Screening the ToxCast Chemical Libraries for Binding to Transthyretin.

Transthyretin (TTR) is one of the serum binding proteins responsible for transport of thyroid hormones (TH) to target tissue and for maintaining the balance of available TH. Chemical binding to TTR and subsequent displacement of TH has been identified as an end point in screening chemicals for potential disruption of the thyroid system. To address the lack of data regarding chemicals binding to TTR, we optimized an in vitro assay utilizing the fluorescent probe 8-anilino-1-napthalenesulfonic acid (ANSA) and the human protein TTR to screen over 1500 chemicals from the U.S. EPA's ToxCast ph1_v2, ph2, and e1k libraries utilizing a tiered approach. Testing of a single high concentration (target 100 µM) resulted in 888 chemicals with 20% or greater activity based on displacement of ANSA from TTR. Of these, 282 chemicals had activity of 85% or greater and were further tested in 12-point concentration-response with target concentrations ranging from 0.015 to 100 µM. An EC50 was obtained for 276 of these 301 chemicals. To date, this is the largest set of chemicals screened for binding to TTR. Utilization of this assay is a significant contribution toward expanding the suite of in vitro assays used to identify chemicals with the potential to disrupt thyroid hormone homeostasis.

In Vitro Screening for ToxCast Chemicals Binding to Thyroxine-Binding Globulin.

Thyroid hormone (TH) carrier proteins play an important role in distributing TH to target tissue as well as maintaining the balance of free versus bound TH in the blood. Interference with the TH carrier proteins has been identified as a potential mechanism of thyroid system disruption. To address the lack of data regarding chemicals binding to these carrier proteins and displacing TH, a fluorescence-based in vitro screening assay was utilized to screen over 1,400 chemicals from the U.S. EPA's ToxCast phase1_v2, phase 2, and e1k libraries for competitive binding to one of the carrier proteins, thyroxine-binding globulin. Initial screening at a single high concentration of 100 µM identified 714 chemicals that decreased signal of the bound fluorescent ligand by 20% or higher. Of these, 297 produced 50% or greater reduction in fluorescence and were further tested in concentration-response (0.004 to 150 µM) to determine relative potency. Ten chemicals were found to have EC50 values <1 µM, 63 < 10 µM, and 141 chemicals between 10 and 100 µM. Utilization of this assay contributes to expanding the number of in vitro assays available for identifying chemicals with the potential to disrupt TH homeostasis. These results support ranking and prioritization of chemicals to be tested in vivo to aid in the development of a framework for predicting in vivo effects from in vitro high-throughput data.

Evaluating potential developmental toxicity of perfluoroalkyl and polyfluoroalkyl substances in Xenopus laevis embryos and larvae.

As part of the US Environmental Protection Agency's perfluoroalkyl and polyfluoroalkyl substances (PFAS) Action Plan, the agency is committed to increasing our understanding of the potential ecological effects of PFAS. The objective of these studies was to examine the developmental toxicity of PFAS using the laboratory model amphibian species Xenopus laevis. We had two primary aims: (1) to understand the developmental toxicity of a structurally diverse set of PFAS compounds in developing embryos and (2) to characterize the potential impacts of perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), and hexafluoropropylene oxide-dimer acid (HFPO-DA a.k.a. GenX), on growth and thyroid hormone-controlled metamorphosis. We employed a combination of static renewal and flow-through exposure designs. Embryos were exposed to 17 structurally diverse PFAS starting at the midblastula stage through the completion of organogenesis (96 h). To investigate impacts on PFOS, PFOA, PFHxS, and HFPO-DA on development and metamorphosis, larvae were exposed from premetamorphosis (Nieuwkoop Faber stage 51 or 54) through pro metamorphosis. Of the PFAS tested in embryos, only 1H,1H,10H,10H-perfluorodecane-1,10-diol (FC10-diol) and perfluorohexanesulfonamide (FHxSA) exposure resulted in clear concentration-dependent developmental toxicity. For both of these PFAS, a significant increase in mortality was observed at 2.5 and 5 mg/L. For FC10-diol, 100% of the surviving embryos were malformed at 1.25 and 2.5 mg/L, while for FHxSA, a significant increase in malformations (100%) was observed at 2.5 and 5 mg/L. Developmental stage achieved was the most sensitive endpoint with significant effects observed at 1.25 and 0.625 mg/L for FC10-diol and FHxSA, respectively. In larval studies, we observed impacts on growth following exposure to PFHxS and PFOS at concentrations of 100 and 2.5 mg/L, respectively, while no impacts were observed in larvae when exposed to PFOA and HFPO-DA at concentration of 100 mg/L. Further, we did not observe impacts on thyroid endpoints in exposed larvae. These experiments have broadened our understanding of the impact of PFAS on anuran development.

