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Disruption of the thyroid hormone (TH) system is connected with diverse adverse health outcomes in wildlife and humans. It is crucial to develop and validate suitable in vitro assays capable of measuring the disruption of the thyroid hormone (TH) system. These assays are also essential to comply with the 3R principles, aiming to replace the ex vivo tests often utilised in the chemical assessment. We compared the two commonly used assays applicable for high throughput screening [Luminol and Amplex UltraRed (AUR)] for the assessment of inhibition of thyroid peroxidase (TPO, a crucial enzyme in TH synthesis) using several cell lines and 21 compounds from different use categories. As the investigated cell lines derived from human and rat thyroid showed low or undetectable TPO expression, we developed a series of novel cell lines overexpressing human TPO protein. The HEK-TPOA7 model was prioritised for further research based on the high and stable TPO gene and protein expression. Notably, the Luminol assay detected significant peroxidase activity and signal inhibition even in Nthy-ori 3-1 and HEK293T cell lines without TPO expression, revealing its lack of specificity. Conversely, the AUR assay was specific to TPO activity. Nevertheless, despite the different specificity, both assays identified similar peroxidation inhibitors. Over half of the tested chemicals with diverse structures and from different use groups caused TPO inhibition, including some widespread environmental contaminants suggesting a potential impact of environmental chemicals on TH synthesis. Furthermore, in silico SeqAPASS analysis confirmed the high similarity of human TPO across mammals and other vertebrate classes, suggesting the applicability of HEK-TPOA7 model findings to other vertebrates., (© 2024. The Author(s).)

Although measurements of blood triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) levels in rodent toxicity studies are useful for detection of antithyroid substances, assays for these measurements are expensive and can show high variability depending on blood sampling conditions. To develop more efficient methods for detecting thyroid disruptors, we compared histopathological and immunohistochemical findings in the thyroid and pituitary glands with blood hormone levels. Six-week-old male and female Sprague-Dawley rats (five rats per group) were treated with multiple doses of the thyroid peroxidase inhibitors propylthiouracil (PTU) and methimazole by gavage for 28 days. Significant decreases in serum T3 and T4 and increases in TSH were observed in the ≥1 mg/kg PTU and ≥3 mg/kg methimazole groups. An increase in TSH was also detected in male rats in the 0.3 mg/kg PTU group. Histopathological and immunohistochemical analyses revealed that follicular cell hypertrophy and decreased T4 and T3 expressions in the thyroid gland were induced at doses lower than doses at which significant changes in serum hormone levels were observed, suggesting that these findings may be more sensitive than blood hormone levels. Significant increases in thyroid weights, Ki67-positive thyroid follicular cell counts, and TSH-positive areas in the pituitary gland were detected at doses comparable with those at which changes in serum T4 and TSH levels were observed, indicating that these parameters may also be useful for evaluation of antithyroid effects. Combining these parameters may be effective for detecting antithyroid substances without relying on hormone measurements., (© 2024 John Wiley & Sons Ltd.)

The selenocysteine-containing enzyme class deiodinases (DIO) consists of three isoforms. DIOs play a role in regulation of thyroid hormone (TH) signaling through the removal of iodide from TH leading to TH that interact with the hypothalamic-pituitary-thyroid (HPT) axis with differing potency. Some gold-containing organic substances are known to inhibit many selenoenzymes, including DIOs. It is, however, unclear whether the Au-containing substances or the Au ions are causing the inhibition. In this study, five organic and inorganic gold substances as well as three gold nanoparticles (AuNPs) were tested for their potential to inhibit DIO1. The enzyme activity was tested using human liver microsomes as an enzyme source and reverse T3 as a substrate; iodide release was measured by the Sandell-Kolthoff method. The three organic gold substances aurothioglucose, auranofin and sodium aurothiomalate inhibited DIO1 with IC 50 between 0.38 and 0.75 μM while their structural analogues lacking the gold ion did not. Likewise, the two tested gold salts, Au(I) and Au(III) chloride, showed a concentration-dependent inhibition of the DIO1 with IC 50 values of 0.95 and 0.57 μM. Further, AuNPs of different sizes (100, 30 and 5 nm diameter) were tested with only the 5 nm AuNPs leading to inhibition with an IC 50 of 8 × 10 14 AuNP/L. This inhibition was not caused by the Au ions released by the AuNP into the incubation media. The exact mechanism of inhibition of DIO1 by 5 nm AuNPs should be further examined. In conclusion, the microsomal DIO1 assay demonstrated the inhibition of DIO1 by gold ions originating from different gold-containing substances, but not by Au released from AuNPs; rather DIO1 is inhibited by 5 nm, but not larger, AuNPs., (Copyright © 2021. Published by Elsevier B.V.)

The enzyme iodothyronine deiodinase type 3 (DIO3) contributes to cancer proliferation by inactivating the tumor-suppressive actions of thyroid hormone (T3). We recently established DIO3 involvement in the progression of high-grade serous ovarian cancer (HGSOC). Here we provide a link between high DIO3 expression and lower survival in patients, similar to common disease markers such as Ki67, PAX8, CA-125, and CCNE1. These observations suggest that DIO3 is a logical target for inhibition. Using a DIO3 mimic, we developed original DIO3 inhibitors that contain a core of dibromomaleic anhydride (DBRMD) as scaffold. Two compounds, PBENZ-DBRMD and ITYR-DBRMD, demonstrated attenuated cell counts, induction in apoptosis, and a reduction in cell proliferation in DIO3-positive HGSOC cells (OVCAR3 and KURAMOCHI), but not in DIO3-negative normal ovary cells (CHOK1) and OVCAR3 depleted for DIO3 or its substrate, T3. Potent tumor inhibition with a high safety profile was further established in HGSOC xenograft model, with no effect in DIO3-depleted tumors. The antitumor effects are mediated by downregulation in an array of pro-cancerous proteins, the majority of which known to be repressed by T3. To conclude, using small molecules that specifically target the DIO3 enzyme we present a new treatment paradigm for ovarian cancer and potentially other DIO3-dependent malignancies., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Deiodinase enzymes are critical for tissue-specific and temporal control of activation or inactivation of thyroid hormones during vertebrate development, including amphibian metamorphosis. We previously screened ToxCast chemicals for inhibitory activity toward human recombinant Type 3 iodothyronine deiodinase enzyme (hDIO3) and subsequently produced Xenopus laevis recombinant dio3 enzyme (Xldio3) with the goals to identify specific chemical inhibitors of Xldio3, to evaluate cross-species sensitivity and explore whether the human assay results are predictive of the amphibian. We identified a subset of 356 chemicals screened against hDIO3 to test against Xldio3, initially at a single concentration (200 μM), and further tested 79 in concentration-response mode. Most chemicals had IC 50 values lower for hDIO3 than for Xldio3 and many had steep Hill slopes (a potential indication of non-specific inhibition). However, eight of the most potent chemicals are likely specific inhibitors, with IC 50 values of 14 μM or less, Hill slopes near -1 and curves not significantly different between species likely due to conservation of catalytically active amino acids. Controlling for assay conditions, human in vitro screening results can be predictive of activity in the amphibian assay. This study lays the groundwork for future studies using recombinant non-mammalian proteins to test cross-species sensitivity to chemicals. DISCLAIMER: The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use., (Published by Elsevier Ltd.)

The iodide recycling enzyme, iodotyrosine deiodinase (IYD), is a largely unstudied molecular mechanism through which environmental chemicals can potentially cause thyroid disruption. This highly conserved enzyme plays an essential role in maintaining adequate levels of free iodide for thyroid hormone synthesis. Thyroid disruption following in vivo IYD inhibition has been documented in mammalian and amphibian models; however, few chemicals have been tested for IYD inhibition in either in vivo or in vitro assays. Presented here are the development and application of a screening assay to assess susceptibility of IYD to chemical inhibition. With recombinant human IYD enzyme, a 96-well plate in vitro assay was developed and then used to screen over 1800 unique substances from the U.S. EPA ToxCast screening library. Through a tiered screening approach, 194 IYD inhibitors were identified (inhibited IYD enzyme activity by 20% or greater at target concentration of 200 μM). 154 chemicals were further tested in concentration-response (0.032-200 μM) to determine IC 50 and rank-order potency. This work broadens the coverage of thyroid-relevant molecular targets for chemical screening, provides the largest set of chemicals tested for IYD inhibition, and aids in prioritizing chemicals for targeted in vivo testing to evaluate thyroid-related adverse outcomes., (Published by Elsevier Ltd.)

