ToxSci Advance Access originally published online on August 23, 2006
Toxicological Sciences 2006 94(1):57-70; doi:10.1093/toxsci/kfl088
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Constitutive Androstane Receptor (CAR) as a Potential Sensing Biomarker of Persistent Organic Pollutants (POPs) in Aquatic Mammal: Molecular Characterization, Expression Level, and Ligand Profiling in Baikal Seal (Pusa sibirica)
* Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama 790-8577, Japan
Baikal Institute of Nature Management, Siberian Branch of Russian Academy of Sciences, Ulan-Ude, Buryatia 670047, Russia
Center for International Cooperation, Ocean Research Institute, The University of Tokyo, Nakano-ku, Tokyo 164-8639, Japan
The Eastern-Siberian Scientific and Production Fisheries Center, "VOSTSIBRYBCENTR," Ulan-Ude, Buryatia 670034, Russia
1To whom correspondence should be addressed at Division of Ecotoxicology, Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan. Fax: +81 89-927-8172. E-mail: iwatah{at}agr.ehime-u.ac.jp.
Received May 26, 2006; accepted August 10, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To characterize the function of constitutive active/androstane receptor (CAR) in aquatic mammals, CAR complementary DNA (cDNA) was cloned from the liver of Baikal seal (Pusa sibirica) from Lake Baikal, Russia, and the messenger RNA (mRNA) expression levels in various tissues/organs of the wild population and the CAR ligand profiles were investigated. The seal CAR cDNA had an open reading frame of 1047 bp encoding 348 amino acids that revealed 74–84% amino acid identities with CARs from rodents and human. The mRNA expression profile of tissues/organs represented that Baikal seal CAR was predominantly expressed in the liver followed by heart and intestine. The expression analysis of hepatic CAR mRNA showed no correlation with expression of cytochrome P450 (CYP) 1A, 1B, 2B, 2C, and 3A-like proteins, indicating that the CAR expression level may not be the sole determinant of the regulation of these CYP expressions in the seal liver. There was no significant correlation between CAR expression and any of the persistent organic pollutants (POPs) levels. Furthermore, we performed an in vitro CAR transactivation assay using MCF-7 cells transfected with Baikal seal CAR expression plasmid and (NR1)3-luciferase reporter gene plasmid. In the transactivation analysis of Baikal seal CAR, neither repression by androstanol and androstenol, nor activation by estrone and estradiol, which are recognized as endogenous ligands for mouse and human CARs, was detected. On the other hand, bile acids such as chenodeoxycholic acid, deoxycholic acid, and lithocholic acid activated the seal CAR as well as mouse CAR. As for exogenous chemicals, the seal CAR was transactivated by a human CAR agonist, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime), but not by a mouse CAR agonist, (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene). In addition, the seal CAR was also activated by polychlorinated biphenyls (PCBs) (Kanechlor-500, International Union of Pure and Applied Chemistry No. PCB153; 2,2',4,4',5,5'-hexachlorobiphenyl and PCB180; 2,2',3,4,4',5,5'-heptachlorobiphenyl), and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT) and its metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE). The seal CAR responded more sensitively to PCBs than the mouse CAR. Based on the results of CAR transactivation assay, the lowest observable effect levels of Kanechlor-500, PCB153, PCB180, p,p'-DDT, and p,p'-DDE in Baikal seal were estimated to be 10, 20, 20, 10, and 10 ppm on wet weight basis, respectively. These results suggest that CAR is conserved in diverse mammalian species including seals. Whereas the seal CAR-mediated gene transcription may potentially be a sensitive response to the exposure of certain POPs, the ligand profile of seal CAR may be different from those of other mammalian CARs. This study indicates that CAR-mediated responses may be useful information to assess the ecotoxicological risk of xenobiotics such as POPs in wildlife but the previous results derived from rodent and human CAR may not be applicable to the risk assessment in wild species.
Key Words: CAR; Baikal seal; biomarker; ligand profile.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Environmental pollution by persistent organic pollutants (POPs) is of great concern worldwide because of their long-term exposure to human and wildlife, and a variety of toxic effects. Aquatic mammals including seals and cetaceans accumulate POPs such as polychlorinated biphenyls (PCBs) and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT) through the food chain (Tanabe et al., 1994
). Since POPs are widely spread in the environment of Lake Baikal, Russia (Iwata et al., 1995), the endemic species and a top predator in the lake, Baikal seals (Pusa sibirica) are greatly contaminated by POPs (Iwata et al., 2004; Nakata et al., 1995, 1997). However, little information is available on the risk and effects of exposure to POPs on the wild population of Baikal seal, due to a limited number of biomarkers of POPs applicable to wild populations, their toxicity and susceptibility (Kim et al., 2002, 2005).
The xenobiotic/drug metabolizing enzymes, cytochrome P450 (CYP), especially CYP1-4 families play a key role in the metabolism of xenobiotics, steroid hormones, and fatty acids (Waxman, 1999).
