Hepatic transporters are involved in the regulation of bile formation and disposition of xenobiotics. The hepatocyte has a polarized plasma membrane with basolateral and apical domains, enabling vectorial movement of endogenous and exogenous compounds from blood into bile. Basolateral sodium-taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptides (Slco/Oatps) are primarily responsible for the Na⁺-dependent and -independent uptake of bile acids from the portal blood, respectively (reviewed by Kullak-Ublick et al., 2000). Furthermore, Oatps also mediate xenobiotic uptake into liver, and in turn, greatly affect drug disposition.

Canalicular secretion of bile components represents the rate-limiting step in bile formation. Bile acids, glutathione conjugates, and xenobiotics are removed from hepatocytes and concentrated into the bile by canalicular efflux transporters in an ATP-dependent manner. These transporters include the breast cancer resistance protein (Bcrp), multidrug resistance protein (Mdr, P-gp), and multidrug resistance-associated protein 2 (Mrp2) (Kullak-Ublick et al., 2004). The multidrug resistance-associated proteins (Mrp1, 3, and 4) are basolateral efflux proteins in the liver (reviewed by Renes et al., 2000b). Induction of efflux transporters occurs in response to cellular distress (Konig et al., 1999; Cherrington et al., 2004; Aleksunes et al., 2005). Mrp1 has widespread tissue distribution and is important for detoxification of xenobiotics by excreting conjugates of glucuronic acid, sulfate, and glutathione (Jedlitschky et al., 1996). Expression levels of mouse Mrp3 and Mrp4 are dramatically up-regulated in response to cholestasis or chemical injury (Soroka et al., 2001; Assem et al., 2004; Aleksunes et al., 2005). It has been hypothesized that Mrp3 and Mrp4 are up-regulated as a compensatory mechanism when canalicular efflux is compromised (Konig et al., 1999; Schuetz et al., 2001). By instead excreting biliary components and xenobiotics into the blood, Mrp3 and Mrp4 can decrease potentially toxic intracellular concentrations of these substrates.

Regulation of hepatic gene expression involves participation of numerous nuclear receptors. The pregnane X receptor, constitutive androstane receptor, and peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that heterodimerize with the retinoid X receptor and enable gene transcription. Recent studies indicate that pregnane X receptor mediates the expression of rodent Mrp2 (Kast et al., 2002) and Oatp1a4 (Staudinger et al., 2003), in addition to human MDR1 (Geick et al., 2001). Furthermore, constitutive androstane receptor activation induces Mrp2–7 mRNA in mouse liver (Maher et al., 2005) and is involved in the regulation of Mrp4 and sulfotransferase2a1 (Sult2a1) (Assem et al., 2004). Whereas peroxisome proliferator treatment alters levels of Mdr2 and cholesterol efflux transporters in the liver, little is known regarding the regulation of hepatic organic anion uptake and efflux transporters by PPARα (Kok et al., 2003a,b).

Peroxisome proliferators (PPs) are a diverse group of chemicals that when administered to rodents increase the size and number of peroxisomes, most prominently in liver and kidney. Treatment with these chemicals also results in significant liver enlargement, and chronic exposure leads to hepatocellular carcinomas. PPs mediate their actions in liver through binding and activating PPAR. Activation of PPARα is responsible for the induction of enzymes involved in uptake, metabolism, and β-oxidation of fatty acids. Another effect of PPs is that they protect the liver against the toxic actions of chemicals such as acetaminophen (Nicholls-Grzemski et al., 1992; Manautou et al., 1994). Whereas the precise mechanism of this hepatoprotective effect is not known, mice lacking the PPARα nuclear receptor are not protected by PPs (Chen et al., 2000). Up-regulation of hepatic transporters could contribute to this by enhancing the disposition of hepatotoxicants and/or oxidative stress and inflammatory mediators involved in liver injury. The present study examines the expression of several basolateral and canalicular hepatic transporters in CD-1 mice in response to the PPARα agonist clofibrate (CFB). To further characterize the regulation of these hepatic transporters, CFB-mediated changes in transporter mRNA levels were assessed in wild-type and PPARα-null mice. Results from this study demonstrate that CFB induces gene and protein expression of Mrp3 and Mrp4 efflux transporters in a PPARα-dependent manner while having little effect on mRNA expression of Ntcp, Oatp1a1, Oatp1a4, and Oatp1b2 uptake transporters.

