How the Xenopus eleutheroembryonic thyroid assay compares to the amphibian metamorphosis assay for detecting thyroid active chemicals.

The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently published as an OECD Test Guideline for detecting chemicals acting on the thyroid axis. However, the OECD validation did not cover all mechanisms that can potentially be detected by the XETA. This study was therefore initiated to investigate and consolidate the applicability domain of the XETA regarding the following mechanisms: thyroid hormone receptor (THR) agonism, sodium-iodide symporter (NIS) inhibition, thyroperoxidase (TPO) inhibition, deiodinase (DIO) inhibition, glucocorticoid receptor (GR) agonism, and uridine 5'-diphospho-glucuronosyltransferase (UDPGT) induction. In total, 22 chemicals identified as thyroid-active or -inactive in Amphibian Metamorphosis Assays (AMAs) were tested using the XETA OECD Test Guideline. The comparison showed that both assays are highly concordant in identifying chemicals with mechanisms of action related to THR agonism, DIO inhibition, and GR agonism. They also consistently identified the UDPGT inducers as thyroid inactive. NIS inhibition, investigated using sodium perchlorate, was not detected in the XETA. TPO inhibition requires further mechanistic investigations as the reference chemicals tested resulted in opposing response directions in the XETA and AMA. This study contributes refining the applicability domain of the XETA, thereby helping to clarify the conditions where it can be used as an ethical alternative to the AMA.

Overlapping action of T3 and T4 during Xenopus laevis development.

Thyroid hormones are involved in many biological processes such as neurogenesis, metabolism, and development. However, compounds called endocrine disruptors can alter thyroid hormone signaling and induce unwanted effects on human and ecosystems health. Regulatory tests have been developed to detect these compounds but need to be significantly improved by proposing novel endpoints and key events. The Xenopus Eleutheroembryonic Thyroid Assay (XETA, OECD test guideline no. 248) is one such test. It is based on Xenopus laevis tadpoles, a particularly sensitive model system for studying the physiology and disruption of thyroid hormone signaling: amphibian metamorphosis is a spectacular (thus easy to monitor) life cycle transition governed by thyroid hormones. With a long-term objective of providing novel molecular markers under XETA settings, we propose first to describe the differential effects of thyroid hormones on gene expression, which, surprisingly, are not known. After thyroid hormones exposure (T3 or T4), whole tadpole RNAs were subjected to transcriptomic analysis. By using standard approaches coupled to system biology, we found similar effects of the two thyroid hormones. They impact the cell cycle and promote the expression of genes involves in cell proliferation. At the level of the whole tadpole, the immune system is also a prime target of thyroid hormone action.

A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka.

Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T3) and thyroxine (T4)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T3, T4, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 µg/L, 1 µg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.

Evaluation of the endocrine activity of surface water samples using aquatic eleuthero-embryos-A comparison with in vitro assays.

Over the previous decade, numerous new approach methodologies (NAMs) have been developed and validated for the detection of endocrine activity of individual chemicals or environmental samples. These NAMs can be largely separated into three categories, in silico tools, in vitro assays, and in vivo assays using organisms or life stages not considered as laboratory animals, each with their own advantages and disadvantages. While in vitro assays provide more mechanistic information, the use of whole organisms such as fish or amphibian embryos provides a more holistic view of the net effects of an environmental sample on hormonal activity. A panel of bioassays was used to test the endocrine activity of several samples from the Danube River at Novi Sad, Serbia. The results of the in vitro assays have been published previously. Here, we present the results of the in vivo assays that were performed at the same time on the same samples. These whole organism assays are based on the use of transgenic fish and amphibian eleuthero-embryos and included the Xenopus Eleuthero-embryo Thyroid Assay (XETA), the Rapid Estrogen ACTivity In Vivo assay (REACTIV), and the Rapid Androgen Disruption Activity Reporter (RADAR) assay. Discrepancies between the different in vitro assays have previously been reported. The results of the in vivo studies also indicate discrepancies between the in vivo and in vitro data with an underestimation of the endocrine activity by the in vitro tests. Therefore, a battery of tests is advised with the initial diagnostic performed with in vivo tests to cover a wider range of modes of action and to allow the appropriate in vitro assay(s) to be selected to confirm the mode of action. PRACTITIONER POINTS: Endocrine activity was quantified in surface water using in vitro and in vivo models. The in vivo results fit with previously reported in vitro results. Higher activity was observed in water samples with in vivo models, which cover a wider range of modes of action. Endocrine activity of surface water samples may be underestimated when measured with in vitro models.

