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TOXICOLOGY

Role of Pituitary Hormones on 17-Ethinylestradiol-Induced Cholestasis in Rat

Molecular Endocrinology Group, Department of Clinical Sciences, University of Las Palmas of Gran Canaria–Canary Institute for Cancer Research, Spain (L.A.H.-H., R.S.-F., L.F.-P.); Molecular Endocrinology Group, Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden (A.F-M., G.N.); Department of Clinical Chemistry, Karolinska Hospital, Stockholm, Sweden (M.A.); and Department of Biotechnology, Kungliga Tekniska Högskolan (Royal Institute of Technology), AlbaNova University Center, Stockholm, Sweden (P.N.)

Received August 30, 2006; accepted November 13, 2006.

	Abstract

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Estrogens cause intrahepatic cholestasis in susceptible women during pregnancy, after administration of oral contraceptives, or during postmenopausal hormone replacement therapy. 17-Ethinylestradiol (EE) is a synthetic estrogen widely used to cause experimental cholestasis in rodents with the aim of examining molecular mechanisms involved in this disease. EE actions on the liver are thought to be mediated by estrogen receptor (ER) and pituitary hormones. We tested this hypothesis by analyzing metabolic changes induced by EE in livers from hypophysectomized (HYPOX) and hypothyroid rats. Microarray studies revealed that the number of genes regulated by EE was increased almost 4-fold in HYPOX rat livers compared with intact males. Little overlap was apparent between the effects of EE in intact and HYPOX rats, demonstrating that pituitary hormones play a critical role in the hepatic effects of EE. Consistently, hypophysectomy protects the liver against induction by EE of serum bilirubin and alkaline phosphatase, two markers of cholestasis and hepatotoxicity and modulates the effects of EE on several genes involved in bile acid homeostasis (e.g., FXR, SHP, BSEP, and Cyp8b1). Finally, we demonstrate a novel mechanism of action of EE through binding and negative regulation of glucocorticoid receptor-mediated transcription. In summary, pituitary- and ER-independent mechanisms contribute to development of EE-induced changes in liver transcriptome. Such mechanisms may be relevant when this model of EE-induced cholestasis is evaluated. The observation that the pharmacological effects of estrogen in liver differ in the absence or presence of the pituitary could be clinically relevant, because different drugs that block actions of pituitary hormones are now available.

Bile acid homeostasis is tightly regulated through the activation of the farnesoid orphan receptor (FXR)/small heterodimer partner (SHP) signaling pathway (Boulias et al., 2005; Kalaany and Mangelsdorf, 2006). FXR, in response to bile acid binding, transiently induces the expression of SHP, and elevated levels of SHP in turn lead to transcriptional repression of genes involved in bile acid synthesis. FXR activation down-regulates Ntcp, which prevents bile acid uptake into hepatocytes, and induces expression of Bsep, which increases bile acid efflux from the liver into bile. Therefore, FXR regulates synthesis and transport of bile acids and prevents their overaccumulation in hepatocytes and hepatic toxicity. The FXR/SHP signaling pathway also underlies the down-regulation of the hepatic fatty acid and triglyceride biosynthesis mediated by sterol regulatory element binding protein-1c (SREBP-1c) (Kalaany and Mangelsdorf, 2006). Consistent with the critical role of FXR in lipid metabolism, FXR-null mice have a proatherogenic lipid profile characterized by increased hepatic and serum cholesterol and triglyceride levels. Interestingly, EE treatment causes changes in gene expression similar to that caused in mice constitutively expressing SHP (SHP-Tg) in the liver (Boulias et al., 2005) and cholic acid (CA)-fed mice (Watanabe et al., 2004).

The effects of estrogens on the liver can be direct, i.e., through the interaction with estrogen receptor (ER) (Yamamoto et al, 2006). Interestingly, ER, unlike other nuclear receptors (e.g., ER, FXR, pregnane X receptor, and constitutive androstane receptor), has been shown to be implicated in the pathogenesis of EE-induced cholestasis in mice (Yamamoto et al., 2006). In addition, indirect mechanisms also play a crucial role because of the influence of estrogens on the secretion of pituitary GH that, in turn, can exert significant effects on liver gene expression related to xenobiotic, bile acid, and lipid metabolism (Wehrenberg and Giustina, 1992; Leung et al., 2004; Waxman and O'Connor, 2006). A gender-differentiated secretion pattern exists in all mammals that leads to gender-dependent differences in xenobiotic clearance (Waxman and O'Connor, 2006), lipid metabolism (Ameen et al., 2004), and bile acid synthesis and transport (Rudling et al., 1999; Simon et al., 2004). However, to what extent pituitary hormones are required for EE effects on genes involved in bile acid/lipid homeostasis remains, in most cases, to be elucidated. In the present study, we provide in vivo evidence that exposure to EE causes significant changes in the liver transcriptome that are associated with altered bile acid/lipid homeostasis. In addition to direct actions through nuclear receptors, EE cross-talking with pituitary-dependent hormones plays a critical role in regulation of hepatic gene expression. We also provide indirect evidence that the effects of EE treatment, which, at least in part, lead to changes in the liver transcriptome, occur through pituitary- and ER-independent mechanisms.

	Materials and Methods

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Materials. TRIzol reagent and SuperScript II were purchased from Invitrogen (Carlsbad, CA). Oligo(dT)₁₅ primers, and cyanine (Cy)-labeled nucleotides were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Recombinant human (rh) GH was kindly donated by Pfizer Laboratories (Madrid, Spain). Unless otherwise indicated, the rest of the products cited in this work were purchased from the Sigma-Aldrich (St. Louis, MO).