Materials and Methods

Chemicals. CFB and corn oil were purchased from Sigma-Aldrich Corporation (St. Louis, MO). All chemicals were of reagent grade or better.

Treatment of Animals. Outbred male CD-1 mice aged 10 to 12 weeks were purchased from Charles River Laboratories (Wilmington, MA). Inbred sv/129 background wild-type (+/+) and PPARα-null (–/–) male mice were provided by Frank Gonzalez (National Institutes of Health, Bethesda, MD). Mice were housed in community cages with free access to water and feed (rodent diet 5001; PMI Feeds, St. Louis, MO). The vivarium maintained a 12-h dark/light cycle with controlled temperature and humidity. Groups of mice (n = 3–6) received daily dosing of CFB (500 mg/kg) or corn oil vehicle (5 ml/kg) i.p. for 10 days. This treatment regimen not only results in peroxisome proliferation but also confers protection against hepatotoxicants such as acetaminophen (Nicholls-Grzemski et al., 1992; Manautou et al., 1994). Livers were removed, snap frozen in liquid nitrogen, and stored at –80°C until assayed. The University of Connecticut Institutional Animal Care and Use Committee approved all experimental animal protocols.

RNA Isolation. Total tissue RNA was extracted using RNAzol B reagent (Tel-test Inc., Friendswood, TX) according to the manufacturer's protocol. RNA integrity was confirmed by agarose gel electrophoresis through visualization of intact 18S and 28S rRNA.

Branched DNA Signal Amplification (bDNA) Assay. Mouse Bcrp, cytochrome P450 4A14, Mdr1a, Mdr1b, Mrp1, Mrp2, Mrp3, Mrp4, Ntcp, Oatp1a1, Oatp1a4, and Oatp2b1 mRNA were measured using the branched DNA signal amplification assay (Quantigene High Volume bDNA Signal Amplification Kit; Genospectra, Fremont, CA). Mouse gene sequences were accessed from GenBank. Multiple oligonucleotide probe sets [capture extender, label extender, and blocker probes] were designed using Probe Designer software, version 1.0 (Bayer Corp., Emeryville, CA) to be specific to a single mRNA transcript. The following probe sets used were described previously: Mdr1a and Mdr1b (Brady et al., 2002); Mrp3 (Cherrington et al., 2003); and Mrp1, Mrp2, Mrp4, Oatp1a1, Oatp1a4, Oatp1b2, Bcrp, and Ntcp (Aleksunes et al., 2005). The probe set for mouse cytochrome P450 4A14 is listed in Supplemental Table 1. Oligonucleotide probes were designed with a melting temperature of approximately 63°C, enabling hybridization conditions to be held constant (i.e., 53°C) during each hybridization step. All probes were submitted to the National Center for Biotechnology Information (Bethesda, MD) for nucleotide comparison by the basic local alignment search tool (BLASTn) to ensure minimal cross-reactivity with other known mouse sequences. Total RNA (1 μg/μl) was added to each well (10 μl/well) of a 96-well plate containing capture hybridization buffer and 100 μl of each diluted probe set. The RNA was allowed to hybridize overnight at 53°C, and subsequent hybridization steps were carried out according to the manufacturer's protocol. Luminescence was measured with a Quantiplex 320 bDNA Luminometer interfaced with Quantiplex Data Management Software (Bayer Diagnostics, Tarreytown, NY). The luminescence for each well was reported as relative light units (RLU) per 10 μg of total RNA.