Electrochemical degradation of 17α-ethinylestradiol: Transformation products, degradation pathways and in vivo assessment of estrogenic activity.

Conventional water treatment methods are not efficient in eliminating endocrine disrupting compounds (EDCs) in wastewater. Electrochemical Advanced Oxidation Processes (eAOPs) offer a promising alternative, as they electro-generate highly reactive species that oxidize EDCs. However, these processes produce a wide spectrum of transformation products (TPs) with unknown chemical and biological properties. Therefore, a comprehensive chemical and biological evaluation of these remediation technologies is necessary before they can be safely applied in real-life situations. In this study, 17α-ethinylestradiol (EE2), a persistent estrogen, was electrochemically degraded using a boron doped diamond anode with sodium sulfate (Na2SO4) and sodium chloride (NaCl) as supporting electrolytes. Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was used for the quantification of EE2 and the identification of TPs. Estrogenic activity was assessed using a transgenic medaka fish line. At optimal operating conditions, EE2 removal reached over 99.9% after 120 min and 2 min, using Na2SO4 and NaCl, respectively. The combined EE2 quantification and in vivo estrogenic assessment demonstrated the overall estrogenic activity was consistently reduced with the degradation of EE2, but not completely eradicated. The identification and time monitoring of TPs showed that the radical agents readily oxidized the phenolic A-ring of EE2, leading to the generation of hydroxylated and/or halogenated TPs and ring-opening products. eAOP revealed to be a promising technique for the removal of EE2 from water. However, caution should be exercised with respect to the generation of potentially toxic TPs.

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.

Effect-based trigger values for in vitro and in vivo bioassays performed on surface water extracts supporting the environmental quality standards (EQS) of the European Water Framework Directive.

Effect-based methods including cell-based bioassays, reporter gene assays and whole-organism assays have been applied for decades in water quality monitoring and testing of enriched solid-phase extracts. There is no common EU-wide agreement on what level of bioassay response in water extracts is acceptable. At present, bioassay results are only benchmarked against each other but not against a consented measure of chemical water quality. The EU environmental quality standards (EQS) differentiate between acceptable and unacceptable surface water concentrations for individual chemicals but cannot capture the thousands of chemicals in water and their biological action as mixtures. We developed a method that reads across from existing EQS and includes additional mixture considerations with the goal that the derived effect-based trigger values (EBT) indicate acceptable risk for complex mixtures as they occur in surface water. Advantages and limitations of various approaches to read across from EQS are discussed and distilled to an algorithm that translates EQS into their corresponding bioanalytical equivalent concentrations (BEQ). The proposed EBT derivation method was applied to 48 in vitro bioassays with 32 of them having sufficient information to yield preliminary EBTs. To assess the practicability and robustness of the proposed approach, we compared the tentative EBTs with observed environmental effects. The proposed method only gives guidance on how to derive EBTs but does not propose final EBTs for implementation. The EBTs for some bioassays such as those for estrogenicity are already mature and could be implemented into regulation in the near future, while for others it will still take a few iterations until we can be confident of the power of the proposed EBTs to differentiate good from poor water quality with respect to chemical contamination.

Comparison of in vitro and in vivo bioassays to measure thyroid hormone disrupting activity in water extracts.

Environmental chemicals can induce thyroid disruption through a number of mechanisms including altered thyroid hormone biosynthesis and transport, as well as activation and inhibition of the thyroid receptor. In the current study six in vitro bioassays indicative of different mechanisms of thyroid disruption and one whole animal in vivo assay were applied to 9 model compounds and 4 different water samples (treated wastewater, surface water, drinking water and ultra-pure lab water; both unspiked and spiked with model compounds) to determine their ability to detect thyroid active compounds. Most assays correctly identified and quantified the model compounds as agonists or antagonists, with the reporter gene assays being the most sensitive. However, the reporter gene assays did not detect significant thyroid activity in any of the water samples, suggesting that activation or inhibition of the thyroid hormone receptor is not a relevant mode of action for thyroid endocrine disruptors in water. The thyroperoxidase (TPO) inhibition assay and transthyretin (TTR) displacement assay (FITC) detected activity in the surface water and treated wastewater samples, but more work is required to assess if this activity is a true measure of thyroid activity or matrix interference. The whole animal Xenopus Embryonic Thyroid Assay (XETA) detected some activity in the unspiked surface water and treated wastewater extracts, but not in unspiked drinking water, and appears to be a suitable assay to detect thyroid activity in environmental waters.