Animal Treatment. All animal experimentation described in this work was conducted in accordance with approved institutional guidelines for the care and use of laboratory animals. Adult (2–3 months old) male Sprague-Dawley rats were used throughout these experiments. Animals were kept under a constant dark/light cycle and in a controlled temperature (21–23°C) environment and had free access to lab chow and tap water throughout the experiment. For generation of hypothyroid (TX) animals, methimazole (0.05%) was added to the drinking water for 4 weeks. Some TX rats were treated with T₃ or rhGH. Thyroid hormones were dissolved in a minimal volume of 0.01 N NaOH and were brought up to the appropriate concentration with sterile saline and administered as a single daily i.p. injection at the dose of 10 µg/kg b.wt. over 7 days. rhGH was administrated as a s.c. injection in sterile physiological saline at a dose of 0.6 mg/kg/day, divided into two injections carried out at 8:00 AM and 8:00 PM. Hypophysectomized (HYPOX) adult male rats were purchased from Charles River Inc. (St. Aubin les Elbeuf, France). These rats received a solution containing 2.03 g of NaCl, 0.0833 g of KCl, 0.0021 g of CaCl₂, 0.0167 g of MgCl₂, and 50 g of glucose//l of drinking water, and they were used for experimentation 21 to 28 days after hypophysectomy. All HYPOX rats were examined at autopsy for traces of remaining pituitaries. A successful removal of the pituitary was evident in all animals that also showed dramatic impairments of somatic growth and weight gain. Some of the HYPOX rats were treated with rhGH by continuous infusion from osmotic minipumps (model 2004; Alza, Mountain View, CA). rhGH was administered to animals at a daily dose of 0.34 mg/kg b.wt. for 1 week, as described previously (Flores-Morales et al., 2001). Where indicated, animals were injected s.c. daily with EE (5 mg/kg/day) dissolved in corn oil for 1 and 5 consecutive days, as described previously (Rodriguez-Garay, 2003). Twenty-four hours (in the case of T₃ and EE) or 12 h (in the case of rhGH) after the last injection, animals were deeply anesthetized with pentobarbital injection and killed by exsanguination. The vehicle-treated animals were sacrificed at the same time with equivalent amounts of the vehicle alone. Blood samples were taken, and serum was obtained and stored at –80°C. Portions of the liver were snap-frozen in liquid nitrogen and stored at –80°C until processed for analysis of mRNA. Sections of liver were fixed in 10% neutral buffered formalin for 48 h, transferred to 70% ethanol, and embedded in paraffin. Subsequently, 5-µm sections were mounted on slides, stained with hematoxylin and eosin, and examined by light microscopy.

RNA Isolation, cDNA Microarray, Probe Preparation, and Hybridization. Four independent hybridizations were performed to compare individual animals from the different experimental groups (four vehicle-treated and four EE-treated rats), for a total of eight analyses. Total RNA was isolated by disrupting tissues in TRIzol reagent with a Polytron PT-2000 homogenizer (Kinematica, Basel, Switzerland) followed by RNA isolation according the manufacturer's instructions. RNA integrity was assessed by ethidium bromide staining followed by resolution on denaturing agarose gels and also by the RNA 6000 LabChip kit using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The cDNA microarrays, containing probes for 6200 rat protein-coding genes, were fabricated essentially as described previously (Stahlberg et al., 2005). The protocol used for probe labeling and purification was already essentially described as well (Stahlberg et al., 2005). The arrays were prehybridized in 1% bovine serum albumin, 5x saline sodium citrate, and 0.1% SDS at 42°C for 1 to 2 h, washed in Milli-Q water, and dried immediately before the probe was applied. The final probe volume was adjusted to 25 µl with hybridization buffer consisting of 3.4x saline sodium citrate, 0.3% SDS, 20 µg of mouse Cot1 DNA (Invitrogen), 20 µg of poly(A) RNA, and 20 µg of yeast tRNA. After being heated at 98°C for 2 min and cooled to room temperature, the labeled probe mix was added to the array and placed in a sealed hybridization chamber (Corning Glassworks, Corning, NY) for 15 to 18 h at 65°C, after which the array was washed and dried.

Data Processing and Analysis. The arrays were scanned using a GenePix scanner (Axon Instruments Inc., Union City, CA). Image analysis was performed using GenePix Pro 6.0 software (Axon Instruments Inc.). Automatic and manual flagging was used to localize absent or very weak spots (<2 times above background), which were excluded from analysis. Fluorescence (Cy5/Cy3) ratios were normalized as described previously (Stahlberg et al., 2005) using a method that takes into account and corrects for intensity-dependent artifacts in the measurements: the locally weighted scatter plot smoother method in the Statistics for Microarray Analysis (Quackenbush, 2002) package (http://www.bioconductor.org). Identification of differentially expressed genes was performed using the significance analysis for microarrays (SAM) statistical technique (Tusher et al., 2001) for each experimental group separately. A q value was assigned for each of the detectable genes in the array. This value is similar to a p value, measuring the lowest false discovery rate (FDR) at which differential expression of a gene is considered significant. In this study, genes with a FDR of <5% were identified as differentially expressed. The raw data and completed list of regulated genes are available as supplemental files and in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/). An additional selection requirement was added to this statistically based criterion, based on absolute changes in the gene expression ratios. A value of 1.5 (50%) (log₂ ratio EE-treated/untreated |0.59|) was chosen to describe ratios as up- or down-regulated, even though smaller changes in gene expression may have important biological consequences.

Functional classification of differentially expressed genes was performed with the Web-based tool eGOn V2.0 (explore GeneOntology, developed at the Norwegian University of Science and Technology; http://www.genetools.microarray.ntnu.no). With the two-sided one-sample binomial test implemented in eGOn, we compared the list of differentially expressed genes to all genes expressed in EE experiments (EE-treated intact plus EE-treated HYPOX rats) to identify significant bias toward specific Gene Ontology (GO) terms. Additionally, we compared two lists of differentially expressed genes to identify differences in GO terms between both EE-treated groups. The regulated genes presented in the tables were grouped on the basis of GO terms, significantly associated (p < 0.05) with the differentially expressed genes, with the help of the Web-based tool DAVID (Database for Annotation and Visualization and Integrated Discovery; http://david.abcc.ncifcrf.gov) (Dennis et al., 2003). A gene can be annotated for several functions, which are not displayed in the tables, where a gene is assigned to only one group. However, the statistical testing in eGOn has taken multiple functions for genes into consideration, which is the reason that the same gene can be found in several of the categories created by eGOn.

Gene Expression Analysis by qRT-PCR. The expression of selected genes from the array experiments were verified using qRT-PCR as described previously (Stahlberg et al., 2005). Two micrograms of RNA were treated with DNase (Invitrogen) before reverse transcription into cDNA by Superscript II (Invitrogen) using oligo(dT) priming. The specificity of the primers (Table 1) and the uniformity of the PCR-generated products were assessed by analyzing the melting curve of the PCR product. The level of an individual mRNA measured in the qRT-PCR was normalized with the level of two estrogen-insensitive housekeeping genes (cyclophilin and ribosomal protein L13). There were four animals per treatment group, and each sample was analyzed in duplicate. The statistical significance of induced or repressed genes from vehicle-treated animals was determined using one-way analysis of variance. Variability is expressed as S.E.M. For graphing purposes, the relative expression levels were scaled so that the expression level of the vehicle-matched control group equalled 1.