Assessing utility of thyroid in vitro screening assays through comparisons to observed impacts in vivo.

To better understand endocrine disruption, the U.S. Environmental Protection Agency's (USEPA) Endocrine Disruptor Screening Program (EDSP) utilizes a two-tiered approach to investigate the potential of a chemical to interact with the estrogen, androgen, or thyroid systems. As in vivo testing lacks the throughput to address data gaps on endocrine bioactivity for thousands of chemicals, in vitro high-throughput screening (HTS) methods are being developed to screen larger chemical libraries. The primary objective of this work was to investigate for how many of the 52 chemicals with weight-of-evidence (WoE) determinations from EDSP Tier 1 screening there are available in vitro HTS data supporting a thyroid impact. HTS data from the USEPA ToxCast program and the EDSP WoE were collected for this analysis. Considering the complexity of endocrine disruption and interpreting HTS data, concordance between in vitro activity and in vivo effects ranges from 58 to 78%. Based on this evaluation, we conclude that the current suite of HTS assays is beneficial for prioritizing chemicals for further inquiry; however, without a more detailed analysis, one cannot conclude whether HTS results are the primary mode-of-action. Furthermore, development of in vitro assays for additional thyroid-relevant molecular initiating events is required to effectively predict in vivo thyroid impacts.

Metabolism of cyclic phenones in rainbow trout in vitro assays.

1. Cyclic phenones are chemicals of interest to the USEPA due to their potential for endocrine disruption to aquatic and terrestrial species.2. Prior to this report, there was very limited information addressing metabolism of cyclic phenones by fish species and the potential for estrogen receptor (ER) binding and vitellogenin (Vtg) gene activation by their metabolites.3. The main objectives of the current research were to characterize rainbow trout (rt) liver slice-mediated in vitro metabolism of model parent cyclic phenones exhibiting disparity between ER binding and ER-mediated Vtg gene induction, and to assess the metabolic competency of fish liver in vitro tests to help determine the chemical form (parent and/or metabolite) associated with the observed biological response.4. GC-MS, HPLC and LC-MS/MS technologies were applied to investigate the in vitro biotransformation of cyclobutyl phenyl ketone (CBP), benzophenone (DPK), cyclohexyl phenyl ketone (CPK) mostly in the absence of standards for metabolite characterization.5. It was concluded that estrogenic effects of the studied cyclic phenones are mediated by the parent chemical structure for DPK, but by active metabolites for CPK. A definitive interpretation was not possible for CBP and CBPOH (alcohol), although a contribution of both structures to gene induction is suspected.

Development of a thyroperoxidase inhibition assay for high-throughput screening.

High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein, we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR), were employed in an end-point assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics, including Z', dynamic range, and activity, using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and Z' score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2',4,4'-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negatives: 2-hydroxy-4-methoxybenzophenone, dibutylphthalate, diethylhexylphthalate, diethylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, and sodium perchlorate. This assay could be used to screen large numbers of chemicals as an integral component of a tiered TH-disruptor screening approach.

