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.

Evidence for Cross Species Extrapolation of Mammalian-Based High-Throughput Screening Assay Results.

High-throughput screening (HTS) and computational technologies have emerged as important tools for chemical hazard identification. The US Environmental Protection Agency (EPA) launched the Toxicity ForeCaster (ToxCast) Program, which has screened thousands of chemicals in hundreds of mammalian-based HTS assays for biological activity. The data are being used to prioritize toxicity testing on those chemicals likely to lead to adverse effects. To use HTS assays in predicting hazard to both humans and wildlife, it is necessary to understand how broadly these data may be extrapolated across species. The US EPA Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS; https://seqapass.epa.gov/seqapass/ ) tool was used to assess conservation of the 484 protein targets represented in the suite of ToxCast assays and other HTS assays. To demonstrate the utility of the SeqAPASS data for guiding extrapolation, case studies were developed which focused on targets of interest to the US Endocrine Disruptor Screening Program and the Organisation for Economic Cooperation and Development. These case studies provide a line of evidence for conservation of endocrine targets across vertebrate species, with few exceptions, and demonstrate the utility of SeqAPASS for defining the taxonomic domain of applicability for HTS results and identifying organisms for suitable follow-up toxicity tests.

Coexistence of Y, W, and Z sex chromosomes in Xenopus tropicalis.

Homomorphic sex chromosomes and rapid turnover of sex-determining genes can complicate establishing the sex chromosome system operating in a given species. This difficulty exists in Xenopus tropicalis, an anuran quickly becoming a relevant model for genetic, genomic, biochemical, and ecotoxicological research. Despite the recent interest attracted by this species, little is known about its sex chromosome system. Direct evidence that females are the heterogametic sex, as in the related species Xenopus laevis, has yet to be presented. Furthermore, X. laevis' sex-determining gene, DM-W, does not exist in X. tropicalis, and the sex chromosomes in the two species are not homologous. Here we identify X. tropicalis' sex chromosome system by integrating data from (i) breeding sex-reversed individuals, (ii) gynogenesis, (iii) triploids, and (iv) crosses among several strains. Our results indicate that at least three different types of sex chromosomes exist: Y, W, and Z, observed in YZ, YW, and ZZ males and in ZW and WW females. Because some combinations of parental sex chromosomes produce unisex offspring and other distorted sex ratios, understanding the sex-determination systems in X. tropicalis is critical for developing this flexible animal model for genetics and ecotoxicology.

Development of the Larval Amphibian Growth and Development Assay: effects of chronic 4-tert-octylphenol or 17ß-trenbolone exposure in Xenopus laevis from embryo to juvenile.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized test guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of the Environment. The LAGDA was designed to evaluate apical effects of chronic chemical exposure on growth, thyroid-mediated amphibian metamorphosis and reproductive development. During the validation phase, two well-characterized endocrine-disrupting chemicals were tested to evaluate the performance of the initial assay design: xenoestrogen 4-tert-octylphenol (tOP) and xenoandrogen 17ß-trenbolone (TB). Xenopus laevis embryos were exposed, in flow-through conditions, to tOP (nominal concentrations: 0.0, 6.25, 12.5, 25 and 50 µg l-1 ) or TB (nominal concentrations: 0.0, 12.5, 25, 50 and 100 ng l-1 ) until 8 weeks post-metamorphosis, at which time growth measurements were taken, and histopathology assessments were made of the gonads, reproductive ducts, liver and kidneys. There were no effects on growth in either study and no signs of overt toxicity, sex reversal or gonad dysgenesis. Exposure to tOP caused a treatment-related decrease in circulating thyroxine and an increase in thyroid follicular cell hypertrophy and hyperplasia (25 and 50 µg l-1 ) during metamorphosis. Müllerian duct development was affected after exposure to both chemicals; tOP exposure caused dose-dependent maturation of oviducts in both male and female frogs, whereas TB exposure caused accelerated Müllerian duct regression in males and complete regression in >50% of the females in the 100 ng l-1 treatment. Based on these results, the LAGDA performed adequately to evaluate apical effects of chronic exposure to two endocrine-active compounds and is the first standardized amphibian multiple life stage toxicity test to date. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Development of the Larval Amphibian Growth and Development Assay: Effects of benzophenone-2 exposure in Xenopus laevis from embryo to juvenile.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of Environment to support risk assessment. The assay is employed as a higher tiered approach to evaluate effects of chronic chemical exposure throughout multiple life stages in a model amphibian species, Xenopus laevis. To evaluate the utility of the initial LAGDA design, the assay was performed using a mixed mode of action endocrine disrupting chemical, benzophenone-2 (BP-2). X. laevis embryos were exposed in flow-through conditions to 0, 1.5, 3.0 or 6.0 mg l-1 BP-2 until 2 months post-metamorphosis. Overt toxicity was evident throughout the exposure period in the 6.0 mg l-1 treatment due to elevated mortality rates and observed liver and kidney pathologies. Concentration-dependent increases in severity of thyroid follicular cell hypertrophy and hyperplasia occurred in larval tadpoles indicating BP-2-induced impacts on the thyroid axis. Additionally, gonads were impacted in all treatments with some genetic males showing both testis and ovary tissues (1.5 mg l-1 ) and 100% of the genetic males in the 3.0 and 6.0 mg l-1 treatments experiencing complete male-to-female sex reversal. Concentration-dependent vitellogenin induction occurred in both genders with associated accumulations of protein in the livers, kidneys and gonads, which was likely vitellogenin and other estrogen-responsive yolk proteins. This is the first study that demonstrates the endocrine effects of this mixed mode of action chemical in an amphibian species and demonstrates the utility of the LAGDA design for supporting chemical risk assessment. Copyright © 2016 John Wiley & Sons, Ltd.

