Nuclear Thyroid Hormone Receptors Research Articles

Analysis of Thyroid Tumorigenesis in Xenograft Mouse Model.

Analysis of thyroid tumorigenesis in xenograft mouse model is important to study human thyroid cancer. Recent studies have made big strides toward understanding the molecular mechanisms by which thyroid hormone nuclear receptors (TR) act to maintain normal cellular functions in growth, differentiation, and development. Despite growing interest, the role of TR in oncogenesis remains to be fully elucidated. Two TR genes give rise to three major TR isoforms: TRα1, TRβ1, and TRβ2. These TR subtypes express in a tissue- and development-dependent manner. Research has been directed at understanding the mechanisms by which TR could mediate aberrant cellular signaling that contributes to oncogenesis, at dissecting possible distinct roles of TR isoforms in oncogenesis, and at the differential susceptibility of target tissues to the oncogenic actions of TR. This chapter gives a brief overview of the current undersatanding of known molecular oncogenic actions of TR. Here, we describe analysis of thyroid tumorigenesis used in interrogating the in vivo oncogenic actions of TR.

Thyroid Hormones and Derivatives: Endogenous Thyroid Hormones and Their Targets.

More than a century after the discovery of L-Thyroxine, the main thyroid hormone secreted solely by the thyroid gland, several metabolites of this iodinated, tyrosine-derived ancestral hormone have been identified. These are utilized as hormones during development, differentiation, metamorphosis, and regulation of most biochemical reactions in vertebrates and their precursor species. Among those metabolites are the thyromimetically active 3,3',5-Triiodo-L-thyronine (T3) and 3,5-Diiodo-L-thronine, reverse-T3 (3,3',5'-Triiodo-L-thyronine) with still unclear function, the recently re-discovered thyronamines (e.g., 3-Iodo-thyronamine), which exert in part T3-antagonistic functions, the thyroacetic acids (e.g., Tetrac and Triac), as well as various sulfated or glucuronidated metabolites of this panel of iodinated signaling compounds. In the blood most of these hydrophobic metabolites are tightly bound to the serum distributor proteins thyroxine binding globulin (TBG), transthyretin (TTR), albumin or apolipoprotein B100. Cellular import and export of these charged, highly hydrophobic amino acid derivatives requires a number of cell-membrane transporters or facilitators such as MCT8 or MCT10 and members of the OATP and LAT families of transporters. Depending on their structure, the thyroid hormone metabolites exert their cellular action by binding and thus modulating the function of various receptors systems (e.g., ανβ3 integrin receptor and transient receptor potential channels (TRPM8) of the cell membrane), in part linked to intracellular downstream kinase signaling cascades, and several isoforms of membrane-associated, mitochondrial or nuclear thyroid hormone receptors (TR), which are members of the c-erbA family of ligand-modulated transcription factors. Intracellular deiodinase selenoenzymes, which obligatory are membrane integrated enzymes, ornithine decarboxylase and monoamine oxidases control local availability of biologically active thyroid hormone metabolites. Inactivation of thyroid hormone metabolites occurs mainly by deiodination, sulfation or glucuronidation, reactions which favor their renal or fecal elimination.

Demonstration of the Receptor Site for Thyroid Hormone on Integrin αvβ3.

Integrin αvβ3 is one of the 24 heterodimeric structural proteins of the plasma membrane of animal cells. The extracellular domain of αvβ3 comprises about 80% of its mass and is devoted largely to interactions with extracellular matrix (ECM) proteins, such as vitronectin and fibronectin, and cell-cell interaction. The binding of specific ECM proteins is conveyed to the cell interior by signal transduction. In the past decade, integrin αvβ3 has been shown to bind small molecules, such as nonprotein hormones-thyroid hormone analogues and dihydrotestosterone-and the stilbene, resveratrol. The αvβ3-thyroid hormone interaction signal generated at the integrin is transduced into intracellular protein trafficking, nucleoprotein phosphorylation, and expression of specific genes. The hormone receptor on the integrin bears no structural homologies with nuclear thyroid hormone receptors (TRs). Because integrin αvβ3 is generously expressed on the surface of cancer cells and rapidly dividing endothelial cells, thyroid hormone actions initiated at the receptor on integrin not unexpectedly relate to cell proliferation, cancer cell survival pathways and to angiogenesis. In this chapter, we present methods for the definition of the receptor, for monitoring certain of its functions and for the downregulation of the gene for the integrin.

Thyroid Hormone Action: The p43 Mitochondrial Pathway.