In vitro screening of per- and polyfluorinated substances (PFAS) for interference with seven thyroid hormone system targets across nine assays.

The US Environmental Protection Agency is evaluating the ecological and toxicological effects of per- and polyfluorinated chemicals. A number of perfluorinated chemicals have been shown to impact the thyroid axis in vivo suggesting that the thyroid hormone system is a target of these chemicals. The objective of this study was to evaluate the activity of 136 perfluorinated chemicals at seven key molecular initiating events (MIE) within the thyroid axis using nine in vitro assays. The potential MIE targets investigated are Human Iodothyronine Deiodinase 1 (hDIO1), Human Iodothyronine Deiodinase 2 (hDIO2), Human Iodothyronine Deiodinase 3 (hDIO3), Xenopus Iodothyronine Deiodinase (xDIO3); Human Iodotyrosine Deiodinase (hIYD), Xenopus Iodotyrosine Deiodinase (xIYD), Human Thyroid Peroxidase (hTPO); and the serum binding proteins Human Transthyretin (hTTR) and Human Thyroxine Binding Globulin (hTBG). Of the 136 PFAS chemicals tested, 85 had sufficient activity to produce a half-maximal effect concentration (EC50) in at least one of the nine assays. In general, most of these PFAS chemicals did not have strong potency towards the seven MIEs examined, apart from transthyretin binding, for which several PFAS had potency similar to the respective model inhibitor. These data sets identify potentially active PFAS chemicals to prioritize for further testing in orthogonal in vitro assays and at higher levels of biological organization to evaluate their capacity for altering the thyroid hormone system and causing potential adverse health and ecological effects.

A biologically based computational model for the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis larvae.

A biologically based computational model was developed to describe the hypothalamic-pituitary-thyroid (HPT) axis in developing Xenopus laevis larvae. The goal of this effort was to develop a tool that can be used to better understand mechanisms of thyroid hormone-mediated metamorphosis in X. laevis and predict organismal outcomes when those mechanisms are perturbed by chemical toxicants. In this report, we describe efforts to simulate the normal biology of control organisms. The structure of the model borrows from established models of HPT axis function in mammals. Additional features specific to X. laevis account for the effects of organism growth, growth of the thyroid gland, and developmental changes in regulation of thyroid stimulating hormone (TSH) by circulating thyroid hormones (THs). Calibration was achieved by simulating observed changes in stored and circulating levels of THs during a critical developmental window (Nieuwkoop and Faber stages 54-57) that encompasses widely used in vivo chemical testing protocols. The resulting model predicts that multiple homeostatic processes, operating in concert, can act to preserve circulating levels of THs despite profound impairments in TH synthesis. Represented in the model are several biochemical processes for which there are high-throughput in vitro chemical screening assays. By linking the HPT axis model to a toxicokinetic model of chemical uptake and distribution, it may be possible to use this in vitro effects information to predict chemical effects in X. laevis larvae resulting from defined chemical exposures.

Cross-species comparison of chemical inhibition of human and Xenopus iodotyrosine deiodinase.

The transition to include in vitro-based data in chemical hazard assessment has resulted in the development and implementation of screening assays to cover a diversity of biological pathways, including recently added assays to interrogate chemical disruption of proteins relevant to thyroid signaling pathways. Iodotyrosine deiodinase (IYD), the iodide recycling enzyme, is one such thyroid-relevant endpoint for which a human-based screening assay has recently been developed and used to screen large libraries of chemicals. Presented here is the development of an amphibian IYD inhibition assay and its implementation to conduct a cross-species comparison between chemical inhibition of mammalian and non-mammalian IYD enzyme activity. The successful development of an amphibian IYD inhibition assay was based on demonstration of sufficient IYD enzyme activity in several tissues collected from larval Xenopus laevis. With this new assay, 154 chemicals were tested in concentration-response to provide a basis for comparison of relative chemical potency to results obtained from the human IYD assay. Most chemicals exhibited similar inhibition in both assays, with less than 25% variation in median inhibition for 120 of 154 chemicals and 85% concordance in categorization of "active" (potential IYD inhibitor) versus "inactive". For chemicals that produced 50% or greater inhibition in both assays, rank-order potency was similar, with the majority of the IC50s varying by less than 2-fold (and all within an order of magnitude). Most differences resulted from greater maximum inhibition or higher chemical potency observed with human IYD. This strong cross-species agreement suggests that results from the human-based assay would be conservatively predictive of chemical effects on amphibian IYD.

