oatp2 protein levels moderately, but only DAS significantly increased oatp2 mRNA levels; PB and PCB 99 had no effect. All three PXR ligands (PCN, SPIRO, and DEX) increased oatp2 protein levels markedly, but only PCN significantly increased oatp2 mRNA levels to a degree comparable with that observed at the protein levels. The two other PXR ligands, SPIRO and DEX, did not increase oatp2 mRNA levels. Treatment with PPAR ligands (CLOF, DEHP, and PFDA), or the ARE/EpRE activators (BHA, EQ, and OLTI) did not affect hepatic oatp2 mRNA levels.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Effects of the prototypical drug-metabolizing enzyme inducers on rat oatp2 mRNA expression. Oatp2 mRNA levels were determined by the bDNA technique after drug-metabolizing enzyme inducer treatment, using an oatp2 gene-specific probe set. All data are expressed as mean ± S.E. for five animals in each group, except for the controls that were combined from the three individual controls after it was determined that they were not statistically different. *, statistical significance from control values (p < 0.05).

Determinations of Rat Hepatic oatp2 mRNA Levels after PXR Ligands Treatment by Northern Blot Analysis. Because changes in oatp2 mRNA levels did not correspond to the changes in oatp2 protein levels, liver oatp2 mRNA content after PXR ligand treatments was also determined by Northern blot analysis. The amount of oatp2 mRNA after treatment with three PXR ligands was examined, because all three PXR ligands robustly increased oatp2 protein levels, but only PCN increased oatp2 mRNA levels, when quantified by the bDNA technique. Northern blotting agreed with the bDNA results and confirmed that PCN was the only PXR ligand that markedly increased oatp2 mRNA content in liver (Fig. 3). Therefore, the results of the Northern blots and bDNA method confirmed that among the PXR ligands, only PCN markedly increased oatp2 mRNA levels, whereas SPIRO and DEX did not significantly alter oatp2 mRNA levels.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Effects of PXR ligands on oatp2 mRNA determined by Northern blot analysis. Total RNA was isolated from rat livers of PXR ligand treatment groups (PCN, spironolactone, and dexamethasone) and corn-oil controls. Northern blot was performed using a oatp2-gene-specific probe set cocktail as described in the Materials and Methods. The internal control for comparison of loading was rat -actin. The upper panel is a Northern blot, which is quantitatively expressed in the lower panel.

Determinations of oatp2 Gene Transcription by hnRNA RT-PCR after PXR Ligand Treatment. Increased mRNA levels can be due to either increased transcription or increased stabilization of the mRNA. The amount of hnRNA, the primary transcripts that reside in the nucleus and give rise to mRNA after post-transcriptional modification, such as splicing and editing, are good indicators of gene transcription (Sharp, 1994). Figure 4 illustrates the relative amount of oatp2 hnRNA in livers from rats treated with either vehicle or the three PXR ligands (PCN, SPIRO, and DEX) by RT-PCR. The primers were designed from the intron regions of rat oatp2, which are present in the primary transcripts but not in mRNA. The results clearly showed that PCN, but not SPIRO and DEX, increased oatp2 hnRNA levels compared with that in corn-oil controls, indicating that PCN induction of oatp2 in liver is, at least partially, due to activation of oatp2 gene expression.

View larger version (77K): [in this window] [in a new window]   Fig. 4.   Effects of PXR ligands on oatp2 gene transcription determined by hnRNA RT-PCT. DNA free total RNA was isolated from livers of PXR ligand-treated groups of rats (PCN, spironolactone, and dexamethasone) and corn-oil controls. RT-PCR primers were designed from oatp2 intron regions, flanking exon 7. Internal control for comparison of loading was rat ribosomal protein L19.

	Discussion

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Transporters are involved in xenobiotic absorption, distribution, and excretion. For some chemicals, uptake into liver is required before they are biotransformed by phase I and II drug-metabolizing enzymes. Subsequently, after the metabolites are formed, they need to be transported out of the cell. Therefore, regulation of transport into cells would alter the kinetics of some xenobiotics. Some export transporters are altered by chemical pretreatment in mice. For example, the phosphatidylcholine transporter, mdr2, was induced by the PPAR ligand, clofibrate (Chianale et al., 1996; Miranda et al., 1997). In isolated rat hepatocytes, the organic anion exporter Mrp2 was induced by vincristin, tamoxifen, and rifampicin (Kauffmann et al., 1997), and by dexamethasone and PCN (Courtois et al., 1999). Another exporter Mrp3 was induced by treatment with phenobarbital (Ogawa et al., 2000).

