Distinct transcriptional profiles in rat thyroid glands after developmental exposure to three in vitro thyroperoxidase inhibiting chemicals.

Thyroperoxidase (TPO) is central in thyroid hormone (TH) synthesis and inhibition can lead to TH deficiency. Many chemicals can inhibit TPO activity in vitro, but how this may manifest in the developing thyroid gland at the molecular level is unclear. Here, we characterized the thyroid gland transcriptome of male rats developmentally exposed to the in vitro TPO-inhibitors amitrole, 2-mercaptobenzimidazole (MBI), or cyanamide by use of Bulk-RNA-Barcoding (BRB) and sequencing. Amitrole exposure caused TH deficiency and 149 differentially expressed genes in the thyroid gland. The effects indicated an activated and growing thyroid gland. MBI caused intermittent changes to serum TH concentrations in a previous study and this was accompanied by 60 differentially expressed genes in the present study. More than half of these were also affected by amitrole, indicating that they could be early effect biomarkers of developmental TH system disruption due to TPO inhibition. Further work to validate the signature is needed, including assessment of substance independency and applicability domain.

The thyroid hormone system disrupting potential of resorcinol in fish.

Environmental pollutants capable of interfering with the thyroid hormone (TH) system increasingly raise concern for both human and environmental health. Recently, resorcinol has received attention as a compound of concern due to its endocrine disrupting properties. It is a known inhibitor of thyroperoxidase (TPO), an enzyme required in TH synthesis, and therapeutic use of resorcinol exposure has led to hypothyroidism in humans. There is limited evidence concerning ecotoxicologically relevant effects of resorcinol in fish. A set of adverse outcome pathways (AOPs) has recently been developed linking thyroid hormone system disruption (THSD) to impaired swim bladder inflation and eye development in fish. In the present study, these AOPs were used to provide the background for testing potential THSD effects of resorcinol in zebrafish eleutheroembryos. We exposed zebrafish eleutheroembryos to resorcinol and assessed TH levels, swim bladder inflation and eye morphology. As a TPO inhibitor, resorcinol is expected to affect TH levels and eye morphology but not swim bladder inflation during embryonic development. Indeed, thyroxine (T4) levels were significantly decreased following resorcinol exposure. In contrast to our hypothesis, swim bladder inflation was impaired at 5 days post fertilization (dpf) and no effects on eye morphology were detected. Therefore, in vitro assays were performed to identify potential additional thyroid hormone system disruption-related mechanisms through which resorcinol may act. Two new mechanisms were identified: TH receptor (TR) antagonism and transthyretin (TTR) binding inhibition. Both of these mechanisms can plausibly be linked to impaired swim bladder inflation and could, therefore, explain the observed effect. Overall, our study contributes to the knowledge of the THSD potential of resorcinol both in vivo in the zebrafish model as well as in vitro.

Optimized methods for measuring competitive binding of chemical substances to thyroid hormone distributor proteins transthyretin and thyroxine binding globulin.

Transthyretin (TTR) and thyroxine-binding globulin (TBG) are two major thyroid hormone (TH) distributor proteins in human plasma, playing important roles in stabilizing the TH levels in plasma, delivery of TH to target tissues, and trans-barrier transport. Binding of xenobiotics to these distributor proteins can potentially affect all these three important roles of distributor proteins. Therefore, fast and cost-effective experimental methods are required for both TTR and TBG to screen both existing and new chemicals for their potential binding. In the present study, the TTR-binding assay was therefore simplified, optimized and pre-validated, while a new TBG-binding assay was developed based on fluorescence polarization as a readout. Seven model compounds (including positive and negative controls) were tested in the pre-validation study of the optimized TTR-binding assay and in the newly developed TBG-binding assay. The dissociation constants of the natural ligand (thyroxine, T4) and potential competitors were determined and compared between two distributor proteins, showing striking differences for perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA).

Development of the integrated fish endocrine disruptor test-Part B: Implementation of thyroid-related endpoints.

