Adult Xenopus laevis frogs were purchased from NASCO (Fort Atkinson, WI, USA). The AB strain was used for the zebrafish experiments. Juvenile and transformer stage sea lampreys were obtained from Acme Lamprey Company (Harrison, ME, USA). All animal studies were performed in conformity with the Public Health Service Policy on Human Care and Use of Laboratory Animals, incorporated in the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals. Vertebrate animal studies were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (hepatocyte studies in African clawed frog, zebrafish, and sea lamprey), the Committee on Animal Use of the University of California at San Diego (bile isolation and purification studies), and Ethics Committee of the Federal University of Santa Catarina (in vivo zebrafish liver studies).
The sources of the chemicals were as follows: n-butyl-p-aminobenzoate, ethyl 3-aminobenzoate, 1,25-(OH)2-vitamin D3, glycocholic acid, taurocholic acid, fexaramine, GW3965, GW4064, phenobarbital (Sigma, St. Louis, MO, USA); 5α-cholic acid, 5α-petromyzonol, 5α-petromyzonol 24-sulfate, 3-ketopetromyzonol sulfate (Toronto Research Chemical, Inc., North York, ON, Canada); TCDD (Cambridge Isotopes, Andover, MA); 1α-hydroxyvitamin D2, 1α-hydroxyvitamin D3 (EMD Chemicals, San Diego, CA, USA); 24(R),25-(OH)2-vitamin D3, 25-hydroxyvitamin D3, Nuclear Receptor Ligand Library (76 compounds known as ligands of various nuclear hormone receptors; BIOMOL International, Plymouth Meeting, PA, USA). 5α-cyprinol 27-sulfate was isolated from Asiatic carp (Cyprinus carpio) bile . 5β-scymnol 27-sulfate was isolated from the bile of Spotted eagle ray (Aetobatus narinari). Bile salts were purified by extraction and Flash column chromatography. Bile alcohol sulfates were chemically deconjugated (e.g., to 5α-cyprinol and 5β-scymnol) using a solution of 2,2-dimethoxypropane:1.0 N HCl, 7:1 v/v, and incubating 2 hours at 37°C, followed by the addition of water and extraction into ether. Completeness of deconjugation and assessment of purity was performed by thin-layer chromatography using known standards. Other than those described above, steroids and bile salts were obtained from Steraloids (Newport, RI, USA).
Maintenance of cell lines
The creation of a HepG2 (human liver) cell line stably expressing the human Na+-taurocholate cotransporter (NTCP; SLC10A1) has been previously reported [29, 53]. HepG2-NTCP cells were grown in modified Eagle's medium-α containing 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were grown at 37°C in 5% CO2. The zebrafish ZFL liver cell line (ATCC) was grown in 50% Leibovitz's L-15 medium with 2 mM L-glutamine, 35% Dulbecco's modified Eagle's medium with 4.5 g/L glucose and 4 mM L-glutamine, 15% Ham's F-12 with 1 mM L-glutamine supplemented with 0.15 g/L sodium bicarbonate, 15 mM HEPES, 10 μg/mL human insulin (Sigma), 50 ng/mL recombinant human epidermal growth factor (Sigma), and 5% fetal bovine serum. ZFL cells were grown at 28°C in room air. The Xenopus laevis A6 kidney cell line (ATCC, Manassus, VA, USA) was grown in 75% NCTC 109 medium, 15% distilled water, and 10% fetal bovine serum at 26°C in 2% CO2. Except as noted above, all media and media supplements for the HepG2, ZFL, and A6 cell lines were obtained from Invitrogen (Carlsbad, CA, USA).