Serum Analysis. Markers of hepatocellular damage and cholestasis, including the enzymes alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase (ALP), and -glutamyltransferase, as well as total cholesterol and total bilirubin were all determined in serum by a dry-slide technique using the Vitros 950 Chemistry System. 7-Hydroxy-4-cholesten-3-one (C4), a validated plasma/serum marker of the hepatic activity of the rate-limiting enzyme in bile acid synthesis CYP7A1, was quantified by high-performance liquid chromatography (Axelson et al., 1991).

Glucocorticoid Receptor Measurement. All steps were carried out at 0 to 4°C. Liver samples were homogenized in a Teflon-glass Potter-Elvehjem homogenizer (B. Braun, Melsungen, Germany) in TMMDSI buffer (50 mM Tris-HCl, 10 mM sodium molybdate, 5 mM magnesium chloride, 2 mM dithiothreitol, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 0.001 mM leupeptin, 0.01 mM aprotinin, and 1 µg/ml soybean trypsin inhibitor, pH 7.5). Homogenates were centrifuged at 17,000g for 15 min. The supernatant was then centrifuged at 105,000g for 1 h to obtain cytosol. For GR measurement, aliquots of cytosol were incubated overnight at 0 to 4°C in duplicate with increasing concentrations (from 0.5 to 50 nM, final concentration) of [³H]DEX. Nonspecific binding for GR activity was measured in parallel incubations with a 200-fold excess of unlabeled DEX. At the end of the incubation period, a suspension of 200 µl of dextran T70-coated charcoal (DCC) (0.08–0.8%, final concentration) in TMMDSI buffer was added, and the samples were shaken, incubated for 10 min at 0 to 4°C, and centrifuged. At this DCC concentration, the nonspecific binding represented <10% of the total binding; higher concentrations of DCC did not yield greater effectiveness. Nonspecific binding showed an excellent linearity in the whole range of concentrations assayed. Aliquots of the supernatant were taken for radioactivity counting. In the control experiments, incubations were carried out in the presence of the vehicle alone (2% ethanol final concentration). No effect of ethanol (1–5%) was observed. Before the binding assay, cytosol was always treated with DCC to remove any steroid that could compete with [³H]DEX for its binding to GR.

Cell Transfection. HepG2 cells were grown to 60% confluence in 60-mm dishes and washed once with phosphate-buffered saline before transfection. Transfection was carried out in serum-free Dulbecco's modified Eagle's medium with N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (Roche Diagnostic, Mannheim, Germany) according to the manufacturer's instructions using MMTV-Luc (1 µg/dish) and cytomegalovirus-GR (0.25 µg/dish) plasmid DNA. MMTV-Luc and cytomegalovirus-GR plasmids were kindly provided by Dr. S. Okret (Karolinska Institute, Stockholm, Sweden). After treatment, the cells were washed with cold phosphate-buffered saline and scraped into 0.25 M Tris HCl, pH 8. After three rounds of freeze-thaw lysis in the presence of Cell Culture Lysis Reagent (Promega, Madison, WI), the extracts were centrifuged to remove cell debris. Luciferase activity from 20 µg of protein was measured in a Fluoroskan Ascent FL (Labsystems, Waltham, MA) by using a Dual-Luciferase Assay System (Promega) according to the manufacturer's instructions. Luciferase activity was normalized for transfection efficiency using the phRL-TK vector as an internal control. Proteins were measured by the Bio-Rad D_c (detergent-compatible) method (Bio-Rad, Hercules, CA), using bovine serum albumin as standard.

Statistical Analysis. The statistical analysis was carried out using GraphPad Prism 4 curve-fitting programs (GraphPad Software, San Diego, CA). Analysis of variance following by post hoc analysis and Student's t test for paired data were used when appropriate. Results are presented as means ± S.E. and are considered significant when p < 0.05.

	Results

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17-Ethinylestradiol-Induced Hepatotoxicity and Cholestasis. To understand how pituitary hormones influence the hepatic actions of EE, we first analyzed the changes in various markers of cholestasis in serum of both intact and HYPOX rats. As shown in Table 2, serum total bilirubin and ALP activities, two markers of cholestasis and hepatotoxicity, were significantly elevated in EE-treated intact rats compared with vehicle-treated rats. Serum total bilirubin was increased 4-fold by hypophysectomy itself, whereas serum ALP was reduced compared with that in vehicle-treated intact rats. Importantly, neither levels of total bilirubin nor ALP activity was further increased by EE in HYPOX rats. As shown in Table 2, serum levels of basal C4, a critical marker of bile acid synthesis (Axelson et al., 1991), was 50% reduced in EE-treated intact rats. The critical role of pituitary hormones to regulate CYP7A1 activity (Rudling et al., 1997) was manifested by a 50% reduction of C4 caused by hypophysectomy itself. Importantly, in the absence of pituitary hormones, 5 days of EE treatment were needed to further reduce serum levels of C4. Serum total cholesterol was significantly decreased in EE-treated intact rats, which is explained by EE influencing the secretion of pituitary GH that in turn induces LDLR expression in liver (Rudling et al., 1997, 1999). The critical role of GH in regulation of cholesterol uptake was manifested by hypercholesterolemia caused by hypophysectomy itself (Table 2). As expected, HYPOX rats were less sensitive to the hypocholesterolemic effects of EE (Rudling et al., 1999). In contrast with intact rats, in the absence of pituitary hormones, 5 days of EE treatment were needed to reduce serum cholesterol, and it was associated with a moderate induction of LDLR mRNA (data not shown), which could be explained by GH-independent regulation of LDLR expression (Horton et al., 2003). This fact contrasts with results in EE-treated intact rats in which estrogen effects on serum cholesterol and LDLR were observed already on day 1. Taken together, these results suggest that in the absence of pituitary hormones liver manifests a different response to EE.

View this table: [in this window] [in a new window]   TABLE 2 Effects of 17-ethinylestradiol treatment on serum components in intact and hypophysectomized male rats Intact and HYPOX male rats were injected s.c. daily with EE (5 mg/kg) for 1 and 5 days. Control rats received the appropriate vehicle (VEH). Twenty four hours after the last injection, serum was extracted, and serum markers were measured as described under Materials and Methods. Data represent a mean ± S.D. from serum samples of at least five individual rats.