Given the vital role of thyroid hormones (THs) in vertebrate development, it is essential to identify chemicals that interfere with the TH system. Whereas, among nonmammalian laboratory animals, fish are the most frequently utilized test species in endocrine disruptor research, for example, in guidelines for the detection of effects on the sex hormone system, there is no test guideline (TG) using fish as models for thyroid-related effects; rather, amphibians are used. Therefore, the objective of the present project was to integrate thyroid-related endpoints for fish into a test protocol combining OECD TGs 229 (Fish Short-Term Reproduction Assay) and 234 (Fish Sexual Development Test). The resulting integrated Fish Endocrine Disruption Test (iFEDT) was designed as a comprehensive approach to covering sexual differentiation, early development, and reproduction and to identifying disruption not only of the sexual and/or reproductive system but also the TH system. Two 85-day exposure tests were performed using different well-studied endocrine disruptors: 6-propyl-2-thiouracil (PTU) and 17α-ethinylestradiol (EE2). Whereas the companion Part A of this study presents the findings on effects by PTU and EE2 on endpoints established in existing TGs, the present Part B discusses effects on novel thyroid-related endpoints such as TH levels, thyroid follicle histopathology, and eye development. 6-Propyl-2-thiouracil induced a massive proliferation of thyroid follicles in any life stage, and histopathological changes in the eyes proved to be highly sensitive for TH system disruption especially in younger life stages. For measurement of THs, further methodological development is required. 17-α-Ethinylestradiol demonstrated not only the well-known disruption of the hypothalamic-pituitary-gonadal axis, but also induced effects on thyroid follicles in adult zebrafish (Danio rerio) exposed to higher EE2 concentrations, suggesting crosstalk between endocrine axes. The novel iFEDT has thus proven capable of simultaneously capturing endocrine disruption of both the steroid and thyroid endocrine systems. Integr Environ Assess Manag 2024;20:830-845. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Deiodinase inhibition impairs the formation of the three posterior swim bladder tissue layers during early embryonic development in zebrafish.

Thyroid hormone system disruption (THSD) negatively affects multiple developmental processes and organs. In fish, inhibition of deiodinases, which are enzymes crucial for (in)activating thyroid hormones (THs), leads to impaired swim bladder inflation. Until now, the underlying mechanism has remained largely unknown. Therefore, the objective of this study was to identify the process during swim bladder development that is impacted by deiodinase inhibition. Zebrafish embryos were exposed to 6 mg/L iopanoic acid (IOP), a model deiodinase inhibitor, during 8 different exposure windows (0-60, 60-120, 24-48, 48-72, 72-96, 96-120, 72-120 and 0-120 h post fertilization (hpf)). Exposure windows were chosen based on the three stages of swim bladder development: budding (24-48 hpf), pre-inflation, i.e., the formation of the swim bladder tissue layers (48-72 hpf), and inflation phase (72-120 hpf). Exposures prior to 72 hpf, during either the budding or pre-inflation phase (or both), impaired swim bladder inflation, while exposure during the inflation phase did not. Based on our results, we hypothesize that DIO inhibition before 72 hpf leads to a local decrease in T3 levels in the developing swim bladder. Gene transcript analysis showed that these TH level alterations disturb both Wnt and hedgehog signaling, known to be essential for swim bladder formation, eventually resulting in impaired development of the swim bladder tissue layers. Improper development of the swim bladder impairs swim bladder inflation, leading to reduced swimming performance. This study demonstrates that deiodinase inhibition impacts processes underlying the formation of the swim bladder and not the inflation process, suggesting that these processes primarily rely on maternal rather than endogenously synthetized THs since TH measurements showed that THs were not endogenously synthetized during the sensitive period.

Cross-species applicability of an adverse outcome pathway network for thyroid hormone system disruption.

Thyroid hormone system disrupting compounds are considered potential threats for human and environmental health. Multiple adverse outcome pathways (AOPs) for thyroid hormone system disruption (THSD) are being developed in different taxa. Combining these AOPs results in a cross-species AOP network for THSD which may provide an evidence-based foundation for extrapolating THSD data across vertebrate species and bridging the gap between human and environmental health. This review aimed to advance the description of the taxonomic domain of applicability (tDOA) in the network to improve its utility for cross-species extrapolation. We focused on the molecular initiating events (MIEs) and adverse outcomes (AOs) and evaluated both their plausible domain of applicability (taxa they are likely applicable to) and empirical domain of applicability (where evidence for applicability to various taxa exists) in a THSD context. The evaluation showed that all MIEs in the AOP network are applicable to mammals. With some exceptions, there was evidence of structural conservation across vertebrate taxa and especially for fish and amphibians, and to a lesser extent for birds, empirical evidence was found. Current evidence supports the applicability of impaired neurodevelopment, neurosensory development (eg, vision) and reproduction across vertebrate taxa. The results of this tDOA evaluation are summarized in a conceptual AOP network that helps prioritize (parts of) AOPs for a more detailed evaluation. In conclusion, this review advances the tDOA description of an existing THSD AOP network and serves as a catalog summarizing plausible and empirical evidence on which future cross-species AOP development and tDOA assessment could build.