Constitutive active/androstane receptor (CAR) plays an important role in the transcriptional activation of multiple xenochemical metabolizing enzymes, CYP2B, 2C, and 3A, and Uridine 5'di-phosphate (UDP)-glucuronosyltransferase 1A1 and cytosolic sulfotransferase 2A1 in response to phenobarbital (PB)–type chemicals including ortho-chlorine substituted PCB congeners and DDTs in rodent species (Assem et al., 2004; Ferguson et al., 2002; Gerbal-Chaloin et al., 2002; Goodwin et al., 2002; Honkakoski et al., 1998; Sueyoshi et al., 1999; Sugatani et al., 2001; Yoshinari et al., 2001). CAR is retained in the cytoplasm in nonchemical exposed cells as a complex with heat shock protein 90 and cytoplasmic CAR retention protein (Kawamoto et al., 1999; Kobayashi et al., 2003; Yoshinari et al., 2003; Zelko et al., 2001). Following treatment with PB-type inducers, CAR is activated and translocated into the nucleus through the phosphorylation/dephosphorylation pathways (Kawamoto et al., 1999; Zelko et al., 2001). CAR forms a heterodimer with retinoid X receptor 
in nucleus and binds to PB-responsive enhancer module (PBREM) located in the 5' upstream promoter region of CAR target genes. Steroid receptor coactivator 1 and peroxisome proliferator-activated receptor 
coactivator-1 enhance the transcription of CAR target genes as coactivator (Forman et al., 1998; Min et al., 2002). The CAR target genes can potentially regulate physiological condition through the metabolism of endogenous substrates such as steroid and thyroid hormones, bile acids, and bilirubin (Huang et al., 2003; Lee et al., 2003; Qatanani et al., 2005; Wagner et al., 2005). In addition, CAR activation is known to lead to hepatocarcinogenesis through the upregulation of Mdm2, which contributes to both increased DNA replication and inhibition of p53-mediated apoptosis (Huang et al., 2005). Therefore, the molecular characterization of CAR and the signaling pathways mediated by CAR in wildlife may provide valuable information on the exposure and risk of PB-type xenochemicals including PCBs and DDTs in the ecosystem. Knowledge of CAR-target gene regulation mechanisms in a variety of animals could have important implications for our understanding of the evolution and physiological roles of CAR. In particular, comparative study is necessary to address questions of functional conservation and divergence of CAR signaling pathways (Iwata et al., 2002). However, studies addressing CAR are limited to certain laboratory animals and cell lines, and little is known on the presence and function of CAR in wildlife (Iwata et al., 2002; Sakai et al., 2004).
In order to understand CAR signaling pathway in terms of POPs exposure in wildlife, this study attempted to isolate and sequence CAR complementary DNA (cDNA) in Baikal seal (P. sibirica). Based on the sequence data of seal CAR cDNA, specific primers and probe were designed and the messenger RNA (mRNA) expression levels were quantified in various tissues and organs by real-time reverse transcription–PCR (RT-PCR) method. In addition, hepatic CAR expression levels were measured and statistically analyzed in relation to the concentrations of POPs and the expression levels of CYPs in the wild population. Furthermore, CAR ligand profiles, especially focusing on the ligand-dependent transactivation using an in vitro reporter gene assay were also investigated, and compared with those of mouse CAR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents.
TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene) and CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime) were purchased from Calbiochem (San Diego, CA) and Biomol (Plymouth Meeting, PA), respectively. Androstanol and estrogens (estrone and 17ß-estradiol) were obtained from Sigma-Aldrich, Inc. (St Louis, MO). Androstenol and bile acids (cholic acid [CA], chenodeoxycholic acid [CDCA], lithocholic acid [LCA], and deoxycholic acid [DCA]) were purchased from Steraloids (Newport, RI) and Wako (Osaka, Japan), respectively. PCB congeners (International Union of Pure and Applied Chemistry No. PCB153; 2,2',4,4',5,5'-hexachlorobiphenyl and PCB180; 2,2',3,4,4',5,5'-heptachlorobiphenyl) and DDT compounds (p,p'-DDT and p,p'-DDE; 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) were obtained from AccuStandard, Inc. (New Haven, CT) and Sigma-Aldrich, Inc., respectively.
Sample collection.
Baikal seals were collected from Lake Baikal, Russia in May-June in 1992 and 2005 under the permission of local government. The tissues and organs were removed on board immediately after the collection, and the subsamples were frozen in liquid nitrogen and stored at –80°C until RNA isolation. Data on the age, sex, and POPs concentrations of Baikal seals collected in 1992 (n = 58) have already been reported in Nakata et al. (1995). Two liver samples collected in 1992 were used for CAR cDNA cloning. Among 58 specimens in the 1992 collection that were employed for organochlorine analyses, 27 specimens including immature and mature animals of both sexes were selected, and CAR mRNA expression levels in their liver samples were quantified. The CAR mRNA levels were subjected to the correlation analysis with expression levels of CYPs and residue levels of POPs. Various tissues and organs including liver, kidney, heart, lung, pancreas, spleen, gonad, small intestine, muscle, cerebrum, cerebellum, hypothalamus, pituitary gland, and medulla oblongata for clarifying tissue/organ distribution profile of CAR mRNA were sampled from two specimens (one mature male and mature female) of 2005 collection.
Cloning of CAR cDNA.