Forkhead box O (FoxO) proteins and thyroid hormone (TH) have well established roles in cardiovascular morphogenesis and remodeling. However, specific role(s) of individual FoxO family members in stress-induced growth and remodeling of cardiomyocytes remains unknown. Here, we report that FoxO1, but not FoxO3, activity is essential for reciprocal regulation of types II and III iodothyronine deiodinases (Dio2 and Dio3, respectively), key enzymes involved in intracellular TH metabolism. We further show that Dio2 is a direct transcriptional target of FoxO1, and the FoxO1-Dio2 axis governs TH-induced hypertrophic growth of neonatal cardiomyocytes in vitro and in vivo. Utilizing transverse aortic constriction as a model of hemodynamic stress in wild-type and cardiomyocyte-restricted FoxO1 knockout mice, we unveil an essential role for the FoxO1-Dio2 axis in afterload-induced pathological cardiac remodeling and activation of TRα1. These findings demonstrate a previously unrecognized FoxO1-Dio2 signaling axis in stress-induced cardiomyocyte growth and remodeling and intracellular TH homeostasis.

During pregnancy, fetal thyroid hormones (THs) are dependent on maternal placental transport and their physiological level is crucial for normal fetal neurodevelopment. Earlier research has shown that Di-(2-ethylhexyl) phthalate (DEHP) disrupts thyroid function and THs homeostasis in pregnant women and fetuses, and affects placental THs transport. However, the underlying mechanisms are poorly understood. The present study, therefore, aimed to systematically investigate the potential mechanisms of DEHP-induced disruption in the placental THs transport using two human placental trophoblastic cells, HTR-8/SVneo cells and JEG-3 cells. While the exposure of DEHP at the doses of 0-400 μM for 24 h did not affect cell viability, we found reduced consumption of T 3 and T 4 in the culture medium of HTR-8/Svneo cells treated with DEHP at 400 μM. DEHP treatment did not affect T 3 uptake and the expression of monocarboxylate transporters 8 (MCT8) and organic anion transporters 1C1 (OATP1C1). However, DEHP significantly inhibited transthyretin (TTR) internalization, down-regulated TTR, deiodinase 2 (DIO2), and thyroid hormone receptors mRNA expression and protein levels, and up-regulated deiodinase 3 (DIO3) protein levels in a dose-dependent manner. These results indicate that DEHP acts on placental trophoblast cells, inhibits its TTR internalization, down-regulates TTR expression and affects the expression of DIO2, DIO3, and thyroid hormone receptor. These may be the mechanisms by which PAEs affects THs transport through placental., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company., (Copyright © 2020 Elsevier Inc. All rights reserved.)

1. A number of chemicals have been shown to produce disruption of the thyroid gland, resulting in reduced thyroid hormone synthesis, by a mechanism involving inhibition of thyroid peroxidase (TPO) activity (EC 1.11.1.8).2. An assay was developed for rat thyroid gland microsomal TPO activity, employing L-tyrosine as the physiological substrate, with analysis of the formation of the 3-iodo-L-tyrosine (3MIT) metabolite by ultra-performance liquid chromatography-mass spectrometry-mass spectrometry.3. Formation of 3MIT was linear with respect to both rat thyroid gland microsomal protein concentration and incubation time, whereas only small quantities of 3,5-diodo-L-tyrosine were formed.4. Studies were performed with nine known TPO inhibitors. The most potent inhibitors were 3-amino-1,2,4-triazole, ethylene thiourea, methimazole and 6-propyl-2-thiouracil which had IC 50 values (i.e. concentration to produce a 50% inhibition of enzyme activity) of 0.059, 0.791, 1.07 and 1.96 μM, respectively, whereas the least potent inhibitor was sodium perchlorate which had an IC 50 value of 13,800 µM.5. For five inhibitors, where literature data were available, the observed IC 50 values obtained in this study employing rat thyroid gland microsomes and L-tyrosine as substrate were similar to those previously reported using the spectrophotometric guaiacol oxidation assay.

Thyroperoxidase (TPO) is an enzyme essential for thyroid hormone (TH) synthesis and a target site for a number of xenobiotics that disrupt TH homeostasis. An in vitro high-throughput screening assay for TPO inhibition, the Amplex UltraRed-TPO (AUR-TPO), has been used to screen the ToxCast chemical libraries for this action. Output from this assay would be most useful if it could be readily translated into an in vivo response, namely a reduction of TH in serum. To this end, the relationship between TPO inhibition in vitro and serum TH decreases was examined in rats exposed to 2 classic TPO inhibitors, propylthiouracil (PTU) and methimazole (MMI). Serum and gland PTU, MMI, and TH levels were quantified using tandem liquid chromatography mass spectrometry. Thyroperoxidase activity was determined in thyroid gland microsomes treated with PTU or MMI in vitro and ex vivo from thyroid gland microsomes prepared from exposed animals. A quantitative model was constructed by contrasting in vitro and ex vivo AUR-TPO results and the in vivo time-course and dose-response analysis. In vitro:ex vivo correlations of AUR-TPO outputs indicated that less than 30% inhibition of TPO in vitro was sufficient to reduce serum T4 by 20%, a degree of regulatory significance. Although further testing of model estimates using other TPO inhibitors is essential for verification of these initial findings, the results of this study provide a means to translate in vitro screening assay results into predictions of in vivo serum T4 changes to inform risk assessment., (Published by Oxford University Press on behalf of the Society of Toxicology 2019. This work is written by US Government employees and is in the public domain in the US.)

This study focused on the effect of kaempferol, catechin, apigenin, sinapinic acid, and extracts from plants (i.e., parsley, cumin, mustard, green tea, and green coffee) on thyroid peroxidase (TPO) and lipoxygenase (LOX) activity, antiradical potential, as well as the result of interactions among them. Catechin, sinapinic acid, and kaempferol acted as a competitive TPO inhibitors, while apigenin demonstrated an uncompetitive mode of inhibitory action. Ethanol extracts from all plants acted as competitive TPO inhibitors, while, after in vitro digestion, TPO activation was found especially in the case of mustard (24%) and cumin (19.85%). Most importantly, TPO activators acted synergistically. The TPO effectors acted as LOX inhibitors. The most effective were potentially bioaccessible compounds from green tea and green coffee (IC 50 = 29.73 mg DW/mL and 30.43 mg DW/mL, respectively). The highest free radical scavenging ability was determined for catechin and sinapinic acid (IC 50 = 78.37 µg/mL and 84.33 µg/mL, respectively) and potentially bioaccessible compounds from mustard (0.42 mg DW/mL) and green coffee (0.87 mg DW/mL). Green coffee, green tea, cumin, and mustard contain potentially bioaccessible TPO activators that also act as effective LOX inhibitors, which indicate their potentially health-promoting effects for people suffering from Hashimoto's disease.

