Role of the Constitutive Androstane Receptor in Xenobiotic-Induced Thyroid Hormone Metabolism
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内分泌学杂志 2005年第3期
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: David D. Moore, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: moore@bcm.tmc.edu.
Abstract
The induction of hepatic drug metabolizing enzymes alters not only the metabolism of the xenobiotic substances that induce them but also the metabolism of various endogenous hormones. The xenobiotic receptor constitutive androstane receptor (CAR) (NR1I3) mediates the well-studied induction of CYP2B genes and other drug-metabolizing enzymes by phenobarbital (PB), an antiepileptic drug that has been shown to alter thyroid hormone (TH) levels. Here we show that CAR is required for PB-mediated disruption of TH homeostasis and the induction of thyroid follicular cell proliferation. Treatment with PB or the more potent and more effective CAR ligand 1, 4-bis-[2-(3, 5,-dichloropyridyloxy)] benzene resulted in universal induction of thyroid hormone glucuronidation and sulfation pathways in a CAR-dependent manner. This resulted in a decrease in serum T4 concentration and a concomitant increase in serum TSH levels. CAR activation also decreased serum T3 levels in mice in which T3 production was blocked. The increase in serum TSH levels resulted in the stimulation of thyroid-follicular cell proliferation. These results highlight the central role of the xenosensor CAR in drug-hormone interactions.
Introduction
METABOLISM PLAYS A key role in regulating the biological activity of many hormones. In the case of thyroid hormones (TH), a number of important metabolic pathways interact to balance synthesis, bioactivation, and deactivation (1). Because drugs, industrial chemicals, and chemicals of environmental importance can increase TH metabolism, it is important to consider the impact these compounds have on TH and thyroid gland homeostasis (2).
TH levels are regulated at the levels of synthesis and secretion by the thyroid gland and also metabolism in the periphery (3). TH are synthesized and secreted under the influence of the hypothalamus-pituitary-thyroid axis. The anterior pituitary gland secretes TSH, which stimulates the production and release of TH by the thyroid gland. TRH is secreted from the hypothalamus and stimulates TSH release. Expression of both TRH and TSH is controlled by a negative-feedback mechanism that is very sensitive to circulating TH levels (4). The main product secreted by the thyroid gland is T4, which is considered a precursor of the bioactive hormone T3. Most of T3 is produced by outer ring deiodination (ORD) of T4 in the peripheral tissues.
The principal pathways of peripheral TH metabolism are deiodination and conjugation to glucuronic acid or sulfate (3). Whereas ORD activates TH by converting T4 to T3, inner ring deiodination (IRD) deactivates TH by converting T4 and T3 to their inactive metabolites 3, 3', 5' triiodothyronine and 3, 3'-diiodothyronine, respectively. There are three deiodinases, designated D1, D2, and D3 (5). D1, which is highly expressed in the liver and believed to regulate systemic TH levels, can both activate and inactivate TH by mediating ORD as well as IRD of T4 and T3. D2 is an activating enzyme in that it can only catalyze the ORD of TH, whereas D3 is an inactivating enzyme; it can only catalyze IRD of TH. Conjugation of TH involves glucuronidation or sulfation of the phenolic hydroxyl group, which increases water solubility and facilitates urinary and biliary clearance (6, 7). Sulfation also promotes the inactivation of TH, particularly T3, because IRD of sulfated T4 and T3 by D1 is accelerated 40- to 200-fold, whereas the ORD of sulfated T4 is completely blocked (8).
The phase II drug-metabolizing enzymes uridine 5'-diphosphate-glucuronosyltransferase (UGT) and sulfotransferase (SULT) mediate the glucuronidation and sulfation of TH (9, 10, 11). Induction of these enzymes by the widely used antiepileptic drug phenobarbital (PB) and other xenobiotics increases TH metabolism and decreases serum TH levels in both animals and humans (12, 13, 14, 15, 16, 17, 18, 19). Moreover, chronic PB treatment causes thyroid tumors in rats (20, 21). This is thought to be a consequence of the decreased serum TH levels, which increase serum TSH and stimulate thyroid growth and ultimately neoplasm (21, 22, 23).
The nuclear hormone receptor constitutive androstane receptor (CAR) mediates the induction of hepatic drug metabolism in response to PB and other xenobiotics (24, 25). Thus, the loss of CAR function in mice results in the complete absence of CYP2B10 induction in response to PB and PB-like inducers in the liver and decreased drug-metabolizing capabilities (26). Recently the list of CAR target genes has been expanded to encompass genes involved in all phases of xenobiotic metabolism, including the CYP3A enzymes (oxidative metabolism phase), UGT1A enzymes as well as SULT2A and SULT1A enzymes (conjugation phase) and members of the multidrug resistance-associated protein family of transporters (transport phase) (27, 28, 29, 30, 31, 32, 33, 34, 35, 36). Many of these genes are also involved in TH metabolism (8, 9, 37, 38, 39).
In the present study, we examine the hypothesis that the negative effects of PB on serum TH levels are dependent on its ability to activate CAR. As expected, activation of CAR by PB or the potent and more effective agonist ligand 1, 4-bis-[2-(3, 5,-dichloropyridyloxy)] benzene (TCPOBOP) led to a decrease in the serum TH levels. This was accompanied by an increase in serum TSH levels and a concomitant increase in thyroid follicular cell proliferation. This effect of CAR is dependent on its ability to induce the expression of both UGT and SULT enzymes that are involved in TH metabolism and excretion.
Materials and Methods
Animals and treatment
Mice carrying the CAR mutation have been described elsewhere (26). Animals used in all experiments were age-matched (8–10 wk old) male mice housed in groups of four or five in plastic microisolator cages at 22 C with 12-h light, 12-h dark cycle and had free access to food and water. All protocols for animal use and euthanasia were approved by the Animal Care Committee at Baylor College of Medicine and were in accordance with National Institutes of Health guidelines. All chemicals were purchased from Sigma (St. Louis, MO). TCPOBOP (3 mg/kg) was administered as a single ip injection dissolved in corn oil 3 d before assays; control animals received an equal volume of corn oil. PB was added to the food at a concentration of 0.05% (wt/wt) (500 ppm), and animals were fed for 7 d. Each treatment group consisted of four to six animals, and all experiments were repeated twice.