For the cDNA cloning of Baikal seal CAR, total RNA was isolated from the livers of two specimens using RNAgent Total RNA Isolation System (Promega, Madison, WI). Poly(A)+ RNA were purified by PolyATtract mRNA Isolation Systems (Promega). Baikal seal CAR cDNA was cloned by RT-PCR method using a specific pair of forward (5'-CTGACTTGTGAGGGCTGCAA-3') and reverse (5'-CAGCTTTGCATACAGAAACC-3') primers which were designed from the highly conserved cDNA sequences of human (accession no. Z30425), rat (AF133095), and mouse (AF009327) CARs. For 5'- and 3'-rapid amplification of cDNA ends (RACE), double-stranded cDNA was synthesized using a Marathon cDNA Amplification Kit (BD Biosciences Clontech, Palo Alto, CA). Gene specific primers designed for 5'-RACE (5'-CAGATCTCCCTTCTCAAGGGAGCAGCGG-3') and 3'-RACE (5'-GCTGGGCCAGGGCTTCTGCCGACAGGAT-3') were used for the amplification of cDNA ends by PCR. The amplified cDNAs were sequenced using ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). CAR amino acid sequences deduced from its cDNA sequence were aligned using CLUSTAL W version 1.7.
Quantification of CAR mRNA expression levels.
For the tissue distribution profile of CAR mRNA, total RNA was isolated from the tissues/organs (liver, kidney, heart, lung, pancreas, spleen, gonad, small intestine, muscle, cerebrum, cerebellum, hypothalamus, pituitary gland, and medulla oblongata) of two Baikal seals (one mature male and mature female) that were collected in 2005 using MagExtracter -RNA- with MFX-2100 (Toyobo, Osaka, Japan) and TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacture's instruction. To clarify the individual variability in hepatic CAR mRNA levels, 27 liver samples from seals collected in 1992 were subjected to the RNA isolation. The isolated RNA was treated using RNeasy Mini Kit (Qiagen, Tokyo, Japan) and RNase-Free DNase Set (Qiagen) to prevent the RNA solution from DNA contamination. The concentration of total RNA was determined, and the quality was evaluated by integrity of 28S and 18S ribosomal RNA (rRNA) bands by electrophoresis on agarose gel. Baikal seal CAR mRNA expression levels were measured by quantitative real-time RT-PCR with One-Step RT-PCR Master Mix Reagent Kit (Applied Biosystems) using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The CAR cDNA fragment was amplified using 5'-AGGACCAGATCTCCCTTCTCAAG-3' and 5'-CGTGTTTGGAGACAGAAAGTGGTA-3' primers designed for the real-time RT-PCR. The amplicon was detected using a specific probe, 5'-CAGCGGTTGAAATCTGCCATATCGC-3'. FAM was used as a reporter dye at 5'-end of probe, and TAMRA as a quencher dye at 3'-end. The primers and probe for determination of hepatic CAR expression levels were used at a final concentration of 300 and 250nM, respectively. As for tissue distribution profile of the seal CAR mRNA, the primers and probe were used at 300 and 50nM, respectively. PCR amplification was performed using the primer and probe set under the following conditions: 30 min at 48°C, 10 min at 95°C, and 40 cycles of 15 s at 94°C and 1 min at 50°C. The 18S rRNA of the same specimen was also determined with Taqman Ribosomal RNA Control Reagents VIC Probe (Applied Biosystems) for the normalization of individual CAR mRNA expression levels.
Expression analysis of CAR, pregnane X receptor, vitamin D receptor, and farnesoid X receptor mRNAs in MCF-7 cells.
In order to confirm the native expression levels of CAR, pregnane X receptor (PXR), vitamin D receptor (VDR), and farnesoid X receptor (FXR) mRNAs in MCF-7 cells used for CAR reporter gene assay, they were semiquantified by RT-PCR. Total RNA was isolated from MCF-7 cells and treated with DNase by the same method as that used for quantification of CAR mRNA expression levels in various tissues/organs of Baikal seals. The partial fragments of native CAR, PXR, VDR, and FXR mRNAs in MCF-7 cells were amplified by RT-PCR using their specific primers (CAR; 5'-TGCAAGGGTTTCTTCAGGAG-3' and 5'-CAATTGTGTAGCGAAGAGGC-3', PXR; 5'-CAAGCGGAAGAAAAGTGAACG-3' and 5'-CTGGTCCTCGATGGGCAAGTC-3', VDR; 5'-ATCACCAAGGACAACCGAC-3' and 5'-TGACCTCAATGGCACTTGAC-3', FXR; 5'-GAAGTGGAACCATACTCGCA-3' and 5'-TGTTGTCGAGGTCACTTGTC-3'). Expression of the target genes were confirmed by agarose gel electrophoresis.
Immunoblot analysis.