Because uptake by transporters is necessary for some xenobiotics for the subsequent biotransformation, a thorough study was conducted on the rat hepatic uptake transporter, oatp2, to determine its induction profile by prototypical drug-metabolizing enzyme inducers. Rat oatp2 is a member of the multispecific organic anion transporting polypeptide family (Noe et al., 1997; Abe et al., 1998). It transports many structurally unrelated compounds, including anions, cations, and neutral compounds (Abe et al., 1998; Eckhardt et al., 1999; Reichel et al., 1999). Among them, cardiac glycosides such as ouabain and digoxin are transported by oatp2 with very high affinity compared with other oatp family members (Noe et al., 1997). Research from this laboratory has shown that the hepatic uptake of cardiac glycosides is increased after treatment of the rats with PCN and phenobarbital (Klaassen, 1974a, 1974b). A study from this laboratory demonstrated that oatp2 was induced by PCN and phenobarbital (Rausch-Derra et al., 2001). Research from this laboratory has also shown that mouse oatp2 induction was abolished in PXR knockout mice after PCN treatment (Staudinger et al., 2001). It was discovered recently that phenobarbital and PCN are the classical ligands for orphan nuclear receptors, CAR and PXR, respectively. However, there is no information of the effects on rat oatp2 of other drug-metabolizing enzyme inducers that also activate gene expression through ligand-activated transcription factor pathways. Elucidation of the regulation of rat oatp2 will certainly help to understand the pharmacokinetics of xenobiotics and to understand and prevent drug-drug interactions. Therefore, the current study was designed to determine the induction profile of rat oatp2 by five classes of prototypical drug-metabolizing enzyme inducers that increase gene expression through ligand-activated transcription factor pathways.

Ligand-activated transcription factor pathways are responsible for the induction of most of the drug-metabolizing enzymes (Waxman, 1999; Fuhr, 2000). AhR, CAR, PXR, PPAR, and ARE/EpRE have been shown to be the major ligand-activated nuclear receptors for induction of CYP1A1/2, CYP2B1/2, CYP3A, CYP4A, and quinone reductase. Recently, liver X receptor (Janowski et al., 1996, 1999; Peet et al., 1998) and farnesoid X receptor (Makishima et al., 1999; Parks et al., 1999; Wang et al., 1999) have been discovered and seem to play important roles in maintaining cholesterol and bile acid homeostasis. However, the regulatory roles of ligand-activated transcription factor pathways on transporters have not been thoroughly examined.

The current study showed that oatp2 protein levels were regulated by several classes of drug-metabolizing enzyme inducers. Oatp2 protein levels were decreased 56 to 72% by AhR ligands, moderately increased by CAR ligands (84-132%), and dramatically increased by PXR ligands (233-363%).

The mechanism by which these drug-metabolizing enzyme inducers alter oatp2 protein levels is not apparent. For example, all three PXR ligands significantly increased oatp2 protein levels, but only PCN increased oatp2 mRNA content in liver. This finding was confirmed by three independent experimental methods (bDNA, Northern blot, and hnRNA RT-PCR). Therefore, measurement of mRNA levels alone may not be an accurate marker for induction of oatp2. In addition, rat hepatic oatp2 mRNA levels do not correlate with the increased levels of rat hepatic oatp2 protein after treatment with prototypical CYP3A inducers, indicating that these chemicals may regulate oatp2 post-transcriptionally. A similar phenomenon has been reported for the differential regulation of CYP3A and P-glycoprotein by CYP 3A inducers. P-glycoprotein is induced by dexamethasone, but not by PCN and triacetyloleandomycin in male rat liver, whereas CYP3A is induced by all three chemicals, indicating the possibility of independent regulation of CYP3A and P-glycoprotein by the CYP3A inducers (Schuetz et al., 1996; Salphati and Benet, 1998).