RNA preparation and Northern blot analysis
Mice were killed and their livers isolated after 3 d (TCPOBOP treatment) or 7 d (PB treatment). Livers were immediately place in liquid nitrogen for later use. Total RNA from pooled livers (pooled liver samples of four to six mice) was isolated using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. Twenty micrograms of RNA from each treatment group was resolved on 1% agarose/2.2 M formaldehyde denaturing gel and transferred to a nylon membrane (Hybond N+, Amersham Biosciences, Piscataway, NJ) by capillary blotting. The probe for UGT1A1 was described previously (33). Other probes were prepared from liver total RNA using SuperScript one-step RT-PCR system (Invitrogen) according to the manufacturer’s instructions. Blots were hybridized using ULTRAHyb solution (Ambion, Austin, TX) with 32P-labeled cDNA probes corresponding to mouse UGT2B1 (bases 50–517 of the published cDNA; GenBank accession no. AK050435), mouse UGT2B5 (bases 2–499 of the published cDNA; GenBank accession no. NM_009467), mouse SULT1A1 (bases 30–505 of the published cDNA; GenBank accession no. NM_133670), mouse 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase (PAPSS)2 (bases 72–690 of the published cDNA; GenBank accession no. NM_011864), and mouse SAT1 (bases 1–640 of the published cDNA; GenBank accession no. NM_174870) at 42 C overnight. The blots were then washed twice with 0.1% sodium dodecyl sulfate/ 2x saline sodium citrate at room temperature followed by one wash with 0.1% sodium dodecyl sulfate/0.1x saline sodium citrate at 55 C. The blots were subsequently reprobed with a radiolabeled ?-actin cDNA (BD Clontech Laboratories Inc., Palo Alto, CA). The intensity of signals was quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The levels of target gene mRNA were normalized with the levels of ?-actin mRNA.
Real-time quantitative PCR analysis
Real-time quantitative PCR (RT-QPCR) analysis was used for genes whose expression levels were below the detection limit of Northern blot analysis. RT-QPCR was performed using ABI PRISM 7700 sequence detection system instrument and software (Applied Biosystems, Inc., Foster City, CA). RNA samples were diluted to 100 ng/μl and treated with RNA-free deoxyribonuclease I (Ambion) according to the manufacturer’s recommendations. Samples were then assayed in triplicate 25-μl reactions using 100 ng of RNA per reaction. Gene-specific primers were used at 3 pmol/reaction, and the gene-specific probe was used at 12.5 pmol/reaction. Primers and dual-labeled probes were designed using Primer Express (version 2.0.0, Applied Biosystems) and synthesized by Sigma-Genosys (The Woodlands, TX). All primers and probes were entered into the NCBI Blast program to ensure specificity. The primers for SULT1C1 (GenBank accession no. NM_026935) were 5'-tgagcagaatggtgatgtagagaag-3' and 5'-tgcccactcaataaaagggtg-3', and the probe was 5'-cggcgaaccatcattcaacaccg-3'. Primers for SULT1E1 (GenBank accession no. NM_023135) were 5'-gaaagggaattataggagactggaaga-3' and 5'-tgctgcttgtagtgctcatcaa-3', and the probe was 5'-cattccagaccaccctgagggaga-3'. Primers for SULT2A1 (GenBank accession no. L27121) were 5'-tcactcggaacttattttgaatggt-3' and 5'-cagccacggacatgctca-3', and the probe was 5'-cctccaaggaaatgttctattcggatcatgg-3'. Fold induction values were calculated by subtracting the mean threshold cycle number for each treatment group from the mean threshold cycle number for the vehicle group and raising 2 to the power of this difference. The rodent glyceraldehyde-3-phosphate dehydrogenase internal control kit (Applied Biosystems) was used for loading control.
Measurements of serum TH levels
Blood was collected from the right retroorbital sinus 24 h after the last treatment and transferred to T-MGA tubes (Terumo Medical Corp., Elkton, MD). Serum was separated by centrifugation at 1200 x g for 10 min. Serum T4, T3, and TSH levels were measured by ELISA performed by the chemistry laboratory of Methodist Hospital (Houston, TX).
Measurement of T3 clearance
Wild-type and CAR null animals were divided into three groups each: control group (corn oil treatment), PB treated [0.05% wt/wt (500 ppm) PB mixed with food], and TCPOBOP treated (3 mg/kg, administered as a single ip injection dissolved in corn oil). Treatments were initiated 3 d (TCPOBOP) and 7 d (PB) before T3 injection. Mice were then given two ip injections (one injection per day) of T3 (100 μg/kg) dissolved in 0.05 N NaOH/PBS for 2 d. Blood was collected from the right retroorbital sinus 24 h after the last treatment to assay for serum T3 and TSH levels. T3 injection suppressed serum TSH levels to less than 0.01 μIU/ml, the detection limit of the assay used. In this experiment, one time point measurement of T3 was used to indicate clearance.
Propylthiouracil (PTU) treatment
All mice were fed a low-iodine diet supplemented with 0.15% PTU (Harlan Tekland Co., Madison, WI) for 4 d. Wild-type and CAR null animals were divided into three groups each: control group (a single corn oil injection on the first day), PB treated [0.05% wt/wt (500 ppm) PB mixed with the low-iodine diet], and TCPOBOP treated (3 mg/kg, administered as a single ip injection dissolved in corn oil on the first day). The PB-treated group also received a single corn oil injection on the first day. On the morning of the fifth day, blood was collected from the right retroorbital sinus and the serum isolated and assayed for T3 levels.
Proliferating cell nuclear antigen (PCNA) immunocytochemistry
Mice were treated with corn oil, PB [0.05% wt/wt (500 ppm) PB mixed with food], or TCPOBOP (3 mg/kg, administered as a single ip injection) for 10 d. On the 11th day, the animals were killed and their thyroid glands isolated. The glands were immediately fixed at room temperature in 10% buffered formalin for at least 2 h. The glands were then dehydrated and embedded in paraffin blocks. Five-micron sections of the thyroid glands were cut and mounted on polylysine-coated slides. The slides were then stained for PCNA using a PCNA staining kit (Zymed Laboratory Inc., San Francisco, CA).
Data presentation and statistics
Values are reported as mean ± SEM. Statistical analyses of the results were performed by one-way ANOVA, followed by Newman-Keuls test for comparisons among multiple groups. Values of P < 0.05 were accepted as statistically significant.
Results
Effect of CAR activation on serum TH
To examine the role of CAR in TH metabolism, serum T4, T3, and TSH levels were measured in wild-type and CAR–/– mice pretreated with PB or TCPOBOP. In the wild-type animals, PB and TCPOBOP decreased serum T4 levels 56 and 64%, respectively, but had no effect in the CAR–/– animals (Fig. 1A). As expected, the decrease in T4 was associated with a concomitant increase (255% for PB and 262% for TCPOBOP) in serum TSH levels in wild-type but not CAR–/– animals (Fig. 1B). As also expected based on their intact negative feedback circuit, there was no significant change in serum T3 levels on treatment with the various drugs (Fig. 1C).
FIG. 1. The effect of CAR activation on serum T4 (A), TSH (B), and T3 (C) levels. Values are mean ± SEM (n = 4–6). CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated. *, Significantly different from vehicle-treated control (P < 0.05); **, P < 0.01.