Effect-based assessment of toxicity removal during wastewater treatment.

Wastewaters contain complex mixtures of chemicals, which can cause adverse toxic effects in the receiving environment. In the present study, the toxicity removal during wastewater treatment at seven municipal wastewater treatment plants (WWTPs) was investigated using an effect-based approach. A battery of eight bioassays was applied comprising of cytotoxicity, genotoxicity, endocrine disruption and fish embryo toxicity assays. Human cell-based CALUX assays, transgenic larval models and the fish embryo toxicity test were particularly sensitive to WWTP effluents. The results indicate that most effects were significantly reduced or completely removed during wastewater treatment (76-100%), while embryo toxicity, estrogenic activity and thyroid disruption were still detectable in the effluents suggesting that some harmful substances remain after treatment. The responsiveness of the bioassays was compared and the human cell-based CALUX assays showed highest responsiveness in the samples. Additionally, the fish embryo toxicity test and the transgenic larval models for endocrine disrupting effects showed high responsiveness at low sample concentrations in nearly all of the effluent samples. The results showed a similar effect pattern among all WWTPs investigated, indicating that the wastewater composition could be rather similar at different locations. There were no considerable differences in the toxicity removal efficiencies of the treatment plants and no correlation was observed with WWTP characteristics, such as process configuration or sludge age. This study demonstrated that a biotest battery comprising of multiple endpoints can serve as a powerful tool when assessing water quality or water treatment efficiency in a holistic manner. Rather than analyzing the concentrations of a few selected chemicals, bioassays can be used to complement traditional methods of monitoring in the future by assessing sum-parameter based effects, such as mixture effects, and tackling chemicals that are present at concentrations below chemical analytical detection limits.

Development of a bioanalytical test battery for water quality monitoring: Fingerprinting identified micropollutants and their contribution to effects in surface water.

Surface waters can contain a diverse range of organic pollutants, including pesticides, pharmaceuticals and industrial compounds. While bioassays have been used for water quality monitoring, there is limited knowledge regarding the effects of individual micropollutants and their relationship to the overall mixture effect in water samples. In this study, a battery of in vitro bioassays based on human and fish cell lines and whole organism assays using bacteria, algae, daphnids and fish embryos was assembled for use in water quality monitoring. The selection of bioassays was guided by the principles of adverse outcome pathways in order to cover relevant steps in toxicity pathways known to be triggered by environmental water samples. The effects of 34 water pollutants, which were selected based on hazard quotients, available environmental quality standards and mode of action information, were fingerprinted in the bioassay test battery. There was a relatively good agreement between the experimental results and available literature effect data. The majority of the chemicals were active in the assays indicative of apical effects, while fewer chemicals had a response in the specific reporter gene assays, but these effects were typically triggered at lower concentrations. The single chemical effect data were used to improve published mixture toxicity modeling of water samples from the Danube River. While there was a slight increase in the fraction of the bioanalytical equivalents explained for the Danube River samples, for some endpoints less than 1% of the observed effect could be explained by the studied chemicals. The new mixture models essentially confirmed previous findings from many studies monitoring water quality using both chemical analysis and bioanalytical tools. In short, our results indicate that many more chemicals contribute to the biological effect than those that are typically quantified by chemical monitoring programs or those regulated by environmental quality standards. This study not only demonstrates the utility of fingerprinting single chemicals for an improved understanding of the biological effect of pollutants, but also highlights the need to apply bioassays for water quality monitoring in order to prevent underestimation of the overall biological effect.

Using short-term bioassays to evaluate the endocrine disrupting capacity of the pesticides linuron and fenoxycarb.

