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.
•Thyroid activity of model compounds and water extracts tested in vitro and in vivo.•Reporter gene assays sensitive to compounds, but unable to detect activity in water.•Whole animal XETA assay most suitable to detect thyroid activity in water extracts.
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 (T 3 or T 4 ), 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.
Diclofenac (DCF) is an environmentally persistent, nonsteroidal anti-inflammatory drug (NSAID) with thyroid disrupting properties. Electrochemical advanced oxidation processes (eAOPs) can efficiently ...remove NSAIDs from wastewater. However, eAOPs can generate transformation products (TPs) with unknown chemical and biological characteristics. In this study, DCF was electrochemically degraded using a boron-doped diamond anode. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry was used to analyze the TPs of DCF and elucidate its potential degradation pathways. The biological impact of DCF and its TPs was evaluated using the Xenopus Eleutheroembryo Thyroid Assay, employing a transgenic amphibian model to assess thyroid axis activity. As DCF degradation progressed, in vivo thyroid activity transitioned from anti-thyroid in non-treated samples to pro-thyroid in intermediately treated samples, implying the emergence of thyroid-active TPs with distinct modes of action compared to DCF. Molecular docking analysis revealed that certain TPs bind to the thyroid receptor, potentially triggering thyroid hormone-like responses. Moreover, acute toxicity occurred in intermediately degraded samples, indicating the generation of TPs exhibiting higher toxicity than DCF. Both acute toxicity and thyroid effects were mitigated with a prolonged degradation time. This study highlights the importance of integrating in vivo bioassays in the environmental risk assessment of novel degradation processes.
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•eAOP degradation of DCF evaluated by LC-QTOF-MS and in vivo thyroid activity assay.•DCF reduces the activity of the thyroid endocrine system in xenopus eleutheroembryos.•Acute toxicity first increases and then decreases throughout the eAOP process.•Unexpected pro-thyroid activity observed in intermediately degraded samples.•Transformation products have a different mode of action than the parent compound.
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.
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•Bioassay battery for water quality assessment assembled and applied to 34 chemicals.•Data mining exercise highlighted lack of available effect data for mixture modeling.•All chemicals active in at least one assay, with bisphenol A active in 70% of assays.•Diversity of possible modes of action advises complementary use of apical endpoints.•Single chemical contribution to observed effect shown by mixture toxicity modeling.
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
Eleutheroembryonic Thyroid Assay (XETA, OECD test guideline no. 248) is one such test. It is based on
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 (T
or T
), 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.
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.
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.
•The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently developed to screen for thyroid activity of chemicals.•XETA results were compared to Amphibian Metamorphosis Assay (AMA) outcomes for the detection of thyroid-active chemicals.•Results were concordant for all selected mechanisms, but the XETA did not detect inhibition of the sodium-iodide symporter.•Thyroperoxidase inhibition requires further mechanistic investigations due to equivocal responses in the XETA.•The XETA and the AMA consistently identified uridine 5′-diphospho-glucuronosyltransferase inducers as thyroid-inactive.
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.
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.
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