Two approaches were used to study the effects of CAR activation on T3 levels in the absence of TSH influence. First, TSH levels were suppressed by injecting the animals with T3 (40, 41, 42), which resulted in a 2-fold increase in serum T3 levels in the vehicle-pretreated wild-type and all CAR–/– animals (Fig. 2A). Activation of CAR by pretreatment with PB or TCPOBOP markedly increased clearance of the exogenously administered T3. This was only seen in wild-type but not CAR–/– animals. Serum T3 levels in the drug-treated wild-type animals were almost the same as their levels before T3 treatment (Fig. 1C).
FIG. 2. The effect of CAR activation on serum T3 levels in animals rendered TSH nonresponsive by exogenous T3 administration (A) or PTU treatment (B). Values are mean ± SEM (n = 4–6). CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated. **, Significantly different from vehicle-treated control (P < 0.01).
The effect of TSH was also blocked by treating animals with PTU, which inhibits the activity of thyroid peroxidase, the enzyme responsible for the synthesis of TH (43, 44, 45). Administration of PB or TCPOBOP to animals on a PTU-supplemented diet led to a dramatic decrease in serum T3 levels in wild-type but not CAR–/– null animals (Fig. 2B). The same effect was also seen in animals when their diet was supplemented with another thyroid peroxidase inhibitor, methimazole (data not shown).
Effect of CAR on expression of phase II TH-conjugating enzymes
To delineate the molecular mechanism of CAR-induced alterations in TH levels, we profiled the expression of hepatic genes involved in TH metabolism. UGT enzymes play a major role in TH metabolism in the liver (13). Several members of the UGT family of enzymes can metabolize T4 as well as T3. T4 is glucuronidated by the phenol/bilirubin UGT (UGT1A1), whereas T3 is glucuronidated by the androsterone UGT (UGT2B1) (9, 37, 46). Induction of these enzymes increases T4 and T3 glucuronidation and their subsequent excretion into bile (47, 48). PB and TCPOBOP treatments induced the expression of several UGTs in wild-type but not CAR–/– animals (Fig. 3). PB induced the expression of UGT1A1 2.8-fold, whereas TCPOBOP induced its mRNA levels by 3.2-fold, compared with vehicle-treated wild-type animals. UGT2B1 was also modestly induced (1.4-fold PB and 1.8-fold TCPOBOP), as was UGT2B5, a C19 steroid-specific UGT (1.7-fold PB and 1.6 TCPOBOP). Because CAR is normally sequestered in the hepatocyte cytoplasm under basal conditions, the basis for the reduction in basal expression of all the assayed UGT genes in the CAR–/– animals is unclear.
FIG. 3. The effect of CAR activation on the expression of UGT enzymes involved in TH metabolism. Northern blot analysis was performed with probes specific for UGT1A1, UGT2B1, and UGT2B5. Hepatic total RNA from vehicle or PB- or TCPOBOP-treated animals was used for this experiment. The values shown below each blot represent fold change relative to vehicle-treated wild-type animals. CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated.
Sulfation is also an important pathway for the irreversible inactivation of TH, and several SULTs sulfate both T4 and T3 (49, 50, 51, 52). Expression analysis of the different TH SULTs revealed a universal induction by CAR (Fig. 4). The phenol SULT (SULT1A1) showed a 2.1-fold and a 1.6-fold induction by PB and TCPOBOP, respectively (Fig. 4A). Surprisingly, the PB-mediated induction of this enzyme seems to be, at least partly, CAR independent. RT-QPCR analysis of three other TH-sulfating enzymes revealed strong induction in the wild-type but not CAR–/– mice. Consistent with previous studies (34, 35, 36), the hydroxysteroid SULT (SULT2A1) was induced 25-fold by PB and 30-fold by TCPOBOP (Fig. 4B). The cytosolic SULT (SULT1C1) was induced 2-fold by PB and 8-fold by TCPOBOP (Fig. 4C), and the estrogen SULT (SULT1E1) was induced 40-fold by PB and more than 500-fold by TCPOBOP (Fig. 4D).
FIG. 4. CAR activation leads to induction of TH sulfating enzymes. Hepatic total RNA from vehicle or PB- or TCPOBOP-treated animals was used for this experiment. SULT1A1 was assayed using Northern blot analysis (A). RT-QPCR was used to study the effects of the different treatments on the mRNA levels of SULT2A1 (B), SULT1C1 (C), and SULT1E1 (D). The values shown below SULT1A1 blot represent fold change relative to vehicle-treated wild-type animals. RT-QPCR values are mean ± SEM (n = 4–6). CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated. *, Significantly different from vehicle-treated control (P < 0.05); **, P < 0.01.
CAR activation of PAPS synthetic enzymes
The sulfation reaction requires the donation of a sulfonyl group from the obligate cosubstrate PAPS. PAPS, the universal sulfate donor in mammals, is rate limiting for the sulfation reactions and is present in limited amounts in the liver (53). The participation of PAPS in the sulfation reactions is dependent on its availability through synthesis, which in turn depends on the availability of sulfate anions and the activity of PAPSS in the liver. Consistent with previous results (34), induction of the different SULTs by CAR is accompanied by induction of PAPSS2, the major PAPSS in the liver (Fig. 5). This induction was seen after PB treatment (2.2-fold) or TCPOBOP (4.3-fold) in wild-type but not CAR–/– mice. Moreover, CAR activation modestly increased transcripts encoding the sulfate anion transporter (SAT)1 (PB 1.3-fold and TCPOBOP 2.3-fold) (Fig. 5). SAT1 is a major sulfate transporter in the liver, and its induction increases cellular uptake of sulfate anions (54, 55).
FIG. 5. Analysis of mRNA levels of enzymes that are essential for the sulfation reactions. Northern blot analysis was performed with probes specific for PAPSS2 and SAT1 (slc26A1). Hepatic total RNA from vehicle or PB- or TCPOBOP-treated animals was used for this experiment. The values shown below each blot represent fold change relative to vehicle-treated wild-type animals. CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated.
Effect of CAR activation on thyroid-follicular cell proliferation
Increased levels of TSH promote thyroid tumors by stimulating thyroid gland growth, and small increases in serum TSH can result in large increases in thyroid-follicular cell proliferation (13, 14). The increase in serum TSH levels is believed to be the mechanism by which PB mediates thyroid hypertrophy and tumor development (21, 56). PCNA immunocytochemistry was performed to assess the effect of CAR activation on proliferation in the thyroid gland (Fig. 6). PCNA-positive thyroid follicular cell nuclei were seen only in the thyroid glands of PB and TCPOBOP-treated wild-type animals. No changes were seen in the thyroid cells of any of the CAR–/– animals. This phenotype correlates with the higher levels of TSH seen in these animals (Fig. 1B). Moreover, there was an obvious decrease in the colloid size in the PB- and TCPOBOP-treated wild-type animals, compared with CAR–/– mice, which is believed to indicate colloid depletion due to TSH stimulation (14).