Several short-term whole-organism bioassays based on transgenic aquatic models are now under validation by the OECD (Organization for Economic Co-operation and Development) to become standardized test guidelines for the evaluation of the endocrine activity of substances. Evaluation of the endocrine disrupting capacity of pesticides will be a domain of applicability of these future reference tests. The herbicide linuron and the insecticide fenoxycarb are two chemicals commonly used in agricultural practices. While numerous studies indicate that linuron is likely to be an endocrine disruptor, there is little information available on the effect of fenoxycarb on vertebrate endocrine systems. Using whole-organism bioassays based on transgenic Xenopus laevis tadpoles and medaka fry we assessed the potential of fenoxycarb and linuron to disrupt thyroid, androgen and estrogen signaling. In addition we used in silico approach to simulate the affinity of these two pesticides to human hormone receptors. Linuron elicited thyroid hormone-like activity in tadpoles at all concentrations tested and, showed an anti-estrogenic activity in medaka at concentrations 2.5mg/L and higher. Our experiments suggest that, in addition to its previously established anti-androgenic action, linuron exhibits thyroid hormone-like responses, as well as acting at the estrogen receptor level to inhibit estrogen signaling. Fenoxycarb on the other hand, did not cause any changes in thyroid, androgen or estrogen signaling at the concentrations tested.

Remyelination by Resident Oligodendrocyte Precursor Cells in a Xenopus laevis Inducible Model of Demyelination.

We have generated a Xenopus laevis transgenic line, MBP-GFP-NTR, allowing conditional ablation of myelin-forming oligodendrocytes. In this transgenic line the transgene is driven by the proximal portion of the myelin basic protein regulatory sequence, specific to mature oligodendrocytes. The transgene protein is formed by the green fluorescent protein reporter fused to the Escherichia coli nitroreductase (NTR) selection enzyme. The NTR enzyme converts the innocuous prodrug metronidazole (MTZ) to a cytotoxin. Ablation of oligodendrocytes by MTZ treatment of the tadpole induced demyelination, and here we show that myelin debris are subsequently eliminated by microglial cells. After cessation of MTZ treatment, remyelination proceeded spontaneously. We questioned the origin of remyelinating cells. Our data suggest that Sox10+ oligodendrocyte precursor cells (OPCs), which are already present in the optic nerve prior to the experimentally induced demyelination, are responsible for remyelination, and this required only minimal (if any) cell division of OPCs. © 2015 S. Karger AG, Basel.

Rapid fluorescent detection of (anti)androgens with spiggin-gfp medaka.

Widespread environmental antiandrogen contamination has been associated with negative impacts on biodiversity and human health. In particular, many pesticides are antiandrogenic, creating a need for robust and sensitive environmental monitoring. Our aim was to develop a sensitive and specific transgenic medaka (Oryzias latipes) model bearing an androgen responsive fluorescent reporter construct for whole organism-based environmental screening of pro- and antiandrogens. We analyzed the 5' regions of the androgen responsive three-spined stickleback (Gasterosteus aculeatus) spiggin genes in silico, revealing conserved blocks of sequence harboring androgen response elements. Identified putative promoters were cloned upstream of GFP. Germinal transgenesis with spg1-gfp led to stable medaka lines. GFP induction was exclusive to the kidney, the site of spiggin protein production in sticklebacks. Significant GFP expression was induced by three or four-day androgen treatment of newly hatched fry, but not by estrogens, mineralocorticoids, glucocorticoids or progestogens. The model responded dose-dependently to androgens, with highest sensitivity to 17MT (1.5 µg/L). In addition to flutamide, the biocides fenitrothion, vinclozolin and linuron significantly inhibited 17MT-induced GFP induction, validating the model for detection of antiandrogens. The spg1-gfp medaka model provides a sensitive, specific, and physiologically pertinent biosensor system for analyzing environmental androgen activity.

Unfertilized Xenopus eggs die by Bad-dependent apoptosis under the control of Cdk1 and JNK.

Ovulated eggs possess maternal apoptotic execution machinery that is inhibited for a limited time. The fertilized eggs switch off this time bomb whereas aged unfertilized eggs and parthenogenetically activated eggs fail to stop the timer and die. To investigate the nature of the molecular clock that triggers the egg decision of committing suicide, we introduce here Xenopus eggs as an in vivo system for studying the death of unfertilized eggs. We report that after ovulation, a number of eggs remains in the female body where they die by apoptosis. Similarly, ovulated unfertilized eggs recovered in the external medium die within 72 h. We showed that the death process depends on both cytochrome c release and caspase activation. The apoptotic machinery is turned on during meiotic maturation, before fertilization. The death pathway is independent of ERK but relies on activating Bad phosphorylation through the control of both kinases Cdk1 and JNK. In conclusion, the default fate of an unfertilized Xenopus egg is to die by a mitochondrial dependent apoptosis activated during meiotic maturation.