Plastics are ubiquitously present in our daily life and some components of plastics are endocrine-disrupting chemicals, such as bisphenol A and phthalates. Herein, we aimed to evaluate the effect of plastic endocrine disruptors on type 1 and type 2 deiodinase activities, enzymes responsible for the conversion of the pro-hormone T4 into the biologically active thyroid hormone T3, both in vitro and in vivo. Initially, we incubated rat liver type 1 deiodinase and brown adipose tissue type 2 deiodinase samples with 0.5 mM of the plasticizers, and the deiodinase activity was measured. Among them, only BPA was capable to inhibit both type 1 and type 2 deiodinases. Then, adult male Wistar rats were treated orally with bisphenol A (40 mg/kg b.w.) for 15 days and hepatic type 1 deiodinase and brown adipose tissue type 2 deiodinase activities and serum thyroid hormone concentrations were measured. In vivo bisphenol A treatment significantly reduced hepatic type 1 deiodinase activity but did not affect brown adipose tissue type 2 deiodinase activity. Serum T4 levels were higher in bisphenol A group, while T3 remained unchanged. T3/T4 ratio was decreased in rats treated with bisphenol A, reinforcing the idea that peripheral metabolism of thyroid hormone was affected by bisphenol A exposure. Therefore, our results suggest that bisphenol A can affect the metabolism of thyroid hormone thus disrupting thyroid signaling., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Objective: Glucocorticoids (GCs) are widely prescribed medications that are well recognized to cause adverse metabolic effects including hyperphagia, obesity, and hyperglycemia. These effects have been recapitulated in a murine model of GC excess, and we hypothesize that they are mediated, in part, through central mechanisms. This study aimed to identify genes in the hypothalamic arcuate nucleus (ARC) that are altered with GC treatment and evaluate their contribution to GC-induced metabolic abnormalities., Methods: Corticosterone (Cort; 75 μg/ml) was administered in the drinking water to male C57Bl/6J mice for 2 days or 4 weeks. Phenotypic analysis of each group was undertaken and central and peripheral tissues were collected for biochemical and mRNA analyses. Arcuate nuclei were isolated by laser capture microdissection and tissue analyzed by RNA-seq., Results: RNA-seq analysis of ARC tissue from 4 week Cort treated mice revealed 21 upregulated and 22 downregulated genes at a time when mice had increased food intake, expansion of adipose tissue mass, and insulin resistance. In comparison, after 2 days Cort treatment, when the main phenotypic change was increased food intake, RNA-seq identified 30 upregulated and 16 downregulated genes. Within the genes altered at 2 days were a range of novel genes but also those known to be regulated by GCs, including Fkbp5, Mt2, Fam107a, as well as some involved in the control of energy balance, such as Agrp, Sepp1, Dio2, and Nmb. Of the candidate genes identified by RNA-seq, type-II iodothyronine deiodinase (Dio2) was chosen for further investigation as it was increased (2-fold) with Cort, and has been implicated in the control of energy balance via the modulation of hypothalamic thyroid hormone availability. Targeted knockdown of Dio2 in the MBH using AAV-mediated CRISPR-Cas9 produced a mild attenuation in GC-induced brown adipose tissue weight gain, as well as a 56% reduction in the GC-induced increase in Agrp. However, this conferred no protection from GC-induced hyperphagia, obesity, or hyperglycemia., Conclusions: This study identified a comprehensive set of genes altered by GCs in the ARC and enabled the selection of key candidate genes. Targeted knockdown of hypothalamic Dio2 revealed that it did not mediate the chronic GC effects on hyperphagia and hyperglycemia., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The aim of this study was to estimate the mode of thyroid peroxidase (TPO) inhibition by polyphenols: Chlorogenic acid, rosmarinic acid, quercetin, and rutin. All the tested polyphenols inhibited TPO; the IC 50 values ranged from 0.004 mM to 1.44 mM (for rosmarinic acid and rutin, respectively). All these pure phytochemical substances exhibited different modes of TPO inhibition. Rutin and rosmarinic acid showed competitive, quercetin-uncompetitive and chlorogenic acid-noncompetitive inhibition effect on TPO. Homology modeling was used to gain insight into the 3D structure of TPO and molecular docking was applied to study the interactions of the inhibitors with their target at the molecular level. Moreover, the type and strength of mutual interactions between the inhibitors (expressed as the combination index, CI) were analyzed. Slight synergism, antagonism, and moderate antagonism were found in the case of the combined addition of the pure polyphenols. Rutin and quercetin as well as rutin and rosmarinic acid acted additively (CI = 0.096 and 1.06, respectively), while rutin and chlorogenic acid demonstrated slight synergism (CI = 0.88) and rosmarinic acid with quercetin and rosmarinic acid with chlorogenic acid showed moderate antagonism (CI = 1.45 and 1.25, respectively). The mixture of chlorogenic acid and quercetin demonstrated antagonism (CI = 1.79). All the polyphenols showed in vitro antiradical ability against 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), ABTS. The highest ability (expressed as IC 50 ) was exhibited by rosmarinic acid (0.12 mM) and the lowest value was ascribed to quercetin (0.45 mM).

Deiodinase enzymes play an essential role in converting thyroid hormones between active and inactive forms by deiodinating the pro-hormone thyroxine (T4) to the active hormone triiodothyronine (T3) and modifying T4 and T3 to inactive forms. Chemical inhibition of deiodinase activity has been identified as an important endpoint to include in screening chemicals for thyroid hormone disruption. To address the lack of data regarding chemicals that inhibit the deiodinase enzymes, we developed robust in vitro assays that utilized human deiodinase types 1, 2, and 3 and screened over 1800 unique chemicals from the U.S. EPA's ToxCast phase 1_v2, phase 2, and e1k libraries. Initial testing at a single concentration identified 411 putative deiodinase inhibitors that produced inhibition of 20% or greater in at least 1 of the 3 deiodinase assays, including chemicals that have not previously been shown to inhibit deiodinases. Of these, 228 chemicals produced enzyme inhibition of 50% or greater; these chemicals were further tested in concentration-response to determine relative potency. Comparisons across these deiodinase assays identified 81 chemicals that produced selective inhibition, with 50% inhibition or greater of only 1 of the deiodinases. This set of 3 deiodinase inhibition assays provides a significant contribution toward expanding the limited number of in vitro assays used to identify chemicals with the potential to interfere with thyroid hormone homeostasis. In addition, these results set the groundwork for development and evaluation of structure-activity relationships for deiodinase inhibition, and inform targeted selection of chemicals for further testing to identify adverse outcomes of deiodinase inhibition., (Published by Oxford University Press on behalf of the Society of Toxicology 2018.)

Current practices for evaluating the cumulative risk of thyroid-active chemical mixtures (perchlorate, thiocyanate, nitrate) focus on the inhibition of thyroidal iodide uptake via the sodium iodide symporter (NIS) as the mode of action for potency equivalence calculations. However, unlike perchlorate, thiocyanate presents additional modes of action within the thyroid that could contribute to the overall thyroid perturbation. We tested the hypothesis of whether assuming a single mode of action of thyroidal iodide uptake inhibition is sufficient for describing the observed dose-response relationship for thiocyanate and its effects on serum thyroxine levels. An interaction model was developed by linking a biologically based dose-response model for iodide and thyroid hormones to a thiocyanate physiologically based pharmacokinetic model. Each model, adapted from the literature, was restructured and recalibrated in a Bayesian framework for the current mode of actions study. For a chronic exposure scenario, NIS inhibition alone was found not to be sufficient to describe the dose-response relationship for thiocyanate. Inclusion of additional modes of action involving iodide flux across the thyroid membrane and inhibition of iodide organification via thyroid peroxidase showed only moderate improvements in capturing the dose-response at environmental thiocyanate doses of exposure and failed to capture trends at very high doses. Our findings emphasize the need for more mechanistic data for chronic exposure scenarios to characterize better the overall dose-response relationship for thiocyanate. Risk assessment approaches for thyroid-active chemical mixtures that rely on NIS inhibition as the single mode of action may over-predict the contribution of thiocyanate to thyroid disruption., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Transcriptomics technologies have been used for risk assessment of chemicals, mainly to predict the modes of action (MOAs) of chemicals or identify biomarkers. Transcriptomics data may also be helpful to understand MOAs of chemicals at the molecular level in more detail. As an example of the known MOAs, there are two MOAs of thyroid toxicity: inhibition of thyroid hormone synthesis ("direct" effect) and hypermetabolism of thyroid hormone by enzyme induction in liver ("indirect" effect). In the present study, global profiles of gene expression were analyzed in rats treated with chemicals acting directly on the thyroid (thyroid peroxidase inhibitors such as propylthiouracil and methimazole) and chemicals acting indirectly on the thyroid (hepatic enzyme inducers such as phenobarbital and pregnenolone-16α-carbonitrile) using microarrays. Using a subtraction method between these two types of chemicals, we identified characteristic gene expression changes on the thyroid hormone synthesis pathway by direct-acting chemicals. Based on the functions of these genes, alterations of their expression seem to indicate the results of thyroid peroxidase inhibition, and might be helpful in more accurate evaluation of MOAs for thyroid toxicity., (© 2018 John Wiley & Sons, Ltd.)

The enzyme iodotyrosine deiodinase (dehalogenase, IYD) catalyzes iodide recycling and promotes iodide retention in thyroid follicular cells. Loss of function or chemical inhibition of IYD reduces available iodide for thyroid hormone synthesis, which leads to hormone insufficiency in tissues and subsequent negative developmental consequences. IYD activity is especially critical under conditions of lower dietary iodine and in low iodine environments. Our objective was to evaluate the toxicological relevance of IYD inhibition in a model amphibian (Xenopus laevis) used extensively for thyroid disruption research. First, we characterized IYD ontogeny through quantification of IYD mRNA expression. Under normal development, IYD was expressed in thyroid glands, kidneys, liver, and intestines, but minimally in the tail. Then, we evaluated how IYD inhibition affected developing larval X. laevis with an in vivo exposure to a known IYD inhibitor (3-nitro-l-tyrosine, MNT) under iodine-controlled conditions; MNT concentrations were 7.4-200 mg/L, with an additional 'rescue' treatment of 200 mg/L MNT supplemented with iodide. Chemical inhibition of IYD resulted in markedly delayed development, with larvae in the highest MNT concentrations arrested prior to metamorphic climax. This effect was linked to reduced glandular and circulating thyroid hormones, increased thyroidal sodium-iodide symporter gene expression, and follicular cell hypertrophy and hyperplasia. Iodide supplementation negated these effects, effectively rescuing exposed larvae. These results establish toxicological relevance of IYD inhibition in amphibians. Given the highly conserved nature of the IYD protein sequence and scarcity of environmental iodine, IYD should be further investigated as a target for thyroid axis disruption in freshwater organisms.