Hepatic microsomal preparation and immunoblot analysis of Baikal seal were performed according to Kubota et al. (2005). Expression levels of individual CYP proteins were determined in the same specimens (n = 27) as those employed for the measurement of CAR mRNA, although only CYP1B1 protein was measured in 26 samples. Approximately 5.0 g of liver sample from each specimen was minced and homogenized in a 10-ml Teflon-glass homogenizer containing a cold buffer (50mM Tris-HCl, 0.15M KCl, adjusted to pH 7.4–7.5 at 25°C). The sample was then centrifuged at 750 x g for 10 min at 4°C. The supernatant was centrifuged at 12,000 x g for 10 min at 4°C. The supernatant was further centrifuged at 105,000 x g for 1.5 h at 4°C. Following centrifugation, the supernatant was removed and the microsomal pellet was resuspended in an equivalent volume of resuspension buffer (50mM Tris-HCl, 1mM EDTA, 1mM dithiothreitol, pH 7.4–7.5, dissolved in a 20% glycerol solution). Microsomes were placed in cryostorage vials, flash frozen in liquid nitrogen, and stored at –80°C until immunoblot analysis. Immunoblot analysis using the microsomal fraction was performed for the quantification of CYP1A, CYP1B, CYP2B, CYP2C, and CYP3A-like protein expression levels in the liver of Baikal seals. Anti-rat CYP1A1, CYP1B1, CYP2C6, and CYP3A2 polyclonal antibodies and anti-dog CYP2B11 polyclonal antibody were used for the detection of seal CYP1A, CYP1B, CYP2C, CYP3A, and CYP2B homologs, respectively. The antibody for CYP1B1 was purchased from BD Gentest (Woburn, MA), and other antibodies were from Daiichikagaku (Tokyo, Japan). Anti-goat or anti-rabbit immunoglobulin G–horseradish peroxidase conjugate was used as the secondary antibody. Microsome standards in which certain CYP protein was expressed in a baculovirus system were obtained from the same companies as antibodies were purchased, and each CYP protein in the microsome was used as a reference standard. Detection of the antibody cross-reactive proteins was performed using highly sensitive enhanced chemiluminescence Western blotting analysis system (Amersham Biosciences, Piscataway, NJ). The intensities of bands were visualized by an imaging analyzer, ChemiDoc, and quantified by Quantity One (Bio-Rad Laboratories, Hercules, CA). Expression levels of the antibody cross-reactive proteins in the liver of individual animals were expressed as a relative value to the staining intensity of the antibody cross-reactive protein in a specimen. Data on the protein expression levels of CYPs were cited from Iwata et al. (2003) and Kim et al. (2005).
Construction of plasmids for CAR reporter gene assay.
The Baikal seal CAR expression plasmid, pcDNA3.2TOPO-BSCAR, and mouse CAR expression plasmid, pcDNA3.2TOPO-mCAR were constructed by inserting full-length Baikal seal and mouse CAR cDNAs into pcDNA3.2/V5/GW/D-TOPO vector, respectively, according to manufacture's instruction (Invitrogen). To construct pGL3-(NR1)3-Luc luciferase reporter plasmid, a complementary oligonucleotide containing three copies of NR1 site sequence (5'-AGAATCTGTACTTTCCTGACCTTGGCAC-3') of PBREM located in the upstream region of mouse Cyp2b10 gene was synthesized and subcloned into KpnI/XhoI sites of the pGL3-Promoter Vector (Promega). The sequence of fragments inserted in constructed plasmids (pcDNA3.2TOPO-BSCAR, pcDNA3.2TOPO-mCAR, and pGL3-(NR1)3-Luc) was confirmed by ABI PRISM 310 Genetic Analyzer.
Cells and transfection assays.
Human breast cancer cell line, MCF-7 cells were kindly provided by Prof. Chung Kyu-Hyuck of Sungkyunkwan University, Korea. Cells were seeded into 24-well plates (54 cells per well) and grown overnight in 5% CO2 incubator at 37°C. Following incubation, the growth media was changed to phenol-red–free Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% charcoal/dextran double-treated fetal bovine serum (CDFBS). After 24 h, MCF-7 was cotransfected with 100 ng of pGL3-(NR1)3-Luc, 300 ng of CAR expression plasmid (pcDNA3.2TOPO-BSCAR or pcDNA3.2TOPO-mCAR), and 10 ng of phRL-TK control vector (Promega) as internal standard using Lipofectamine (Invitrogen) and Plus reagent (Invitrogen), and incubated at 37°C for 4 h. Cells were then washed by phenol-red–free DMEM and incubated in 10% CDFBS-DMEM treated with various concentrations of each test chemical at 37°C for 24 h. Luciferase activity was then measured using a Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase activities were normalized against Renilla luciferase activities of an internal control phRL-TK vector, and determined from at least three independent transfections.
Residue levels of POPs.
Residue levels of POPs including p,p'-DDE, p,p'-DDT, and PCBs in the blubber of Baikal seal were identified and quantified by high resolution gas chromatograph-electron capture detector (Hewlett-Packard: 5890 Series II, Palo Alto, CA) equipped with moving needle-type injection port. The concentrations of p,p'-DDE and p,p'-DDT were quantified from the peak area of the sample to that of the corresponding external standard. The PCB standard used for quantification was an equivalent mixture of Kanechlors 300, 400, 500, and 600. Total PCB concentrations were calculated by adding the concentrations of individually resolved peaks. Data on the POPs concentrations in the blubber of Baikal seals were cited from Nakata et al. (1995).
Statistical analysis.