Characterization of the Mechanistic Linkages Between Iodothyronine Deiodinase Inhibition and Impaired Thyroid-Mediated Growth and Development in Xenopus laevis Using Iopanoic Acid.

Iodothyronine deiodinases (DIO) are key enzymes that influence tissue-specific thyroid hormone levels during thyroid-mediated amphibian metamorphosis. Within the larger context of evaluating chemicals for thyroid system disrupting potential, chemical activity toward DIOs is being evaluated using high-throughput in vitro screening assays as part of U.S. EPA's ToxCast program. However, existing data gaps preclude any inferences between in vitro chemical inhibition of DIOs and in vivo outcomes relevant to ecological risk assessment. This study aimed to generate targeted data in a laboratory model species (Xenopus laevis) using a model DIO inhibitor, iopanoic acid (IOP), to characterize linkages between in vitro potency, in vivo biochemical responses, and adverse organismal outcomes. In vitro potency of IOP toward DIOs was evaluated using previously developed in vitro screening assays, which showed concentration-dependent inhibition of human DIO1 (IC50: 97 µM) and DIO2 (IC50: 231 µM) but did not inhibit human or X. laevis DIO3 under the assay conditions. In vivo exposure of larval X. laevis to 0, 2.6, 5.3, and 10.5 µM IOP caused thyroid-related biochemical profiles in the thyroid gland and plasma consistent with hyperthyroxinemia but resulted in delayed metamorphosis and significantly reduced growth in the highest 2 exposure concentrations. Independent evaluations of dio gene expression ontogeny, together with existing literature, supported interpretation of IOP-mediated effects resulting in a proposed adverse outcome pathway for DIO2 inhibition leading to altered amphibian metamorphosis. This study highlights the types of mechanistic data needed to move toward predicting in vivo outcomes of regulatory concern from in vitro bioactivity data.

Thyroid-stimulating hormone (TSH): measurement of intracellular, secreted, and circulating hormone in Xenopus laevis and Xenopus tropicalis.

Thyroid-stimulating hormone (TSH) is an important regulator of the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis. To evaluate the role of this hormone on developing tadpoles, immunologically-based Western blots and sandwich ELISAs were developed for measuring intracellular (within pituitaries), secreted (ex vivo pituitary culture), and circulating (serum) amounts. Despite the small size of the tadpoles, these methods were able to easily measure intracellular and secreted TSH, and circulating TSH was measurable in situations where high levels were induced. The method was validated after obtaining a highly purified and enriched TSH sample using anti-TSH-ß antibodies conjugated to magnetic beads. Subsequent mass-spectrometric analysis of the bands from SDS-PAGE and Western procedures identified the presence of amino acid sequences corresponding to TSH subunits. The purified sample was also used to prepare standard curves for quantitative analysis. The Western and ELISA methods had limits of detection in the low nanogram range. While the majority of the developmental work for these methods was done with X. laevis, the methods also detected TSH in Xenopus tropicalis. To our knowledge this is the first report of a specific detection method for TSH in these species, and the first to measure circulating TSH in amphibians. Examples of the utility of the methods include measuring a gradual increase in pituitary TSH at key stages of development, peaking at stages 58-62; the suppression of TSH secretion from cultured pituitaries in the presence of thyroid hormone (T4); and increases in serum TSH following thyroidectomy.