The possibility that several pathways are involved in the multiplicity of thyroid hormone physiological influences led to searches for the occurrence of T3 extra nuclear receptors. The existence of a direct T3 mitochondrial pathway is now well established. The demonstration that TRα1 mRNA encodes not only a nuclear thyroid hormone receptor but also two proteins imported into mitochondria with molecular masses of 43 and 28kDa has provided new clues to understand the pleiotropic influence of iodinated hormones.The use of a T3 photo affinity label derivative (T3-PAL) allowed detecting two mitochondrial T3 binding proteins. In association with western blots using antibodies raised against the T3 nuclear receptor TRα1, mitochondrial T3 receptors were identified as truncated TRα1 forms. Import and in organello transcription experiments performed in isolated mitochondria led to the conclusion that p43 is a transcription factor of the mitochondrial genome, inducing changes in the mitochondrial/nuclear crosstalk. In vitro experiments indicated that this T3 mitochondrial pathway affects cell differentiation, apoptosis, and transformation. Generation of transgenic mice demonstrated the involvement of this mitochondrial pathway in the determination of muscle phenotype, glucose metabolism, and thermogenesis.

Deiodinases share an evolutionarily conserved thyroid hormone-binding motif.

Human and animal thyroid hormone (TH) plasma carriers (THPC, n=426), cell surface TH transporters (CSTT, n=8,691), and TH nuclear receptors (TR, n=624) share a 49-position long TH-binding motif. Particularly conserved are positions 22-26, where a 5-residue TH-binding motif was originally detected. We extended our research to the 488 sequences of human and animal selenodeiodinases (dio-1, dio-2, dio-3). In the 10,229 sequences (THPC+CSTT+TR+deiodinases), 15/49 positions are very highly and another 18/49 highly conserved. The very highly conserved 5-residue core TH-binding motif is now W/F/Y, L/I/V/M, I/V/M/L, P, L/V/I/M. Concerning deiodinases, of the 13 conserved residues in their active centers (F121-E214, F128-E228, F139-E233), 11 (dio-1/dio-3) or 10 (dio-2) fall within the 49-position motif. Conserved in the consensus from THPC, CSTT and TR are F129/F136/F147 (position 11). Other matches of deiodinases concerned the nonapolipoprotein THPC: of particular interest, P135/P146 (dio-2/dio-3; position 9), E155/D162/E173 (position 28), S160/S167/S178 (position 33), W163/W170/W178 (position 36). The shared TH-binding domain, which is likely the product of an ancestor gene, may explain why diverse plasma and cell-associated proteins interact with TH.

Posttranslational Modification of Thyroid Hormone Nuclear Receptor by Sumoylation.

Thyroid hormone has a broad range of biological effects, both during development and in the adult. Nuclear thyroid hormone action is mediated by thyroid hormone receptor (TR) α and β. Thyroid hormone also has nongenomic actions at the membrane, which are less well characterized. Both TRα and TRβ undergo posttranslational modification, including phosphorylation, acetylation, and recently identified sumoylation. These posttranslational modifications have been shown to influence thyroid hormone signaling by altering TR DNA binding, TR interaction with cofactors, and TR-mediated transcription. The best characterized modification, with respect to gene regulation, is sumoylation, which plays an important role in nutrient regulators-mediated gene expression. We present an approach to study posttranslational modification of TR by small ubiquitin-like modifier (SUMO), a process referred to as sumoylation.

Posttranslational Modification of Thyroid Hormone Nuclear Receptor by Phosphorylation.

TR phosphorylation promotes TR binding to DNA and heterodimerization with RXR. TRβ phosphorylation is induced by thyroid hormone on the cell membrane and phosphorylation by extracellular signal-regulated kinases (ERK), presumably at serine 142. TRα1 N-termini harbors two phosphorylation sites at serine 12 and serine 28/29. Serine 12 is phosphorylated by casein 2 and serine 28/29 by protein kinase A. Mutation analysis of TRα2 identified 2 serine sites, S472 and S473, as the substrates for casein kinase II. Phosphorylated TRα2 does not bind to DNA and dephosphorylated TRα binds to DNA and antagonizes TRα1 binding. Phosphorylation of TR is critical for TR function and T3 signaling and approaches to detection and analysis of phosphorylated TR are described.