The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rutabaga sprouts belong to the Brassicaceae family and may exert a negative influence on thyroid function, because they are a rich in glucosinolates. These sprouts are also valuable source of iodine (6.5 ± 0.6 μg/100 g of fresh weight). Sprouts were tested in a long-term experiment with young male rats as an element of their diet, combined with two models of hypothyroidism, the first - deficit of iodine and the second - sulfadimethoxine ingestion as a pharmacological agent caused inhibition of thyroid peroxidase. Evaluations were performed for the serum TSH and thyroid hormones together with analyzes of thyroid histopathology, cytosolic glutathione peroxidase (GPX1), thioredoxin reductase in the thyroid, plasma GPX3 and CAT, erythrocyte GPX1. Rutabaga sprouts' intake by healthy rats did not cause any harmful effect on their health, including thyroid function. For animals with hypothyroidism, rutabaga sprouts enhanced the adverse effect of iodine deficiency or ingestion of sulfadimethoxine on the organism. According to the results obtained for young male rats thyroid function, the interpretation of data for human exposure to rutabaga sprouts has to be avoided. Furthermore, unless new scientific data confirms a lack of the negative effect of brassica sprouts on thyroid function in human, they should not be excluded from the group of goitrogenic products., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Thyroid hormones (THs) play key roles in modulating the overall metabolism of the body, protein synthesis, fat metabolism, neuronal and bone growth, and cardiovascular as well as renal functions. In this review, we discuss on the thyroid hormone synthesis and activation, thyroid hormone receptors (TRs) and mechanism of action, applications of thyroid hormone analogues, particularly the compounds that are selective ligands for TRβ receptors, or enzyme inhibitors for the treatment of thyroidal disorders with a specific focus on thyroid peroxidase and iodothyronine deiodinases. We also discuss on the development of small-molecule deiodinase mimetics and their mechanism of deiodination, as these compounds have the potential to regulate the thyroid hormone levels., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Adequate levels of thyroid hormone (TH) are needed for proper brain development, deficiencies may lead to adverse neurologic outcomes in humans and animal models. Environmental chemicals have been linked to TH disruption, yet the relationship between developmental exposures and decline in serum TH resulting in neurodevelopmental impairment is poorly understood. The present study developed a quantitative adverse outcome pathway where serum thyroxin (T4) reduction following inhibition of thyroperoxidase in the thyroid gland are described and related to deficits in fetal brain TH and the development of a brain malformation, cortical heterotopia. Pregnant rats were exposed to 6-propylthiouracil (PTU 0, 0.1, 0.5, 1, 2, or 3 parts per million [ppm]) from gestational days 6-20, sequentially increasing PTU concentrations in maternal thyroid gland and serum as well as in fetal serum. Dams exposed to 0.5 ppm PTU and higher exhibited dose-dependent decreases in thyroidal T4. Serum T4 levels in the dam were significantly decreased with exposure to 2 and 3 ppm PTU. In the fetus, T4 decrements were first observed at a lower dose of 0.5 ppm PTU. Based on these data, fetal brain T4 levels were estimated from published literature sources, and quantitatively linked to increases in the size of the heterotopia present in the brains of offspring. These data show the potential of in vivo assessments and computational descriptions of biologic responses to predict the development of this structural brain malformation and use of quantitative adverse outcome pathway approach to evaluate brain deficits that may result from exposure to other TH disruptors., (Published by Oxford University Press on behalf of the Society of Toxicology 2017. This work is written by US Government employees and is in the public domain in the US.)

Inflation of the posterior and/or anterior swim bladder is a process previously demonstrated to be regulated by thyroid hormones. We investigated whether inhibition of deiodinases, which convert thyroxine (T4) to the more biologically active form, 3,5,3'-triiodothyronine (T3), would impact swim bladder inflation. Two experiments were conducted using a model deiodinase inhibitor, iopanoic acid (IOP). First, fathead minnow embryos were exposed to 0.6, 1.9, or 6.0 mg/L or control water until 6 d postfertilization (dpf), at which time posterior swim bladder inflation was assessed. To examine anterior swim bladder inflation, a second study was conducted with 6-dpf larvae exposed to the same IOP concentrations until 21 dpf. Fish from both studies were sampled for T4/T3 measurements and gene transcription analyses. Incidence and length of inflated posterior swim bladders were significantly reduced in the 6.0 mg/L treatment at 6 dpf. Incidence of inflation and length of anterior swim bladder were significantly reduced in all IOP treatments at 14 dpf, but inflation recovered by 18 dpf. Throughout the larval study, whole-body T4 concentrations increased and T3 concentrations decreased in all IOP treatments. Consistent with hypothesized compensatory responses, deiodinase-2 messenger ribonucleic acid (mRNA) was up-regulated in the larval study, and thyroperoxidase mRNA was down-regulated in all IOP treatments in both studies. These results support the hypothesized adverse outcome pathways linking inhibition of deiodinase activity to impaired swim bladder inflation. Environ Toxicol Chem 2017;36:2942-2952. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America., (Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

Graves' disease (GD) and Hashimoto's disease (HD) are well known autoimmune thyroid diseases (AITDs), and the severity and intractability of AITDs varies among patients. Thyroid peroxidase (TPO) is a thyroid-specific antigen. The levels of anti-thyroid peroxidase antibody (TPOAb) were higher in patients with HD and may be associated with thyroid destruction. In this study, we genotyped eight single nucleotide polymorphisms (SNPs) in the TPO gene to clarify the association of TPO gene polymorphisms with the development, severity and intractability of AITD. We genotyped TPO rs2071399G/A, rs2071400C/T, rs2071402A/G, rs2071403A/G, rs1126799C/T, rs1126797T/C, rs732609A/C, and rs2048722A/G polymorphisms in 145 patients with GD, 147 patients with HD and 92 healthy controls by PCR-RFLP method. TPO rs2071400 T carriers (CT + TT genotypes) were more frequent in AITD, GD, and HD patients (p=0.0079, 0.0041, and 0.0488, respectively). The TPO rs2071403 GG genotype was more frequent in AITD, GD, and HD patients (p=0.0227, 0.0465, and 0.0305, respectively). There was no significant association between the SNPs and the prognosis of AITD. Serum levels of TPOAb were significantly higher in AITD patients with TPO rs2071400 T carriers (CT + TT genotypes) than in those with the CC genotype (p=0.0295), and were also significantly higher in AITD patients with TPO rs2048722 T carriers (CT + TT genotypes) than in those with the CC genotype (p=0.0056). In conclusion, TPO rs2071400 and rs2071403 polymorphisms were associated with the development of HD and GD, but not with the prognosis. Moreover, TPO rs2071400 and rs2048722 polymorphisms were associated with the serum levels of TPOAb.