Selenium, Iodine and Iron-Essential Trace Elements for Thyroid Hormone Synthesis and Metabolism.

The adequate availability and metabolism of three essential trace elements, iodine, selenium and iron, provide the basic requirements for the function and action of the thyroid hormone system in humans, vertebrate animals and their evolutionary precursors. Selenocysteine-containing proteins convey both cellular protection along with H2O2-dependent biosynthesis and the deiodinase-mediated (in-)activation of thyroid hormones, which is critical for their receptor-mediated mechanism of cellular action. Disbalances between the thyroidal content of these elements challenge the negative feedback regulation of the hypothalamus-pituitary-thyroid periphery axis, causing or facilitating common diseases related to disturbed thyroid hormone status such as autoimmune thyroid disease and metabolic disorders. Iodide is accumulated by the sodium-iodide-symporter NIS, and oxidized and incorporated into thyroglobulin by the hemoprotein thyroperoxidase, which requires local H2O2 as cofactor. The latter is generated by the dual oxidase system organized as 'thyroxisome' at the surface of the apical membrane facing the colloidal lumen of the thyroid follicles. Various selenoproteins expressed in thyrocytes defend the follicular structure and function against life-long exposure to H2O2 and reactive oxygen species derived therefrom. The pituitary hormone thyrotropin (TSH) stimulates all processes required for thyroid hormone synthesis and secretion and regulates thyrocyte growth, differentiation and function. Worldwide deficiencies of nutritional iodine, selenium and iron supply and the resulting endemic diseases are preventable with educational, societal and political measures.

Amphibian toxicity testing for identification of thyroid disrupting chemicals.

Both amphibian metamorphosis assay (AMA) and larval amphibian growth and development assay (LAGDA) can detect thyroid-mediated modality and adversity on the basis of morphological changes during the thyroid hormone-dependent metamorphosis. They are used for identification of thyroid hormone system disrupting chemicals (TDCs) for non-target organisms or the environment. The EU Guidance recommends that the AMA and the LAGDA should be used to address sufficient investigation of the thyroid-mediated modality and adversity, respectively. In the EU discussions over identification of TDCs, the necessity of using LAGDA as a follow-up of positive results of the AMA has been questioned because of the overlap between the endpoints and the exposure of both tests. This study analyzed similarities, differences, and sensitivity of these two assays in detection of TDCs. For agonists and most of antagonists of the hypothalamic-pituitary-thyroid (HPT) axis, both AMA and LAGDA can detect the thyroid-mediated modality and adversity. The LAGDA, as a follow-up of the positive results of the AMA, may not be needed because the results of AMA are considered enough for identification of TDCs. For chemicals like inhibitors of iodotyrosine deiodinase, the LAGDA is considered necessary for identification of TDCs because the thyroid-mediated adversity cannot be detected until Nieuwkoop and Faber (NF) stage 62. Incorporation of mechanistic endpoints into existing test guidelines and the use of Xenopus Eleutheroembryo Thyroid Assay (XETA), extended amphibian metamorphosis assay (EAMA) and adverse outcome pathways (AOPs) for testing and identification of TDCs are further discussed.

Tentative Application of a Streamlined Protocol to Determine Organ-Specific Regulations of Deiodinase 1 and Dehalogenase Activities as Readouts of the Hypothalamus-Pituitary-Thyroid-Periphery-Axis.

In animal studies, both in basic science and in toxicological assessment of potential endocrine disruptors, the state of the thyroid hormone (TH) axis is often described and defined exclusively by the concentrations of circulating THs and TSH. Although it is known that the local, organ-specific effects of THs are also substantially regulated by local mechanisms such as TH transmembrane transport and metabolism of TH by deiodinases, such endpoint parameters of the axis are rarely assessed in these experiments. Currently developed in vitro assays utilize the Sandell-Kolthoff reaction, a photometric method of iodide determination, to test the effect of chemicals on iodotyrosine and iodothyronine deiodinases. Furthermore, this technology offers the possibility to determine the iodine content of various sample types (e.g., urine, ex vivo tissue) in a simple way. Here, we measured deiodinase type 1 and iodotyrosine dehalogenase activity by means of the Sandell-Kolthoff reaction in ex vivo samples of hypo- and hyperthyroid mice of two age groups (young; 3 months and old; 20 months). In thyroid, liver and kidney, organ-specific regulation patterns emerged across both age groups, which, based on this pilot study, may serve as a starting point for a deeper characterization of the TH system in relevant studies in the future and support the development of Integrated Approach for Testing and Assessment (IATA).