Effects of Hypophysectomy on Gene Expression Profiling Regulated by 17-Ethinylestradiol. We next used microarray (6200 gene probes)-based gene expression profiling to compare the hepatic response to EE treatment in intact and HYPOX rats. As assessed using SAM statistics, a FDR of <5% and a mean ratio of log₂ > |0.59|, we identified a consolidated list of genes that were differentially regulated by EE in intact (Supplemental File 1) and HYPOX (Supplemental File 2) rats. Figure 1A shows that 126 of those genes regulated by EE in intact rats increased in abundance by >50%, whereas 149 decreased to the same extent. Comparative analysis of genes with altered expression levels from both intact and HYPOX rats revealed considerable reduction after hypophysectomy of the effects induced by EE in intact rats. This was a general phenomenon that affected most of the EE-regulated genes (Fig. 1B). Accordingly, in intact rat livers, the average expression changes (log₂) across four independent hybridizations were 1.06 ± 0.06 and –1.19 ± 0.06 for induced and repressed genes, respectively, whereas the average fold regulation for the same set of genes in the absence of pituitary hormones was close to 0 (Fig. 1C). These differences were highly significant (P < 0.0001) and demonstrate an important role for the pituitary in the hepatic response to EE in normal rats. The lack of regulation generally observed in HYPOX animals for the subset of genes identified as EE-regulated in intact animals (Fig. 1C) could not be attributed to diminished sensitivity to EE treatment in HYPOX rats as has previously been suggested (Norstedt et al., 1981; Parini et al., 1997; Yamamoto et al., 2006). Interestingly, we found that the number of genes induced and repressed by EE injection was increased almost 4-fold in the HYPOX rat livers (Fig. 1A), a model in which major mediators of EE action on liver are absent (i.e., GH) or drastically reduced (i.e., ER). Furthermore, most of genes regulated by EE in HYPOX rats were not regulated in intact rats (Fig. 1A). Finally, we analyzed whether there were statistically significant differences between the distributions of genes into GO categories when we compared the effects of EE in the presence or in the absence of pituitary hormones. Interestingly, we detected significant differences (P < 0.005) in the distribution into two GO categories: lipid metabolism (GO: 0006629) and lipid biosynthesis (GO: 0008610). These results show that little overlap is apparent between the effects of EE in intact and HYPOX rats, indicating that pituitary hormones are quantitative and qualitative determinants of the hepatic response to EE.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Effects of hypophysectomy on gene expression profiling regulated by 17-ethinylestradiol. Intact and HYPOX male rats were injected s.c. with EE (5 mg/kg) for 1 day. Control rats received the appropriate vehicle (VEH). Twenty-four hours after the last injection, livers were removed and snap-frozen in liquid nitrogen, and differently expressed genes in the livers were identified by cDNA microarrays as described under Materials and Methods. The analysis is based on the SAM statistical technique, and differentially expressed genes were discovered using a FDR < 5% and a mean ratio of log2 > |0.59|. A, the number of EE-regulated genes in the liver of intact () and HYPOX (– – –) rats 24 h after EE injection. The overlapping area shows genes for which expression was altered by EE in both intact and HYPOX rats. B, individual genes are arranged along the x-axis according to the value order of decreases and increases in gene expression measured in EE-treated intact rats. The y-axis shows the log 2 ratio of the transcript signals in EE-treated intact and HYPOX rats. C, box plot shows a statistical evaluation of the differences in the mean expression changes induced by EE in HYPOX rats for the set of genes induced and repressed by EE treatment in intact rats. The lines connect the medians, the boxes cover the 25th to 75th percentiles, and the minimal and maximal values are shown by the ends of the bars. ***, p < 0.0001 in comparison to EE-treated HYPOX rats.

EE is extensively metabolized in rat liver by CYP3A, CYP2C, and UDP-glucuronosyl-transferase 1A1 (Wang et al., 2004). Therefore, the differences observed in the absence of pituitary hormones could be secondary, at least in part, to differential metabolism of EE. We found that, unlike UDP-glucuronosyl-transferase 1A1, several CYP3A isozymes were differently regulated in intact (Supplemental File 1) and HYPOX (Supplemental File 2) rats. In intact rats, CYP3A9 was 3-fold induced by EE, whereas CYP3A2 was down-regulated. However, these CYP genes were not regulated by EE in HYPOX rats. These findings suggest differences in EE metabolism in both models. However, the quantitative and qualitative differences demonstrated in our study make it unlikely that the differences between the effects of EE on intact and HYPOX male rats were attributed only to a dose effects due to changes in EE disposition (i.e., differences in EE metabolism or EE concentration in livers from intact and HYPOX rats).