Background: Verification of new reagent lots is a required laboratory task. The Clinical and Laboratory Standards Institute (CLSI) EP26-A guideline provides a lot-to-lot verification protocol to detect significant changes in test performance. The aim of this study was to compare the performance of EP26-A with our laboratory reagent lot verification protocol., Methods: Prospective evaluations for two reagent lots each for thyroid stimulating hormone (TSH), thyroglobulin (Tg), thyroxine (T4), triiodothyronine (T3), free triiodothyronine (fT3), and thyroid peroxidase antibody (TPOAb) were performed. The laboratory's lot verification process included evaluation of 20 patient samples with the current and new lots and acceptability based on a predefined criteria. For EP26-A, method imprecision data and critical differences based on previously defined lot-to-lot consistency goals were used to define sample size requirements and rejection limits., Results: EP26-A required the following number of samples: 23 for TSH, 17 for Tg, 33 for T4, 31 for T3, 48 for fT3, and 1 for TPOAb. Our current protocol and EP26-A were in agreement in 9 of the 12 (75%) paired verifications. Of the 3 discrepant verifications, Tg and TSH reagent lots were rejected by EP26-A due to significant differences at medical decision points; whereas TPOAb was rejected by the current laboratory protocol., Conclusions: The EP26-A protocol arrived at the same conclusions as our protocol in 75% of the evaluations and required more samples for 4 of the 6 analytes tested. Challenges associated with determining rejection limits and the need for increased sample sizes may be critical factors that limit the utility of EP26-A., (Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Polybrominated diphenyl ethers (PBDEs) increase flame resistance in consumer goods, but these compounds and their hydroxylated derivatives (OH-BDEs) impair normal thyroid function. Halogen bonding (XB) of PBDEs to an active site selenocysteine may prevent iodothyronine deiodinase(Dio)-catalyzed activation/deactivation of thyroid hormone (TH) derivatives. In this study, we compare the strength of the XB interactions of TH derivatives, iodine-based contrast agents and PBDEs/OH-BDEs with a methylselenolate model of the Dio active site using density functional theory calculations. The strength of the XB interaction depends upon the acceptor halide, the position of the halide, the number of ring substituents, and the proximity of hydroxyl groups to the XB site. The weaker Se⋅⋅⋅Br interactions relative to Se⋅⋅⋅I interactions are consistent with a model of competitive inhibition that blocks binding of THs at elevated PBDE/OH-BDE concentrations. XB interactions were generally more favorable at ortho and meta positions and in substrates with more electron-withdrawing substituents. PBDEs/OH-BDEs that mimic the binding behavior of THs, that is, containing ortho and meta bromides and adjacent hydroxyl groups, may be the most effective inhibitors. Highly-brominated PBDEs/OH-BDEs have comparable interaction energies to THs and may undergo debromination. These results may also suggest that XB strength must exceed a threshold value in order for PBDEs/OH-BDEs to undergo nucleophilic attack by Dio., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Iodine is essential for thyroid hormone synthesis and is an important regulator of thyroid function. Chronic iodine deficiency leads to hypothyroidism, but iodine excess also impairs thyroid function causing hyperthyroidism, hypothyroidism, and/or thyroiditis. This study aimed to investigate the underlying mechanisms by which exposure to chronic iodine excess impairs pituitary-thyroid axis function., Methods: Male Wistar rats were treated for two months with NaI (0.05% and 0.005%) or NaI+NaClO 4 (0.05%) dissolved in drinking water. Hormone levels, gene expression, and thyroid morphology were analyzed later., Results: NaI-treated rats presented high levels of iodine in urine, increased serum thyrotropin levels, slightly decreased serum thyroxine/triiodothyronine levels, and a decreased expression of the sodium-iodide symporter, thyrotropin receptor, and thyroperoxidase mRNA and protein, suggesting a primary thyroid dysfunction. In contrast, thyroglobulin and pendrin mRNA and protein content were increased. Kidney and liver deiodinase type 1 mRNA expression was decreased in iodine-treated rats. Morphological studies showed larger thyroid follicles with higher amounts of colloid and increased amounts of connective tissue in the thyroid of iodine-treated animals. All these effects were prevented when perchlorate treatment was combined with iodine excess., Conclusions: The present data reinforce and add novel findings about the disruption of thyroid gland function and the compensatory action of increased thyrotropin levels in iodine-exposed animals. Moreover, they draw attention to the fact that iodine intake should be carefully monitored, since both deficient and excessive ingestion of this trace element may induce pituitary-thyroid axis dysfunction.

The potencies of resorcinol, 6-propylthiouracil (PTU) and methimazole (MMI) for inducing developmental toxicity and neurotoxicity were compared in pregnant rats, regarded as valid model for human thyroid toxicity. Profound differences on maternal thyroid hormone levels (THs), maternal toxicity as well as developmental and neurotoxicity sequelae occurred. Resorcinol affected none of those end points. PTU and MMI caused significant effects. Therapy with either PTU or MMI during the first trimester of human pregnancy can cause reductions of maternal THs, accompanied by disruptions of prenatal development. Clinical MMI studies show sporadic evidence of teratogenic effects, with equivocal relation to thyroid peroxidase (TPO) inhibition. In recent decades no MMI associated prenatal toxicity has been reported, an outcome possibly related to carefully managed therapy. Orally administered resorcinol was rapidly absorbed, metabolized and excreted and was undetectable in the thyroid. In contrast, PTU or MMI accumulated. Resorcinol's potency to inhibit TPO was profoundly lower than that of PTU or MMI. Quantum chemical calculations may explain low resorcinol reactivity with TPO. Thus, distinctions in the target organ and the TPO inhibitory potency between these chemicals are likely contributing to different reductions of maternal THs levels and affecting the potency to cause developmental toxicity and neurotoxicity., (Copyright © 2016. Published by Elsevier Inc.)

The herbicide acetochlor is widely used and detected in the environment and biota, and has been suspected to disrupt the thyroid endocrine system, but underlying mechanisms have not yet been clarified. In the present study, zebrafish larvae (7 days post-fertilization) were exposed to a series concentration of acetochlor (0, 1, 3, 10, 30, 100 and 300 µg l(-1) ) within a 14-day window until 21 days post-fertilization. Thyroid hormones and mRNA expression profiles of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis were analyzed. Exposure to the positive control, 3,5,3'-triiodothyronine (T3 ), altered the mRNA expression, suggesting that the HPT axis in the critical window of zebrafish responded to chemical exposure and could be used to evaluate the effects of chemicals on the thyroid endocrine system. The mRNA expressions of genes involved in thyroid hormone synthesis (tshβ, slc5a5 and tpo) were upregulated significantly with acetochlor treatment, which might be responsible for the increased thyroxine concentrations. The downregulation of genes related to thyroid hormone metabolism (dio1 and ugt1ab) and transport (ttr) in zebrafish larvae exposed to acetochlor might further explain the increased thyroxine levels and decreased T3 levels. The mRNA expression of the thyroid hormone receptor (trα) was also upregulated upon acetochlor exposure. Results suggested that acetochlor altered mRNA expression of the HPT axis-related genes and changed the whole body thyroid hormone levels in zebrafish larvae. It demonstrated that acetochlor could cause endocrine disruption of the thyroid system by simulating the biological activity of T3 . Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

High-throughput screening for potential thyroid-disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the U.S. Environmental Protection Agency ToxCast screening assay portfolio. To fill 1 critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast phase I and II chemical libraries, comprised of 1074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single-concentration screen were retested in concentration-response. Due to high false-positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed 2 additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using guaiacol as a substrate to confirm the activity profiles of putative TPO inhibitors. This effort represents the most extensive TPO inhibition screening campaign to date and illustrates a tiered screening approach that focuses resources, maximizes assay throughput, and reduces animal use., (Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.)

Some pharmaceuticals and environmental chemicals bind the thyroid peroxidase (TPO) enzyme and disrupt thyroid hormone production. The potential for TPO inhibition is a function of both the binding affinity and concentration of the chemical within the thyroid gland. The former can be determined through in vitro assays, and the latter is influenced by pharmacokinetic properties, along with environmental exposure levels. In this study, a physiologically based pharmacokinetic (PBPK) model was integrated with a pharmacodynamic (PD) model to establish internal doses capable of inhibiting TPO in relation to external exposure levels predicted through exposure modeling. The PBPK/PD model was evaluated using published serum or thyroid gland chemical concentrations or circulating thyroxine (T4) and triiodothyronine (T3) hormone levels measured in rats and humans. After evaluation, the model was used to estimate human equivalent intake doses resulting in reduction of T4 and T3 levels by 10% (ED10) for 6 chemicals of varying TPO-inhibiting potencies. These chemicals were methimazole, 6-propylthiouracil, resorcinol, benzophenone-2, 2-mercaptobenzothiazole, and triclosan. Margin of exposure values were estimated for these chemicals using the ED10 and predicted population exposure levels for females of child-bearing age. The modeling approach presented here revealed that examining hazard or exposure alone when prioritizing chemicals for risk assessment may be insufficient, and that consideration of pharmacokinetic properties is warranted. This approach also provides a mechanism for integrating in vitro data, pharmacokinetic properties, and exposure levels predicted through high-throughput means when interpreting adverse outcome pathways based on biological responses., (Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.)

Purpose: Chemokines play an important role in the pathogenesis of autoimmune thyroid diseases. Platelet factor 4 (PF4, CXCL4) released from activated platelets is a chemokine. However, its clinical importance in autoimmune thyroiditis remains unknown. This study is intended to determine circulating levels of PF4 levels in patients with autoimmune thyroiditis (AIT)., Methods: Circulating levels of PF4 were measured in 34 consecutive patients with newly diagnosed AIT and 18 euthyroid controls. Among AIT group, 16 patients were euthyroid and 18 had subclinic hypothyroidism. Controls and individuals with AIT were similar in terms of age., Results: Serum levels of PF4 were comparable in patients with AIT and in controls. Among patients with AIT, PF4 was significantly lower in those with subclinical hypothyroidism than in euthyroid individuals (p = 0.001). In correlation analysis, PF4 was negatively correlated with TSH (r = -0.663, p = 0.000) and positively correlated with free T4 (r = 0.428, p = 0.012). There was not any significant correlation between PF4 and AbTPO, AbTg., Conclusion: The present study demonstrated for the first time that circulating PF4 levels are decreased in subclinically hypothyroid AIT. This result draws attention to the circulating PF4 levels in subclinically hypothyroid AIT and may shed light on further researches at this topic.