FIG. 6. Effects of CAR activation on thyroid follicular cell proliferation. Photomicrographs of representative thyroid tissue from corn oil-, PB-, or TCPOBOP-treated animals labeled with PCNA and counterstained with hematoxylin. PCNA-positive thyroid follicular cell nuclei appear black and are indicated with arrows. CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated.
Discussion
Hormone levels are regulated not only by synthesis and secretion but also by catabolism and elimination. Thus, the hepatic-endocrine axis is an important component in the homeostatic control of a number of hormones. The induction of hepatic microsomal enzymes alters the metabolism of the xenobiotics and also the metabolism of various endogenous compounds, including TH (23). A number of previous studies have identified CAR as a regulator of a variety of enzymes involved in TH catabolism (27, 28, 29, 30, 31, 32, 33, 34, 35, 36), and one recent study (36) indicated that activation of CAR by fasting increases TH breakdown. For unknown reasons, we have not observed CAR activation in response to such a stress. However, the current studies confirm and significantly extend the potential role of CAR in TH metabolism, and we propose a multistep mechanism for xenobiotic suppression of TH levels and subsequent promotion of thyroid follicular cell proliferation (Fig. 7) (13, 14, 21).
FIG. 7. Proposed mechanism of thyroid tumor promotion mediated by CAR. CAR activation by xenobiotics such as PB and TCPOBOP induces several enzymes involved in TH metabolism. This leads to increased hepatic disposition of T4 and T3, which reduces their serum levels. To compensate for this decrease in serum T4 and T3 levels, serum TSH concentration increases, which stimulates thyroid follicular cell proliferation and ultimately neoplasia.
The perturbation of TH balance in xenobiotic exposed mice is thought to be due to enhanced metabolism and excretion of these hormones via induction of UGT and SULT enzymes (8, 13). Induction of UGT1A1 and UGT2B1 increases the glucuronidation of T4 and T3, respectively, and their excretion into bile (9, 37, 57). It is believed that glucuronidation is the main pathway for T4, and to a lesser extent T3, metabolism. In this study, we show that the induction of UGT1A1 and UGT2B1 by PB and TCPOBOP is CAR dependent. This presumably contributes to the marked decrease in T4 levels on CAR activation. We also found that CAR induces UGT2B5 expression. The activity of this enzyme in TH metabolism has not been elucidated but is closely related to human UGT2B7, an enzyme that metabolizes TH (58).
Sulfation is a major mechanism in the regulation of TH levels and activity (1, 8). Sulfation prevents the binding of T3 to its receptors and facilitates excretion of both T4 and T3 into bile and urine (3, 59). Additionally, T3 sulfate is subject to accelerated degradation because sulfation facilitates the IRD of T3 by D1. Sulfation also facilitates the inactivating IRD of T4 by D1, whereas the activating ORD of this prohormone is completely blocked (1). Our results suggest that CAR functions as a global regulator of sulfation by controlling the production of not only a number of SULT enzymes but also the obligate cosubstrate, PAPS and the sulfate transporter SAT1. The induction of PAPSS2 is of particular importance because it is estimated that the entire liver content of PAPS can be consumed in less than 2 min if its synthesis is stopped (53).
It has been assumed that the maintenance of normal T3 levels in PB-treated animals is due to increased serum TSH (13, 60), and our results demonstrate that neutralization of TSH effects leads to an obvious decrease in serum T3 in the xenobiotic-treated mice. Particularly because the ability of PB to promote thyroid growth and neoplasia is attenuated by suppression of TSH via readministration of T4 or T3 (21), we believe that the increase in thyroid-follicular cell proliferation upon CAR activation is secondary to the increase in TH metabolism and the compensatory increase in TSH levels.
The ability of CAR activation to alter TH levels has significant clinical importance. Several studies have shown that patients treated with PB have increased TH metabolism and reduced serum T4 levels, and hypothyroid patients require an increase in their thyroid replacement therapy dosage after treatments with antiepileptic drugs, such as the CAR activator phenytoin (12, 13, 14, 15, 16, 17, 18, 19, 61, 62, 63). Surprisingly, the decrease in serum T4 in these patients is often not associated with a clear compensatory increase in TSH. It has been suggested that a slight increase in TSH levels in earlier studies could not be detected due to the sensitivity of the assay used (64), and more recent studies on the effects of antiepileptic drugs including PB have shown higher levels of TSH (65). This has been linked to an increase in the incidence of initial clinical signs of thyroid hyperplasia and the frequency of thyroid goiter (66).
In children exposed to the polychlorinated biphenyl PCB-153, an effective CAR activator, levels of the environmental pollutant were negatively correlated with plasma T4 and T3 levels and positively correlated with TSH levels (67, 68, 69). This is consistent with the higher incidence of thyroid hypertrophy in children exposed to such chemicals (70). Interestingly, recent results suggest that CAR expression is higher in females than males (71), and the correlations of TH alterations with both PCB levels (67) and PB and phenytoin treatments (72) had a stronger effect in women than men.
Several studies have suggested a key role for CAR in drug-hormone interactions (73, 74). Epileptics taking PB and oral contraceptives (e.g. ethynylestradiol) have a 25-fold higher risk of pill failure caused by the increased rate of estrogen metabolism (75, 76, 77), and the induction of UGT1A1 and SULT1E1, two major enzymes involved in estrogen metabolism, may contribute to this effect. Changes in hormone levels may also affect drug metabolism. For example, hypothyroid women who are being treated with thyroxine often need higher doses when they are pregnant (78, 79, 80). It has been suggested that this is due to estrogen induction of thyroxine binding globulin (TBG) expression (81). However, patients with mutations in the TBG gene are euthyroid and show normal levels of T4 and T3 (82). Consistent with this, a recent case was described in which despite low baseline TBG levels and blunted pregnancy-associated TBG induction, the patient’s absolute and relative pregnancy-associated increases in thyroxine replacement dosage mirrored those found in non-TBG-deficient, hypothyroid patients (83). Moreover, the increase in serum TBG levels reaches a plateau 6–12 wk into pregnancy (84), whereas the increase in T4 requirements persists throughout pregnancy. Thus, increased TBG concentration may not be the key determinant for the increase in thyroxine requirement in pregnancy. An alternative explanation is that higher estrogen and progesterone levels during pregnancy may activate CAR or its closest relative, pregnane X receptor, and increase TH metabolism (85, 86). Consistent with this, serum levels of anticonvulsants as well as other drugs decrease during pregnancy by as much as 40%, apparently due to an increase their hepatic metabolism (77, 87, 88, 89, 90). It is believed that this is a consequence of the higher estrogen levels in pregnant women because similar effects have been reported for nonpregnant women on oral estrogen contraceptives (91, 92).
Acknowledgments
We thank Dr. Ann Marie Zavacki (Brigham and Women’s Hospital/Harvard Medical School, Boston, MA) for technical advice.