Statistical analysis was performed using SPSS (version 12.0, SPSS Japan, Inc., Tokyo, Japan). Mann-Whitney's U-test was employed to detect gender and growth-stage differences in CAR mRNA expression levels. Spearman's rank correlation coefficient was used to measure the strength of the association between hepatic CAR mRNA expression levels and POPs residue revels or hepatic CYP protein expression levels. The experimental data from reporter gene assays were analyzed by Levene's test to check the homogeneity of variance. Differences in reporter gene activities between control and chemical exposure groups were analyzed by an analysis of variance (ANOVA) followed by Dunnett's post hoc test. The 50% effective concentration (EC50) or 50% inhibition concentration (IC50) of each chemical examined was calculated using SigmaPlot (version 9.0, Systat, Inc., Richmond, CA). Statistical significance was regarded as p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Identification of CAR cDNA in Baikal Seal
The full-length CAR cDNA of Baikal seal had an open reading frame of 1047 bp that encodes 348 amino acid residues with a predicted molecular mass of 39.4 kDa (Fig. 1). The C-terminus included 142 bp of 3'-untranslated region with poly (A)+ tail. Comparison of the deduced amino acid sequence of CAR from the seal with those from other mammalian species showed high identities with CARs from northern fur seal (96%), human (84%), monkey (83%), rat (77%), and mouse (74%), whereas lower identities were found with PXR (36
37%) and VDR (31 32%) of human and rodents (Fig. 2). DNA binding domain (DBD) and ligand binding/dimerization domain (LBD), which include activation function-2 transactivation domain at the C-terminus of Baikal seal CAR showed higher amino acid identities (DBD; 85–99%, LBD; 71–95%) with other mammalian CARs (Fig. 2). In addition, P-box in DBD, which plays a critical role for binding to response element such as PBREM located in the promoter region of CAR target genes (Baes et al., 1994), represented a perfect identity among mammalian CARs (Fig. 3).
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Tissue Expression Profile of CAR mRNA
Tissue distribution of CAR mRNA expression in Baikal seal was determined by a real-time RT-PCR with specific primers and a probe. The seal CAR mRNA expression was the highest in the liver and slightly expressed in heart, small intestine, muscle, kidney, and brain (Fig. 4). The expression levels in lung, pancreas, spleen, and gonad, were traceable. This CAR mRNA expression pattern was similar to the results from mouse and human CAR (Baes et al., 1994
; Choi et al., 1997).
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Relationships between Hepatic CAR and CYPs Expression Levels
From the result of CAR tissue expression profile, we found that liver is the major tissue in which CAR is present in Baikal seals. Therefore, hepatic CAR mRNA expression levels in the individual seals were determined by a real-time RT-PCR. As a result, there was no significant relationship of the hepatic CAR mRNA expression levels with sex and age in Baikal seals (data not shown). In addition, no significant correlation was observed between hepatic CAR mRNA and CYP1A, 1B, 2B, 2C, and 3A-like protein expression levels in Baikal seal (Fig. 5). Among these CYP subfamilies analyzed, CYP2B, 2C, and 3A subfamilies are well known as target genes for CAR in rodents and human (Ferguson et al., 2002
; Gerbal-Chaloin et al., 2002; Goodwin et al., 2002; Honkakoski et al., 1998; Sueyoshi et al., 1999; Yoshinari et al., 2001). Furthermore, no significant correlation was detected between POPs including PCBs and DDTs concentration and hepatic CAR mRNA expression levels in Baikal seal (Fig. 6), although these chemicals are known to induce the expression of hepatic CYP2B, 2C, and 3A enzymes in rodents and human (Craft et al., 2002; Dai et al., 2001; Nims and Lubet, 1995; Vezina et al., 2004; Wyde et al., 2003; You et al., 1999).
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CAR Transactivation by Endogenous and Xenobiotic Chemicals
To investigate ligand profiles in Baikal seal CAR, we attempted the transactivation analysis using a luciferase reporter gene assay. Before starting the reporter gene assay, we initially confirmed mRNA expression of endogenous CAR, PXR, VDR, and FXR in MCF-7 using RT-PCR, because these receptors are functionally similar and potentially activate NR1 site (Guo et al., 2003
; Handschin and Meyer, 2005). As a result, VDR was endogenously expressed in MCF-7 cells, and PXR was only at the trace levels. Neither of endogenous CAR nor FXR was detected (Fig. 7).
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In contrast to classical nuclear receptors, rodent and human CARs are known to have a constitutive activity in the absence of ligands in cells transfected with CAR expression plasmid (Baes et al., 1994
; Choi et al., 1997). As well as the activity of CARs in other mammalian species, transfection of Baikal seal CAR also enhanced the luciferase activity in cells without ligand treatment (Fig. 8).
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Endogenous ligands, androstanol, and androstenol, which are recognized as antagonists of the CAR constitutive activity in mammalian species (Forman et al., 1998
; Jyrkkärinne et al., 2003; Moore et al., 2000), showed no repression in CAR of Baikal seal, although mouse CAR–mediated transcriptional activity was repressed in this study (Fig. 9A). Other endogenous compounds such as estrogens and bile acids were also investigated to compare CAR ligand profiles between Baikal seal and mouse. Estrone and estradiol, both of which have been verified as rodent CAR agonists (Kawamoto et al., 2000), also activated mouse CAR in this assay, but not Baikal seal CAR (Fig. 9B). Nonresponsiveness to these estrogens in Baikal seal CAR was similar to that of human CAR (Kawamoto et al., 2000). As for bile acids, both Baikal seal and mouse CARs were activated by CDCA, DCA, and LCA, whereas no activation was recorded by CA (Fig. 9C).