Plasmids containing human VDR, zebrafish PXR, human organic anion transporting polypeptide (SLC21), as well as the reporter constructs tk-UAS-Luc and CYP3A4-PXRE-Luc, and 'empty' vectors pCDNA, PsG5, and PM2 were generously provided by SA Kliewer, JT Moore, and LB Moore (GlaxoSmithKline, Research Triangle Park, NC, USA). Mouse VDR (IMAGE clone 3710866) and pCMV-sport6 vectors were obtained from Invitrogen (Carlsbad, CA). The expression vectors were either full-length receptors (i.e., containing both a DBD and LBD; hVDR and mVDR) or GAL4/VDR chimeras that contain only the LBD of the VDR (xlVDR, zfVDR, lampVDR, ciVDR/PXR). For the full-length expression vectors, the reporter plasmid was CYP3A4-PXRE-Luc, a construct that contains a promoter element from CYP3A4 (recognized by VDR DBDs) driving luciferase expression. For the GAL4/LBD expression constructs, the reporter plasmid was tk-UAS-Luc, which contains GAL4 DNA binding elements driving luciferase expression. xlVDR was cloned by RT-PCR from total RNA extracted from the frog A6 cell line. zfVDR was cloned by RT-PCR from total RNA extracted from the ZFL liver cell line. lampVDR was cloned by PCR from a full-length sea lamprey VDR clone, described as the 'insertless' full-length cDNA , generously provided by G.K. Whitfield (University of Arizona College of Medicine, Tucson, AZ, USA). The LBDs of xlVDR (amino acid residues 90–422) [Genbank: U91846], zfVDR (amino acid residues 121–453) [Genbank: AF164512], and lampVDR (amino acid residues 92–406) [Genbank: AY249863] were inserted into the pM2-GAL4 vector to create GAL4/LBD chimeras. Details of the cloning of the ciVDR/PXR are being described in a separate report. The ciVDR/PXR LBD construct contains amino acid residues 57–391 [Genbank: BR000137].
Co-transfections and transactivation assays
The basic methodology for the luciferase reporter assays in 96-well format was as follows. On day 1, cells were seeded onto 96-well white opaque plates at 30,000 cells/well. On day 2, the medium was exchanged, and cells were transfected using calcium phosphate precipitation. Ligand activation of VDRs was determined by a luciferase-based functional assay using the HepG2-NTCP cells as previously described . For hVDR, 3.5 ng/well of VDR plasmid was co-transfected with 30 ng/well of the reporter CYP3A4-PXRE-Luc and 20 ng/well of pSV-μ-galactosidase (Promega, Madison, WI, USA). For mVDR, 4.5 ng/well of VDR plasmid was co-transfected with 45 ng/well of the CYP3A4-PXRE-Luc reporter and 20 ng/well of β-galactosidase. For xlVDR and ciVDR/PXR, 67 ng/well of VDR plasmid was co-transfected with 100 ng/well of the reporter tk-UAS-Luc and 20 ng/well of β-galactosidase. For zfVDR and lampVDR, 100 ng/well of VDR plasmid was co-transfected with 150 ng/well tk-UAS-Luc and 20 ng/well of β-galactosidase. For zebrafish PXR, 75 ng/well of VDR plasmid was co-transfected with 100 ng/well tk-UAS-Luc and 20 ng/well of β-galactosidase.
For experiments involving sulfated bile salts or steroids, human SLC21 was co-transfected at 10 ng/well to facilitate compound uptake. On day 3, the cells were washed with Hanks' buffered salt solution (Invitrogen) and then exposed to medium containing the ligands or vehicle to be tested. The medium utilized charcoal-dextran-treated fetal bovine serum (Hyclone, Logan, UT, USA) to reduce background activation. Each drug concentration was performed at least in quadruplicate and repeated in separate experiments for a total of at least three times. For screening experiments, at least three concentrations of each drug were tested. On day 4, the cells were washed with Hanks' buffered salt solution and then exposed to 150 μL lysis buffer (Reporter Lysis Buffer, Promega). Separate aliquots were taken for measurement of β-galactosidase activity (Promega) and luciferase activity (Steady-Glo, Promega).