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Address all correspondence and requests for reprints to: David D. Moore, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: moore@bcm.tmc.edu.
Abstract
The induction of hepatic drug metabolizing enzymes alters not only the metabolism of the xenobiotic substances that induce them but also the metabolism of various endogenous hormones. The xenobiotic receptor constitutive androstane receptor (CAR) (NR1I3) mediates the well-studied induction of CYP2B genes and other drug-metabolizing enzymes by phenobarbital (PB), an antiepileptic drug that has been shown to alter thyroid hormone (TH) levels. Here we show that CAR is required for PB-mediated disruption of TH homeostasis and the induction of thyroid follicular cell proliferation. Treatment with PB or the more potent and more effective CAR ligand 1, 4-bis-[2-(3, 5,-dichloropyridyloxy)] benzene resulted in universal induction of thyroid hormone glucuronidation and sulfation pathways in a CAR-dependent manner. This resulted in a decrease in serum T4 concentration and a concomitant increase in serum TSH levels. CAR activation also decreased serum T3 levels in mice in which T3 production was blocked. The increase in serum TSH levels resulted in the stimulation of thyroid-follicular cell proliferation. These results highlight the central role of the xenosensor CAR in drug-hormone interactions.
Introduction
METABOLISM PLAYS A key role in regulating the biological activity of many hormones. In the case of thyroid hormones (TH), a number of important metabolic pathways interact to balance synthesis, bioactivation, and deactivation (1). Because drugs, industrial chemicals, and chemicals of environmental importance can increase TH metabolism, it is important to consider the impact these compounds have on TH and thyroid gland homeostasis (2).
TH levels are regulated at the levels of synthesis and secretion by the thyroid gland and also metabolism in the periphery (3). TH are synthesized and secreted under the influence of the hypothalamus-pituitary-thyroid axis. The anterior pituitary gland secretes TSH, which stimulates the production and release of TH by the thyroid gland. TRH is secreted from the hypothalamus and stimulates TSH release. Expression of both TRH and TSH is controlled by a negative-feedback mechanism that is very sensitive to circulating TH levels (4). The main product secreted by the thyroid gland is T4, which is considered a precursor of the bioactive hormone T3. Most of T3 is produced by outer ring deiodination (ORD) of T4 in the peripheral tissues.
The principal pathways of peripheral TH metabolism are deiodination and conjugation to glucuronic acid or sulfate (3). Whereas ORD activates TH by converting T4 to T3, inner ring deiodination (IRD) deactivates TH by converting T4 and T3 to their inactive metabolites 3, 3', 5' triiodothyronine and 3, 3'-diiodothyronine, respectively. There are three deiodinases, designated D1, D2, and D3 (5). D1, which is highly expressed in the liver and believed to regulate systemic TH levels, can both activate and inactivate TH by mediating ORD as well as IRD of T4 and T3. D2 is an activating enzyme in that it can only catalyze the ORD of TH, whereas D3 is an inactivating enzyme; it can only catalyze IRD of TH. Conjugation of TH involves glucuronidation or sulfation of the phenolic hydroxyl group, which increases water solubility and facilitates urinary and biliary clearance (6, 7). Sulfation also promotes the inactivation of TH, particularly T3, because IRD of sulfated T4 and T3 by D1 is accelerated 40- to 200-fold, whereas the ORD of sulfated T4 is completely blocked (8).
The phase II drug-metabolizing enzymes uridine 5'-diphosphate-glucuronosyltransferase (UGT) and sulfotransferase (SULT) mediate the glucuronidation and sulfation of TH (9, 10, 11). Induction of these enzymes by the widely used antiepileptic drug phenobarbital (PB) and other xenobiotics increases TH metabolism and decreases serum TH levels in both animals and humans (12, 13, 14, 15, 16, 17, 18, 19). Moreover, chronic PB treatment causes thyroid tumors in rats (20, 21). This is thought to be a consequence of the decreased serum TH levels, which increase serum TSH and stimulate thyroid growth and ultimately neoplasm (21, 22, 23).
The nuclear hormone receptor constitutive androstane receptor (CAR) mediates the induction of hepatic drug metabolism in response to PB and other xenobiotics (24, 25). Thus, the loss of CAR function in mice results in the complete absence of CYP2B10 induction in response to PB and PB-like inducers in the liver and decreased drug-metabolizing capabilities (26). Recently the list of CAR target genes has been expanded to encompass genes involved in all phases of xenobiotic metabolism, including the CYP3A enzymes (oxidative metabolism phase), UGT1A enzymes as well as SULT2A and SULT1A enzymes (conjugation phase) and members of the multidrug resistance-associated protein family of transporters (transport phase) (27, 28, 29, 30, 31, 32, 33, 34, 35, 36). Many of these genes are also involved in TH metabolism (8, 9, 37, 38, 39).
In the present study, we examine the hypothesis that the negative effects of PB on serum TH levels are dependent on its ability to activate CAR. As expected, activation of CAR by PB or the potent and more effective agonist ligand 1, 4-bis-[2-(3, 5,-dichloropyridyloxy)] benzene (TCPOBOP) led to a decrease in the serum TH levels. This was accompanied by an increase in serum TSH levels and a concomitant increase in thyroid follicular cell proliferation. This effect of CAR is dependent on its ability to induce the expression of both UGT and SULT enzymes that are involved in TH metabolism and excretion.
Materials and Methods
Animals and treatment
Mice carrying the CAR mutation have been described elsewhere (26). Animals used in all experiments were age-matched (8–10 wk old) male mice housed in groups of four or five in plastic microisolator cages at 22 C with 12-h light, 12-h dark cycle and had free access to food and water. All protocols for animal use and euthanasia were approved by the Animal Care Committee at Baylor College of Medicine and were in accordance with National Institutes of Health guidelines. All chemicals were purchased from Sigma (St. Louis, MO). TCPOBOP (3 mg/kg) was administered as a single ip injection dissolved in corn oil 3 d before assays; control animals received an equal volume of corn oil. PB was added to the food at a concentration of 0.05% (wt/wt) (500 ppm), and animals were fed for 7 d. Each treatment group consisted of four to six animals, and all experiments were repeated twice.
