Thyroid Hormone Receptor Antagonist Research Articles

Human Biomonitoring of Environmental Chemicals among Elderly in Wuhan, China: Prioritizing Risks Using EPA's ToxCast Database.

In line with the "healthy aging" principle, we aim to assess the exposure map and health risks of environmental chemicals in the elderly. Blood samples from 918 elderly individuals in Wuhan, China, were analyzed using the combined gas/liquid-mass spectrometry technology to detect levels of 118 environmental chemicals. Cluster analysis identified exposure profiles, while risk indexes and bioanalytical equivalence percentages were calculated using EPA's ToxCast database. The detection rates for 87 compounds exceeded 70%. DEHP, DiBP, naphthalene, phenanthrene, DnBP, pyrene, anthracene, permethrin, fluoranthene, and PFOS showed the highest concentrations. Fat-soluble pollutants varied across lifestyles. In cluster 2, which was characterized by higher concentrations of fat-soluble substances, the proportion of smokers or drinkers was higher than that of nonsmokers or nondrinkers. Pesticides emerged as the most active environmental chemicals in peroxisome proliferator-activated receptor gamma antagonist, thyroid hormone receptor (TR) antagonist, TR agonist, and androgen receptor (AR) agonist activity assays. Additionally, PAEs and polycyclic aromatic hydrocarbons played significant roles as active contaminants for the corresponding targets of AR antagonists and estrogen receptor alpha. We proposed a list of priority pollutants linked to endocrine-disrupting toxic effects in the elderly, which may provide the groundwork for further research into environmental etiology.

Daily exposure to low concentrations Tetrabromobisphenol A interferes with the thyroid hormone pathway in HepG2 cells

Tetrabromobisphenol A (TBBPA) is a flame retardant that adversely affects the environment and human health. The present study exposed HepG2 cells to low concentrations of TBBPA daily to investigate the changes in gene regulation, mainly related to pathways associated with the endocrine system. The quantitative polymerase chain reaction (qPCR) confirmed that prolonged exposure gradually activated the thyroid hormone and parathyroid hormone signaling pathways. The expression levels of genes related to the thyroid hormone signaling pathway were upregulated (1.15−8.54 times) after five generations of exposure to 1 and 81 nM TBBPA. Furthermore, co-exposure to 81 nM TBBPA and 0.5 nM thyroid hormone receptor antagonist for five generations significantly reduced the expression of thyroid hormone and parathyroid hormone receptors. Meanwhile, 81 nM TBBPA inhibited the activation of the Ras pathway and downregulated Ras gene expression level (3.7 times), indicating the association between the toxic effect and thyroid hormone receptors. Additionally, our experiments revealed that the thyroid hormone pathway regulated the induction of the Ras signaling pathway by TBBPA. The study thus proves that daily exposure to TBBPA interferes with the thyroid hormone signaling pathway and subsequently the endocrine system.

Thyroid endocrine disruption and hepatotoxicity induced by bisphenol AF: Integrated zebrafish embryotoxicity test and deep learning

Bisphenol AF (BPAF) is an emerging contaminant prevalent in the environment as one of main substitutes of bisphenol A (BPA). It was found that BPAF exhibited estrogenic effects in zebrafish larvae in our previous study, while little is known about its effects on the thyroid and liver. A 7 d zebrafish embryotoxicity test was conducted to study the potential thyroid disruption and hepatotoxicity of BPAF. BPAF decreased levels of thyroid hormones and deiodinases but increased expressions of transthyretin at 12.5 and 125 μg/L after 7 d exposure, indicating that both the metabolism and transport of thyroid hormones were perturbed. The thyroid hormone receptor (TR) levels decreased significantly upon exposure to ≥12.5 μg/L BPAF, implying that BPAF acts as a TR antagonist, which coincided well with the prediction from the Direct Message Passing Neural Network. The liver impairment (mainly cell necrosis of hepatocytes) and apoptosis were triggered by 125 μg/L and ≥12.5 μg/L BPAF respectively, accompanied by the increased activities of caspase 3 and caspase 9. Thus BPAF might not be a safe alternative to BPA given the thyroid and liver toxicity. DMPNN appears useful to screen for thyroid disrupting activity from molecular structures.