Xenopus laevis and human type 3 iodothyronine deiodinase enzyme cross-species sensitivity to inhibition by ToxCast chemicals.

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 IC50 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 IC50 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.

In vitro screening for chemical inhibition of the iodide recycling enzyme, iodotyrosine deiodinase.

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 IC50 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.

Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment.

Ecological risk assessors face increasing demands to assess more chemicals, with greater speed and accuracy, and to do so using fewer resources and experimental animals. New approaches in biological and computational sciences may be able to generate mechanistic information that could help in meeting these challenges. However, to use mechanistic data to support chemical assessments, there is a need for effective translation of this information into endpoints meaningful to ecological risk-effects on survival, development, and reproduction in individual organisms and, by extension, impacts on populations. Here we discuss a framework designed for this purpose, the adverse outcome pathway (AOP). An AOP is a conceptual construct that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome at a biological level of organization relevant to risk assessment. The practical utility of AOPs for ecological risk assessment of chemicals is illustrated using five case examples. The examples demonstrate how the AOP concept can focus toxicity testing in terms of species and endpoint selection, enhance across-chemical extrapolation, and support prediction of mixture effects. The examples also show how AOPs facilitate use of molecular or biochemical endpoints (sometimes referred to as biomarkers) for forecasting chemical impacts on individuals and populations. In the concluding sections of the paper, we discuss how AOPs can help to guide research that supports chemical risk assessments and advocate for the incorporation of this approach into a broader systems biology framework.

Targeted Pathway-based In Vivo Testing Using Thyroperoxidase Inhibition to Evaluate Plasma Thyroxine as a Surrogate Metric of Metamorphic Success in Model Amphibian Xenopus laevis.

Chemical safety evaluation is in the midst of a transition from traditional whole-animal toxicity testing to molecular pathway-based in vitro assays and in silico modeling. However, to facilitate the shift in reliance on apical effects for risk assessment to predictive surrogate metrics having characterized linkages to chemical mechanisms of action, targeted in vivo testing is necessary to establish these predictive relationships. In this study, we demonstrate a means to predict thyroid-related metamorphic success in the model amphibian Xenopus laevis using relevant biochemical measurements during early prometamorphosis. The adverse outcome pathway for thyroperoxidase inhibition leading to altered amphibian metamorphosis was used to inform a pathway-based in vivo study design that generated response-response relationships. These causal relationships were used to develop Bayesian probabilistic network models that mathematically determine conditional dependencies between biochemical nodes and support the predictive capability of the biochemical profiles. Plasma thyroxine concentrations were the most predictive of metamorphic success with improved predictivity when thyroid gland sodium-iodide symporter gene expression levels (a compensatory response) were used in conjunction with plasma thyroxine as an additional regressor. Although thyroid-mediated amphibian metamorphosis has been studied for decades, this is the first time a predictive relationship has been characterized between plasma thyroxine and metamorphic success. Linking these types of biochemical surrogate metrics to apical outcomes is vital to facilitate the transition to the new paradigm of chemical safety assessments.

Screening the ToxCast Phase 1, Phase 2, and e1k Chemical Libraries for Inhibitors of Iodothyronine Deiodinases.

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.

Phenone, Hydroxybenzophenone, and Branched Phenone ER Binding and Vitellogenin Agonism in Rainbow Trout In Vitro Models.

Phenones and hydroxy benzophenones are widely used as UV radiation filters, and in the manufacturing of insecticides and pharmaceuticals. Understanding the estrogenic potential these chemicals is of interest to the US Environmental Protection Agency and other international environmental organizations. The current study sequentially combined complementary in vitro rainbow trout estrogen receptor (rtER) binding and liver slice vitellogenin (Vtg) mRNA induction assays in the context of a defined ER-mediated adverse outcome pathway (AOP). Cyclic phenones, branched phenones, and hydroxybenzophenones bound to rtER with relative potency ranging from no affinity to high binding affinity of 0.11%, and many induced Vtg gene expression in rt liver slices. In addition, cyclohexylphenylketone which did not bind rtER binding in cytosol was biotransformed within liver tissue to a chemical that induced Vtg expression.