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In addition to endogenous compounds, xenobiotics including environmental contaminants were also investigated for CAR-mediated transactivation. TCPOBOP, which is known to be a model compound as mouse CAR specific agonist (Tzameli et al., 2000
), showed no induction of luciferase gene in Baikal seal CAR expressed cells (Fig. 10A). On the other hand, Baikal seal CAR activation was induced in response to a human CAR agonist, CITCO (Maglich et al., 2003) in a dose-dependent manner, whereas mouse CAR showed a slight activation (Fig. 10A).
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As for environmental contaminants, a technical PCB mixture, Kanechlor-500 induced Baikal seal CAR–mediated transcriptional activity in a dose-dependent manner. Mouse CAR activation was also induced by PCBs, but the fold induction of transcription of luciferase gene is lower (Fig. 10B) and the lowest observable effect level (LOEL) is larger than that of seal CAR (Table 1). Furthermore, transactivation by the major PCB congeners, PCB153 and PCB180, which are the well-known CYP2B inducers in laboratory animals (Connor et al., 1995
; Craft et al., 2002; Vezina et al., 2004), was also investigated. Baikal seal CAR was activated by both PCB congeners in a dose-dependent manner but the significant elevation of the transactivation was detected at more than 20 ppm for both congeners (Fig. 10C). As for mouse CAR, both PCB153 and PCB180 induced the activities only at the highest level of exposure, 50 ppm (Fig. 10C). In case of DDT compounds, both of Baikal seal and mouse CARs were activated at more than 10 ppm by p,p'-DDE and p,p'-DDT exposure (Fig. 10D). In both species, treatment by p,p'-DDT or p,p'-DDE at 50 ppm concentration showed a tendency to decrease the activity.
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We also investigated CAR activation by pyrethroid pesticides such as permethrin, cypermethrin, and fenvalerate. These compounds are known to induce CYP2B and 3A through PXR-mediated pathway (Heder et al., 2001
; Lemaire et al., 2004). Among these pesticides, permethrin and cypermethrin activated only mouse CAR but not seal CAR (Fig. 10E). In this study, CAR-independent reporter gene activities using no CAR transfected cells were examined for each compound (data not shown). When the cells were treated with endogenous ligands including estrone, estradiol, CDCA, DCA, and LCA, the activities in no CAR transfected cells were increased only at the highest concentration. As for exogenous chemicals, PCBs (a technical mixture, PCB153 and PCB180), and DDT compounds (p,p'-DDT and p,p'-DDE) induced the activities at more than 10, 50, 50, 10, and 10 ppm, respectively. However, these CAR-independent activities were lower than those in Baikal seal or mouse CAR transfected cells.
EC50 or IC50 of each chemical was estimated using fold induction values from the CAR transactivation assay. In mouse CAR, IC50 values of androstanol and androstenol were 0.056 and 0.064µM, respectively. These estimated values in the present study were 10-fold lower than those (0.40 or 0.84µM for androstanol and 0.40µM for androstanol) in previous reports (Forman et al., 1998; Moore et al., 2000). As for the xenobiotic chemicals, EC50 values of CITCO treatment in Baikal seal CAR was 0.20µM. The EC50 value of TCPOBOP in mouse CAR was estimated to be 0.014µM. As for other chemicals tested, no significant value was obtained (p > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, CAR cDNA was cloned and sequenced in Baikal seal. Our previous study (Sakai et al., 2004
) succeeded in isolating CAR cDNA from the liver of northern fur seal (Callorhinus ursinus). These results indicate that CARs are conserved among a variety of mammalian species including seals. The two amino acids, Arg90 and Arg91 at the C-terminal extension of DBD, are known to be involved in the formation of a CAR-DNA binding in human (Frank et al., 2003). A recent study has reported that xenochemical response signal region comprising leucine rich sequences (L/MXXLXXXL) near the C-terminus of CAR regulates the xenochemical-induced nuclear translocation of CAR (Zelko et al., 2001). These amino acid residues were also well conserved in Baikal seal CAR (Fig. 3). These results indicate that Baikal seal CAR functions as a transcription factor as human and mouse CARs. In contrast, LBD of Baikal seal CAR showed lower amino acid identities (71–95%) with those of other mammalian CARs when compared with amino acid identities of DBD (85–99%) and full-length (74–96%) sequences (Fig. 2). This implies that ligand profile of the CAR may be potentially diversified among mammalian species.
Tissue distribution profile of CAR showed that Baikal seal CAR mRNA was predominantly expressed in the liver as well as mouse and human CARs (Fig. 4). This result suggests that the liver may be a primary target tissue on CAR signaling pathways in mammalian species. As CAR plays a central role of transcriptional regulation of certain CYP expression in rodents and human livers, relationship between expression levels of CAR and CYPs in the liver of wild Baikal seal population was examined. The result revealed that there was no significant correlation between expression levels of hepatic CAR mRNA and each CYP protein (Fig. 5). This appeared to be different from the result of study on human CAR expression which showed a significant positive correlation between hepatic CAR and CYP2B6 mRNA expression levels (Chang et al., 2003). This result was also in contrast with the result of aryl hydrocarbon receptor (AHR)-CYP1A signaling pathway using the same specimens as this study, there was a significant positive correlation between hepatic AHR and its target gene, CYP1A expression (Kim et al., 2005).