Thyroid hormone system disrupting chemicals.

The thyroid hormone system is a main target of endocrine disruptor compounds (EDC) at all levels of its intricately fine-tuned feedback regulation, synthesis, distribution, metabolism and action of the 'prohormone' thyroxine and its active metabolites. Apart from classical antithyroid effects of EDC on the gland, the majority of known and suspected effects occurs at the pre-receptor control of T3 ligand availability to T3 receptors exerting ligand modulated thyroid hormone action. Tissue-, organ- and cell-specific expression and function of thyroid hormone transporters, deiodinases, metabolizing enzymes and T3-receptor forms, all integral components of the system, may mediate adverse EDC effects. Established evidence from nutritional, pharmacological and molecular genetic studies clearly support the functional, biological, and clinical relevance of these targets. Iodine-containing thyroid hormones and the organization of this system are highly conserved during evolution from primitive aquatic life forms, amphibia, birds throughout all vertebrates including humans. Mechanistic studies from various animal experimental models strongly support cause-effect relationships upon EDC exposure, hazards and adverse effects of EDC across various species. Retrospective case-control, cohort and population studies linking EDC exposure with epidemiological data on thyroid hormone-related (dys-)functions provide clear evidence that human development, especially of the fetal and neonatal brain, growth, differentiation and metabolic processes in adult and aging humans are at risk for adverse EDC effects. Considering that more than half of the world population still lives on inadequate iodine supply, the additional ubiquitous exposure to EDC and their mixtures is an additional threat for the essential thyroid hormone system, the health of the human population and their future progenies, animal life forms and our global environment.

Effects of the tributyltin on the blood parameters, immune responses and thyroid hormone system in zebrafish.

Tributyltin (TBT) is a widely used organotin compound around the world and was frequently detected in surface waters, which would pose risk to aquatic organisms. However, the mechanisms of TBT-induced toxicity is not full clear. The present study investigated the effects of the tributyltin (TBT) on the blood parameters, immune responses and thyroid hormone system in zebrafish. Fish were exposed to sublethal concentrations of TBT (10 ng/L, 100 ng/L and 300 ng/L) for 6 weeks. The effects of long-term exposure to TBT on blood parameters (NH3, ammonia; GLU, glucose; TP, total proteins; CK, creatine kinase; ALT, alanine aminotransferase; AST, aspartate aminotransferase), immune responses (Lys, lysozyme; IgM, immunoglobulin M) and some indexes related thyroid hormone system (T3, 3,5,3'-triiodothyronine; T4, thyroxine) were measured in zebrafish, as well as the expression of genes related to immune responses and thyroid hormone system. Based on the results, the physiological-biochemical responses was significantly enhanced with an increase in TBT concentration, reflected by the abnormal blood indices, dysregulation of endocrine system and immunotoxicity in zebrafish under TBT stress. The present study greatly extends our understanding of adverse effects of TBT on aquatic organisms.

Removing Critical Gaps in Chemical Test Methods by Developing New Assays for the Identification of Thyroid Hormone System-Disrupting Chemicals-The ATHENA Project.

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.

Effects of isoflavones on breast tissue and the thyroid hormone system in humans: a comprehensive safety evaluation.

Isoflavones are secondary plant constituents of certain foods and feeds such as soy, linseeds, and red clover. Furthermore, isoflavone-containing preparations are marketed as food supplements and so-called dietary food for special medical purposes to alleviate health complaints of peri- and postmenopausal women. Based on the bioactivity of isoflavones, especially their hormonal properties, there is an ongoing discussion regarding their potential adverse effects on human health. This review evaluates and summarises the evidence from interventional and observational studies addressing potential unintended effects of isoflavones on the female breast in healthy women as well as in breast cancer patients and on the thyroid hormone system. In addition, evidence from animal and in vitro studies considered relevant in this context was taken into account along with their strengths and limitations. Key factors influencing the biological effects of isoflavones, e.g., bioavailability, plasma and tissue concentrations, metabolism, temporality (pre- vs. postmenopausal women), and duration of isoflavone exposure, were also addressed. Final conclusions on the safety of isoflavones are guided by the aim of precautionary consumer protection.