Periodic Reporting for period 3 - ATHENA (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies)
Berichtszeitraum: 2022-01-01 bis 2023-06-30
The thyroid hormone system maintains biological functions such as growth and homeostasis. Critical during development is the delivery of thyroid hormones at the right concentration and the right time. Thyroid hormone system-disrupting chemicals (THSDCs) can interfere with these processes in many different ways. In pregnant women, THSDCs can decrease circulating thyroid hormone, which leads to lower levels reaching the foetus. This can be particularly damaging in the first trimester when the foetus cannot produce its own thyroid hormone and relies on the mother for this essential supply, as it could lead to impaired psychomotor development and lowered IQ.
The ATHENA project researches how brain development in utero is affected by THSDCs which disrupt the maternal thyroid hormone system.
There are several points within the thyroid hormone system where THSDCs can interact and disrupt the system. Partners within the ATHENA consortium focus on different areas within the thyroid hormone system to develop test methods to identify THSDCs, delineate their mechanism of action to construct an AOP network and to inform a comprehensive testing strategy.
The ATHENA project aims to develop new methods for incorporation into existing OECD test guidelines for the identification of THSDCs. Further tests with new endpoints need to be established to close critical gaps in the test guidelines.
The ATHENA project researches how brain development in utero is affected by THSDCs which disrupt the maternal thyroid hormone system.
There are several points within the thyroid hormone system where THSDCs can interact and disrupt the system. Partners within the ATHENA consortium focus on different areas within the thyroid hormone system to develop test methods to identify THSDCs, delineate their mechanism of action to construct an AOP network and to inform a comprehensive testing strategy.
The ATHENA project aims to develop new methods for incorporation into existing OECD test guidelines for the identification of THSDCs. Further tests with new endpoints need to be established to close critical gaps in the test guidelines.
Associations of exposure to EDCs with thyroid function in pregnant women are determined by leveraging already available human epidemiological data from the longitudinal SELMA study and the Generation R study. We have explored associations with phenolic compounds, phthalates, perfluorinated chemicals, PCBs and hexachlorobenzene and found that thyroid hormone levels were changed by a variety of mechanisms.
We further investigated chemicals with structural similarities. Most EDCs that were associated with lower child IQ were also identified as gestational THSDCs. The first results of ongoing analyses indicate that altered maternal thyroid hormone is associated with behavioral problems and a lower child IQ.
To understand how THSDCs interfere with brain development and neural stem cell decisions during brain development, we are developing innovative 3D in vitro test methods. Two models are under development: human cerebral organoids and mouse neurospheres. Our efforts to scale up the assays were successful, and we used machine learning technology to accelerate time-consuming measurements.
We are developing high-throughput screening HTS and QSAR to determine how THSDC can to alter the concentrations of thyroid hormones in systemic circulation by acting on non-receptor targets. At present no HTS assays to screen potential THSDCs. To date we have screened large and small molecule libraries of up to 70,000 compounds using a non-radioactive semiautomatic HTS platform based on the Sandell-Kolthoff reaction.
Based on an improved understanding of thyroid hormone transport between mother and foetus which is essential for brain development, we aim to identify new thyroid hormone transmembrane transporters (THTT) in physiological barriers and evaluate their suitability for HTS assays. We have identified THTT capable of transporting iodothyronines and sulfated iodothyronines across the placenta, from mother to fetus, and vice versa. We have investigated the inhibition of THTT (OATP1C1 and OAT4) in cell lines stably overexpressing these transporters and screened >30 chemicals.
We are developing in vivo assays for downstream effects of THSDCs on brain development. We have provided a preliminary study protocol for the evaluation of neurodevelopmental endpoints of thyroid hormone action, based on results from in vivo studies using potent TPO inhibitors. We have followed up on this work by performing a developmental toxicity study with two chemicals that exert their THSD action through the induction of liver enzymes that affect thyroid hormones. We have also continued our search for new biomarkers by performing transcriptomic analyses of hippocampi from offspring exposed to TPO inhibitors during early development.
The Xenopus Eleutheroembryonic Thyroid Assay (XETA) protocol was published as an OECD test guideline in June 2019. Work on this assay aims to automate this existing assay to reduce the number of animals used for chemical testing and increase the speed of testing. To identify additional endpoints and biomarkers, transcriptomic experiments were performed and candidate genes identified.
The “pregnancy hormone” human chorionic gonadotropin (hCG) stimulates the thyroid gland in early pregnancy to produce sufficient thyroid hormone to ensure healthy brain development; it can also be affected by EDCs. We have completed analysis of human datasets from the SELMA study in which we investigated associations of EDCs with maternal hCG concentrations. These results show that these EDCs have the potential to decrease and disrupt hCG production and secretion by the placenta.
We are developing a testing strategy for THSDCs based on in vitro testing methods for molecular initiating events that have a strong impact on perturbing the thyroid hormone system. In the course of this work, it has become clear that several key events on which multiple pathways converge are currently not covered by adequate test methods. We have also initiated work to fill these gaps by targeted gene expression studies. We have also completed a systematic review of gestational exposures to test chemicals in which hormone profiles in both dams and offspring were recorded. The findings are relevant to the development of a testing strategy. We have now begun work on preparing the ground for submitting new in vitro test methods to the ECVAM modular process
The ATHENA project hopes to build strategies for thyroid disruptors for international regulation and harmonisation with non-EU trading blocs. Considerations include the differences in the regulatory management of substances deemed to be endocrine disruptors and preference for risk versus hazard assessments. We have conducted an in-depth analysis of international approaches to identifying and regulating THSDCs by way of detailed case studies of benthiavalicarb, mancozeb and triclosan.