Peripubertal Bisphenol A Exposure Imparts Detrimental Age-Related Changes in Body Composition, Cognition, and Hydrogen Sulfide Production Capacities.

Aims:Peripubertal endocrine disruption has immediate and lifelong consequences on health, cognition, and lifespan. Disruption comes from dietary, environmental, and pharmaceutical sources. The plasticizer Bisphenol A (BPA) is one such endocrine disrupting chemical. However, it is unclear whether peripubertal BPA exposure incites long-lasting physiological, neuro-cognitive, and/or longevity-related metabolic impairments. Catabolism of cysteine via transsulfuration enzymes produces hydrogen sulfide (H2S), a redox-modulating gasotransmitter causative to endocrine and metabolic homeostasis and improved cognitive function with age. As thyroid hormone (TH) regulates hepatic H2S production and BPA is a TH receptor antagonist, we hypothesized that BPA exposure during peripubertal development impairs metabolic and neuro-cognitive/behavioral endpoints in aged mice, in part, due to altered peripheral and/or localized H2S production and redox status.Results:To test this, male C57BL/6J mice at 5 weeks of age were orally exposed daily for 5 weeks to 250 μg BPA/kg, defined as low dose group (LD BPA), or 250 mg BPA/kg, defined as high dose group (HD BPA). Both LD and HD BPA exposure decreased lean mass and increased fat mass accompanied by decreased serum total TH at advanced ages. In addition, LD BPA had an anxiogenic effect whereas HD BPA caused cognitive deficits. Notably, HD BPA disrupted tissue-specific H2S production capacities and/or protein persulfidation, with the former negatively correlated with memory deficits and oxidative stress.Innovation and Conclusion:These findings provide a potential mechanism of action for acute and long-term health impacts of BPA-induced peripubertal endocrine disruption and bolster the need for improved monitoring and limitation of adolescent BPA exposure. Antioxid. Redox Signal. 36, 1246–1267.

Identification of thyroid hormone/thyroid hormone receptor interaction based on aptamer-assisted protein-induced fluorescence enhancement

We herein describe a novel method to identify thyroid hormone (TH)/thyroid hormone receptor (TR) interaction, termed aptamer-assisted protein-induced fluorescence enhancement (AptPIFE). In this method, a detection probe consisting of an RNA strand incorporating TH-specific aptamer and a Cy3-labeled DNA strand holds TH in close proximity to Cy3. The corresponding TR then binds to the TH near Cy3, consequently stimulating Cy3 to emit a significantly enhanced fluorescence through PIFE phenomenon. Based on this simple yet efficient design principle, we successfully identified the interaction of TH with TR within 10 min, down to 0.37 pM with excellent specificity. The practical and robust applicability of this method was also successfully validated by properly screening TR antagonists and reliably quantifying TH present in real clinical serum samples from patients with hyperthyroidism and healthy volunteers.

Investigation of NH3 as a selective thyroid hormone receptor modulator in larval zebrafish (Danio rerio)

Thyroid hormones (THs) are essential for normal vertebrate development and diverse environmental chemicals are hypothesized to cause developmental toxicity by disrupting TH-mediated signaling. The larval zebrafish (Danio rerio) is an emerging in vivo model of developmental TH disruption; however, the effects of TR antagonism have not yet been studied in zebrafish. NH3, generally considered a potent and specific thyroid hormone receptor (TR) antagonist, has been used in rodents and Xenopus laevis to characterize phenotypes of TR antagonism. The objective of this study is to determine the effects of NH3 on endpoints previously determined to be TH-sensitive in larval zebrafish, specifically teratology and mortality, photomotor behavior, and mRNA expression of TH signaling genes. Zebrafish embryos were exposed to NH3 via static waterborne exposure at concentrations ranging from 0.001 to 10 μM beginning at 6 h post-fertilization (hpf) through 5 days post fertilization (dpf). Significant mortality and teratogenesis was observed at 3, 4, and 5 dpf in zebrafish exposed to NH3 at 10 μM. At concentrations that did not cause significant mortality, NH3 did not exert a consistent antagonistic effect on photomotor behavior assays or mRNA expression when administered alone or in the presence of exogenous T4. Rather, depending on the NH3 concentration and larval age NH3 decreased or increased swimming triggered by transition from light to dark. Similarly, inconsistent antagonistic and agonistic effects on mRNA expression of TH signaling genes were noted following treatment with NH3 alone. NH3 did inhibit T4 (30 nM)-induced gene expression; however, this was only consistently observed at a concentration of NH3 (10 μM) that also caused significant mortality. Collectively, these results suggest that NH3 does not act solely as a TR antagonist in larval zebrafish, but instead exhibits complex modulatory effects on TR activity. These data support the hypothesis that NH3 is a selective thyroid hormone receptor modulator. Further studies of NH3 interactions with the zebrafish thyroid hormone receptor are required to characterize the activity of NH3 in target tissues of the larval zebrafish at the molecular level, highlighting the importance of characterizing NH3 effects in specific models of TH-disruption to better interpret its actions in mechanistic screens of environmental chemicals for TH action.