Western Blot Analysis of Hepatic Transporter Proteins. Livers were homogenized in ST buffer (10 mM Tris base and 150 mM sucrose, pH 7.5) and centrifuged at 100,000g for 1 h at 4°C. Membrane preparations were generated by resuspending the resulting pellet in ST buffer and stored at –80°C until assayed. Protein concentrations were determined by the Lowry method (Lowry et al., 1951) using the Bio-Rad DC Protein Assay kit (Hercules, CA). Uniform amounts of proteins (40 μg for Mrp3 and 4, 30 μg for P-gp and β-actin, or 60 μg for Bcrp/lane) were resolved on 8% (Mrp3, Mrp4, P-gp, and β-actin) or 10% (Bcrp) SDS-polyacrylamide electrophoresis slab gels using a 4% stacking gel followed by electrotransfer to a PVDF-Plus membrane (Micron Separations, Westborough, MA). Immunochemical detection of hepatic transporters was carried out using purified antibodies raised against Mrp3 (M₃II-2), Mrp4 (M₄I-10), P-gp (C219), β-actin (ab8227), and Bcrp (Bxp-53) followed by a secondary peroxidase-conjugated IgG. Mrp3, Mrp4, and Bcrp primary antibodies were provided by George Scheffer (VU Medical Center, Amsterdam, The Netherlands). P-gp and β-actin primary antibodies were purchased from Abcam, Inc. (Cambridge, MA). Polyvinylidene difluoride membranes were blocked in 1% (Mrp3, Mrp4, and Bcrp) or 5% (β-actin) nonfat dry milk/phosphate-buffered saline-Tween 20. P-gp blots were blocked in 5% nonfat dry milk/Tris-buffered saline-Tween 20. All polyvinylidene difluoride membranes were blocked for 1 h. Primary antibodies were diluted 1:2000 (Mrp3, Mrp4, and P-gp) or 1:5000 (Bcrp and β-actin) in blocking solution and incubated for 1 h. Immunostained blots were then incubated with peroxidase-conjugated antibodies and diluted 1:2000 in blocking buffer for an additional hour. The peroxidase-conjugated secondary antibodies were directed against rat (Mrp3, Mrp4, and Bcrp) (A9037; Sigma), mouse (P-gp) (A4416; Sigma), or rabbit (β-actin) (A4914; Sigma). Immunoreactive bands were detected using the ECL Chemiluminescent kit (Amersham Life Science, Arlington Heights, IL). Proteins were visualized by exposure to Fuji Medical X-ray film (Stamford, CT). The immunoreactive intensity of proteins was quantified using a PDI Image Analyzer (Protein and DNA ImageWare System; PDI, Inc., Huntington Station, NY). Equivalent protein loading was confirmed by immunostaining blots for β-actin.

Immunofluorescent Analysis of Hepatic Transporter Proteins. Mouse livers were embedded in Optimal Cutting Temperature compound and brought to –20°C. Cryosections (6 μm) were thaw-mounted onto Superfrost glass slides (Fisher Scientific, Pittsburgh, PA) and stored at –80°C under dehumidified conditions until use. Tissue sections were fixed with 4% paraformaldehyde for 5 min. Sections were blocked with 5% goat serum/phosphate-buffered saline with 0.1% Triton X-100 for 1 h and then incubated with either M₃II-2, M₄I-10, or Bxp-53 primary antibodies diluted 1:100 in 5% goat serum/PBS with Triton X-100 for 2 h at room temperature. Antibody preparations were filtered through 0.22-μm membranes (Fisher Scientific, Pittsburgh, PA) before use. Sections were washed three times in PBS with Triton X-100 and incubated for 1 h at room temperature with goat anti-rat AlexiFluor 488 IgG (A-11006; Invitrogen, Carlsbad, CA) diluted 1:200 in 5% goat serum/PBS with Triton X-100. Slides were washed in PBS and then rinsed twice with distilled deionized water. The sections were air-dried and mounted with Prolong Gold with DAPI (Invitrogen). Sections were visualized and images were captured using a Leica SP2 Spectral Confocal microscope (Leica Microsystems AG, Wetzlar, Germany) and analyzed with an excitation wavelength of 488 nm. Negative control staining was performed by incubating cryosections without primary antibody.