Differential transcriptome regulation by 3,5-T2 and 3\u2032,3,5-T3 in brain and liver uncovers novel roles for thyroid hormones in tilapia

Although 3,5,3′-triiodothyronine (T3) is considered to be the primary bioactive thyroid hormone (TH) due to its high affinity for TH nuclear receptors (TRs), new data suggest that 3,5-diiodothyronine (T2) can also regulate transcriptional networks. To determine the functional relevance of these bioactive THs, RNA-seq analysis was conducted in the cerebellum, thalamus-pituitary and liver of tilapia treated with equimolar doses of T2 or T3. We identified a total of 169, 154 and 2863 genes that were TH-responsive (FDR < 0.05) in the tilapia cerebellum, thalamus-pituitary and liver, respectively. Among these, 130, 96 and 349 genes were uniquely regulated by T3, whereas 22, 40 and 929 were exclusively regulated by T2 under our experimental paradigm. The expression profiles in response to TH treatment were tissue-specific, and the diversity of regulated genes also resulted in a variety of different pathways being affected by T2 and T3. T2 regulated gene networks associated with cell signalling and transcriptional pathways, while T3 regulated pathways related to cell signalling, the immune system, and lipid metabolism. Overall, the present work highlights the relevance of T2 as a key bioactive hormone, and reveals some of the different functional strategies that underpin TH pleiotropy.

Temperature induced variation in gene expression of thyroid hormone receptors and deiodinases of European eel (Anguilla anguilla) larvae

Thyroid hormones (THs) are key regulators of growth, development, and metabolism in vertebrates and influence early life development of fish. TH is produced in the thyroid gland (or thyroid follicles) mainly as T4 (thyroxine), which is metabolized to T3 (3,5,3′-triiodothyronine) and T2 (3,5-diiodothyronine) by deiodinase (DIO) enzymes in peripheral tissues. The action of these hormones is mostly exerted by binding to a specific nuclear thyroid hormone receptor (THR). In this study, we i) cloned and characterized thr sequences, ii) investigated the expression pattern of the different subtypes of thrs and dios, and iii) studied how temperature affects the expression of those genes in artificially produced early life history stages of European eel (Anguilla anguilla), reared in different thermal regimes (16, 18, 20 and 22 °C) from hatch until first-feeding. We identified 2 subtypes of thr (thrα and thrβ) with 2 isoforms each (thrαA, thrαB, thrβA, thrβB) and 3 subtypes of deiodinases (dio1, dio2, dio3). All thr genes identified showed high similarity to the closely related Japanese eel (Anguilla japonica). We found that all genes investigated in this study were affected by larval age (in real time or at specific developmental stages), temperature, and/or their interaction. More specifically, the warmer the temperature the earlier the expression response of a specific target gene. In real time, the expression profiles appeared very similar and only shifted with temperature. In developmental time, gene expression of all genes differed across selected developmental stages, such as at hatch, during teeth formation or at first-feeding. Thus, we demonstrate that thrs and dios show sensitivity to temperature and are involved in and during early life development of European eel.

Thyroid hormone inhibits growth of hepatoma cells through induction of miR-214

Thyroid hormone (TH) plays a role in regulating the metabolic rate, heart functions, muscle control and maintenance of bones. 3,3′5-tri-iodo-L-thyronine (T3) displays high affinity to nuclear thyroid hormone receptors (TRs), which mediate most TH actions. Recent studies have shown hypothyroidism in patients with an increased risk of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs), a class of non-protein-coding RNA, are suggested to control tumor growth by interacting with target genes. However, the clinical significance of T3/TR-regulated miRNAs in tumors has yet to be established. In the current study, miRNA expression profile screening was performed using SYBR Green-Based qRT-PCR array in TR-overexpressing HepG2 cells. miR-214-3p, which is expressed at low levels in HCC, was stimulated upon T3 application. The 3′UTR luciferase reporter assay confirmed that the proto-oncogene serine/threonine-protein kinase, PIM-1, is a miR-214-3p target. PIM-1 was decreased upon treatment with miR-214-3p or T3 stimulation. PIM-1 was highly expressed in HCC, and the effect of PIM-1 on cell proliferation might be mediated by the inhibition of p21. Furthermore, the T3-induced suppression of cell proliferation was partially rescued upon miR-214-3p knockdown. Our data demonstrate that T3 induces miR-214-3p expression and suppresses cell proliferation through PIM-1, thus contributing to the inhibition of HCC tumor formation.

Potential Role of Thyroid Receptor β Agonists in the Treatment of Hyperlipidemia.