Conformational Changes of Thyroid Receptors in Response to Antagonists.

Thyroid hormone receptors (TRs) play a critical role in human development, growth, and metabolism. Antagonists of TRs offer an attractive strategy to treat hyperthyroidism without the disadvantage of a delayed onset of drug action. While it is challenging to examine the atomistic behavior of TRs in a laboratory setting, computational methods such as molecular dynamics (MD) simulations have proven their value to elucidate ligand-induced conformational changes in nuclear receptors. Here, we performed MD simulations of TRα and TRβ complexed to their native ligand triiodothyronine (T3) as well as several antagonists. Based on the examination of 27 μs MD trajectories, we showed how binding of these compounds influences various structural features of the receptors including the helicity of helices 3 and 10 as well as the location of helix-12. Helices 3 and 12 are known to mediate coactivator association required for downstream signaling, suggesting these changes to be the molecular basis for TR antagonism. A mechanistic analysis of the trajectories revealed an allosteric pathway between H3 and H12 to be responsible for the conformational adaptations. Even though a mechanistic understanding of conformational adaptations triggered by TR antagonists is important for the development of novel therapeutics, they have not been previously examined in detail as it was done here.

Toxicity testing and endocrine disrupting chemicals.

Regulatory agencies around the world depend on standardized testing approaches to evaluate environmental chemicals for endocrine disrupting properties. The US Environmental Protection Agency (EPA) has developed a two-tiered testing approach within its Endocrine Disruptor Screening Program (EDSP). The eleven Tier 1 and three Tier 2 EDSP assays can be used to identify chemicals that act as agonists or antagonists of estrogen receptor, androgen receptor, or thyroid hormone receptor, or chemicals that interfere with steroidogenesis. Additional assays have been developed in the context of Tox21, and others have been validated by the OECD. In spite of the availability of validated toxicity tests, problems have been identified with the approaches and methods used to identify endocrine disrupting chemicals (EDCs). This chapter will provide an overview of several of these issues including: (1) The way an EDC is defined by an agency impacts whether a specific test can be used to determine if a chemical is an EDC. This is especially important when considering which assays examine outcomes that are considered "adverse effects." (2) Some assumptions about the validated studies used to identify EDCs may not be true (e.g., their reproducibility has been questioned). (3) Many of the validated assays are less sensitive than other methods that have not yet been validated. Ultimately, these and other problems contribute to the current landscape, where testing approaches have failed to protect the public from known EDCs. The chapter concludes with a review of approaches that have been taken to improve current guideline studies.

Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway.

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TRα and TRβ), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.

Thyroid-Disrupting Activities of Groundwater from a Riverbank Filtration System in Wuchang City, China: Seasonal Distribution and Human Health Risk Assessment

The recombinant thyroid hormone receptor (TR) gene yeast assay was used to evaluate thyroid disruption caused by groundwater from the riverbank filtration (RBF) system in Wuchang City, China. To investigate seasonal fluctuations, groundwater was collected during three seasons. Although no TR agonistic activity was found, many water samples exhibited TR antagonistic activity. The bioassay-derived amiodarone hydrochloride (AH) equivalents ranged from 2.99 to 274.40 μg/L. Water samples collected from the riverbank filtration system during the dry season had higher TR antagonistic activity. All samples presented adverse 3,3′,5-triiodo-L-thyronine (T3) equivalent levels, ranging from −2.00 to −2.12 μg/kg. Following exposure to water samples with substantial TR antagonist activity, predicted hormonal changes in humans of different gender and age ranged from 0.65 to 1.48 μg/kg of T3, being 47% to 231% of normal. No obvious difference was found between genders or among age groups. Overall, the results revealed that the RBF system could remove the thyroid-disrupting chemicals in the river water to some extent. Considering the varying degrees of risk to human health, further treatment is needed to remove the potential thyroid-disrupting chemicals in pumping water after riverbank filtration to ensure drinking water safety.