Statistical Analysis. Results are expressed as means ± standard error of the mean (S.E.) for three to six mice per treatment group. Relative mRNA and protein amounts were compared using a Student's t test. Differences were considered significant at p < 0.05.

Results

Uptake and Efflux Transporter mRNA Levels in Response to CFB Treatment. The mRNA levels of several basolateral and canalicular hepatic efflux transporters were assessed in male CD-1 mice after daily administration of CFB for 10 days (Fig. 1). The mRNA expression of basolateral efflux transporters Mrp3 and Mrp4 was up-regulated 150 and 110%, respectively, by CFB treatment compared with controls. Likewise, CFB up-regulated mRNA expression of the canalicular efflux transporters Bcrp (60%) and Mdr1a (90%). No significant changes in mRNA levels were detected for Mrp1, Mrp2, and Mdr1b. The mRNA level of mouse Ntcp, the primary bile salt uptake carrier, was not altered in response to CFB treatment. Furthermore, mRNA levels of the Na⁺-independent uptake transporters Oatp1a1, Oatp1a4, and Oatp1b2 were unchanged by CFB. Significant induction of cytochrome P450 4A14 mRNA was observed, which confirmed PPARα activation by CFB treatment (data not shown).

Hepatic Transporter Protein Expression in Response to CFB Treatment. To determine whether the observed changes in CFB-mediated mRNA levels translated into altered hepatic protein levels, Western blot analysis of Bcrp, Mrp3, Mrp4, P-gp, and β-actin was performed (Fig. 2). Administration of CFB increased expression of the canalicular efflux transporter Bcrp by 70% of control. The basolateral efflux proteins Mrp3 and Mrp4 were also significantly up-regulated by CFB to 250 and 180% of control values, respectively. CFB increased P-gp levels (140% of control), confirming previous observations that PPARα agonists induce P-gp expression (Chianale et al., 1996; Kok et al., 2003a). Western blot immunostaining for β-actin confirmed equivalent protein loading. Western blot analysis demonstrated that changes in mRNA levels induced by CFB translated into increased levels of these transporters in CD-1 mice.

Regulation of Hepatic Transporter Gene and Protein Expression in Response to CFB Treatment by PPARα. The mRNA levels of efflux transporters were determined in both wild-type and PPARα-null mice after 10 days of CFB administration. As seen in CD-1 mice, treatment with CFB increased mRNA levels of Bcrp, Mdr1a, Mrp3, and Mrp4 in livers from wild-type mice. These changes were not observed in PPARα-null mice (Fig. 3). Interestingly, PPARα-null mice had a significantly higher constitutive level of Mrp3 mRNA. As expected, induction of cytochrome P450 4A14 mRNA levels (75-fold) occurred after CFB treatment in the wild-type mice but not in PPARα knockout mice (data not shown). Similar induction of protein expression (approximately doubling) was seen in CFB-treated sv/129 wild-type mice for Mrp3, Mrp4, and P-gp (Fig. 4). However, the higher constitutive mRNA levels for Mrp3 in PPARα-null mice did not translate into increased protein levels. The lack of P-gp induction by CFB in PPARα-null mice confirmed previous reports (Kok et al., 2003a). Bcrp protein tended to increase in wild-type mice receiving CFB, although this was not statistically significant. This is in contrast to the CD-1 mouse data, where CFB significantly induced both Bcrp mRNA levels and protein levels. However, PPARα seems to play a role in the basal expression of Bcrp, because protein expression is significantly reduced in PPARα-null mice. Expression of β-actin confirmed equivalent protein loading.

In silico analysis (Podvinec et al., 2002) of mouse Mrp3, Mrp4, and Bcrp 5′-flanking regions indicate the presence of multiple putative peroxisome proliferator response elements in the promoter region of these genes (Supplemental Table 2). This supports transcriptional regulation as the likely mechanism for transport protein induction. In summary, protein expression data indicate that Mrp3 and Mrp4 are regulated by CFB in a PPARα-dependent manner and that the constitutive expression of Bcrp in liver is regulated in part by PPARα.