CAR regulates expression of xenobiotic/drug metabolizing enzymes including phase I (CYPs), II, and III (Assem et al., 2004; Maglich et al., 2002; Wei et al., 2002; Xie et al., 2000), and also transcription of a variety of genes involved in basic biological processes such as energy metabolism and gluconeogenic enzymes in the liver of rodents (Kodama et al., 2004; Ueda et al., 2002). If the basic function of CAR is conserved among mammalian species, the seal CAR may also play roles in the regulation of diverse genes expression in the liver. Therefore, the present study suggests that hepatic CAR expression levels would not be the sole determinant of the regulation of these CYPs expression in Baikal seal. Apart from CAR, PXR also mediates CYP2B, 2C, and 3A gene expression in human (Gerbal-Chaloin et al., 2002; Goodwin et al., 1999, 2001, 2002). Thus, regulatory mechanisms of these CYP isozymes shared by both CAR and PXR may lead to a vague relationship of CAR and these CYP expression levels.
No significant correlation was detected between CAR mRNA and POPs concentrations in Baikal seal (Fig. 6). This result indicates that transcriptional regulation of Baikal seal CAR may not be influenced by POPs exposure. It has been suggested that CAR transcription is regulated by glucocorticoid receptor (GR) through glucocorticoid response element in the distal region of human CAR promoter (Pascussi et al., 2003). Johansson et al. (1998) indicated that GR-ligand interaction and ligand-dependent GR activation are competed by methyl sulfone metabolites of PCB congeners in a ligand binding assay and a GR transfected reporter gene assay, respectively. Although the concentrations of methyl sulfone metabolites of PCBs are not determined in Baikal seal population, other seal species such as harbor seals (Phoca vitulina), gray seals (Halichoerus grypus), and ringed seals (Pusa hispida) collected from Swedish coastline accumulate methyl sulfone PCBs at levels that inhibit GR-mediated transcriptional activation (Haraguchi et al., 1992). Therefore, the PCB metabolites may be involved in regulation of CAR transcription in the liver of Baikal seals.
In the transactivation assay, reporter gene activities were enhanced by the treatment of some bile acids and environmental contaminants even in cells with no CAR transfection (data not shown). We also attempted to measure the endogenous CAR, VDR, PXR, and FXR mRNA expression in MCF-7 cells by RT-PCR. As a result, we detected endogenous VDR and PXR but not CAR and FXR mRNA expression (Fig. 7). Thus, these endogenous receptors may contribute to induce the reporter gene activities in no CAR expressed cells. This result is supported by several recent studies showing that VDR and PXR are activated by bile acids in transactivation assays, and can bind to the same type of DR4 response element as that used in this study (Krasowski et al., 2005; Quack and Carlberg, 2000; Xie et al., 2000). However, the transcriptional activities in no CAR transfected cells were lower than those in Baikal seal or mouse CAR transfected cells. In addition, mouse CAR activities repressed by a mouse CAR antagonist, androstenol were recovered following the treatment of chemicals which are known to be mouse CAR agonists such as estrone, estradiol, CDCA, DCA, LCA, PCBs, PCB153, PCB180, p,p'-DDT, and p,p'-DDE (data not shown). Therefore, we concluded that the reporter gene activities that we measured were mostly CAR-dependent reactions.
As suggested from relatively low amino acid identities in CAR LBD among mammalian species (Fig. 2), CAR ligand profiles exhibited marked differences between Baikal seal and mouse in the present transactivation assay (Figs. 9 and 10). Ligand-dependent activities of CAR were compared between Baikal seal and mouse in terms of LOEL for each chemical examined (Table 1). No repression of the seal CAR transactivation by androstanol and androstenol observed here was strikingly different from the results obtained for the mouse CAR in the present and previous studies (Fig. 9A). A recent study using X-ray crystallography analysis indicates that some amino acid residues in CAR LBD are critical for CAR-androstenol binding in mouse, and also specific nonpolar interaction positioned at Leu212 among these amino acid residues is essential for androstenol binding in mouse CAR (Shan et al., 2004). In Baikal seal CAR, the corresponding amino acid was a polar amino acid, Cys202. However, amino acid residue at the corresponding position in human CAR, which is also deactivated by androstenol, was also Cys, indicating that Cys is not the only critical amino acid residue. Comparison of amino acid residues in the ligand binding pocket between human and seal CAR indicated that only two differences at the position of Val164 and Leu226 in seal CAR were found. Therefore, not only Cys202 but also other two amino acid residues may be involved in nonantagonistic response by androstenol in Baikal seal CAR.
Although Baikal seal CAR was not repressed by androsta(e)nol, some bile acids, such as CDCA, DCA, and LCA activated the seal CAR at 100µM as well as mouse CAR (Fig. 9C). The concentrations of these bile acids were much higher than normal concentration (a few µM) in the liver of human (Fischer et al., 1996), whereas comparable levels of these bile acids were reported in intestinal fluid in human (McJunkin et al., 1981). However, some abnormal condition including cholestatic liver disease and hepatic cirrhosis may cause higher concentrations of bile acids in the body. Therefore, these bile acids may be candidates as endogenous ligands for CARs like other bile acid receptors, PXR and FXR. On the other hand, there was no significant CAR activation in the cells exposed by CA (Fig. 9C). This result was different from the case of study in which CA deactivated mouse CAR transfection in CV-1 cells (Moore et al., 2002). The reason still remains unclear but the different cell lines and experimental procedures may be responsible for the different results.