Effects of 17-Ethinylestradiol on Bile Acid Genes in Hypophysectomized Rats. To further understand the role of pituitary hormones in EE-induced cholestasis, we focused on examining the expression of key hepatic genes involved in bile acid homeostasis. The effects of EE were analyzed by using qRT-PCR assays from individual RNA samples at day 1 and day 5 after EE administration to intact or HYPOX rats. We first examined the expression of genes involved in bile acid synthesis. As seen in Fig. 2A, the mRNA expression levels of CYP7A1, the enzyme that catalyzes the rate-limiting first step of the classic bile acid synthetic pathway, as well as those of CYP8B1 and CYP27A1, were markedly decreased after 24 h of EE administration to intact rats. Consistent with the critical role of pituitary hormones on bile acid synthesis (Rudling et al., 1997), hypophysectomy decreased the mRNA levels of CYP7A1 and CYP27A mRNA expression. Unexpectedly, EE treatment of HYPOX rats increased the mRNA expression levels of CYP8B1 and CYP27A, suggesting that, at least in part, hypophysectomy protects the liver against the inhibitory effects of EE on bile acid synthesis. We next examined the mRNA expression levels of the transporters Ntcp, Mrp2, and Bsep. As seen in Fig. 2B, all of these were decreased after EE administration to intact rats. However, EE treatment of HYPOX rats, although transient, increased the mRNA level of Bsep twice, whereas it failed to decrease nRNA levels of Ntcp and Mrp2. Finally, we examined the expression of FXR and SHP, critical regulators of bile acid synthesis and transport (Sinal et al., 2000; Watanabe et al., 2004; Boulias et al., 2005; Kalaany and Mangelsdorf, 2006). As shown in Fig. 2C, the steady-state mRNA level of FXR was markedly decreased by EE administration to intact rats. In contrast, EE induced SHP mRNA expression, which was associated with functional consequences of SHP activity (Fig. 2A), such as decreased expression of genes involved in bile acid synthesis and transport. Paradoxically, Bsep, a gene that is induced by the FXR/SHP signaling pathway (Sinal et al., 2000; Watanabe et al., 2004; Boulias et al., 2005; Kalaany and Mangelsdorf, 2006), was down-regulated after EE administration to intact rats. Consistent with the critical role of pituitary hormones on ER mRNA expression in liver, hypophysectomy caused a marked down-regulation of this nuclear receptor (data not shown) and protected the liver against the inducing effects of EE on SHP mRNA, which can be explained by a critical role of ER in SHP induction by estrogens (Lai et al., 2003). Taken together, these results indicate that 1) pituitary hormones are critical regulators of bile acid homeostasis, regulating expression of genes involved in synthesis, transport, and transcription, and 2) the absence of pituitary hormones, at least in part, protects the liver against the effects of EE on genes involved in bile acid homeostasis. Interestingly, we have shown that EE down-regulated the mRNA expression levels of ER in a time-dependent manner (from 1 to 5 days) (unpublished data), which could explain why some effects of EE are reversible [e.g., CYP7A1 and SREBP-1c (see below)]. In support of this hypothesis are the recent findings that the ER is responsible for repressing bile acid synthesis and transport in EE-treated mice (Yamamoto et al., 2006). However, from our results we cannot discard the possibility that other mechanisms (e.g., adaptive responses under conditions of drastic changes in lipid/bile acid metabolism) may also contribute to reverse the effects of EE.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Effects of 17-ethinylestradiol on bile acid genes in hypophysectomized rats. Intact and HYPOX male rats were injected s.c. daily with EE (5 mg/kg) for 1 and 5 days. Control rats received the appropriate vehicle (VEH). Twenty-four hours after the last injection, livers were removed, and mRNA levels for genes involved in bile acid synthesis (A), transport (B), and transcription (C) in each individual animal were quantified by qRT-PCR. The values obtained were normalized to cyclophilin and are expressed as the fold change compared with vehicle-treated animals. Bars represent means ± S.E. from RNA samples of at least four individual rats. The mean expression of each gene in intact animals receiving vehicle treatment is defined as 1, with all other expression values reported relative to this level. *, p < 0.05; **, p < 0.01; ***, p < 0.001, for comparison with vehicle-treated intact rats. +, p < 0.05; ++, p < 0.01; +++, p < 0.001, for comparison with vehicle-treated HYPOX rats.

Effects of 17-Ethinylestradiol on Bile Acid Genes in Hypothyroid Male Rats. Next, we analyzed the effects of EE on genes involved in bile acid homeostasis in TX male rats, a model characterized by a drastic decrease of thyroid hormones and GH. As seen in Fig. 3A, T₃ treatment decreased the expression of CYP7A1, CYP8B1, and CYP27A1 with respect to vehicle-treated TX rats. When TX rats were treated with two daily injections of GH to mimic the male GH secretory pattern, CYP7A1 and (more drastically) CYP8B1 were down-regulated, but GH, unlike T₃, did not decrease the CYP27A1 mRNA level. These findings indicate that, at least in part, some of the effects of thyroid hormones on the expression of these genes were due to GH. We next examined the mRNA expression levels of transporters Bsep, Mrp2, and Ntcp. As seen in Fig. 3B, T₃ and GH down-regulated Bsep and Ntcp, whereas both hormones fully restored Mrp2 expression levels with respect to vehicle-treated TX rats. Consistent with the hypothesis that the absence of some pituitary hormones protects the liver against the inhibitory effect of EE on bile acid genes, we observed that this estrogen did not decrease CYP7A1, CYP27A1 (Fig. 3A), Bsep (Fig. 3B), or SHP (Fig. 3C) in TX rats, whereas CYP8B1 and Ntcp were still down-regulated. Although T₃ and GH caused similar effects on Bsep, Ntcp, and Mrp2 in TX male rats (Fig. 3B), these hormones exerted opposite actions on nuclear receptors FXR and SHP. Interestingly, GH treatment of TX rats induced expression of FXR mRNA, whereas T₃ did not (Fig. 3C). In contrast, T₃ decreased the SHP mRNA level unlike GH. These findings support a delicate balance between T₃ and GH on regulation of bile acid homeostasis.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Effects of 17-ethinylestradiol on bile acid genes in hypothyroid rats. TX male rats were injected with one daily dose of T3 (10 µg/kg for 7 days i.p.), two daily doses of GH (0.6 mg/kg/day for 7 days s.c.), or one daily dose of EE (5 mg/kg for 5 days s.c.). Control rats received the appropriate vehicle (VEH). Twenty-four hours (in the case of T3 and EE) or 12 h (in the case of GH) after the last injection, livers were removed, and mRNA levels for genes involved in bile acid synthesis (A), transport (B), and transcription (C) in each individual animal were quantified by qRT-PCR as described under Materials and Methods. The values obtained were normalized to cyclophylin and are expressed as fold change compared with vehicle-treated animals. Bars represent mean ± S.E. from RNA samples of at least four individual rats. The mean expression of each gene in intact animals receiving vehicle treatment is defined as 1, with all other expression values reported relative to this level. *, p < 0.05; **, p < 0.01; ***, p < 0.001, for comparison to vehicle-treated intact rats. ++, p < 0.01; +++, p < 0.001, for comparison with vehicle-treated TX rats.