Limited Chemical Structural Diversity Found to Modulate Thyroid Hormone Receptor in the Tox21 Chemical Library.

Background:Thyroid hormone receptors (TRs) are critical endocrine receptors that regulate a multitude of processes in adult and developing organisms, and thyroid hormone disruption is of high concern for neurodevelopmental and reproductive toxicities in particular. To date, only a small number of chemical classes have been identified as possible TR modulators, and the receptors appear highly selective with respect to the ligand structural diversity. Thus, the question of whether TRs are an important screening target for protection of human and wildlife health remains.Objective:Our goal was to evaluate the hypothesis that there is limited structural diversity among environmentally relevant chemicals capable of modulating TR activity via the collaborative interagency Tox21 project.Methods:We screened the Tox21 chemical library (8,305 unique structures) in a quantitative high-throughput, cell-based reporter gene assay for TR agonist or antagonist activity. Active compounds were further characterized using additional orthogonal assays, including mammalian one-hybrid assays, coactivator recruitment assays, and a high-throughput, fluorescent imaging, nuclear receptor translocation assay.Results:Known agonist reference chemicals were readily identified in the TR transactivation assay, but only a single novel, direct agonist was found, the pharmaceutical betamipron. Indirect activation of TR through activation of its heterodimer partner, the retinoid-X-receptor (RXR), was also readily detected by confirmation in an RXR agonist assay. Identifying antagonists with high confidence was a challenge with the presence of significant confounding cytotoxicity and other, non-TR-specific mechanisms common to the transactivation assays. Only three pharmaceuticals—mefenamic acid, diclazuril, and risarestat—were confirmed as antagonists.Discussion:The results support limited structural diversity for direct ligand effects on TR and imply that other potential target sites in the thyroid hormone axis should be a greater priority for bioactivity screening for thyroid axis disruptors. https://doi.org/10.1289/EHP5314

Thyroid hormone receptor function in maturing ovine cardiomyocytes.

Plasma thyroid hormone (tri-iodo-l-thyronine; T3 ) concentrations rise near the end of gestation and is known to inhibit proliferation and stimulate maturation of cardiomyocytes before birth. Thyroid hormone receptors are required for the action of thyroid hormone in fetal cardiomyocytes. Loss of thyroid hormone receptor (TR)α1 abolishes T3 signalling via extracellular signal-related kinase and Akt in fetal cardiomyocytes. The expression of TRα1 and TRβ1 in ovine fetal myocardium increases with age, although TRα1 levels always remain higher than those of TRβ1. Near term fetal cardiac myocytes are more sensitive than younger myocytes to thyroid receptor blockade by antagonist, NH3, and to the effects of TRα1/α2 short interfering RNA. Although T3 is known to abrogate ovine cardiomyocyte proliferation stimulated by insulin-like growth factor 1, this effect is mediated via the genomic action of thyroid hormone receptors, with little evidence for non-genomic mechanisms. We have previously shown that the late-term rise in tri-iodo-l-thyronine (T3 ) in fetal sheep leads to the inhibition of proliferation and promotion of maturation in cardiomyocytes. The present study was designed to determine whether these T3 -induced changes are mediated via thyroid hormone receptors (TRs) or by non-genomic mechanisms. Fetal cardiomyocytes were isolated from 102±3 and 135±1days of gestational age (dGA) sheep (n=7 per age; term ∼145dGA). Cells were treated with T3 (1.5nm), insulin-like growth factor (IGF)-1 (1μgmL-1 ) or a combination in the presence of TR antagonist NH3 (100nm) or following short interfering RNA (siRNA) knockdown of TRα1/α2. Proliferation was quantified by 5-bromo-2'-deoxyuridine (BrdU) uptake (10μm). Western blots measured protein levels of extracellular signal-related kinase (ERK), Akt, TRα1/β1 and p21. Age specific levels of TRα1/β1 were measured in normal hearts from fetuses [95dGA (n=8), 135dGA (n=7)], neonates (n=8) and adult ewes (n=7). TRα1 protein levels were consistently >50% more than TRβ1 at each gestational age (P<0.05). T3 reduced IGF-1 stimulated proliferation by ∼50% in 100dGA and by ∼75% in 135dGA cardiomyocytes (P<0.05). NH3 blocked the T3 +IGF-1 reduction of BrdU uptake without altering the phosphorylation of ERK or Akt at both ages. NH3 did not suppress T3 -induced p21 expression in 100dGA cardiomyocytes in 135dGA cardiomyocytes, NH3 alone reduced BrdU uptake (-28%, P<0.05), as well as T3 -induced p21 (-75%, P<0.05). In both ages, siRNA knockdown of TRα1/α2 blocked the T3 +IGF-1 reduction of BrdU uptake and dramatically reduced ERK and Akt signalling in 135dGA cardiomyocytes. In conclusion, TRs are required for normal proliferation and T3 signalling in fetal ovine cardiomyocytes, with the sensitivity to TR blockade being age-dependent.