Global gene expression during early differentiation of Xenopus (Silurana) tropicalis gonad tissues.

African clawed frog Xenopus sp. is used extensively for developmental biology and toxicology research. Amid concerns of environmental pollutants disrupting endocrine systems and causing altered reproductive development in wildlife, eco-toxicology research has led to a focus on linking molecular initiating events to population-level effects. As such, efforts to better understand reproductive development at the molecular level in these model species are warranted. To that end, transcriptomes were characterized in differentiating Xenopus tropicalis gonad tissues at Nieuwkoop and Faber (NF) stage 58 (pro-metamorphosis), NF66 (completion of metamorphosis), 1week post-metamorphosis (1WPM), and 2weeks post-metamorphosis (2WPM). Differential expression analysis between tissue types at each developmental stage revealed a substantial divergence of ovary and testis transcriptomes starting between NF58 and NF66; transcriptomes continued to diverge through 2WPM. Generally, testis-enriched transcripts were expressed at relatively constant levels, while ovary-enriched transcripts were up-regulated within this developmental period. Functional analyses of differentially expressed transcripts allowed linkages to be made between their putative human orthologues and specific cellular processes associated with differentiating gonad tissues. In ovary tissue, genetic programs direct germ cells through meiosis to the diplotene stage when maternal mRNAs are transcribed and trafficked to oocytes for translation following fertilization. In the testis, gene expression is consistent with connective tissue development, tubule formation, and germ cell support (Leydig and Sertoli cells). This dataset exhibited remarkable consistency with transcript profiles previously described in gonad tissues across species, and emphasizes the universal importance of certain transcripts for germ cell development and preparation of these tissues for reproduction.

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.

A method for the determination of genetic sex in the fathead minnow, Pimephales promelas, to support testing of endocrine-active chemicals.

Certain endocrine-active toxicants have been reported to completely sex reverse both male and female individuals in amphibian, avian, fish, invertebrate, and reptile species, resulting in a phenotype indistinguishable from unaffected individuals. Detection of low-level sex reversal often requires large numbers of organisms to achieve the necessary statistical power, especially in those species with predominantly genetic sex determination and cryptic/homomorphic sex chromosomes. Here we describe a method for determining the genetic sex in the commonly used ecotoxicological model, the fathead minnow (Pimephales promelas). Analysis of amplified fragment length polymorphisms (AFLP) in a spawn of minnows resulted in detection of 10 sex-linked AFLPs, which were isolated and sequenced. No recombination events were observed with any sex-linked AFLP in the animals examined (n=112). A polymerase chain reaction (PCR) method was then developed that determined the presence of one of these sex-linked polymorphisms for utilization in routine toxicological testing. Analyses of additional spawns from our in-house culture indicate that fathead minnows utilize a XY sex determination strategy and confirm that these markers can be used to genotype sex; however, this method is currently limited to use in laboratory studies in which breeders possess a defined genetic makeup. The genotyping method described herein can be incorporated into endocrine toxicity assays that examine the effects of chemicals on gonad differentiation.

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.

Control of pituitary thyroid-stimulating hormone synthesis and secretion by thyroid hormones during Xenopus metamorphosis.