To facilitate more reliable cross-species comparisons, complete concentration-response curves for ligands were determined in the same microplate as determination of response to a maximal activator. This allows for determination of relative efficacy, ε defined as the maximal response to test ligand divided by maximal response to a reference maximal activator (note than ε can exceed 1). The maximal activators and their concentrations were as follows: hVDR, xlVDR, zfVDR – 1 μM calcitriol (BIOMOL); mVDR and lampVDR – 0.5 μM calcitriol; ciVDR/PXR – 6-formylindolo- [3,2-b]-carbazole 20 μM; zebrafish PXR – 20 μM 5α-androstan-3α-ol. All comparisons to maximal activators were done within the same microplate. Luciferase data were normalized to the internal β-galactosidase control and represent means ± SD of the assays.
Zebrafish primary hepatocyte cultures
Culture of adult zebrafish primary liver cells was adapted from a procedure published by Collodi and colleagues . Zebrafish are sacrificed with MS-222 (Sigma-Aldrich, 0.05% v/v) and immersed in 0.5% bleach diluted in LDF media (50% Leibovitz L-15, 35% Dulbecco's Modified Essential Medium, 15% Ham's F-12, 15 mM HEPES, 0.015% sodium bicarbonate) for 1 min. The livers are microdissected and immersed in 0.5% bleach for 2 min, and then rinsed three times with media. The tissues are then placed in cold LDF media containing 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin. The tissues are minced to 1 mm3 pieces and washed in Hanks' Balanced Salts (HBS) with calcium and magnesium, spun at 500 g for 5 minutes at 4°C. The minceate is digested with 0.25% trypsin for 5 min at room temperature in a 15 mL conical tube. The tube is gently inverted several times to facilitate cell dissociation. The dissociated cells with trypsin are transferred to a tube containing LDF with 5% fetal bovine serum. Fresh trypsin is then added to the remaining liver pieces for 5 min at room temperature and the dissociated cells combined with the other tube of dissociated cells. The total dissociated minceates are spun at 500 g for 5 minutes at 4°C. The supernatant is removed, cells are resuspended in LDF with fetal bovine serum, and spun again at 500 g for 5 minutes at 4°C. The supernatant is again removed and the cells resuspended in 67% LDF containing 5% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 50 ng/mL human recombinant growth factor, and 10 μg/mL human insulin. The cells are then plated into 24-well plates (5 × 105 cells/well) or 96-well plates (9 × 104 cells/well) plates that have been previously coated with Matrigel diluted 1:47 in LDF (BD Biosciences, San Jose, CA). The cells are cultured at 26°C in ambient atmosphere.
Zebrafish in vivo transcription studies
Adult zebrafish were purchased from a commercial fish supplier and acclimatized for at least two-weeks in 20 L tanks with flow-through freshwater at 22°C. A single dose of 15 mg/kg of 5α-cyprinol, 5β-cyprinol 27-sulfate, 5α-scymnol or 5β-scymnol-27 sulfate, dissolved in saline, was injected intraperitoneally. A control group was injected with saline. After 48 hr, the animals were decapitated and livers of three fish (n = 5 each group) were removed, pooled and weighed, totalizing 15 fish per group. Fish were not fed throughout the experimental period. The numbers of animals used were the minimum necessary to demonstrate consistent effects.