A comparison of endocrine disruption potential of nonylphenol ethoxylate, vanillin ethoxylate, 4-n-nonylphenol and vanillin in vitro

The widely used surfactant nonylphenol ethoxylate (NPEO) and its raw material 4-n-nonylphenol (4-n-NP), as well as its degradation products, are recognized as endocrine disrupting chemicals. The USA Environmental Protection Agency (EPA) released an assessment that looked for safe alternatives to NPEO. Vanillin ethoxylate (VAEO) is a novel substitute for NPEO and is quite similar to NPEO in structure; there is a risk that it has similar endocrine disrupting effects to NPEO. However, their effects on various nuclear hormone receptors have not been thoroughly examined. In this study, the effects of NPEO, VAEO, 4-n-NP and Vanillin on the estrogen receptor α (ERα), androgen receptor (AR), thyroid hormone receptor (TR), retinoic X receptor β (RXRβ) and estrogen-related receptor γ (ERRγ) were determined and compared using a battery of recombined yeast strains expressing β-galactosidase. The results showed that NPEO and 4-n-NP acted as significant antagonists of ER, AR, TR and ERRγ. In addition, 4-n-NP also had antagonistic activity toward RXRβ. Moreover, VAEO was shown to be a very weak antagonist of TR and ERRγ, and Vanillin had no interaction with any nuclear receptors. For the first time, it was found that NPEO had AR, TR and ERRγ antagonistic effects and that 4-n-NP was an antagonist of RXRβ. The in vitro data indicated that NPEO, 4-n-NP and VAEO have the potential to act as endocrine disruptors involving more than one nuclear hormone receptor, but VAEO has much lower endocrine disrupting potential than NPEO. Thus, it is critical to find safe substitutes for NPEO and a substitute of NPEO with structural analogues should be carried out with caution. Furthermore, to look for preferable alternatives for NPEO, more in vivo and in vitro studies of the alternatives concerning endocrine disruption are needed, especially in vitro studies need to involve various target points, not only focus on their effects on ER but also take other nuclear hormone receptor pathways into consideration.

Bisphenols and Thyroid Hormone

In recent decades, attention has been directed toward the effects of bisphenol A (BPA) on human health. BPA has estrogenic activity and is regarded as a representative endocrine disruptor. In addition, mounting evidence indicates that BPA can disrupt thyroid hormone and its action. This review examined human epidemiological studies to investigate the association between BPA exposure and thyroid hormone levels, and analyzed in vivo and in vitro experiments to identify the causal relationship and its mechanism of action. BPA is involved in thyroid hormone action not only as a thyroid hormone receptor antagonist, but also through several other mechanisms. Since the use of bisphenols other than BPA has recently increased, we also reviewed the effects of other bisphenols on thyroid hormone action.