Immunofluorescent Analysis of Liver Mrp3, Mrp4, and Bcrp after CFB Treatment. Tissue localization of hepatic Mrp3 and Mrp4 was analyzed in sv/129 wild-type and PPARα-null mice after vehicle or CFB treatment. Confocal scanning microscopy was used to capture representative images from cryosections stained for Mrp3 or Mrp4 (Fig. 5). Images of wild-type control tissue stained for Mrp3 revealed basolateral staining that is more pronounced in centrilobular areas. CFB treatment increased the intensity of Mrp3 staining throughout the liver lobule in wild type but not in PPARα-null mice. Expression of Mrp4 is nearly undetectable in normal mouse liver using the M₄I-10 antibody. Confirmation of the M₄I-10 antibody specificity has been previously demonstrated using Mrp4-null mice (Leggas et al., 2004). Whereas Mrp4 staining was negligible in vehicle-treated wild-type mice, CFB administration markedly increased basolateral staining of Mrp4 exclusively in the centrilobular region of the liver. Mrp4 staining was confined to one or two layers of cells around the central vein. Expression of Mrp4 was undetectable in CFB-treated PPARα-null mice. Similar to the patterns observed with Western blot analysis, immunofluorescent images of Mrp3 and Mrp4 suggest a regulatory role for PPARα. Mrp3- and Mrp4-immunostained liver slices from vehicle-treated PPARα-null mice closely resembled staining intensity and distribution of the CFB-treated PPARα-null livers (data not shown). Images of Bcrp stained livers were also generated for wild-type and PPARα-null mice (Supplemental Fig. 1). Administration of CFB did not alter the expression or localization of Bcrp, regardless of genotype. It is noteworthy that PPARα-null mice had dramatically lower constitutive expression of hepatic Bcrp than wild type, indicating a role for PPARα in the basal expression of this transporter.

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Fig. 1.

Effect of CFB treatment on hepatic transporter mRNA expression. Total RNA was isolated from livers of male CD-1 mice treated with CFB or vehicle. RNA was analyzed for hepatic transporter expression using the bDNA assay. Data are expressed as RLU per 10 μg of total RNA ± S.E. Asterisks (*) denote significant differences from control (p < 0.05).

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Fig. 2.

Western blot analysis of hepatic Bcrp, Mrp3, Mrp4, P-gp, and β-actin after CFB administration in CD-1 mice. Membrane preparations from individual liver samples were analyzed using specific antibodies to these proteins after SDS-polyacrylamide gel electrophoresis and Western blotting as described under Materials and Methods. The optical density (O.D.) of immunoreactive bands was quantified with a PDI image analyzer and expressed as mean O.D. × mm²± S.E. Statistical differences (p < 0.05) are marked by asterisks (*).

Discussion

Given the mounting evidence that nuclear receptors are critically involved in the regulation of transporters, the present study investigates the expression and regulation of hepatic uptake and efflux transporters by the PPARα agonist CFB. Several transporters are known to be regulated by PPARα, including the cholesterol efflux regulatory protein (Knight et al., 2003), adrenoleukodystrophy-related protein (Fourcade et al., 2001), and Mdr2 (Kok et al., 2003a). Recent findings suggest that PPARα agonism induces the mRNA levels of Mrp3 (Maher et al., 2005). The data presented in this study demonstrate that CFB administration enhances gene and protein expression of the hepatic efflux transporters Bcrp, Mrp3, and Mrp4. Moreover, up-regulation of Mrp3 and Mrp4 is dependent upon activation of PPARα.

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Fig. 3.

Effect of CFB treatment on hepatic transporter mRNA expression in wild-type and PPARα-null mice. Male wild-type and PPARα-null mice were dosed with 500 mg/kg i.p. CFB or corn oil vehicle daily for 10 days. Total hepatic RNA was isolated and analyzed by the bDNA assay. Values are expressed as RLU per 10 μg of total RNA ± S.E. Asterisks (*) denote a statistical difference from control, and daggers (†) indicate a statistical difference from PPARα genotype (p < 0.05).

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Fig. 4.