Expression of Genes Associated with Fatty Acid Homeostasis. Because the FXR/SHP signaling pathway underlies the down-regulation of SREBP-1c target genes (Kalaany and Mangelsdorf, 2006; Watanabe et al., 2006), we analyzed the expression level of this transcription factor in this model of EE-induced cholestasis. Administration of EE to intact rats down-regulated expression of SREBP-1c (Fig. 4) and SREBP-1c target genes (e.g., fatty acid synthase, ATP citrate lyase, stearoyl-Co A desaturase 1, and fatty acid desaturase 2) (see Supplemental File 1). Because SREBP-1c is induced by LXR (Kalaany and Mangelsdorf, 2006), we investigated the possibility that EE treatment also regulates LXR mRNA expression. However, the level of LXR mRNA was not affected by EE, either in intact or in HYPOX rats (data not shown), indicating that it is unlikely that changes in the expression levels of LXR would be responsible for the lower expression of SREBP-1c in EE-treated rats. PPAR, a critical regulator of fatty acid oxidation (Beaven and Tontonoz, 2006), was also down-regulated by EE treatment to intact rats (Fig. 4). Interestingly, EE administration to HYPOX rats still repressed SREBP-1c, whereas PPAR was induced instead, suggesting that fatty acid metabolism is regulated differently by EE in the absence of pituitary hormones. Interestingly, the effect of EE administration on SREBP-1c, but not on PPAR, was reversed after 5 days in both intact and HYPOX rats. This could be explained by adaptive responses under conditions of drastic changes in lipid/bile acid metabolism caused by EE. These results taken together with our microarray analysis (see above) support the hypothesis that there are significant effects of EE on fatty acid metabolism and that effects on lipid metabolism and lipid biosynthesis are significantly different when pituitary hormones are eliminated.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Effects of 17-ethinylestradiol on fatty acid genes in hypophysectomized rats. Intact and HYPOX male rats were injected s.c. daily with EE (5 mg/kg) for 1 and 5 days. Control rats received the appropriate vehicle (VEH). Twenty-four hours after last injection, livers were removed and mRNA levels for SREPB-1c and PPAR in each individual animal were quantified by qRT-PCR as described under Materials and Methods. The values obtained were normalized to cyclophilin and are expressed as the fold change compared with vehicle-treated animals. Bars represent means ± S.E. from RNA samples of at least four individual rats. The mean expression of each gene in intact animals receiving vehicle treatment is defined as 1, with all other expression values reported relative to this level. *, p < 0.05, for comparison with vehicle-treated intact rats. +, p < 0.05, for comparison with vehicle-treated HYPOX rats.

Interaction of 17-Ethinylestradiol with Glucocorticoid Receptor Contributes to the Modulation of Liver Gene Expression. Because the mRNA expression levels of several genes involved in bile acid synthesis (e.g., CYP7A1) and transport (e.g., Ntcp, Bsep, and Mrp2) are induced by glucocorticoids (Pandak et al., 1997; Simon et al., 2004; Eloranta et al., 2006), we hypothesized that EE administration to rats would result in interaction with GR-mediated transcription. First, we analyzed the effects of EE treatment on mRNA expression levels of GR and tyrosine aminotransferase (TAT), a well-known GR target gene. As shown in Fig. 5, A and B, EE administration to intact rats reduced the mRNA expression levels of GR and TAT, respectively. Second, we tested whether the effects of EE on GR-dependent genes could be caused by a direct interaction of EE with GR. As seen in Fig. 5C, unlike 17-estradiol, diethylstilbestrol, or mestranol, a methyl derivative of EE, EE interacts with relatively low affinity (K_i = 0.15 ± 0.02 µM) with cytosolic GR. Finally, we showed that EE interaction with GR modulated GR-dependent transcription by carrying out transient transfection of HepG2 cells with the reporter gene MMTV-Luc in the presence of the GR expression vector. Then, the cells were either pretreated with 5 µM EE for 30 min or received no pretreatment before addition of the potent glucocorticoid DEX (from 2 to 50 nM). After 24 h of DEX treatment, cell extracts were prepared and assayed for luciferase activity. As shown in Fig. 5D, in the presence of EE, the EC₅₀ for DEX shifted from 2.9 ± 0.4 to 6.3 ± 0.9 nM (P = 0.003). Taken together, these results indicate that EE interaction with GR contributes to a liver gene expression profile indicative of glucocorticoid antagonism.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Effects of 17-ethinylestradiol on glucocorticoid receptor-mediated transcription. Intact rats were injected s.c. daily with EE (5 mg/kg) for 1 and 5 days. Control rats received the appropriate vehicle (VEH). Twenty-four hours after the last injection, livers were removed, and mRNA levels for GR (A) and TAT (B) in each individual animal were quantified by qRT-PCR as described under Materials and Methods. The values obtained were normalized to cyclophilin and are expressed as the fold change compared with vehicle-treated animals. Bars represent means ± S.E. from RNA samples of at least four individual rats. The mean expression of each gene in intact animals receiving vehicle treatment is defined as 1, with all other expression values reported relative to this level. **, p < 0.01; ***, p < 0.001, for comparison with vehicle-treated intact rats. +, p < 0.05, for comparison to vehicle-treated HYPOX rats. C, liver cytosol was incubated with [3H]DEX (50 nM) in the absence (vehicle) or in the presence of 12 increasing concentrations of unlabeled inhibitor (from 10 nM to 50 µM). Incubations were continued overnight at 0 to 4°C. The samples were then treated with DCC and counted as described under Materials and Methods. Each curve represents the mean of the duplicates from a single experiment representative of at least four separate experiments. D, serum-starved HepG2 cells were transiently cotransfected with an expression vector for GR (0.25 µg) and MMTV-Luc reporter (1 µg) as described under Materials and Methods. Cells were treated with DEX (from 1 to 50 nM) for 24 h in the presence of dimethyl sulfoxide (VEH, ) or 5 µMEE (). Luciferase values were normalized to those obtained for cotransfected Renilla luciferase and expressed as the fold activation compared with vehicle (dimethyl sulfoxide)-treated cells transfected with the same expression vector and reporter. Results represent the means ± S.E. of triplicate experiments. **, p < 0.01, compared with vehicle-treated cells.