Classification of thyroid hormone receptor agonists and antagonists using statistical learning approaches

In silico models are presented for modeling and predicting thyroid hormone receptor (TR) agonists and antagonists. A data set consisting of 258 compounds is used in the present work. The C4.5, random forest (RF) and support vector machine (SVM) statistical methods were used for evaluation. The performance of the quantitative structure-activity relationships was further validated with fivefold cross-validation and an independent external test set. The C4.5 model is slightly weak, and the prediction accuracies of the agonists and antagonists are 93.2 and 57.8% for cross-validation, respectively, averaging 83.1% of correctly classified compounds in the test set. The RF model possesses an average prediction accuracy of 84.0 and 87.1% for the cross-validation and external validation, respectively. Furthermore, the overall prediction accuracy and the external prediction accuracy are 96.6 and 97.2%, respectively, for the SVM model. The results would validate the reliability of the derived models, further demonstrating that RF and SVM models are useful tools capable of classifying TR-binding ligands as agonists or antagonists.

Extension of the carcinogen dose–response database for threshold of toxicological concern

Various synthetic compounds are frequently discharged into the environment via human activities. Among them, certain contaminants may disrupt normal physiological functions of wildlife and humans via interactions with nuclear receptors. To protect human health and the environment, it is important to detect environmental ligands for human nuclear receptors. In this study, yeast-based reporter gene assays were used to investigate the occurrence of xenobiotic ligands for retinoid X receptors (RXR) and thyroid hormone receptors (TR) in the aquatic environment of Taiwan. Experimental results revealed that RXR agonist/antagonist activity was detected in river water and sediment samples. In particular, high RXR agonist/antagonist activity was found in the samples collected near river mouths. Additionally, few samples also elicited significant TR antagonist activity. Our findings show that the aquatic environment of Taiwan was contaminated with RXR and TR ligands. Further study is necessary to identify these xenobiotic RXR and TR agonists and antagonists.

Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and metabolism. Recent studies have implicated TH signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we demonstrated that antithyroid drug treatment and targeting iodothyronine deiodinases (DIOs) to suppress cellular tri-iodothyronine (T3) production or increase T3 degradation preserves cones. In this work, we investigated the effectiveness of inhibition of the TH receptor (TR). Two genes, THRA and THRB, encode TRs; THRB2 has been associated with cone viability. Using TR antagonists and Thrb2 deletion, we examined the effects of TR inhibition. Systemic and ocular treatment with the TR antagonists NH-3 and 1-850 increased cone density by 30-40% in the Rpe65-/- mouse model of Leber congenital amaurosis and reduced the number of TUNEL+ cells. Cone survival was significantly improved in Rpe65-/- and Cpfl1 (a model of achromatopsia with Pde6c defect) mice with Thrb2 deletion. Ventral cone density in Cpfl1/Thrb2-/- and Rpe65-/- /Thrb2-/- mice was increased by 1- to 4-fold, compared with age-matched controls. Moreover, the expression levels of TR were significantly higher in the cone-degeneration retinas, suggesting locally elevated TR signaling. This work shows that the effects of antithyroid treatment or targeting DIOs were likely mediated by TRs and that suppressing TR protects cones. Our findings support the view that inhibition of TR locally in the retina is a therapeutic strategy for retinal degeneration management.-Ma, H., Yang, F., Butler, M. R., Belcher, J., Redmond, T. M., Placzek, A. T., Scanlan, T. S., Ding, X.-Q. Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

The essential role of thyroid hormone (TH) signaling in mammalian development warrants the examination of man-made chemicals for its disruption. Among vertebrate species, the molecular components of TH signaling are highly conserved, including the thyroid hormone receptors (TRs), their heterodimer binding partners the retinoid-X receptors (RXRs), and their DNA recognition sequences (TREs). This molecular conservation allows examination of potential TH disruption in the tractable, in vivo model system of amphibian metamorphosis. Metamorphosis requires TH signaling for both instigation and progression, and it provides dramatic and well-characterized phenotypes involving different cell fates. Here we describe a quantitative, precocious-metamorphosis assay suite we developed using one-week post-fertilization (PF) Xenopus laevis tadpoles in order to assess disruption of TH signaling. Tadpoles at this developmental stage (Nieuwkoop-Faber (NF)-48) are competent to respond to TH hormone, although not yet producing TH, along many metamorphic pathways, and they are uniform in size. This allowed us to quantify changes in morphology associated with natural metamorphosis (e.g. gill and tail resorption, brain expansion, and craniofacial remodeling) after five days of treatment. Using the same tadpoles from morphological measurements, we quantified a 20-fold increase in TH-induced cellular proliferation in the rostral head region by whole-mount immunocytochemistry. At the molecular level, we used F3-generation tadpoles from a transgenic X. laevis line, which expresses luciferase under the control of a native TRE, to assess the ability of compounds to disrupt TR function. The luciferase reporter showed over 10-fold activation by physiologic concentrations of TH. We used the synthetic TR antagonist NH-3 to demonstrate the feasibility of our assay suite to measure inhibition of TH activity at the level of the receptor. Finally, we assessed the capabilities of suspected TH-disrupting chemicals tetrabrominated diphenyl ether 47 (BDE-47) and tetrabromobisphenol A (TBBPA). We found that BDE-47 displays general toxicity rather than TH disruption, as it did not increase brain width nor affect the TRE-luciferase reporter. However, TBBPA, a suspected TR antagonist, although not effective in antagonizing cell proliferation, significantly inhibited the TRE-luciferase reporter, suggesting that it bears closer scrutiny as a TH disruptor. Overall the assay suite has important advantages over the classical tadpole metamorphosis assays with respect to the uniformity of animal size, small test volume, reproducibility, and short test period. The assays are performed before endogenous TH production and free feeding start, which further reduces complexity and variability.