To study the transcription of CYP3C1, ABCB5, PXR, GAPDH and β-actin genes in zebrafish, initial fragments of these genes were identified (Genbank: AW202769, Genbank: BQ284593, Genbank: AAM22215, Genbank: BC083506, Genbank: AF057040, respectively). The PCR primers for CYP3C1 were designed using MacVector Software and primers for MDR1 were designed using Primer 3 Software (Whitehead Institute for Biomedical Research; ). Primer pairs for amplifying CYP3C1 fragment were 5'-TTGAGGAGCGGTGGTGAGCATTAG-3' (sense) and 5'-TGGAGAGAGTGAACTTCGGATTCG-3' (antisense) and for amplifying ABCB5 were 5'-CAGAGTGGGCAGACGTACAA-3' (sense) and 5'-TTCGCAGCAGTAAGCAGAAA-3' (antisense). The PCR primers for PXR were 5'-ATGCGGCGACAAATCTACTGGC-3' (sense) and 5'-TGTGAAGTGTGGCAGAGAGGTG-3' (antisense), for amplifying GAPDH were 5'-CCTCCAAGGAGTAGATGTGACC-3' (sense) and 5' GCAGAGGACTTTTATTCCATCG 3' (antisense) and for β-actin were 5'-CGACCCAGACATCAGGGAGTG-3' (sense) and 5'-GTCCAGGGCCACATAGCA-3' (antisense). RNA was isolated using Trizol (Invitrogen) according to manufacturer's instructions. Purity and concentration of RNA of each sample were verified at 260 nm. The RNA integrity was checked by non-denaturing gel electrophoresis using 1 μg of total RNA in each sample.
Two micrograms of total RNA were reversed transcribed using Ominiscript RT Kit (Qiagen, Valencia, CA, USA). cDNA concentration was checked at 260 nm, and the amount adjusted to 1 μg to perform semi-quantitative RT-PCR. The expected size of the PXR, Cyp3C1, abcb5, GAPDH and β-actin fragments were 577, 477, 218, 234 and 550 bp, respectively. Biotools DNA polymerase (Biotools B&M Labs, S.A., Madrid, Spain) kit was used for the PCR reaction using a thermocycler Personal Mastercycler (Eppendörf) with the following PCR program: 5 cycles of 94°C for 5 s, 72°C for 35 s; 5 cycles of 94°C for 5 s, 70°C for 10 s, 72°C for 35 s; and 23, 24, 35, 15 and 24 cycles (PXR, Cyp3C1, abcb5, GAPDH and β-actin, respectively) of 94°C for 5 s, 61, 54, 48, 49 and 61°C for 10 s (PXR, Cyp3C1, abcb5, GAPDH and β-actin, respectively), and 72°C for 35 s. The PXR, CYP3C1, ABCB5 and GAPDH transcript levels were quantified using Gel-Quant™ (Multiplexed Biotechologies) and β-actin was used to normalize the data. The Shapiro-Wilk W test was used to evaluate normality. When data were parametric the analysis was performed using Student t test; otherwise Mann Whitney U test was applied. Differences were considered significant at the 95% confidence level.
Sea lamprey primary hepatocyte cultures
Culture of sea lamprey primary liver cells was adapted from procedures published by Ma and Collodi . Transformer stage sea lampreys are sacrificed with MS-222 (Sigma-Aldrich, 0.05% v/v) and immersed in 0.5% bleach diluted in LDF media (50% Leibovitz L-15, 35% Dulbecco's Modified Essential Medium, 15% Ham's F-12, 15 mM HEPES, 0.015% sodium bicarbonate) for 1 min. The livers are microdissected and immersed in 0.5% bleach for 2 min, and then rinsed three times with media. The tissues are then placed in cold LDF media containing 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin. The tissues are minced to 1 mm3 pieces and washed in Hanks' Balanced Salts (HBS) with calcium and magnesium, and spun at 500 g for 5 minutes at 4°C. The minceate is digested with 0.25% trypsin for 5 min at room temperature in a 15 mL conical tube. The tube is gently inverted several times to facilitate cell dissociation. The dissociated cells with trypsin are transferred to a tube containing LDF with 10% fetal bovine serum. Fresh trypsin is then added to the remaining liver pieces for 5 min at room temperature and the dissociated cells combined with the other tube of dissociated cells. The total dissociated minceates are spun at 500 g for 5 minutes at 4°C. The supernatant is removed, cells are resuspended in LDF with 10% fetal bovine serum, and spun again at 500 g for 5 minutes at 4°C. The supernatant is again removed and the cells resuspended in 67% LDF containing 5% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 50 ng/mL human recombinant growth factor, and 10 μg/mL human insulin. The cells are then plated into 24-well plates (5 × 105 cells/well) or 96-well plates (9 × 104 cells/well) plates that have been previously coated with Matrigel diluted 1:47 in LDF (BD Biosciences, San Jose, CA). The cells are cultured at 18°C in ambient atmosphere.