Western blot analysis of liver Bcrp, Mrp3, Mrp4, P-gp, and β-actin after CFB administration in wild-type and PPARα-null mice. Membrane preparations from individual liver samples were analyzed using specific antibodies to these proteins after SDS-polyacrylamide gel electrophoresis and Western blotting as described under Materials and Methods. Results are expressed as mean optical density O.D. × mm²± S.E. Statistical differences (p < 0.05) from vehicle and wild type are marked by asterisks (*) and daggers (†), respectively.

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Fig. 5.

Immunofluorescent analysis of Mrp3 and Mrp4 after CFB treatment. Hepatic cryosections were prepared from sv/129 wild-type and PPARα-null mice dosed with either CFB (500 mg/kg i.p.) or vehicle daily for 10 days. The sections were stained with anti-Mrp3 or -Mrp4 antibodies as described under Materials and Methods. Representative liver sections were generated by scanning confocal microscopy, with CV and PV depicting central and portal veins, respectively.

It is worth noting the contradictory observations of Bcrp protein expression by CFB in CD-1 and sv/129 wild-type mice. Whereas CFB significantly up-regulated Bcrp mRNA levels in both strains, protein levels were only elevated in CD-1 mice. A possible explanation for the discrepancy may be attributed to regulatory mechanisms. Recently, expression of hepatic Bcrp was linked to testosterone levels in mice (Tanaka et al., 2005). Further studies are required to determine whether hormonal differences between mouse strains are sufficient to explain the observed differences in Bcrp expression. Alternatively, strain differences in Bcrp expression may result from splicing or polymorphic variants of the nuclear receptor or PPARα coregulators. Regarding the localization of Mrp3, its distribution in human liver has been primarily described in the centrilobular region (Scheffer et al., 2002). We also observed intense Mrp3 localization in the centrilobular region of control mouse liver. However, CFB treatment seems to induce Mrp3 protein expression throughout the liver lobule. The broad distribution of Mrp3 may illustrate its overall importance in the liver's response to xenobiotics, providing enhanced transport of substrates beyond the centrilobular region. We also report novel centrilobular localization of Mrp4 induction after CFB treatment. Given the low liver expression of Mrp4 in naive mice, it has been suggested to have a minor role in normal hepatic transport (Maher et al., 2005). Previous reports indicate that administration of CFB for 4 days (500 mg/kg) did not alter Mrp4 mRNA expression (Maher et al., 2005). These results are in contrast to the present findings, where treatment with CFB for 10 days significantly up-regulated in Mrp4 gene expression. Differences in Mrp4 gene expression may be explained by a longer duration of treatment (10 days instead of 4 days) or by the use of different strains of mice. The present study used CD-1 and sv/129 background mice, whereas Maher et al. (2005) used C57BL/6 mice.

It should be emphasized that Western blot analysis under-represents the relative magnitude of Mrp4 induction by CFB because these changes are highly localized to a few cell layers, as shown in Fig. 5. Many hepatotoxicants such as acetaminophen and carbon tetrachloride primarily target the centrilobular region of the liver. Dramatic induction of Mrp4 in hepatocytes immediately surrounding the central vein may significantly alter disposition of toxic mediators within these cells. Likewise, CFB-mediated induction of Mrp3 in the midzonal and periportal regions may influence disposition. Regional increases in Mrp3 may enhance efflux in areas away from the site of injury, which could limit the chemical burden of centrilobular hepatocytes undergoing regeneration and tissue repair. Collectively, induction of hepatic Mrp3 and Mrp4 through PPARα activation may provide another compensatory mechanism to mediate efflux of diverse substrates from livers with peroxisomal proliferation.