Thyroid Hormone Receptor Antagonists: From Environmental Pollution to Novel Small Molecules.

Thyroid hormone receptors (TRs) are nuclear receptors which control transcription, and thereby have effects in all cells within the body. TRs are an important regulator in many basic physiological processes including development, growth, metabolism, and cardiac function. The hyperthyroid condition results from an over production of thyroid hormones resulting in a continual stimulation of thyroid receptors which is detrimental for the patient. Therapies for hyperthyroidism are available, but there is a need for new small molecules that act as TR antagonists to treat hyperthyroidism. Many compounds exhibit TR antagonism and are considered detrimental to health. Some drugs in the clinic (most importantly, amiodarone) and environmental pollution exhibit TR antagonist properties and thus have the potential to induce hypothyroidism in some people. This chapter provides an overview of novel small molecules that have been specifically designed or screened for their TR antagonist activity as novel treatments for hyperthyroidism. While novel compounds have been identified, to date none have been developed sufficiently to enter clinical trials. Furthermore, a discussion on other sources of TR antagonists is discussed in terms of side effects of current drugs in the clinic as well as environmental pollution.

Triiodothyronine regulates cell growth and survival in renal cell cancer.

Triiodothyronine plays an important role in the regulation of kidney cell growth, differentiation and metabolism. Patients with renal cell cancer who develop hypothyreosis during tyrosine kinase inhibitor (TKI) treatment have statistically longer survival. In this study, we developed cell based model of triiodothyronine (T3) analysis in RCC and we show the different effects of T3 on renal cell cancer (RCC) cell growth response and expression of the thyroid hormone receptor in human renal cell cancer cell lines from primary and metastatic tumors along with human kidney cancer stem cells. Wild-type thyroid hormone receptor is ubiquitously expressed in human renal cancer cell lines, but normalized against healthy renal proximal tube cell expression its level is upregulated in Caki-2, RCC6, SKRC-42, SKRC-45 cell lines. On the contrary the mRNA level in the 769-P, ACHN, HKCSC, and HEK293 cells is significantly decreased. The TRβ protein was abundant in the cytoplasm of the 786-O, Caki-2, RCC6, and SKRC-45 cells and in the nucleus of SKRC-42, ACHN, 769-P and cancer stem cells. T3 has promoting effect on the cell proliferation of HKCSC, Caki-2, ASE, ACHN, SK-RC-42, SMKT-R2, Caki-1, 786-0, and SK-RC-45 cells. Tyrosine kinase inhibitor, sunitinib, directly inhibits proliferation of RCC cells, while thyroid hormone receptor antagonist 1-850 (CAS 251310‑57-3) has less significant inhibitory impact. T3 stimulation does not abrogate inhibitory effect of sunitinib. Renal cancer tumor cells hypostimulated with T3 may be more responsive to tyrosine kinase inhibition. Moreover, some tumors may be considered as T3-independent and present aggressive phenotype with thyroid hormone receptor activated independently from the ligand. On the contrary proliferation induced by deregulated VHL and or c-Met pathways may transgress normal T3 mediated regulation of the cell cycle.