RNA preparation and Northern blot analysis
Mice were killed and their livers isolated after 3 d (TCPOBOP treatment) or 7 d (PB treatment). Livers were immediately place in liquid nitrogen for later use. Total RNA from pooled livers (pooled liver samples of four to six mice) was isolated using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. Twenty micrograms of RNA from each treatment group was resolved on 1% agarose/2.2 M formaldehyde denaturing gel and transferred to a nylon membrane (Hybond N+, Amersham Biosciences, Piscataway, NJ) by capillary blotting. The probe for UGT1A1 was described previously (33). Other probes were prepared from liver total RNA using SuperScript one-step RT-PCR system (Invitrogen) according to the manufacturer’s instructions. Blots were hybridized using ULTRAHyb solution (Ambion, Austin, TX) with 32P-labeled cDNA probes corresponding to mouse UGT2B1 (bases 50–517 of the published cDNA; GenBank accession no. AK050435), mouse UGT2B5 (bases 2–499 of the published cDNA; GenBank accession no. NM_009467), mouse SULT1A1 (bases 30–505 of the published cDNA; GenBank accession no. NM_133670), mouse 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase (PAPSS)2 (bases 72–690 of the published cDNA; GenBank accession no. NM_011864), and mouse SAT1 (bases 1–640 of the published cDNA; GenBank accession no. NM_174870) at 42 C overnight. The blots were then washed twice with 0.1% sodium dodecyl sulfate/ 2x saline sodium citrate at room temperature followed by one wash with 0.1% sodium dodecyl sulfate/0.1x saline sodium citrate at 55 C. The blots were subsequently reprobed with a radiolabeled ?-actin cDNA (BD Clontech Laboratories Inc., Palo Alto, CA). The intensity of signals was quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The levels of target gene mRNA were normalized with the levels of ?-actin mRNA.
Real-time quantitative PCR analysis
Real-time quantitative PCR (RT-QPCR) analysis was used for genes whose expression levels were below the detection limit of Northern blot analysis. RT-QPCR was performed using ABI PRISM 7700 sequence detection system instrument and software (Applied Biosystems, Inc., Foster City, CA). RNA samples were diluted to 100 ng/μl and treated with RNA-free deoxyribonuclease I (Ambion) according to the manufacturer’s recommendations. Samples were then assayed in triplicate 25-μl reactions using 100 ng of RNA per reaction. Gene-specific primers were used at 3 pmol/reaction, and the gene-specific probe was used at 12.5 pmol/reaction. Primers and dual-labeled probes were designed using Primer Express (version 2.0.0, Applied Biosystems) and synthesized by Sigma-Genosys (The Woodlands, TX). All primers and probes were entered into the NCBI Blast program to ensure specificity. The primers for SULT1C1 (GenBank accession no. NM_026935) were 5'-tgagcagaatggtgatgtagagaag-3' and 5'-tgcccactcaataaaagggtg-3', and the probe was 5'-cggcgaaccatcattcaacaccg-3'. Primers for SULT1E1 (GenBank accession no. NM_023135) were 5'-gaaagggaattataggagactggaaga-3' and 5'-tgctgcttgtagtgctcatcaa-3', and the probe was 5'-cattccagaccaccctgagggaga-3'. Primers for SULT2A1 (GenBank accession no. L27121) were 5'-tcactcggaacttattttgaatggt-3' and 5'-cagccacggacatgctca-3', and the probe was 5'-cctccaaggaaatgttctattcggatcatgg-3'. Fold induction values were calculated by subtracting the mean threshold cycle number for each treatment group from the mean threshold cycle number for the vehicle group and raising 2 to the power of this difference. The rodent glyceraldehyde-3-phosphate dehydrogenase internal control kit (Applied Biosystems) was used for loading control.
Measurements of serum TH levels
Blood was collected from the right retroorbital sinus 24 h after the last treatment and transferred to T-MGA tubes (Terumo Medical Corp., Elkton, MD). Serum was separated by centrifugation at 1200 x g for 10 min. Serum T4, T3, and TSH levels were measured by ELISA performed by the chemistry laboratory of Methodist Hospital (Houston, TX).
Measurement of T3 clearance
Wild-type and CAR null animals were divided into three groups each: control group (corn oil treatment), PB treated [0.05% wt/wt (500 ppm) PB mixed with food], and TCPOBOP treated (3 mg/kg, administered as a single ip injection dissolved in corn oil). Treatments were initiated 3 d (TCPOBOP) and 7 d (PB) before T3 injection. Mice were then given two ip injections (one injection per day) of T3 (100 μg/kg) dissolved in 0.05 N NaOH/PBS for 2 d. Blood was collected from the right retroorbital sinus 24 h after the last treatment to assay for serum T3 and TSH levels. T3 injection suppressed serum TSH levels to less than 0.01 μIU/ml, the detection limit of the assay used. In this experiment, one time point measurement of T3 was used to indicate clearance.
Propylthiouracil (PTU) treatment
All mice were fed a low-iodine diet supplemented with 0.15% PTU (Harlan Tekland Co., Madison, WI) for 4 d. Wild-type and CAR null animals were divided into three groups each: control group (a single corn oil injection on the first day), PB treated [0.05% wt/wt (500 ppm) PB mixed with the low-iodine diet], and TCPOBOP treated (3 mg/kg, administered as a single ip injection dissolved in corn oil on the first day). The PB-treated group also received a single corn oil injection on the first day. On the morning of the fifth day, blood was collected from the right retroorbital sinus and the serum isolated and assayed for T3 levels.
Proliferating cell nuclear antigen (PCNA) immunocytochemistry
Mice were treated with corn oil, PB [0.05% wt/wt (500 ppm) PB mixed with food], or TCPOBOP (3 mg/kg, administered as a single ip injection) for 10 d. On the 11th day, the animals were killed and their thyroid glands isolated. The glands were immediately fixed at room temperature in 10% buffered formalin for at least 2 h. The glands were then dehydrated and embedded in paraffin blocks. Five-micron sections of the thyroid glands were cut and mounted on polylysine-coated slides. The slides were then stained for PCNA using a PCNA staining kit (Zymed Laboratory Inc., San Francisco, CA).
Data presentation and statistics
Values are reported as mean ± SEM. Statistical analyses of the results were performed by one-way ANOVA, followed by Newman-Keuls test for comparisons among multiple groups. Values of P < 0.05 were accepted as statistically significant.
Results
Effect of CAR activation on serum TH
To examine the role of CAR in TH metabolism, serum T4, T3, and TSH levels were measured in wild-type and CAR–/– mice pretreated with PB or TCPOBOP. In the wild-type animals, PB and TCPOBOP decreased serum T4 levels 56 and 64%, respectively, but had no effect in the CAR–/– animals (Fig. 1A). As expected, the decrease in T4 was associated with a concomitant increase (255% for PB and 262% for TCPOBOP) in serum TSH levels in wild-type but not CAR–/– animals (Fig. 1B). As also expected based on their intact negative feedback circuit, there was no significant change in serum T3 levels on treatment with the various drugs (Fig. 1C).
FIG. 1. The effect of CAR activation on serum T4 (A), TSH (B), and T3 (C) levels. Values are mean ± SEM (n = 4–6). CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated. *, Significantly different from vehicle-treated control (P < 0.05); **, P < 0.01.