PCN produced the largest increase in oatp2 protein and mRNA levels. Because levels of hnRNA were increased after PCN treatment, the increase in oatp2 mRNA levels appears to be due to an increase in oatp2 gene transcription. PCN is a potent ligand for rodent PXR and activation of the PXR activates CYP3A gene expression in several species, including mouse (Kliewer et al., 1998), rat (Zhang et al., 1999), rabbit (Savas et al., 2000), and human (Lehmann et al., 1998; Jones et al., 2000). Recently, PXR was shown to play a pivotal role in rifampicin-mediated induction of P-glycoprotein, the gene product of MDR1 (Geick et al., 2001). Oatp2 is not only increased by PCN in adult rats but also during postnatal development, which strongly suggests that oatp2 is a potential PXR targeted gene. The mechanism by which PCN increases oatp2 expression can only be understood after thorough studies of the oatp2 promoter. The increase of oatp2 protein levels by two other PXR ligands, spironolactone and dexamethasone, does not appear to be due to increased transcription of the rat oatp2 gene. It is speculated that some post-translational effects such as a decrease in oatp2 protein turnover may be mediated through an (some) unknown PXR targeted gene(s), or may be mediated through unknown PXR-independent pathway(s) that is (are) activated by PCN, but not by spironolactone and dexamethasone.

The CAR ligands phenobarbital, diallyl sulfide, and PCB 99 all moderately increased oatp2 protein levels (84-132%). However, only diallyl sulfide significantly increased hepatic rat oatp2 mRNA levels. Phenobarbital has been shown to induce CYP2B1/2 via activation of the CAR (Honkakoski et al., 1998; Kawamoto et al., 1999; Sueyoshi et al., 1999; Wei et al., 2000). Although CAR and PXR are both ligand-activated orphan nuclear receptors, the mechanism by which CAR and PXR ligands induce CYP2B1/2 and CYP3A, respectively, is different (Sueyoshi and Negishi, 2001). CAR resides in the cytosol, and migrates into the nucleus upon binding of its ligands (Sueyoshi and Negishi, 2001). The receptor-ligand complex then dimerizes with RXR and binds to response element(s) of the gene(s) that it regulates, which results in gene activation (Kawamoto et al., 1999; Sueyoshi and Negishi, 2001). Cross-talk between CAR and PXR has been reported, in that CAR ligands have been shown to be weak PXR ligands and induce CYP3A (Xie et al., 2000). In the present study, all CAR ligands increased oatp2 protein levels but not mRNA levels, indicating that post-translational effects, mediated by activation of either CAR or PXR by CAR ligands, possibly resulted in a decreased turnover of oatp2 protein. Also, CAR- or PXR-independent pathways cannot be excluded.

In the present study, oatp2 protein levels but not the mRNA levels were decreased by AhR ligands (TCDD, I-3-C, BNF, and PCB 126). Although AhR ligands are known for their induction of CYP1A1/2 and CYP1B, they down-regulate a number of genes, such as the human estrogen receptor (White and Gasiewicz, 1993). AhR ligand down-regulation of oatp2 protein could have significant effects, which may result in increased plasma concentrations of oatp2 substrates, many of them important endogenous chemicals or pharmaceuticals. The PPAR ligands and the ARE/EpRE activators had minimal effects on oatp2 protein levels, except for the PPAR ligand, DEHP, which increased oatp2 protein levels significantly. DEHP also induces biological effects that occur independent of the PPAR (Melnick, 2001), therefore, it is possible that DEHP increases oatp2 protein levels through pathways that are independent of PPAR.

In conclusion, the present study provides evidence that some prototypical drug-metabolizing enzyme inducers that act through ligand-activated transcription factor pathways regulates rat oatp2 protein levels. The induction profile of oatp2 protein by these prototypical drug-metabolizing enzyme inducers mimics that of CYP3A. However, rat hepatic oatp2 mRNA levels after treatment with microsomal enzyme inducers generally do not parallel the alteration of oatp2 protein levels. The highest induction of oatp2 was seen after PCN treatment, indicating that oatp2 is a potential PXR-targeted gene. The mechanism by which other drug-metabolizing enzyme inducers regulate oatp2 appears to occur at the post-translational level. In addition, altered protein levels of rat oatp2 after some microsomal enzyme inducer treatment indicates that drug-drug interactions may occur at the hepatic uptake level.