Purpose: Technetium-99m (Tc-99m) thyroid scintigraphy is a well known diagnostic tool that shows the entire gland in a single image. We aimed to evaluate its additive diagnostic value in subclinical autoimmune thyroid disease (S-AITD) in systemic lupus erythematosus (SLE) patients., Methods: We investigated 100 systemic lupus erythematosus (SLE) patients without overt thyroid involvement (eight men and 92 women; mean age 40±6.5 years) and 50 age and sex matched controls. All were subjected to thyroid evaluation using anti-thyroglobulin (anti-TG) and anti-thyroid peroxidase (anti-TPO) antibodies; hormones (FT3; FT4 and TSH) and Tc-99m thyroid scintigraphy., Results: 14/100 (14%) and none (0%) were positive for S-AITD in SLE and control groups, respectively (P = 0.0001). They were classified by thyroid scintigraphy and hormonal profile into 2/14 Hashimoto; 10/14 atrophic thyroiditis and 2/14 Graves' disease. Anti-TPO was elevated in 12 SLE cases, while anti-TG was elevated in only 2/14 (P = 0.0001). Thyroid scintigraphy showed statistically significant associations with FT4, TSH and anti-TPO., Conclusion: Tc-99m thyroid scintigraphy may have an additional diagnostic role in S-AITD among SLE patients, with an impact on patient management. This potential needs to be further evaluated in a larger series on a multicenter basis., (© The Author(s) 2015.)

Iodothyronine deiodinases are a family of enzymes that remove specific iodine atoms from one of the two aromatic rings in thyroid hormones (THs). They thereby fine-tune local TH concentrations and cellular TH signaling. Deiodinases catalyze a remarkable biochemical reaction, i.e., the reductive elimination of a halogenide from an aromatic ring. In metazoans, deiodinases depend on the rare amino acid selenocysteine. The recent solution of the first experimental structure of a deiodinase catalytic domain allowed for a reappraisal of the many mechanistic and mutagenesis data that had been accumulated over more than 30 years. Hence, the structure generates new impetus for research directed at understanding catalytic mechanism, substrate specificity, and regulation of deiodinases. This review will focus on structural and mechanistic aspects of iodothyronine deiodinases and briefly compare these enzymes with dehalogenases, which catalyze related reactions. A general mechanism for the selenium-dependent deiodinase reaction will be described, which integrates the mouse deiodinase 3 crystal structure and biochemical studies. We will summarize further, sometimes isoform-specific molecular features of deiodinase catalysis and regulation, and we will then discuss available compounds for modulating deiodinase activity for therapeutic purposes., (© 2015 Society for Endocrinology.)

Background: Deiodinases (DIO1, 2, and 3) are key enzymes in thyroid hormone (TH) activation and inactivation with impact on energy metabolism, development, cell differentiation, and a number of other physiological processes. The three DIO isoenzymes thus constitute sensitive rate-limiting components within the TH axis, prone to dysregulation by endocrine disruptive compounds or disease state. In animal models and cell culture experiments, they serve as readout for local TH status and disarrangement of the hormonal axis. Furthermore, some human diseases are characterized by apparent deiodinase dysregulation (e.g., the low triiodothyronine syndrome in critical illness). Consequently, these enzymes are targets of interest for the development of pharmacological compounds with modulatory activities. Until now, the portfolio of inhibitors for these enzymes is limited. In the clinics, the DIO1-specific inhibitor propylthiouracil is in use for treatment of severe hyperthyroidism. Other well-known inhibitors (e.g., iopanoic acid or aurothioglucose) are nonselective and block all three isoenzymes. Furthermore, DIO3 was shown to be a potential oncogenic gene, which is strongly expressed in some tumors and might, in consequence, protect tumor tissue form differentiation by TH. With respect to its role in tumorigenesis, specific inhibitors of DIO3 as a potential target for anticancer drugs would be highly desirable. To this end, a flexible and convenient assay for high-throughput screening is needed. We recently described a nonradioactive screening assay, utilizing the classic Sandell-Kolthoff reaction as readout for iodide release from the substrate molecules. While we used murine liver as enzyme source, the assay was limited to murine DIO1 activity testing. Here, we describe the use of recombinant proteins as enzyme sources within the assay, expanding its suitability from murine Dio1 to human DIO1, DIO2, and DIO3., Methods: As proof-of-concept, deiodination reactions catalyzed by these recombinant enzymes were monitored with various nonradioactive substrates and confirmed by liquid chromatography-tandem mass spectrometry., Results: The contrast agent and known DIO inhibitor iopanoic acid was characterized as readily accepted substrate by DIO2 and Dio3. In a screening approach using established endocrine disrupting compounds, the natural food ingredient genistein was identified as a further DIO1-specific inhibitor, while xanthohumol turned out to potently block the activity of all three isoenzymes., Conclusions: A rapid nonradioactive screening method based on the Sandell-Kolthoff reaction is suitable for identification of environmental, nutritive and pharmacological compounds modulating activities of human deiodinase enzymes.

Polybrominated diphenyl ether (PBDE) flame retardants are endocrine disruptors and suspected neurodevelopmental toxicants. While the direct mechanisms of neurodevelopmental toxicity have not been fully elucidated, it is conceivable that alterations in thyroid hormone levels in the developing brain may contribute to these effects. Cells within the brain locally convert thyroxine (T4) to the biologically active triiodothyronine (T3) through the action of the selenodeiodinase type 2 iodothyronine deiodinase (DIO2). Previous studies have demonstrated that PBDEs can alter hepatic deiodinase activity both in vitro and in vivo; however, the effects of PBDEs on the deiodinase isoforms expressed in the brain are not well understood. Here, we studied the effects of several individual PBDEs and hydroxylated metabolites (OH-BDEs) on DIO2 activity in astrocytes, a specialized glial cell responsible for production of more than 50% of the T3 required by the brain. Primary human astrocytes and H4 glioma cells were exposed to individual PBDEs or OH-BDEs at concentrations up to 5 μM. BDE-99 decreased DIO2 activity by 50% in primary astrocyte cells and by up to 80% in the H4 cells at doses of ≥500 nM. 3-OH-BDE-47, 6-OH-BDE-47, and 5'-OH-BDE-99 also decreased DIO2 activity in cultured H4 glioma cells by 45-80% at doses of approximately 1-5 μM. Multiple mechanisms appear to contribute to the decreased DIO2 activity, including weakened expression of DIO2 mRNA, competitive inhibition of DIO2, and enhanced post-translational degradation of DIO2. We conclude that decreases in DIO2 activity caused by exposure to PBDEs may play a role in the neurodevelopmental deficits caused by these toxicants.

Growth hormone (GH) regulates whole body metabolism, and physical exercise is the most potent stimulus to induce its secretion in humans. The mechanisms underlying GH secretion after exercise remain to be defined. The aim of this study was to elucidate the role of estrogen and pituitary type 1 deiodinase (D1) activation on exercise-induced GH secretion. Ten days after bilateral ovariectomy, animals were submitted to 20 min of treadmill exercise at 75% of maximum aerobic capacity and tissues were harvested immediately or 30 min after exercise. Non-exercised animals were used as controls. A significant increase in D1 activity occurred immediately after exercise (~60%) in sham-operated animals and GH was higher (~6-fold) 30 min after exercise. Estrogen deficient rats exhibited basal levels of GH and D1 activity comparable to those found in control rats. However, after exercise both D1 activity and serum GH levels were blunted compared to sedentary rats. To understand the potential cause-effect of D1 activation in exercise-induced GH release, we pharmacologically blocked D1 activity by propylthiouracil (PTU) injection into intact rats and submitted them to the acute exercise session. D1 inhibition blocked exercise-induced GH secretion, although basal levels were unaltered. In conclusion, estrogen deficiency impairs the induction of thyroid hormone activating enzyme D1 in the pituitary, and GH release by acute exercise. Also, acute D1 activation is essential for exercise-induced GH response.

In animals, long-term feeding with peanut (Arachis hypogaea) seed coats causes hypertrophy and hyperplasia of the thyroid gland. However, to date there have been no detailed studies. Here, we explored the thyroidal effects of dietary peanut seed coats (PSC) in rats. The PSC has high levels of pro-goitrogenic substances including phenolic and other cyanogenic constituents. The PSC was mixed with a standard diet and fed to rats for 30 and 60 days, respectively. Animals fed with the PSC-supplemented diet showed a significant increase in urinary excretion of thiocyanate and iodine, thyroid enlargement, and hypertrophy and/or hyperplasia of thyroid follicles. In addition, there was inhibition of thyroid peroxidase (TPO) activity, 5'-deiodinase-I (DIO1) activity, and (Na+-K+)-ATPase activity in the experimental groups of rats as compared to controls. Furthermore, the PSC fed animals exhibited decreased serum circulating total T4 and T3 levels, severe in the group treated for longer duration. These data indicate that PSC could be a novel disruptor of thyroid function, due to synergistic actions of phenolic as well as cyanogenic constituents.