Growth Hormone and 17-Ethinylestradiol Have Overlapping Effects on Gene Expression Profiles in Liver. Because rat liver expressed relatively low levels of ER, it has been proposed that regulation of lipid metabolism by estrogens is mainly mediated by GH (Norstedt et al., 1981; Parini et al., 1997; Rudling et al., 1999). It is well known that estrogens can potentially have an impact on GH-regulated genes in liver through 1) "feminizing" the secretion of pituitary GH (Wehrenberg and Giustina, 1992) and 2) interacting with GH-dependent signaling pathways (Leung et al., 2004). To evaluate the extent of interaction between GH and EE, we next analyzed the similarities in gene expression changes induced by 1 week of GH continuous infusion ("female pattern") in HYPOX male rats in comparison with those induced by 24 h of EE treatment in intact rats. The lists of significantly expressed genes from both experiments were cross-referenced, and all genes were filtered using the same criteria. To ensure that our selection criteria were not biasing our results, we removed the fold change cutoff but retained the significance cutoff and FDR of <5%. Of the 6200 genes sampled on the cDNA microarray, 195 common genes were significantly regulated by EE as well as by GH (Supplemental File 3). To determine the statistical significance of any overlapping genes, we performed a Spearman rank correlation test on the 195 common genes regulated by two hormones. Although there was a low positive correlation (r = 0.3875), it was very significant (P = 0.0001), indicating that the transcript profiles regulated by EE and GH had a significant overlap. Accordingly, 70% of the genes regulated by GH were regulated in the same direction by EE. However, 30% of genes regulated by GH were regulated in an opposite direction by EE (Supplemental File 3). Remarkably, some of these genes (e.g., acyl-CoA synthetase long-chain family member 4, hydroxysteroid 11-dehydrogenase 1, hydroxysteroid 17-dehydrogenase 12, and stearoyl-Co A desaturase 1) are involved in lipid metabolism, suggesting that many of the EE effects in this pathway are independent of GH.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
We have shown that EE-induced cholestasis is associated with rapid changes in several biological pathways, including fatty acid synthesis and xenobiotic metabolism. Hypophysectomy, at least in part, protects the liver against the repressive effect of EE on genes involved in bile acid homeostasis. In addition to direct actions through nuclear receptors, EE cross-talks with pituitary hormones, with GH and T₃ playing a critical role. We also provide evidence that the effects of EE on the liver transcriptome can be exerted through pituitary- and ER-independent mechanisms such as inhibition of GR transcriptional activity.

The ability of EE to down-regulate genes that control bile acid synthesis and transport explains why EE-treated intact rats are unable to efficiently take up bile acids from the blood and why there is a reduced clearance of bile acids from the liver, as well as reduced bile acid synthesis (Rodriguez-Garay, 2003). The intrahepatic accumulation of bile acids upon EE treatment could lead to the activation of FXR and the subsequent induction of SHP (Boulias et al., 2005), which could explain the transcriptional repression of genes involved in bile acid metabolism. Direct transcriptional induction of SHP by estrogen-activated ER has also been demonstrated (Lai et al., 2003), and similar results are observed upon EE administration to intact rats. Indeed, livers from EE-treated intact rats showed a transcriptional profile similar to that of both CA-fed mice (Watanabe et al., 2004) and SHP-Tg mice (Boulias et al., 2005). Interestingly, EE was not capable of inducing SHP in HYPOX (or in TX) rats in which hormone-induced cholestasis is reduced, supporting a key role for SHP. However, EE actions cannot be fully explained by SHP activation. For example, Bsep is repressed by EE in our study and in mice (Yamamoto et al., 2006) but was up-regulated by bile acid activation of the FXR/SHP signaling pathway (Boulias et al., 2005). Additional mechanisms for EE actions are discussed below.

Somewhat surprising was our finding that a large number of genes were still regulated by EE in HYPOX rats, indicating that the effects of EE on liver cannot be fully explained by ER- or pituitary-dependent mechanisms. Recently, it was shown that EE, but not 17-estradiol, transcriptional activity can be mediated by FXR and PXR in HepG2 cells (Yamamoto et al., 2006). Here, we showed that EE binds the GR and represses its transcriptional activity. EE has been used in this and previous studies at a dose of 5 mg/kg b.wt. Assuming that EE was evenly distributed throughout the rat tissues and not degraded or excreted, the plasma concentration would be close to 15 µM. The effect of EE on lipid metabolism begins to be detected at 0.05 mg/kg (Boverhof et al., 2004), corresponding to 150 nM, which is the K_d of EE for GR (Fig. 5). Therefore, this EE concentration is enough to interact with GR in vivo. However, this concentration is still significantly higher than the resting plasma level of estrogen in humans (0.6 nM) or rats (0.1–10 nM), but comparable with levels found in human placenta (Diczfalusy, 1969). Lower concentrations of EE, however, could conceivably have significant effects if administered orally or over longer periods of time (Turgeon et al., 2004). Because glucocorticoids positively regulate transcription of key hepatic enzymes involved in bile acid/lipid homeostasis (Simon et al., 2004; Eloranta et al., 2006), our observations indicate that the interaction of EE with GR most likely contributes to the gene expression patterns indicative of defective bile acid/lipid metabolism.

The expression levels of several LXR target genes (e.g., CYP7A1, CYP8B1, SREBP-1c, and FAS) (Figs. 2 and 4 and Supplemental File 1) were down-regulated by EE treatment in intact rats, suggesting an involvement of this nuclear receptor in EE actions. Because the level of LXR mRNA was not significantly affected by EE, a decreased synthesis of intrahepatic oxysterols should be responsible for the lower activity of the LXR signaling pathway (Horton et al., 2003). It is known that EE administration to intact rats causes an increase in cholesterol uptake together with a marked inhibition of cholesterol conversion to bile acids (Rodriguez-Garay, 2003). Therefore, a reduction in cholesterol conversion to bile acids can contribute to a reduction of intrahepatic oxysterol levels and subsequently the activity of LXR. In addition, we showed that EE reduced expression of PPAR mRNA. Based on previous studies showing that continuous GH administration reduced (Greenhalgh et al., 2005) and glucocorticoids induced (Desvergne and Wahli, 1999) the expression of PPAR mRNA in rat liver, we can speculate that the regulation of GH secretion by EE as well as the GR antagonism seen in this study could contribute to down-regulate PPAR mRNA in intact rat liver.

This picture changes dramatically in HYPOX rats. From a metabolic point of view, hypophysectomy itself results in many alterations (Friedman et al., 1970; Rudling et al., 1997): reduced cholesterol synthesis, LDLR expression, CYP7A1 activity, fecal bile acid excretion, and hepatic fatty acid synthesis and increased total liver cholesterol. As expected, we showed that HYPOX rats were less sensitive to the hypocholesterolemic effects of EE. However, hypophysectomy prevented the inhibitory effects of EE on several genes involved in lipid/bile acid homeostasis. Similarly to EE-treated intact rats, the administration of EE to HYPOX rats down-regulated the mRNA levels of SREBP-1c and CYP7A1, whereas PPAR, CYP8B1, CYP27A, and Bsep were instead up-regulated. Because EE is a powerful inducer of CYP8B1 whose activity determines the ratio between CA and chenodeoxycholic acid (and hence the overall hydrophobicity of the bile), our findings could be interpreted as the effects of EE increasing bile acid hydrophobicity in HYPOX rats. However, this possibility seems unlikely as CYP8B1 is transcriptionally down-regulated by hydrophobic but not hydrophilic bile acids (Vlahcevic et al., 2000). Although it deserves future research, these findings indicate that partial induction of bile acid synthesis (i.e., CYP8B1 and CYP27A) and excretion (i.e., Bsep) together with increased fatty acid oxidation (i.e., increased PPAR activity) and reduced fatty acid synthesis (i.e., reduced SREBP-1c activity) can contribute to the effects of EE in HYPOX rats.