We used ex vivo and in vivo experiments with Xenopus laevis tadpoles to examine the hypothesis that the set-point for negative feedback on pituitary thyroid-stimulating hormone (TSH) synthesis and secretion by thyroid hormones (THs) increases as metamorphosis progresses to allow for the previously documented concomitant increase in serum TH concentrations and pituitary TSH mRNA expression during this transformative process. First, pituitaries from climactic tadpoles were cultured for up to 96 h to characterize the ability of pituitary explants to synthesize and secrete TSHß in the absence of hypothalamic and circulating hormones. Next, pituitary explants from tadpoles NF stages 54-66 were exposed to physiologically-relevant concentrations of THs to determine whether stage-specific differences exist in pituitary sensitivity to negative feedback by THs. Finally, in vivo exposures of tadpoles to THs were conducted to confirm the results of the ex vivo experiments. When pituitaries from climactic tadpoles were removed from the influence of endogenous hormones, TSHß mRNA expression increased late or not at all whereas the rate of TSHß secreted into media increased dramatically, suggesting that TSH secretion, but not TSH mRNA expression, is under the negative regulation of an endogenous signal during the climactic stages of metamorphosis. Pituitaries from pre- and prometamorphic tadpoles were more sensitive to TH-induced inhibition of TSHß mRNA expression and secretion than pituitaries from climactic tadpoles. The observed decrease in sensitivity of pituitary TSHß mRNA expression to negative feedback by THs from premetamorphosis to metamorphic climax was confirmed by in vivo experiments in which tadpoles were reared in water containing THs. Based on the results of this study, a model is proposed to explain the seemingly paradoxical, concurrent rise in serum TH concentrations and pituitary TSH mRNA expression during metamorphosis in larval anurans.

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.

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.

Characterization of thyroid hormone transporter expression during tissue-specific metamorphic events in Xenopus tropicalis.

Thyroid hormone (TH) induces the dramatic morphological and physiological changes that together comprise amphibian metamorphosis. TH-responsive tissues vary widely with developmental timing of TH-induced changes. How larval tadpole tissues are able to employ distinct metamorphic programs in a developmental stage- and TH-dependent manner is still unknown. Recently, several proteins capable of transporting TH have been identified. TH action and metabolism occurs primarily intracellularly, highlighting the importance of TH transporters. We examined the hypothesis that TH transporter expression and tissue distribution play an important role in mediating TH-induced metamorphic events. Xenopus tropicalis homologs for known TH transporting OATP, MCT and LAT family proteins were identified and gene specific qRT-PCR primers were developed. Total RNA was extracted from tissues representing three unique developmental fates including: growth/differentiation (hind limb), death/resorption (gill, tail) and remodeling (brain, liver, kidney). For growing and resorbing tissues, results showed the general trend of low initial expression levels of MCT8 and MCT10 transporters, followed by a several-fold increase of expression as the tissue undergoes TH-dependent metamorphic changes. The expression pattern in remodeling tissues was less uniform: a general decrease in transporter expression was observed in the liver, while the kidney and brain exhibited a range of expression patterns for several TH transporters. Collectively, these developmental expression patterns are consistent with TH transporting proteins playing a role in the effects of TH in peripheral tissues.

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.

Reproductive maturation of the tropical clawed frog: Xenopus tropicalis.

The tropical clawed frog, Xenopus tropicalis, is a relatively new model species being used in developmental biology and amphibian toxicology studies. In order to increase our understanding of reproductive maturation and the role of steroid hormones in X. tropicalis, we collected baseline reproductive data in this species from metamorphosis to adulthood. One cohort of frogs was maintained for 42 weeks post-metamorphosis (PM) with endpoints representative of important reproductive parameters collected at 1- or 2-week intervals. These endpoints were then correlated to titers of either estradiol or testosterone. Male frogs exhibited nuptial pads, starting at 8 weeks (PM) when measureable concentrations of circulating testosterone (5.3 ng/mL plasma) first appeared. Testosterone concentrations remained above this level at all later time points, but were highly variable among individuals. Testes sizes in males reached their peak at 22 weeks PM (21 mg) with sperm counts peaking at the same time (25 million sperm/male). In females, estradiol becomes elevated in the blood at 16 weeks PM (1.5 ng/mL plasma) which corresponds with the presences of vitellogenin (4.4 mg/mL plasma), vitellogenic oocytes in the ovary, ovarian growth, and oviduct growth. Vitellogenic oocytes increased in number up to 15,000 per female at 30 weeks PM and accounted for 75% of the total number of oocytes present in the ovary. The ovary and oviducts continued to grow in mass until 30 weeks PM at which point they had reached sizes of 3.6g and 0.8 g, respectively. These data indicate that male and female X. tropicalis reach reproductive maturation at 22 and 30 weeks PM, respectively. Results from this study are valuable for the design of amphibian toxicology assays and increase our understanding of the reproductive biology of this relatively new model species.

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.