A simple and rapid luminometric assay for the detection of chemical inhibitors of human thyroid peroxidase (hTPO) activity was developed and validated with 10 model compounds. hTPO was derived from the human thyroid follicular cell line Nthy-ori 3-1 and its activity was quantified by measuring the oxidation of luminol in the presence of hydrogen peroxide (H2O2), which results in the emission of light at 428 nm. In this assay,hTPO activity was shown to be inhibited by 5 known TPO inhibitors and not inhibited by 5 non-inhibitors. Similar results were obtained with porcine TPO (pTPO).The inhibition of hTPO by the model compounds was also tested with guaiacol and Ampliflu Red as alternative indicator substrates. While all substrates allowed the detection of pTPO activity and its inhibition, only the Ampliflu Red and luminol-based methods were sensitive enough to allow the quantification of hTPO activity from Nthy-ori 3-1 cell lysates. Moreover, luminol gave results with a narrower 95% confidence interval and therefore more reliable data.Whole extracts of fast-growing Nthy-ori 3-1 cells circumvent the need for animal-derived thyroid organs,thereby reducing costs, eliminating potential contamination and providing the possibility to study human instead of porcine TPO. Overall, the application of luminol and Nthy-ori 3-1 cell lysate for the detection of the disruption of hTPO activity was found to represent a valuable in vitro alternative and a possible candidate for inclusion within a high throughput integrated testing strategy for the detection of compounds that potentially interfere with normal thyroid function in vivo.

Background: It is unclear to what extent uremic toxins participate in inflammatory responses and the activities of deiodinases, as well as the effects of deiodinases on inflammatory cytokines., Materials and Methods: Hepatocellular carcinoma cell lines (HepG2) were transfected with small interfering ribonucleic acid (siRNA) specific for deiodinase type 1 (DIO1) and cultured with or without uremic toxins. The mRNA expression of DIO1, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α was detected by quantitative real-time PCR. The presence of selenoprotein M (SelM) and DIO1 was assessed by western blotting. Sonicate deiodinase activities in HepG2 cells were measured by a dithiothreitol-stimulated assay. The NF-κB, AP-1 and CREB-1 inflammatory signal pathways were confirmed by EMSA., Results: After culturing for 24 h, the mRNA expression of DIO1 was significantly decreased by the specific siRNA (reduced by 76%, P = 0.0002). Uremic toxins significantly increased the mRNA expression (P < 0.01) of IL-1β, IL-6 and TNF-α and inhibited DIO1 mRNA expression (P < 0.01) compared with controls. Suppression of DIO1 by siRNA significantly decreased the mRNA expression of IL-1β and IL-6 (P < 0.05) but not TNF-α (P = 0.093). Uremic toxins and specific siRNA synchronously reduced the protein expression of SelM and DIO1., Conclusions: Uremic toxins activate the expression of inflammatory cytokines. The major findings of this study indicate that the uremic toxins, more than inflammatory cytokines, play direct inhibitory roles in DIO1 enzyme activity, which then provides a negative feedback to the growing accumulation of inflammatory cytokines.

This study aimed to investigate the thyroid peroxidase antibody (TPOAb) positive rate and how TPOAb affects thyroid function between 7 and 12 weeks of gestation. Six hundred and eleven cases of healthy primipara were enrolled in this study between September 2010 and June 2011, and all of them were given a regular prenatal check in Beijing Friendship Hospital. By detecting the levels of thyroid stimulating hormone (TSH), free thyroxine (FT4), and TPOAb between 7 to 12 weeks gestation, we developed an early thyroid function normal reference interval during pregnancy. Additionally, we analyzed the TPOAb-positivity cutoff value, the positive rate, and the affect of TSH and FT4. We found that the TPOAb median values and changes in the range were 38.9 (6.4-1300+) mU/L. By establishing an 7-12 week gestational-specific reference standard for pregnancy, we set a positive cutoff value of 206.77 mU/L according to the 90th percentile TPOAb. The TPOAb positive rate was 10.8% (66/611). Regression analysis showed that, during gestation between 7 and 12 weeks, the titers of TPOAb and TSH correlate positively and negatively with FT4, β = 1.406 and 0.820, respectively [95%CI (1.349-1.413, 0.805-0.835), P = 0.000]. Pregnant TPOAb positive women are more likely to suffer from markedly increased TSH levels, with increased risk being 3-4-fold greater than in a normal pregnancy.

Many synthetic chemicals have been identified as environmental contaminants with activity to disrupt normal function of the thyroid hormone system. Thyroid hormones play important roles in growth, development, differentiation, and basal metabolic homeostasis, as well as in brain development in human fetus and children, and thyroid dysfunction can have very serious consequences, including mental retardation. Environmental chemicals may affect thyroid hormone action in multiple ways, including reduced thyroid hormone synthesis owing to direct toxicity at the thyroid gland, interaction with thyroid hormone receptors and transporters such as transthyretin, and disturbance of thyroid hormone metabolism (e.g., glucuronidation, sulfation and deiodination). In addition, iodotyrosine deiodinase, which is involved in iodide salvage by catalyzing deiodination of iodinated by-products of thyroid hormone production, was recently identified as a possible new target for disruption of thyroid hormone homeostasis by environmental halogenated chemicals. This topic, after briefly summarizing findings on the thyroid hormone-disrupting action of environmental chemicals in mammals, focuses on the effects of environmental halogenated chemicals on iodotyrosine deiodinase activity.

As Th22 subsets are identified, their involvement in the pathogenesis of numerous autoimmune diseases has become apparent. In this study, we investigated differentiation of Th22 cells in the autoimmune thyroid diseases including Hashimoto's thyroiditis (HT) and Graves' disease (GD). Besides, we also explored the involvement of Th22 cells in an iodine-induced autoimmune thyroiditis (AIT) model (i.e., NOD.H-2(h4) mice). In HT patients, we showed the level of circulating Th22 cells correlated with the level of serum IL-22, and was significantly higher than in GD patients and healthy control subjects. Levels of serum IL-6, a major Th22 cell differentiation effector, were also higher in HT, and correlated with Th22 cells concentration. Peripheral blood mononuclear cells isolated from HT patients produced larger amounts of IL-6 in vitro than did those isolated from other groups. Furthermore, unlike those from GD patients, T lymphocytes from HT patients showed an enhanced differentiation in vitro into Th22 cells in the presence of recombinant IL-6 and TNF-α. In addition, levels of circulating Th22 cells and titers of thyroid peroxidase antibody were positively correlated in HT patients. In NOD.H-2(h4) mice, higher numbers of Th22 cells were observed in the spleens of the AIT group, while splenocytes of this group also produced larger amounts of IL-6 and IL-22 in vitro compared with the control. Intra-thyroid infiltrating IL-22+ lymphocytes were significantly increased in mice of the AIT group compared with the control. Our results indicate that Th22 cells may contribute to the pathogenesis of HT.