Thyroid hormones have important effects on cellular development, growth, and metabolism and are necessary for the healthy function of almost all tissues. Hyperthyroid patients with excess thyroid hormone levels experience tachycardia, fatigue, muscle wasting, and osteoporosis. However, although high thyroid hormone levels have adverse effects, efforts have been made to harness the beneficial effects, such as reduced serum low-density lipoprotein (LDL) cholesterol levels, elevated basal metabolic rate, and weight loss. Thyroid hormones interact with nuclear thyroid hormone receptors (TRs), and cholesterol levels are reduced through TRβ, whereas extrahepatic adverse actions are primarily connected to TRα. Thus, to develop a useful compound for clinical use, efforts have been focusing on developing compounds with isomer-specific functions based on the structure of thyroid hormones, i.e., thyromimetics that are liver and/or TRβ specific. In this short review, we discuss the development of the early thyromimetics that enabled, through modern molecular techniques, the progress towards improved design of TRβ-selective thyromimetics. We also address the early promise shown in human clinical trials and the current status of these drugs and other emerging compounds.

Methylcytosine dioxygenase TET3 interacts with thyroid hormone nuclear receptors and stabilizes their association to chromatin

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily that act as ligand-dependent transcription factors. Here we identified the ten-eleven translocation protein 3 (TET3) as a TR interacting protein increasing cell sensitivity to T3. The interaction between TET3 and TRs is independent of TET3 catalytic activity and specifically allows the stabilization of TRs on chromatin. We provide evidence that TET3 is required for TR stability, efficient binding of target genes, and transcriptional activation. Interestingly, the differential ability of different TRα1 mutants to interact with TET3 might explain their differential dominant activity in patients carrying TR germline mutations. So this study evidences a mode of action for TET3 as a nonclassical coregulator of TRs, modulating its stability and access to chromatin, rather than its intrinsic transcriptional activity. This regulatory function might be more general toward nuclear receptors. Indeed, TET3 interacts with different members of the superfamily and also enhances their association to chromatin.

Molecular Basis of Nongenomic Actions of Thyroid Hormone.

Nongenomic actions of thyroid hormone are initiated by the hormone at receptors in the plasma membrane, in cytoplasm, or in mitochondria and do not require the interaction of nuclear thyroid hormone receptors (TRs) with their primary ligand, 3,5,3'-triiodo-l-thyronine (T3). Receptors involved in nongenomic actions may or may not have structural homologies with TRs. Certain nongenomic actions that originate at the plasma membrane may modify the state and function of intranuclear TRs. Reviewed here are nongenomic effects of the hormone-T3 or, in some cases, l-thyroxine (T4)-that are initiated at (a) truncated TRα isoforms, e.g., p30 TRα1, (b) cytoplasmic proteins, or (c) plasma membrane integrin αvβ3. p30 TRα1 is not transcriptionally competent, binds T3 at the cell surface, and consequently expresses a number of important functions in bone cells. Nongenomic hormonal control of mitochondrial respiration involves a TRα isoform, and another truncated TRα isoform nongenomically regulates the state of cellular actin. Cytoplasmic hormone-binding proteins involved in nongenomic actions of thyroid hormone include ketimine reductase, pyruvate kinase, and TRβ that shuttle among intracellular compartments. Functions of the receptor for T4 on integrin αvβ3 include stimulation of proliferation of cancer and endothelial cells (angiogenesis) and regulation of transcription of cancer cell survival pathway genes. T4 serves as a prohormone for T3 in genomic actions of thyroid hormone, but T4 is a hormone at αvβ3 and more important to cancer cell function than is T3. Thus, characterization of nongenomic actions of the hormone has served to broaden our understanding of the cellular roles of T3 and T4.

Anti-androgenic mechanisms of Bisphenol A involve androgen receptor signaling pathway.

We have shown Bisphenol A (BPA) acts as an androgen receptor (AR) antagonist in the previous study. However, the mechanisms underlying anti-androgenic effects of BPA remain unclear. The objective of this study was to explore whether the AR signaling was involved in AR antagonism of BPA. The Cell Counting Kit-8 (CCK-8) assay and Real-Time Cell Analysis (RTCA) iCELLigence system were applied to analyze the mouse Sertoli cell TM4 proliferation. The mammalian two-hybrid assays were performed to investigate the effects of BPA on the AR amino- and carboxyl-terminal regions (N/C) interaction and the interactions of the AR with steroid receptor coactivator-1 (SRC-1), co-repressors including silencing mediator for thyroid hormone receptors (SMRT) and nuclear receptor co-repressor (NCoR). BPA exposure resulted in decreased TM4 cell proliferation. BPA inhibited the AR N/C interaction significantly. Furthermore, BPA enhanced the interactions of AR-SMRT and AR-NCoR significantly. In conclusion, these data suggest BPA inhibits Sertoli cell proliferation due to its anti-androgenic actions. The mechanisms responsible for AR antagonism of BPA involve inhibiting the AR N/C interaction and enhancing the interactions of AR-SMRT and AR-NCoR. The data uncover novel anti-androgenic mechanisms by which BPA antagonizes AR signaling, contributing to Sertoli cell proliferation suppression and male reproductive toxicology.