Xenopus laevis primary hepatocyte cultures
Xenopus laevis primary hepatocytes were cultured by a protocol adapted from a published report . Frogs are sacrificed with MS-222, rinsed with 70% ethanol, and the liver lobes perfused via the heart, initially with 375 mL Barth (88 mM NaCl, 1 mM K2SO4, and 10 mM HEPES-NaOH, pH 7.4) containing 0.82 mM MgCl2 and 0.1 mg/mL heparin, and then with 25 mL Barth containing 2.22 mM Ca(NO3)2, 2.74 mM CaCl2, and 200 U/mL type I collagenase (Worthington Chemicals, Lakewood, NJ). Livers are minced to fine pieces in 12.5 mL Barth-collagenase solution and then incubated for 10 min at room temperature, periodically disaggregating the liver pieces with a Pasteur pipette. The liver cells are then placed on a shaking incubator for 5 min at room temperature, followed by another 10 min of periodic pipetting to complete disaggregation. Cells are then filtered through a 130 μm Nitex nylon mesh (Fisher Scientific), following by spinning at 500 g for 5 min at 4°C. The supernatant is then aspirated and diluted to 50 mL with Barth plus MgCl2 and then allowed to settle while cooled in ice for 30 min. The supernatant is removed, and the cellular pellet is resuspended and subjected to one more round of nylon mesh filtration, dilution in Barth plus MgCl2, and settling while cooled in ice. The supernatant is removed and the remaining cells are washed once with Barth plus MgCl2 and then centrifuged at 500 g for 5 min at 4°C. The cells are then resuspended in 0.6 × Coon's Modified Ham's F-12 medium (Invitrogen) supplemented with 0.1 × Barth (88 mM NaCl, 1 mM K2SO4, 10 mM HEPES), 200 U/mL penicillin G, 100 μg/mL streptomycin sulfate, 2 mM glutamine, 0.2% glucose, and 10 μg/mL bovine insulin (Calbiochem), with the final pH adjusted to 7.5. The cells are plated at a density of 2.5–3.0 × 104/mL in 24-well plates that have been previously coated with Matrigel diluted 1:47 in cell culture medium. Cells are cultured at 20°C in ambient atmosphere.
Cells were incubated with 20 μM 7-ethoxyresorufin (Sigma) in a buffer composed of 50 mM Tris, 0.1 M NaCl, pH 7.8. The production of resorufin was assessed by fluorescence measurement with an excitation wavelength of 530 nm and an emission wavelength of 590 nm, with standards of resorufin made up to determine molar production of resorufin. Reactions were stopped with 1.5 volumes of cold methanol. Measurements were normalized to total protein concentration (BioRad, Hercules, CA, USA).
HPLC analysis of testosterone metabolites
Testosterone metabolites were identified and quantitated by HPLC. 100 μL of sample, calibration standard, or quality material standard were pipetted into separate microcentrifuge tubes and mixed with 100 μL of methanol. Samples were vortexed and centrifuged at 13,000 rpm for 4 min. The supernatant was then injected into the HPLC.
The samples were separated using an isocratic mobile phase of 60% methanol/40% water on a Lichrospher 100 RP-18 (5 μm, 250 × 4 mm) column (Agilent Technologies, Santa Clara, CA, USA). The flow rate was 1.2 mL/min and total run time was 25 min. The samples were detected by UV absorbance at 242 nm. The retention times for the testosterone metabolites were as follows: 6α-testosterone (4.29 min), 15α-testosterone (4.76 min), 7α-testosterone (5.10 min), 6β-testosterone (5.59 min), 16α-testosterone (6.16 min), 16β-testosterone (8.18 min), 2α-testosterone (8.78 min), and testosterone (18.5 min). Measurements were normalized to total protein concentration (BioRad).