The aim of this study was to investigate the possible influence of halogenated compounds on thyroid hormone metabolism via inhibition of iodotyrosine deiodinase (IYD) activity. The structure-activity relationships of 44 halogenated compounds for IYD-inhibitory activity were examined in vitro using microsomes of HEK-293 T cells expressing recombinant human IYD. The compounds examined were 17 polychlorinated biphenyls (PCBs), 15 polybrominated diphenyl ethers (PBDEs), two agrichemicals, five antiparasitics, two pharmaceuticals and three food colorants. Among them, 25 halogenated phenolic compounds inhibited IYD activity at the concentration of 1×10(-4)M or 6×10(-4)M. Rose bengal was the most potent inhibitor, followed by erythrosine B, phloxine B, benzbromarone, 4'-hydroxy-2,2',4-tribromodiphenyl ether, 4-hydroxy-2,3',3,4'-tetrabromodiphenyl ether, 4-hydroxy-2',3,4',5,6'-pentachlorobiphenyl, 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl ether, triclosan, and 4-hydroxy-2,2',3,4',5-pentabromodiphenyl ether. However, among PCBs and PBDEs without a hydroxyl group, including their methoxylated metabolites, none inhibited IYD activity. These results suggest that halogenated compounds may disturb thyroid hormone homeostasis via inhibition of IYD, and that the structural requirements for IYD-inhibitory activity include halogen atom and hydroxyl group substitution on a phenyl ring., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Context: Increasing numbers of women are being treated with l-thyroxine in pregnancy for mild thyroid dysfunction because of its association with impaired neuropsychological development in their offspring and other adverse obstetric outcomes. However, there are limited data to indicate whether treatment should be continued outside of pregnancy., Objectives: We aimed to determine whether subclinical hypothyroidism and maternal hypothyroxinemia resolve postdelivery., Design, Setting, and Participants: A total of 523 pregnant healthy women with no known thyroid disorders were recruited during routine antenatal care and provided blood samples at 28 weeks of pregnancy and at a mean of 4.9 years postpregnancy., Main Outcome Measures: TSH, free T₄, free T₃, and thyroid peroxidase antibody levels were measured in serum taken in pregnancy and at follow-up., Results: Subclinical hypothyroidism in pregnancy (TSH >3 mIU/L) was present in 65 of 523 (12.4%) women. Of these, 49 (75.4%) women had normal thyroid function postpregnancy; 16 of 65 (24.6%) had persistent high TSH (TSH >4.5 mIU/L postpregnancy) with 3 women receiving l-thyroxine treatment. A total of 44 of 523 (8.4%) women had isolated maternal hypothyroxinemia in pregnancy (free T₄ <10th centile and TSH ≤3 mIU/L). Only 2 of 44 (4.5%) had TSH >4.5 mIU/L outside pregnancy. Of the women with subclinical hypothyroidism in pregnancy with antibody measurements available, those with thyroid peroxidase antibodies in pregnancy were more likely to have persistently elevated TSH or be receiving l-thyroxine replacement after pregnancy (6 of 7 [86%] vs 10 of 57 [18%], P < .001)., Conclusions: The majority of cases of subclinical hypothyroidism in pregnancy are transient, so treatment with l-thyroxine in these patients should be reviewed because it may not be warranted after pregnancy.

Thyroperoxidase (TPO), the enzyme that catalyzes the synthesis of thyroid hormone, is a known target for thyroid-disrupting chemicals. In vivo toxicological evidence supporting TPO-inhibition as one molecular-initiating event that leads to thyroid disruption is derived largely from rat models; however, a significant fraction of research on the inhibition of TPO by xenobiotics has been conducted using porcine TPO. The current work tested the hypothesis that porcine and rat thyroid microsomes exposed to TPO-inhibiting chemicals would demonstrate different responses in a guaiacol oxidation assay. A primary objective of this work is to establish the degree of concordance between rat and porcine TPO inhibition data. Microsomes were isolated from both rat and pig thyroid glands, and the guaiacol oxidation assay was performed for a training set of 12 chemicals, including previously reported TPO inhibitors, thyroid-disrupting chemicals thought to perturb other targets, and several previously untested chemicals, to determine the relative TPO inhibition responses across species. Concentration-response curves were derived for methimazole (MMI), dibutylphthalate (DBP), diethylhexylphthalate (DEHP), diethylphthalate (DEP), 3,5-dimethylpyrazole-1-methanol (DPM), iopanoic acid (IOA), 2-mercaptobenzothiazole (MBT), sodium perchlorate (PERC), p-nonylphenol (PNP), 4-propoxyphenol (4POP), 6-propylthiouracil (PTU), and triclosan (TCS). MMI, PTU, MBT, DPM, 4POP, and at extremely high concentrations, PERC, inhibited TPO activity. Results demonstrated a strong qualitative concordance of response between the two species. All chemicals that inhibited TPO in porcine microsomes also inhibited TPO in rat microsomes. Hill model-derived IC50 values revealed approximate 1.5- to 50-fold differences in relative potency to MMI between species for positive chemicals. DPM, MBT, 4POP, and PTU exhibited greater relative potency to MMI using rat TPO versus porcine TPO, but rank order potency for inhibition was similar for the other test chemicals, with: PTU>MBT>DPM>4POP>PERC for rat TPO and MBT>PTU>DPM>4POP>PERC for porcine TPO. These data support the extrapolation of porcine TPO data to potential thyroid-disrupting activity in rodent models to evaluate TPO-inhibiting chemicals., (Published by Elsevier Ireland Ltd.)

Context: Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology, and several studies reported its association with thyroid autoimmune disorders. No study has evaluated longitudinally the incidence of new cases of thyroid autoimmunity and dysfunction in patients with SSc., Objective: The purpose of this study was to evaluate the incidence of new cases of clinical and subclinical thyroid dysfunction in a wide group of women with SSc vs an age- and sex-matched control group from the same geographic area. DESIGN AND PATIENTS OR OTHER PARTICIPANTS: After exclusion of sclerodermic patients with thyroid dysfunction (n = 55) at the initial evaluation, the appearance of new cases of thyroid disorders was evaluated in 179 patients and 179 matched control subjects, with similar iodine intake (median follow-up 73 months in patients with SSc vs 94 months in control subjects)., Results: A high incidence (P < .05) of new cases of hypothyroidism, thyroid dysfunction, anti-thyroperoxidase antibody positivity, and appearance of a hypoechoic thyroid pattern in sclerodermic patients (15.5, 21, 11, and 14.6 of 1000 patients per year; respectively) vs that in control subjects was shown. A logistic regression analysis showed that in patients with SSc, the appearance of hypothyroidism was related to a borderline high initial TSH level, anti-thyroperoxidase antibody positivity, and a hypoechoic and small thyroid., Conclusions: Our study shows a high incidence of new cases of hypothyroidism and thyroid dysfunction in female sclerodermic patients. Female sclerodermic patients, who are at high risk (a borderline high [even if in the normal range] TSH value, anti-thyroperoxidase antibody positivity, and a hypoechoic and small thyroid) should have periodic thyroid function follow-up.

Maternal thyroxine (T4) plays an essential role in fetal brain development, and even mild and transitory deficits in free-T4 in pregnant women can produce irreversible neurological effects in their offspring. Women of childbearing age are daily exposed to mixtures of chemicals disrupting the thyroid gland function (TGFDs) through the diet, drinking water, air and pharmaceuticals, which has raised the highest concern for the potential additive or synergic effects on the development of mild hypothyroxinemia during early pregnancy. Recently we demonstrated that zebrafish eleutheroembryos provide a suitable alternative model for screening chemicals impairing the thyroid hormone synthesis. The present study used the intrafollicular T4-content (IT4C) of zebrafish eleutheroembryos as integrative endpoint for testing the hypotheses that the effect of mixtures of TGFDs with a similar mode of action [inhibition of thyroid peroxidase (TPO)] was well predicted by a concentration addition concept (CA) model, whereas the response addition concept (RA) model predicted better the effect of dissimilarly acting binary mixtures of TGFDs [TPO-inhibitors and sodium-iodide symporter (NIS)-inhibitors]. However, CA model provided better prediction of joint effects than RA in five out of the six tested mixtures. The exception being the mixture MMI (TPO-inhibitor)-KClO4 (NIS-inhibitor) dosed at a fixed ratio of EC10 that provided similar CA and RA predictions and hence it was difficult to get any conclusive result. There results support the phenomenological similarity criterion stating that the concept of concentration addition could be extended to mixture constituents having common apical endpoints or common adverse outcomes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Thyroxine (T₄) needs to be converted to 3,5,3'-triiodothyronine (T₃) by iodothyronine deiodinase to exert its biological activity. Recent studies revealed the presence of type 2 iodothyronine deiodinase (D2) in human thyroid tissue, human skeletal muscle and other tissues, suggesting that D2 is involved in maintaining plasma T₃ level in human. Tumor necrosis factor α (TNFα) is an inflammatory cytokine of which production is elevated in patients with nonthyroidal illness. Although several lines of evidence suggest the causal role of TNFα in nonthyroidal illness, detailed nature of the effect of TNFα on D2 remains unclear. In the present study, we identified D2 activity and D2 mRNA in TCO-1 cells, which were derived from human anaplastic thyroid carcinoma, and studied the mechanisms involved in the regulation of D2 expression by TNFα. The characteristics of the deiodinating activity in TCO-1 cells were compatible with those of D2 and Northern analysis demonstrated that D2 mRNA was expressed in TCO-1cells. D2 activity and D2 mRNA expression were rapidly increased by dibutyryl cAMP ((Bu)₂cAMP). TNFα showed an inhibitory effect on (Bu)₂cAMP-stimulated D2 activity in spite of little effect on (Bu)₂cAMP-stimulated D2 mRNA expression. MG132, a proteasome inhibitor abolished TNFα suppression of D2 activity whereas BAY11-7082 or 6-amino-4-(4-phenoxyphenylethylamino) quinazoline, inhibitors of nuclear factor-κB (NF-κB) failed to attenuate the effect of TNFα on D2 activity. These data suggest that a posttranslational mechanism through proteasomal degradation but not NF-κB activation is involved in the suppression of D2 by TNFα.