Triiodothyronine promotes the proliferation of epicardial progenitor cells through the MAPK/ERK pathway

Thyroid hormone has important functions in the development and physiological function of the heart. The aim of this study was to determine whether 3,5,3′-Triiodothyronine (T3) can promote the proliferation of epicardial progenitor cells (EPCs) and to investigate the potential underlying mechanism. Our results showed that T3 significantly promoted the proliferation of EPCs in a concentration- and time-dependent manner. The thyroid hormone nuclear receptor inhibitor bisphenol A (100 μmol/L) did not affect T3's ability to induce proliferation. Further studies showed that the mRNA expression levels of mitogen-activated protein kinase 1 (MAPK1), MAPK3, and Ki67 in EPCs in the T3 group (10 nmol/L) increased 2.9-, 3-, and 4.1-fold, respectively, compared with those in the control group (P < 0.05). In addition, the mRNA expression of the cell cycle protein cyclin D1 in the T3 group increased approximately 2-fold compared with the control group (P < 0.05), and there were more EPCs in the S phase of the cell cycle (20.6% vs. 12.0%, P < 0.05). The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway inhibitor U0126 (10 μmol/L) significantly inhibited the ability of T3 to promote the proliferation of EPCs and to alter cell cycle progression. This study suggested that T3 significantly promotes the proliferation of EPCs, and this effect may be achieved through activation of the MAPK/ERK signaling pathway.

Thyroid hormone regulation of intestinal epithelial stem cell biology

The gastrointestinal tract is a well-characterized target of thyroid hormones and thyroid hormone nuclear receptors TRs, as extensively described in the literature. The paradigm is its important remodelling in amphibians during thyroid hormone-dependent metamorphosis. Interestingly, several studies have described the conservation of this hormonal signal during intestinal development in mammals. Additional data suggested that it may also play a role in intestinal homeostasis, stem cell physiology and progenitor commitment as well as in tumour development. It is worth underlining that in the mammalian intestine the functionality of the TRα1 receptor is coordinated and integrated with other signalling pathways, such as Wnt and Notch, specifically at the level of stem/progenitor cell populations. Here, we summarize these data and concepts and discuss this new role for thyroid hormones and the TRα1 receptor in the biology of intestinal epithelial precursor cells.

Mitochondrial T3 receptor and targets

The demonstration that TRα1 mRNA encodes a nuclear thyroid hormone receptor and two proteins imported into mitochondria with molecular masses of 43 and 28 kDa has brought new clues to better understand the pleiotropic influence of iodinated hormones. If p28 activity remains unknown, p43 binds to T3 responsive elements occurring in the organelle genome, and, in the T3 presence, stimulates mitochondrial transcription and the subsequent synthesis of mitochondrial encoded proteins. This influence increases mitochondrial activity and through changes in the mitochondrial/nuclear cross talk affects important nuclear target genes regulating cell proliferation and differentiation, oncogenesis, or apoptosis. In addition, this pathway influences muscle metabolic and contractile phenotype, as well as glycaemia regulation. Interestingly, according to the process considered, p43 exerts opposite or cooperative effects with the well-known T3 pathway, thus allowing a fine tuning of the physiological influence of this hormone.

Dissecting the role of regulators of thyroid hormone availability in early brain development: Merits and potential of the chicken embryo model

Thyroid hormones (THs) are important mediators of vertebrate central nervous system (CNS) development, thereby regulating the expression of a wide variety of genes by binding to nuclear TH receptors. TH transporters and deiodinases are both needed to ensure appropriate intracellular TH availability, but the precise function of each of these regulators and their coaction during brain development is only partially understood. Rodent knockout models already provided some crucial insights, but their in utero development severely hampers research regarding the role of TH regulators during early embryonic stages. The establishment of novel gain- and loss-of-function techniques has boosted the position of externally developing non-mammalian vertebrates as research models in developmental endocrinology. Here, we elaborate on the chicken as a model organism to elucidate the function of TH regulators during embryonic CNS development. The fast-developing, relatively big and accessible embryo allows easy experimental manipulation, especially at early stages of brain development. Recent data on the characterisation and spatiotemporal expression pattern of different TH regulators in embryonic chicken CNS have provided the necessary background to dissect the function of each of them in more detail. We highlight some recent advances and important strategies to investigate the role of TH transporters and deiodinases in various CNS structures like the brain barriers, the cerebellum, the retina and the hypothalamus. Exploiting the advantages of this non-classical model can greatly contribute to complete our understanding of the regulation of TH bioavailability throughout embryonic CNS development.