Two approaches were used to study the effects of CAR activation on T3 levels in the absence of TSH influence. First, TSH levels were suppressed by injecting the animals with T3 (40, 41, 42), which resulted in a 2-fold increase in serum T3 levels in the vehicle-pretreated wild-type and all CAR–/– animals (Fig. 2A). Activation of CAR by pretreatment with PB or TCPOBOP markedly increased clearance of the exogenously administered T3. This was only seen in wild-type but not CAR–/– animals. Serum T3 levels in the drug-treated wild-type animals were almost the same as their levels before T3 treatment (Fig. 1C).
FIG. 2. The effect of CAR activation on serum T3 levels in animals rendered TSH nonresponsive by exogenous T3 administration (A) or PTU treatment (B). Values are mean ± SEM (n = 4–6). CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated. **, Significantly different from vehicle-treated control (P < 0.01).
The effect of TSH was also blocked by treating animals with PTU, which inhibits the activity of thyroid peroxidase, the enzyme responsible for the synthesis of TH (43, 44, 45). Administration of PB or TCPOBOP to animals on a PTU-supplemented diet led to a dramatic decrease in serum T3 levels in wild-type but not CAR–/– null animals (Fig. 2B). The same effect was also seen in animals when their diet was supplemented with another thyroid peroxidase inhibitor, methimazole (data not shown).
Effect of CAR on expression of phase II TH-conjugating enzymes
To delineate the molecular mechanism of CAR-induced alterations in TH levels, we profiled the expression of hepatic genes involved in TH metabolism. UGT enzymes play a major role in TH metabolism in the liver (13). Several members of the UGT family of enzymes can metabolize T4 as well as T3. T4 is glucuronidated by the phenol/bilirubin UGT (UGT1A1), whereas T3 is glucuronidated by the androsterone UGT (UGT2B1) (9, 37, 46). Induction of these enzymes increases T4 and T3 glucuronidation and their subsequent excretion into bile (47, 48). PB and TCPOBOP treatments induced the expression of several UGTs in wild-type but not CAR–/– animals (Fig. 3). PB induced the expression of UGT1A1 2.8-fold, whereas TCPOBOP induced its mRNA levels by 3.2-fold, compared with vehicle-treated wild-type animals. UGT2B1 was also modestly induced (1.4-fold PB and 1.8-fold TCPOBOP), as was UGT2B5, a C19 steroid-specific UGT (1.7-fold PB and 1.6 TCPOBOP). Because CAR is normally sequestered in the hepatocyte cytoplasm under basal conditions, the basis for the reduction in basal expression of all the assayed UGT genes in the CAR–/– animals is unclear.
FIG. 3. The effect of CAR activation on the expression of UGT enzymes involved in TH metabolism. Northern blot analysis was performed with probes specific for UGT1A1, UGT2B1, and UGT2B5. Hepatic total RNA from vehicle or PB- or TCPOBOP-treated animals was used for this experiment. The values shown below each blot represent fold change relative to vehicle-treated wild-type animals. CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated.
Sulfation is also an important pathway for the irreversible inactivation of TH, and several SULTs sulfate both T4 and T3 (49, 50, 51, 52). Expression analysis of the different TH SULTs revealed a universal induction by CAR (Fig. 4). The phenol SULT (SULT1A1) showed a 2.1-fold and a 1.6-fold induction by PB and TCPOBOP, respectively (Fig. 4A). Surprisingly, the PB-mediated induction of this enzyme seems to be, at least partly, CAR independent. RT-QPCR analysis of three other TH-sulfating enzymes revealed strong induction in the wild-type but not CAR–/– mice. Consistent with previous studies (34, 35, 36), the hydroxysteroid SULT (SULT2A1) was induced 25-fold by PB and 30-fold by TCPOBOP (Fig. 4B). The cytosolic SULT (SULT1C1) was induced 2-fold by PB and 8-fold by TCPOBOP (Fig. 4C), and the estrogen SULT (SULT1E1) was induced 40-fold by PB and more than 500-fold by TCPOBOP (Fig. 4D).
FIG. 4. CAR activation leads to induction of TH sulfating enzymes. Hepatic total RNA from vehicle or PB- or TCPOBOP-treated animals was used for this experiment. SULT1A1 was assayed using Northern blot analysis (A). RT-QPCR was used to study the effects of the different treatments on the mRNA levels of SULT2A1 (B), SULT1C1 (C), and SULT1E1 (D). The values shown below SULT1A1 blot represent fold change relative to vehicle-treated wild-type animals. RT-QPCR values are mean ± SEM (n = 4–6). CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated. *, Significantly different from vehicle-treated control (P < 0.05); **, P < 0.01.
CAR activation of PAPS synthetic enzymes
The sulfation reaction requires the donation of a sulfonyl group from the obligate cosubstrate PAPS. PAPS, the universal sulfate donor in mammals, is rate limiting for the sulfation reactions and is present in limited amounts in the liver (53). The participation of PAPS in the sulfation reactions is dependent on its availability through synthesis, which in turn depends on the availability of sulfate anions and the activity of PAPSS in the liver. Consistent with previous results (34), induction of the different SULTs by CAR is accompanied by induction of PAPSS2, the major PAPSS in the liver (Fig. 5). This induction was seen after PB treatment (2.2-fold) or TCPOBOP (4.3-fold) in wild-type but not CAR–/– mice. Moreover, CAR activation modestly increased transcripts encoding the sulfate anion transporter (SAT)1 (PB 1.3-fold and TCPOBOP 2.3-fold) (Fig. 5). SAT1 is a major sulfate transporter in the liver, and its induction increases cellular uptake of sulfate anions (54, 55).
FIG. 5. Analysis of mRNA levels of enzymes that are essential for the sulfation reactions. Northern blot analysis was performed with probes specific for PAPSS2 and SAT1 (slc26A1). Hepatic total RNA from vehicle or PB- or TCPOBOP-treated animals was used for this experiment. The values shown below each blot represent fold change relative to vehicle-treated wild-type animals. CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated.
Effect of CAR activation on thyroid-follicular cell proliferation
Increased levels of TSH promote thyroid tumors by stimulating thyroid gland growth, and small increases in serum TSH can result in large increases in thyroid-follicular cell proliferation (13, 14). The increase in serum TSH levels is believed to be the mechanism by which PB mediates thyroid hypertrophy and tumor development (21, 56). PCNA immunocytochemistry was performed to assess the effect of CAR activation on proliferation in the thyroid gland (Fig. 6). PCNA-positive thyroid follicular cell nuclei were seen only in the thyroid glands of PB and TCPOBOP-treated wild-type animals. No changes were seen in the thyroid cells of any of the CAR–/– animals. This phenotype correlates with the higher levels of TSH seen in these animals (Fig. 1B). Moreover, there was an obvious decrease in the colloid size in the PB- and TCPOBOP-treated wild-type animals, compared with CAR–/– mice, which is believed to indicate colloid depletion due to TSH stimulation (14).