Survivin increased vascular development during Xenopus ontogenesis.

Survivin is a member of the inhibitor of apoptosis proteins (IAP) family. These proteins contain one to three zinc-binding motifs termed bacculoviral IAP-binding repeats (BIRs). Survivin contains a single BIR motif. Contrary to other members that directly interact with caspases and inhibit apoptosis, Survivin is believed to have both antiapoptotic and proliferative functions. In mammals, Survivin is not detected in most adult tissues except in endothelial cells of newly formed capillaries and large blood vessels. Importantly, Survivin is highly expressed in all common human cancers. To gain a better view of Survivin expression and function during development, we used the amphibian Xenopus developmental model. We show that the genomes of X. laevis, X. tropicalis, Zebrafish, fugu pufferfish, and rainbow trout encode two different Survivin genes (Su1 and Su2), contrary to mammalian genomes, which encode a single one. In X. laevis, these two genes have a differential spatiotemporal transcription pattern. Transgenic expression of Su1 leads to an enlargement of tadpole's blood vessels with an increase in the number of endothelial cells. This effect requires a functional BIR domain and the p34/cdc2 phosphorylation site. It does not seem to rely on the antiapoptotic activity of Su1 as it is not observed in tadpoles overexpressing other antiapoptotic factors such as XIAP or BclXL. We conclude that Su1 ubiquitous gain of function leads directly or indirectly to an increase in blood vessels size via the proliferation of endothelial cells.

Apoptosis of tail muscle during amphibian metamorphosis involves a caspase 9-dependent mechanism.

The climax of amphibian metamorphosis is marked by thyroid hormone-dependent tadpole tail resorption, implicating apoptosis of multiple cell types, including epidermal cells, fibroblasts, nerve cells, and muscles. The molecular cascades leading to and coordinating the death of different cell types are not fully elucidated. It is known that the mitochondrial pathway, and in particular the Bax and XR11 genes, regulates the balance between apoptosis and survival in muscle. However, the down-stream factors modulated by changes in mitochondrial permeability have not been studied in a functional context. To investigate further the mitochondrial-dependent pathway, we analyzed the regulation and the role of caspase 9 in Xenopus tadpoles. We report that caspase 9 mRNA is expressed in the tail before metamorphosis and increases before and during climax. Similarly, at the protein level, the production of active forms of caspase 9 increases in muscle tissue as metamorphosis progresses. To assess the functional role of caspase 9, we designed a dominant-negative protein. Overexpression of this dominant-negative abrogates both Bax-induced cell death in vitro and muscle apoptosis in vivo during natural metamorphosis. These findings consolidate a model of metamorphic muscle death that directly implicates the mitochondrial pathway and the apoptosome.

Polo-like kinase confers MPF autoamplification competence to growing Xenopus oocytes.

During oogenesis, the Xenopus oocyte is blocked in prophase of meiosis I. It becomes competent to resume meiosis in response to progesterone at the end of its growing period (stage VI of oogenesis). Stage IV oocytes contain a store of inactive pre-MPF (Tyr15-phosphorylated Cdc2 bound to cyclin B2); the Cdc25 phosphatase that catalyzes Tyr15 dephosphorylation of Cdc2 is also present. However, the positive feedback loop that allows MPF autoamplification is not functional at this stage of oocyte growth. We report that when cyclin B is overexpressed in stage IV oocytes, MPF autoamplification does not occur and the newly formed cyclin B-Cdc2 complexes are inactivated by Tyr15 phosphorylation, indicating that Myt1 kinase remains active and that Cdc25 is prevented to be activated. Plx1 kinase (or polo-like kinase), which is required for Cdc25 activation and MPF autoamplification in full grown oocytes is not expressed at the protein level in small stage IV oocytes. In order to determine if Plx1 could be the missing regulator that prevents MPF autoamplification, polo kinase was overexpressed in stage IV oocytes. Under these conditions, the MPF-positive feedback loop was restored. Moreover, we show that acquisition of autoamplification competence does not require the Mos/MAPK pathway.