Activation of PPARα may also represent an important signaling pathway for regulating bile acid homeostasis. Metabolic stress such as fasting initiates a cascade of PPARα-dependent events, including induction of bile acid synthesis and transporters involved in trafficking phospholipids and cholesterol into the bile (Kok et al., 2003b). Although Mrp3 and Mrp4 have lower affinity for bile acids than canalicular efflux transporters, they may also facilitate the efflux of hepatic bile acid levels by moving their conjugates to the blood for renal excretion. Previous studies demonstrate modulation of hepatic transporters by endogenous ligands and xenobiotics possibly representing a coordinated, hepatoprotective response by the liver. Lipopolysaccharide-induced cholestasis causes mRNA induction of Mrp isoforms with concurrent down-regulation of uptake transporters in rats (Cherrington et al., 2004). Furthermore, our laboratory has observed similar patterns of mouse transporter gene regulation in response to acetaminophen and carbon tetrachloride treatment (Aleksunes et al., 2005). Administration of these toxicants reduces the expression of uptake transporters while enhancing efflux transporters. This may potentially limit the buildup of toxic products within hepatocytes during liver injury and regeneration.

Differential regulation of hepatic transporters may play a role in peroxisome proliferator-mediated hepatoprotection. Pretreatment with peroxisome proliferators completely protects mice from several model toxicants, including acetaminophen (Manautou et al., 1994; Nicholls-Grzemski et al., 1996). Whereas the mechanism of protection is unknown, CFB does not modulate bioactivation or conjugative pathways for acetaminophen (Manautou et al., 1994; Nicholls-Grzemski et al., 2000). Although CFB does not alter the biliary or urinary disposition of acetaminophen or its metabolites (Chen et al., 2000), induction of hepatic efflux transporters may expedite vectorial excretion of toxic or inflammatory byproducts resulting from toxicant exposure. Treatment with a toxic dose of acetaminophen results in increased oxidative stress, leading to lipid peroxidation (Adamson and Harman, 1993). Ultimately, lipid peroxidation results in formation of the toxic aldehyde 4-hydroxynonenal, which is a substrate for several Mrp transporters (Renes et al., 2000a; Ji et al., 2002). Enhanced basolateral excretion of 4-hydroxynonenal and possibly other oxidative stress mediators by Mrp transporters might reduce their intracellular concentrations, thus providing a protective mechanism for the liver.

Inflammation secondary to a toxic challenge can exacerbate liver toxicity (reviewed by Ganey and Roth, 2001). Modulation of inflammatory mediators such as prostaglandins and eicosanoids are associated with alterations in the severity of acetaminophen toxicity (Ben-Zvi et al., 1990; Culo et al., 1995). Altered disposition of these inflammatory mediators by hepatic efflux transporters may provide a mechanism for hepatoprotection. Our current understanding of transporter substrate specificity supports this hypothesis because leukotriene C4 and prostaglandins are substrates for Mrp3 and Mrp4, respectively (Zeng et al., 2000; Reid et al., 2003). As an increasing number of transporter substrates are identified, we may gain greater appreciation for the role of hepatic transporters in mediating and/or mitigating toxic insults from endogenous substrates and xenobiotics. Whereas it is unlikely that induction of hepatic efflux transporters is solely responsible for peroxisome proliferator-mediated protection, it may be a contributing factor to the adaptive cellular response induced by CFB.

Functional studies would improve our understanding of the role of transporters after PPARα activation and its relationship to hepatoprotection. However, CFB is known to induce a very large number of drug-metabolizing enzymes in the liver. Therefore, in vivo disposition studies assessing the functional outcome of changes in transport protein expression by CFB can be confounded by simultaneous changes in multiple biotransformation pathways.

In conclusion, treatment with the PPARα agonist CFB induces hepatic mRNA levels of the efflux transporters Bcrp, Mrp3, and Mrp4. Treatment-related induction of Mrp3 and Mrp4 mRNA levels correlated with increased protein levels. Furthermore, localization and induction of Mrp3 and Mrp4 were confirmed by immunohistochemical techniques. Whereas several efflux transporters are up-regulated in response to CFB, none of the hepatic uptake transporters examined were modulated. The dependence of hepatic efflux transporter gene and protein expression on PPARα was confirmed using PPARα-null mice. These findings provide novel evidence for PPARα-dependent regulation of Mrp3 and Mrp4, underscoring the importance for further understanding of the role of nuclear receptors in hepatic transporter regulation.