Our gene expression analysis indicated the existence of a significant overlapping in the hepatic actions of GH and EE for the regulation of a subset of genes involved in lipid and xenobiotic metabolisms. We and others have shown that EE can induce changes in GH secretion, which are reflected in changes in liver transcriptome (Leung et al., 2004; Stahlberg et al., 2005). GH is known to increase the expression of ER in male liver, which could also contribute to the observed expression changes. However, EE administration to male rats caused a rapid and time-dependent (from 1 to 5 days) decrease in ER mRNA (data not shown), which makes it unlikely that the effects of EE were secondary to GH induction of liver ER. Through the study carried out in HYPOX and TX male rats, we provide further evidence that the interaction of EE with GH and/or thyroid hormones is relevant in the regulation of bile acid synthesis and transport. We showed that EE administration to TX rats did not decrease expression of CYP7A1 or CYP27A1, whereas CYP8B1 was down-regulated, an effect caused by T₃ (which is a known inhibitor of CYP8B1 expression in TX rats) as well (Andersson et al., 1999). The hormone response elements for ER and thyroid hormones receptor are very similar (Zhu et al., 1996), suggesting that ER may interact at the promoter region of the genes under regulation by thyroid hormones. This possibility seems unlikely, as no thyroid hormone response elements have been identified in the promoter region of CYP8B1 (Andersson et al., 1999). The promoter analysis of the Cyp8b1 gene has also failed to identify ER response elements (Yamamoto et al., 2006). Interestingly, when TX rats were treated with GH, CYP7A1 and (more drastically) CYP8B1 were reduced with respect to vehicle-treated TX rats, whereas CYP27A1 was not affected. Therefore, it is likely that stimulation of pituitary GH secretion by EE treatment also contributes to down-regulation of CYP8B1. Similar to EE-treated HYPOX rats, EE administration to TX rats failed to decrease Bsep mRNA levels, whereas Ntcp was still down-regulated with respect to vehicle-treated TX rats. However, T₃ (as well as GH) down-regulated mRNA expression levels of both transporters. Finally, we showed that hypothyroidism caused a marked decrease of the FXR mRNA level and GH, unlike T₃, restored this level, which indicates that GH is a crucial regulator of liver FXR. In contrast, T₃, unlike GH, reduced SHP mRNA expression in TX male rats. Similar to HYPOX rats, EE was not capable of inducing the expression levels of SHP, which could be explained by a significant decrease of ER in TX male rats (data not shown).

Collectively, our findings indicate that EE induces changes in bile acid/lipid metabolism through functional interactions with GH, T₃, and glucocorticoids. Direct mechanisms include interaction with liver ER and GR. Indirect mechanisms can involve the actions of GH. EE can also act through ER- and pituitary-independent mechanisms, which are caused by adaptive responses under conditions of drastic changes in lipid/bile acid metabolism. The gene expression changes and the mechanisms we have described provide a framework to evaluate the model of EE-induced cholestasis. The observation that the pharmacological effects of estrogen in liver are different in the absence or presence of the pituitary could be relevant, because different drugs are now available that block the actions of pituitary hormones (Heaney and Melmed, 2004). Therefore, the combined use of these drugs with estrogens may lead to clinically relevant drug interactions.

	Acknowledgements

 
This article is dedicated to the memory of Professors Dr. Isidoro del Rio-Lozano and Dr. Felipe Sánchez de la Cuesta y Alarcón, two excellent researchers and better friends who contributed with their work to the field of pharmacology in Spain. The authors thank Antonio Martin and Carlos Mateo-Díaz for animal management and initial experiments, respectively.

	Footnotes

 
This research was supported by grants to L.F.-P. from the Ministerio de Sanidad y Consumo (FIS 1/1000), the Ministerio de Ciencia y Tecnología (PETRI1995-0711 and SAF2003-02117), and Universidad de Las Palmas de Gran Canaria-Pfizer Spain (CN-78/02-05045). A.F.-M. was supported by Vetenskapsrådet and Wallenberg Consortium North (Sweden). L.H.-H. and R.S.-F. are recipients of predoctoral fellowships from the Ministerio de Educación, Cultura y Deporte (AP2001-3499) and Ministerio de Ciencia y Tecnología (SAF2003-02117), respectively.

L.A.H.-H. and A.F.-M. contributed equally to this work. Therefore, they should be considered interchangeably as the first author.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.113209.

ABBREVIATIONS: EE, 17-ethinylestradiol; BSIF, bile-salt-independent fraction of the bile flow; BSDF, bile-salt-dependent fraction of the bile flow; Ntcp, Na⁺ taurocholate cotransporter protein; Bsep, bile salt export pump; FXR, farnesoid X receptor; SHP, small heterodimer partner; SREBP-1c, sterol regulatory element-binding protein-1c; CA, cholic acid; ER, estrogen receptor; GH, growth hormone; Cy, cyanine; rh, recombinant human; TX, hypothyroid; T₃, triiodothyronine; HYPOX, hypophysectomized; SAM, significance analysis for microarrays; FDR, false discovery rate; GO, Gene Ontology; qRT, quantitative real-time; PCR, polymerase chain reaction; ALP, alkaline phosphatase; CYP7A1, cholesterol 7-hydroxylase; C4, 7-hydroxy-4-cholesten-3-one; GR, glucocorticoid receptor; DEX, dexamethasone; DCC, dextran T70-coated charcoal; MMTV, mouse mammary tumor virus; Luc, luciferase; LDLR, low-density lipoprotein receptor; Mrp2, multidrug resistance protein 2; CYP8B1, cholesterol 12-hydroxylase; CYP27A1, sterol 27-hydroxylase; LXR, liver X receptor; PPAR, peroxisome proliferator-activated receptor; TAT, tyrosine aminotransferase.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Leandro Fernández-Pérez, Molecular Endocrinology Group, University of Las Palmas of G.C., Faculty of Health Sciences, Department of Clinical Sciences, Dr. Pasteur s/n, 35016–Las Palmas of G.C., Canary Islands, Spain. E-mail: lfernandez{at}dcc.ulpgc.es

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