ATHENA together with SCREENED took the role of coordinators of the EURION Cluster. Meetings for the Cluster project coordinators were held every 2 months and the annual Cluster meeting was organised online in January 2022, with several hundred participants. There was also a meeting with stakeholders and a joint workshop with the JRC, plus a joint meeting with ATHENA, ERGO and EnDpoiNTs, which was an outcome of the annual Cluster meeting, which led to further cross-project collaborations.
We further investigated chemicals with structural similarities. Most EDCs that were associated with lower child IQ were also identified as gestational THSDCs. The first results of ongoing analyses indicate that altered maternal thyroid hormone is associated with behavioral problems and a lower child IQ.
To understand how THSDCs interfere with brain development and neural stem cell decisions during brain development, we are developing innovative 3D in vitro test methods. Two models are under development: human cerebral organoids and mouse neurospheres. Our efforts to scale up the assays were successful, and we used machine learning technology to accelerate time-consuming measurements.
We are developing high-throughput screening HTS and QSAR to determine how THSDC can to alter the concentrations of thyroid hormones in systemic circulation by acting on non-receptor targets. At present no HTS assays to screen potential THSDCs. To date we have screened large and small molecule libraries of up to 70,000 compounds using a non-radioactive semiautomatic HTS platform based on the Sandell-Kolthoff reaction.
Based on an improved understanding of thyroid hormone transport between mother and foetus which is essential for brain development, we aim to identify new thyroid hormone transmembrane transporters (THTT) in physiological barriers and evaluate their suitability for HTS assays. We have identified THTT capable of transporting iodothyronines and sulfated iodothyronines across the placenta, from mother to fetus, and vice versa. We have investigated the inhibition of THTT (OATP1C1 and OAT4) in cell lines stably overexpressing these transporters and screened >30 chemicals.
We are developing in vivo assays for downstream effects of THSDCs on brain development. We have provided a preliminary study protocol for the evaluation of neurodevelopmental endpoints of thyroid hormone action, based on results from in vivo studies using potent TPO inhibitors. We have followed up on this work by performing a developmental toxicity study with two chemicals that exert their THSD action through the induction of liver enzymes that affect thyroid hormones. We have also continued our search for new biomarkers by performing transcriptomic analyses of hippocampi from offspring exposed to TPO inhibitors during early development.
The Xenopus Eleutheroembryonic Thyroid Assay (XETA) protocol was published as an OECD test guideline in June 2019. Work on this assay aims to automate this existing assay to reduce the number of animals used for chemical testing and increase the speed of testing. To identify additional endpoints and biomarkers, transcriptomic experiments were performed and candidate genes identified.
The “pregnancy hormone” human chorionic gonadotropin (hCG) stimulates the thyroid gland in early pregnancy to produce sufficient thyroid hormone to ensure healthy brain development; it can also be affected by EDCs. We have completed analysis of human datasets from the SELMA study in which we investigated associations of EDCs with maternal hCG concentrations. These results show that these EDCs have the potential to decrease and disrupt hCG production and secretion by the placenta.
We are developing a testing strategy for THSDCs based on in vitro testing methods for molecular initiating events that have a strong impact on perturbing the thyroid hormone system. In the course of this work, it has become clear that several key events on which multiple pathways converge are currently not covered by adequate test methods. We have also initiated work to fill these gaps by targeted gene expression studies. We have also completed a systematic review of gestational exposures to test chemicals in which hormone profiles in both dams and offspring were recorded. The findings are relevant to the development of a testing strategy. We have now begun work on preparing the ground for submitting new in vitro test methods to the ECVAM modular process
The ATHENA project hopes to build strategies for thyroid disruptors for international regulation and harmonisation with non-EU trading blocs. Considerations include the differences in the regulatory management of substances deemed to be endocrine disruptors and preference for risk versus hazard assessments. We have conducted an in-depth analysis of international approaches to identifying and regulating THSDCs by way of detailed case studies of benthiavalicarb, mancozeb and triclosan.
ATHENA together with SCREENED took the role of coordinators of the EURION Cluster. Meetings for the Cluster project coordinators were held every 2 months and the annual Cluster meeting was organised online in January 2022, with several hundred participants. There was also a meeting with stakeholders and a joint workshop with the JRC, plus a joint meeting with ATHENA, ERGO and EnDpoiNTs, which was an outcome of the annual Cluster meeting, which led to further cross-project collaborations.
The ATHENA project aims to improve understanding of the mechanism of action of different THSDCs and gain insight into predicted outcomes. The test methods established in the ATHENA project will undergo the pre-validation process for incorporation into the OECD test guidelines, together with considerations for international harmonisation. We hope to cover gaps and provide a comprehensive testing strategy for THSDCs to identify chemicals that have disrupting properties, as a lack of identification means that exposure to harmful chemicals will not be eradicated.