Flurbiprofen hydroxylation assay
Flurbiprofen hydroxylation activity was measured in intact cultured hepatocytes as an index for CYP2C9 activity. The formation of 4-hydroxyflurbiprofen was measured with reverse-phase HPLC adapted from a previously published method . 100 μL of sample aliquot was diluted with methanol (1:1, v/v) and injected onto a Supelcosil LC-18 column (4.9 × 150 mm, 5 μm) with a mobile phase of 0.02 mol/L potassium phosphate, pH 3.0 buffer/water (60:40) at a flow rate of 1.2 ml/min. Quantification of 4-hydroxyflurbiprofen was done with fluorescence detection (Waters 2475) at 260 nm excitation and 320 nm emission wavelength. Measurements were normalized to total protein concentration (BioRad).
The following sequences were used for phylogenetic analysis (some links are from the Ensembl database ): human VDR [Genbank: NM_000376], rhesus monkey VDR [Ensembl: ENSMMUT00000009414], cow VDR [Ensembl: ENSBTAT00000021832], dog VDR [Ensembl: ENCAFT00000014497], mouse VDR [Genbank: NM_009504], chicken VDR [Genbank: AF011356], Japanese quail VDR [Genbank: U12641], Xenopus laevis VDR [Genbank: U91849], fugu VDR [Ensembl: NEWSINFRUT00000138841], bastard halibut [Genbank: AB037674], zebrafish VDR [Genbank: AF164512], medaka VDR [Ensembl: ENSORLT00000001311], sea lamprey VDR [Genbank: AY249863], Ciona intestinalis VDR/PXR [Genbank: BR000137], human CAR [Genbank: NM_005122], rhesus CAR [Genbank: AY116212], cow CAR [Ensembl: ENSBTAT00000012145], dog CAR [Ensembl: ENSCAFT00000020528], Baikal seal [Genbank: AB109553], mouse CAR [Genbank: NM_009803], pig CAR [Genbank: AB214979], opossum CAR [Ensembl: ENSMODT00000006393], human PXR [Genbank: AF061056], rhesus monkey PXR [Genbank: AF454671], cow PXR [Ensembl: ENSBTAT00000026059], mouse PXR [Genbank: AF031814], rabbit PXR [Genbank: AF188476], opossum PXR [Ensembl: ENSMODT00000023109], chicken PXR [Genbank: AF276753], Xenopus laevis PXRα [Genbank: BC041187], Xenopus tropicalis PXR [Ensembl: ENSXETT00000039109], fugu PXR [Ensembl: NEWSINFRUT00000171584], medaka PXR [Ensembl: ENSORLT00000022473], Tetraodon nigriviridis PXR [Ensembl: GSTENT00026021001], zebrafish PXR [Genbank: AF454674, Genbank: AF502918], ixotid tick (Amblyomma americanum) ecdysone receptor [Genbank: AF020187], and purple sea urchin (Strongylocentrotus purpuratus) liver × receptor [Genbank: XM_774904]. Sequences were aligned using ClustalW  and Tcoffee software  and manually adjusted as needed. Phylogeny was inferred using parsimony analysis by PAUP*4.0-beta for UNIX/LINUX (Sinauer Associates, Sunderland, MA, USA) with the ixotid tick ecdysone receptor used as the outgroup. A heuristic search of 100 replicates of random addition plus tree-bisection-reconnection branch swapping was used; to estimate support, 10,000 bootstrap replicates were analyzed. Branch labels indicate bootstrap percentages. The results of the phylogenetic analysis are shown in Figure 4.