Evaluating Chemicals for Thyroid Disruption: Opportunities and Challenges with in Vitro Testing and Adverse Outcome Pathway Approaches.

BACKGROUND: Extensive clinical and experimental research documents the potential for chemical disruption of thyroid hormone (TH) signaling through multiple molecular targets. Perturbation of TH signaling can lead to abnormal brain development, cognitive impairments, and other adverse outcomes in humans and wildlife. To increase chemical safety screening efficiency and reduce vertebrate animal testing, in vitro assays that identify chemical interactions with molecular targets of the thyroid system have been developed and implemented. OBJECTIVES: We present an adverse outcome pathway (AOP) network to link data derived from in vitro assays that measure chemical interactions with thyroid molecular targets to downstream events and adverse outcomes traditionally derived from in vivo testing. We examine the role of new in vitro technologies, in the context of the AOP network, in facilitating consideration of several important regulatory and biological challenges in characterizing chemicals that exert effects through a thyroid mechanism. DISCUSSION: There is a substantial body of knowledge describing chemical effects on molecular and physiological regulation of TH signaling and associated adverse outcomes. Until recently, few alternative nonanimal assays were available to interrogate chemical effects on TH signaling. With the development of these new tools, screening large libraries of chemicals for interactions with molecular targets of the thyroid is now possible. Measuring early chemical interactions with targets in the thyroid pathway provides a means of linking adverse outcomes, which may be influenced by many biological processes, to a thyroid mechanism. However, the use of in vitro assays beyond chemical screening is complicated by continuing limits in our knowledge of TH signaling in important life stages and tissues, such as during fetal brain development. Nonetheless, the thyroid AOP network provides an ideal tool for defining causal linkages of a chemical exerting thyroid-dependent effects and identifying research needs to quantify these effects in support of regulatory decision making. https://doi.org/10.1289/EHP5297.

Evaluating Iodide Recycling Inhibition as a Novel Molecular Initiating Event for Thyroid Axis Disruption in Amphibians.

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.

Screening the ToxCast Phase 1 Chemical Library for Inhibition of Deiodinase Type 1 Activity.

Thyroid hormone (TH) homeostasis is dependent upon coordination of multiple key events including iodide uptake, hormone synthesis, metabolism, and elimination, to maintain proper TH signaling. Deiodinase enzymes catalyze iodide release from THs to interconvert THs between active and inactive forms, and are integral to hormone metabolism. The activity of deiodinases has been identified as an important endpoint to include in the context of screening chemicals for TH disruption. To begin to address the potential for chemicals to inhibit these enzymes an adenovirus expression system was used to produce human deiodinase type 1 (DIO1) enzyme, established robust assay parameters for nonradioactive determination of iodide release by the Sandell-Kolthoff method, and employed a 96-well plate format for screening chemical libraries. An initial set of 18 chemicals was used to establish the assay, along with the known DIO1 inhibitor 6-propylthiouracil as a positive control. An additional 292 unique chemicals from the EPA's ToxCast phase 1_v2 chemical library were screened. Chemicals were initially screened at a single high concentration of 200 µM to identify potential DIO1 inhibitors. There were 50 chemicals, or 17% of the TCp1_v2 chemicals tested, that produced >20% inhibition of DIO1 activity. Eighteen of these inhibited DIO1 activity >50% and were further tested in concentration-response mode to determine IC50s. This work presents an initial effort toward identifying chemicals with potential for affecting THs via inhibition of deiodinases and sets the foundation for further testing of large chemical libraries against DIO1 and the other deiodinase enzymes involved in TH function.