FIG. 6. Effects of CAR activation on thyroid follicular cell proliferation. Photomicrographs of representative thyroid tissue from corn oil-, PB-, or TCPOBOP-treated animals labeled with PCNA and counterstained with hematoxylin. PCNA-positive thyroid follicular cell nuclei appear black and are indicated with arrows. CO, Corn oil treated; PB, phenobarbital treated; TC, TCPOBOP treated.
Discussion
Hormone levels are regulated not only by synthesis and secretion but also by catabolism and elimination. Thus, the hepatic-endocrine axis is an important component in the homeostatic control of a number of hormones. The induction of hepatic microsomal enzymes alters the metabolism of the xenobiotics and also the metabolism of various endogenous compounds, including TH (23). A number of previous studies have identified CAR as a regulator of a variety of enzymes involved in TH catabolism (27, 28, 29, 30, 31, 32, 33, 34, 35, 36), and one recent study (36) indicated that activation of CAR by fasting increases TH breakdown. For unknown reasons, we have not observed CAR activation in response to such a stress. However, the current studies confirm and significantly extend the potential role of CAR in TH metabolism, and we propose a multistep mechanism for xenobiotic suppression of TH levels and subsequent promotion of thyroid follicular cell proliferation (Fig. 7) (13, 14, 21).
FIG. 7. Proposed mechanism of thyroid tumor promotion mediated by CAR. CAR activation by xenobiotics such as PB and TCPOBOP induces several enzymes involved in TH metabolism. This leads to increased hepatic disposition of T4 and T3, which reduces their serum levels. To compensate for this decrease in serum T4 and T3 levels, serum TSH concentration increases, which stimulates thyroid follicular cell proliferation and ultimately neoplasia.
The perturbation of TH balance in xenobiotic exposed mice is thought to be due to enhanced metabolism and excretion of these hormones via induction of UGT and SULT enzymes (8, 13). Induction of UGT1A1 and UGT2B1 increases the glucuronidation of T4 and T3, respectively, and their excretion into bile (9, 37, 57). It is believed that glucuronidation is the main pathway for T4, and to a lesser extent T3, metabolism. In this study, we show that the induction of UGT1A1 and UGT2B1 by PB and TCPOBOP is CAR dependent. This presumably contributes to the marked decrease in T4 levels on CAR activation. We also found that CAR induces UGT2B5 expression. The activity of this enzyme in TH metabolism has not been elucidated but is closely related to human UGT2B7, an enzyme that metabolizes TH (58).
Sulfation is a major mechanism in the regulation of TH levels and activity (1, 8). Sulfation prevents the binding of T3 to its receptors and facilitates excretion of both T4 and T3 into bile and urine (3, 59). Additionally, T3 sulfate is subject to accelerated degradation because sulfation facilitates the IRD of T3 by D1. Sulfation also facilitates the inactivating IRD of T4 by D1, whereas the activating ORD of this prohormone is completely blocked (1). Our results suggest that CAR functions as a global regulator of sulfation by controlling the production of not only a number of SULT enzymes but also the obligate cosubstrate, PAPS and the sulfate transporter SAT1. The induction of PAPSS2 is of particular importance because it is estimated that the entire liver content of PAPS can be consumed in less than 2 min if its synthesis is stopped (53).
It has been assumed that the maintenance of normal T3 levels in PB-treated animals is due to increased serum TSH (13, 60), and our results demonstrate that neutralization of TSH effects leads to an obvious decrease in serum T3 in the xenobiotic-treated mice. Particularly because the ability of PB to promote thyroid growth and neoplasia is attenuated by suppression of TSH via readministration of T4 or T3 (21), we believe that the increase in thyroid-follicular cell proliferation upon CAR activation is secondary to the increase in TH metabolism and the compensatory increase in TSH levels.
The ability of CAR activation to alter TH levels has significant clinical importance. Several studies have shown that patients treated with PB have increased TH metabolism and reduced serum T4 levels, and hypothyroid patients require an increase in their thyroid replacement therapy dosage after treatments with antiepileptic drugs, such as the CAR activator phenytoin (12, 13, 14, 15, 16, 17, 18, 19, 61, 62, 63). Surprisingly, the decrease in serum T4 in these patients is often not associated with a clear compensatory increase in TSH. It has been suggested that a slight increase in TSH levels in earlier studies could not be detected due to the sensitivity of the assay used (64), and more recent studies on the effects of antiepileptic drugs including PB have shown higher levels of TSH (65). This has been linked to an increase in the incidence of initial clinical signs of thyroid hyperplasia and the frequency of thyroid goiter (66).
In children exposed to the polychlorinated biphenyl PCB-153, an effective CAR activator, levels of the environmental pollutant were negatively correlated with plasma T4 and T3 levels and positively correlated with TSH levels (67, 68, 69). This is consistent with the higher incidence of thyroid hypertrophy in children exposed to such chemicals (70). Interestingly, recent results suggest that CAR expression is higher in females than males (71), and the correlations of TH alterations with both PCB levels (67) and PB and phenytoin treatments (72) had a stronger effect in women than men.
Several studies have suggested a key role for CAR in drug-hormone interactions (73, 74). Epileptics taking PB and oral contraceptives (e.g. ethynylestradiol) have a 25-fold higher risk of pill failure caused by the increased rate of estrogen metabolism (75, 76, 77), and the induction of UGT1A1 and SULT1E1, two major enzymes involved in estrogen metabolism, may contribute to this effect. Changes in hormone levels may also affect drug metabolism. For example, hypothyroid women who are being treated with thyroxine often need higher doses when they are pregnant (78, 79, 80). It has been suggested that this is due to estrogen induction of thyroxine binding globulin (TBG) expression (81). However, patients with mutations in the TBG gene are euthyroid and show normal levels of T4 and T3 (82). Consistent with this, a recent case was described in which despite low baseline TBG levels and blunted pregnancy-associated TBG induction, the patient’s absolute and relative pregnancy-associated increases in thyroxine replacement dosage mirrored those found in non-TBG-deficient, hypothyroid patients (83). Moreover, the increase in serum TBG levels reaches a plateau 6–12 wk into pregnancy (84), whereas the increase in T4 requirements persists throughout pregnancy. Thus, increased TBG concentration may not be the key determinant for the increase in thyroxine requirement in pregnancy. An alternative explanation is that higher estrogen and progesterone levels during pregnancy may activate CAR or its closest relative, pregnane X receptor, and increase TH metabolism (85, 86). Consistent with this, serum levels of anticonvulsants as well as other drugs decrease during pregnancy by as much as 40%, apparently due to an increase their hepatic metabolism (77, 87, 88, 89, 90). It is believed that this is a consequence of the higher estrogen levels in pregnant women because similar effects have been reported for nonpregnant women on oral estrogen contraceptives (91, 92).
Acknowledgments
We thank Dr. Ann Marie Zavacki (Brigham and Women’s Hospital/Harvard Medical School, Boston, MA) for technical advice.
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