Resistance to Thyroid Hormone due to Heterozygous Mutations in Thyroid Hormone Receptor Alpha.

Thyroid hormone (TH) acts via nuclear thyroid hormone receptors (TRs). TR isoforms (TRα1, TRα2, TRβ1, TRβ2) are encoded by distinct genes (THRA and THRB) and show differing tissue distributions. Patients with mutations in THRB, exhibiting resistance within the hypothalamic-pituitary-thyroid axis with elevated TH and nonsuppressed thyroid-stimulating hormone (TSH) levels, were first described decades ago. In 2012, the first patients with mutations in THRA were identified. Scope of this review: This review describes the clinical and biochemical characteristics of patients with resistance to thyroid hormone alpha (RTHα) due to heterozygous mutations in THRA. The genetic basis and molecular pathogenesis of the disorder together with effects of levothyroxine treatment are discussed. The severity of the clinical phenotype of RTHα patients seems to be associated with the location and type of mutation in THRA. The most frequent abnormalities observed include anemia, constipation, and growth and developmental delay. In addition, serum (F)T3 levels can be high-normal to high, (F)T4 and rT3 levels normal to low, while TSH is normal or mildly raised. Despite heterogeneous consequences of mutations in THRA, RTHα should be suspected in subjects with even mild clinical features of hypothyroidism together with high/high-normal (F)T3, low/low-normal (F)T4, and normal TSH.

Effects of Gadolinium-Based Contrast Agents on Thyroid Hormone Receptor Action and Thyroid Hormone-Induced Cerebellar Purkinje Cell Morphogenesis.

Gadolinium (Gd)-based contrast agents (GBCAs) are used in diagnostic imaging to enhance the quality of magnetic resonance imaging or angiography. After intravenous injection, GBCAs can accumulate in the brain. Thyroid hormones (THs) are critical for the development and functional maintenance of the central nervous system. TH actions in brain are mainly exerted through nuclear TH receptors (TRs). We examined the effects of GBCAs on TR-mediated transcription in CV-1 cells using transient transfection-based reporter assay and TH-mediated cerebellar Purkinje cell morphogenesis in primary culture. We also measured the cellular accumulation and viability of Gd after representative GBCA treatments in cultured CV-1 cells. Both linear (Gd-diethylene triamine pentaacetic acid-bis methyl acid, Gd-DTPA-BMA) and macrocyclic (Gd-tetraazacyclododecane tetraacetic acid, Gd-DOTA) GBCAs were accumulated without inducing cell death in CV-1 cells. By contrast, Gd chloride (GdCl3) treatment induced approximately 100 times higher Gd accumulation and significantly reduced the number of cells. Low doses of Gd-DTPA-BMA (10−8 to 10−6M) augmented TR-mediated transcription, but the transcription was suppressed at higher dose (10−5 to 10−4M), with decreased β-galactosidase activity indicating cellular toxicity. TR-mediated transcription was not altered by Gd-DOTA or GdCl3, but the latter induced a significant reduction in β-galactosidase activity at high doses, indicating cellular toxicity. In cerebellar cultures, the dendrite arborization of Purkinje cells induced by 10−9M T4 was augmented by low-dose Gd-DTPA-BMA (10−7M) but was suppressed by higher dose (10−5M). Such augmentation by low-dose Gd-DTPA-BMA was not observed with 10−9M T3, probably because of the greater dendrite arborization by T3; however, the arborization by T3 was suppressed by a higher dose of Gd-DTPA-BMA (10−5M) as seen in T4 treatment. The effect of Gd-DOTA on dendrite arborization was much weaker than that of the other compounds. These results indicate that exposure to specific GBCAs may, at least in part, cause toxic effects in the brain by disrupting the action of THs on TRs. The toxic effects of GBCAs may depend on the chemical structure of GBCA and the dose. Thus, it is very important to choose appropriate GBCAs for imaging to prevent adverse side effects.