It is known that expression levels of CYP2B and CYP3A are enhanced by exposure of POPs such as PCBs and DDTs (Craft et al., 2002; Medina-Diaz and Elizondo, 2005; Nims and Lubet, 1995). In addition, treatment by POPs including a certain PCB congener (PCB164; 2,3,3',4',5',6-hexachlorobiphenyl) and DDT compounds (1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane and p,p'-DDE) induced CYPs expression through CAR-mediated pathway in rodent (Sueyoshi et al., 1999; Wyde et al., 2003). The present study demonstrated that CAR plays an important role as xenosensor in Baikal seal as well as rodents and human CARs. Baikal seal CAR was more prominently activated by PCBs than mouse CAR (Fig. 10B and 10C). Transactivation of Baikal seal CAR was induced by more than 10 ppm of PCBs (KC-500) at statistically significant level, while mouse CAR was slightly activated at more than 30 ppm (Table 1). These results indicate that Baikal seal may be more sensitive to PCB exposure than mouse. In case of DDTs, Baikal seal CAR, as well as mouse CAR, was significantly activated by p,p'-DDT and p,p'-DDE at more than 10 ppm (Fig. 10D).
In order to evaluate whether or not these concentrations, at which Baikal seal CAR was activated, are environmentally relevant, we attempted to compare these CAR-activating concentrations with those in wild Baikal seal population. Even though the hepatic concentrations of PCBs and DDTs were not given in previous reports, Watanabe et al. (1999) showed that the residue levels on lipid weight basis of PCBs and DDTs in the tissues other than brain of Caspian seal (Pusa caspica) are almost similar. Assuming that the tissue distributions of PCBs and DDTs are dependent on the lipid content in Baikal seal as well as in Caspian seal, we estimated the hepatic concentrations of PCBs and DDTs from their blubber concentrations in Baikal seals using the following equation:
 |
and Iwata et al. (2004), respectively. It is reported that PCBs and DDTs concentrations in the blubber of Baikal seals ranged from 3.5 to 64 ppm and 4.9 to 160 ppm on lipid weight basis, respectively (Nakata et al., 1995). As a result, estimated hepatic concentrations of PCBs and DDTs in Baikal seals were 0.086 to 3.1 ppm and 0.12 to 4.0 ppm on wet weight basis, respectively. The results imply that the hepatic concentrations of individual compounds in Baikal seal populations may be lower than the levels at which CAR was activated in the transactivation assay. However, sum of concentrations of PCBs and DDTs is closed to 10 ppm, and is almost equivalent to LOEL for each chemical including PCBs, p,p'-DDT and p,p'-DDE (Table 1). Furthermore, previous reports demonstrated that not only PCBs and DDTs but also other organochlorine pesticides such as chlordanes and hexachlorocyclohexanes were accumulated in Baikal seals (Nakata et al., 1995, Tanabe et al., 2003). Therefore, if a mixture of POPs additively contributes to the CAR transactivation, even if the concentration level of each compound was low, total concentrations of POPs could exceed the levels at which seal CAR is activated.
In contrast to PCBs and DDTs, TCPOBOP and pyrethroid pesticides were activators only for mouse CAR. A recent study indicates that Thr350 in mouse CAR is a critical site for TCPOBOP activities (Suino et al., 2004). The amino acid residue at the corresponding position of Baikal seal CAR was Met (Fig. 3). Replacement of this amino acid may be a reason for nonresponse of seal CAR to this chemical.
Although no direct evidence is available on CAR activation associated with toxic effects, Mdm2 gene expression that is known to be involved in formation of human hepatocellular carcinoma was shown to be induced by CAR-dependent pathway (Huang et al., 2005). Baikal seal populations have been chronically exposed by multiple CAR activators such as PCBs and DDTs. The residue levels of a mixture of POPs may reach the levels at which CAR is activated in Baikal seal population. Therefore, accumulation of POPs in this seal would be a trigger to elicit a variety of biological responses such as CYPs induction and tumor promotion via CAR signaling pathway.
In summary, the present study demonstrated that CAR is conserved among mammalian species. Our results also suggested that the seal CAR-mediated gene transcription may potentially be a sensitive biological response to evaluate the exposure and effects of POPs. In addition, the ligand profile of seal CAR appeared to be different from those of other mammalian CARs, implying that the results derived from experiments focusing on CARs of rodents and human could not simply be extrapolated into CAR-mediated responses in wildlife.
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ACKNOWLEDGMENTS |
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The authors wish to thank Prof. An. Subramanian, Ehime University, for critical reading of this manuscript. This study was supported by Grants-in-Aid for Scientific Research (A) (Nos. 17208030 and 16201014) and (B) (No. 13480170) from Japan Society for the Promotion of Science, and for Scientific Research on Priority Areas (A) (No. 13027101). Financial assistance was also provided by "21st Century COE Program" from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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REFERENCES |
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