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Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies

Periodic Reporting for period 1 - ATHENA (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies)

Berichtszeitraum: 2019-01-01 bis 2020-06-30

Endocrine disrupting chemicals (EDCs) are found to be ubiquitous in the environment; these chemicals are added to various materials and products such as plastics, pesticides and flame retardants but ultimately end up in the environment and food chain and can accumulate in the human body, causing negative health consequences. EDCs disrupt the normal functioning of hormonal systems by mimicking hormones or blocking their natural action in the body. From amongst the different hormonal systems, the ATHENA project is interested in the thyroid hormone system and how brain development in utero is affected by EDCs which act on the thyroid system, called thyroid hormone system disrupting chemicals (THDCs). For the first few weeks of gestation, the foetus is unable to produce thyroid hormone and relies on the mother for its essential supply. It is well-established that maternal hypothyroidism (thyroid under-function) can lead to impaired psychomotor development and lowered IQ in offspring. It has been shown that THDCs in the maternal system (for example certain pesticides and flame-retardant chemicals) can decrease circulating thyroid hormone, with lower levels reaching the foetus and leading to the same negative outcome. Over-production of thyroid hormone also has detrimental effects. There are several points within the thyroid hormone system where THDCs can act to disrupt the system; partners within the ATHENA consortium will focus on different areas within the thyroid hormone system to develop test methods to identify THDCs and their mechanism of action. An improvement of test methods is urgently required; the available test methods only provide a minimal assessment of thyroid hormone system disruption which is inadequate and leaves several gaps in the available test methods for protection of human health against THDCs. The overall objective of the ATHENA consortium is to develop test methods for the identification of chemicals that disrupt the thyroid hormone system. The focus of test development will be to capture the consequences of maternal thyroid hormone deficiency or over-production on the developing brain. Work will focus on understanding disruption of local maternal thyroid hormone by inhibition of enzymes which are involved in thyroid hormone synthesis and metabolism and cell membrane transporters, by development of predictive methods and screening of compound libraries. The consequence of maternal hypothyroidism on the foetus due to interference of the delivery of thyroid hormones across the placenta, the blood brain barrier (BBB) and the blood cerebrospinal fluid barrier (BCSFB), will also be studied.
The results of experimental work will allow us to construct a network of the thyroid system that can explain how maternal exposure to THDCs can lead to adverse neurodevelopmental effects. It will allow us to formulate a comprehensive testing strategy which will be positioned for international harmonisation to protect against chemicals that are harmful to brain development in foetal life.
In the first 18 months of the project, the ATHENA consortium achieved the following aims:

• Using existing epidemiological data from two large studies, found that exposure to thyroid system disrupting chemicals in pregnant women is associated with disruption of hypothalamic-pituitary-thyroid axis and exploited these results as a published manuscript (Derakhshan et al Environment International 133 (2019) 105123). There are further manuscripts in preparation.

• Progress in the development of 3D cell models of brain development and neural stem cell division: a human organoid model with a defined panel of neuronal markers covering different stages of maturation and the various cell populations within the organoids, and mouse neurospheres, for which pharmacological assessment of specific TH-dependent endpoints and neural stem cell proliferation has been studied.

• Smaller studies of several thyroid system disrupting compounds have been performed in rats and mice. These studies were dose-selection studies performed to ascertain thyroid hormone system disruption and clarify potential toxicities when compounds were given to pregnant animals. The results from these studies form the basis for large developmental toxicity studies where effects on the thyroid hormone system and brain development will be examined in detail. One major endpoint in these studies will be the formation of heterotopia - the occurrence of misplaced neurons in a defined structure. Heterotopia are linked to thyroid hormone system disruption in rats and in humans they have been correlated to seizure and epilepsy.

• Progress in test method development for a non-radioactive high throughput assay for screening interactions at two non-receptor targets, which has also generated data for QSAR modelling contributing to the development and validation of new QSAR models for inhibition of deiodinases and cell transporter (DIO2, DIO3, DEHAL1 and OATP1C1). A model is also being generated for another transporter (NIS) involved in thyroid hormone synthesis.

• Work has progressed towards robotisation of the Xenopus Eleutheroembryonic Thyroid Assay (XETA), an existing method in the OECD test guidelines. The assay has been refined and validated for medium throughput and will progress to high throughput format. There has also been progress towards development of strategies for establishing the mode of action of THDCs to elucidate their targets in the body and provide information about specific molecular initiating events (MIE).

• Cellular models are being established for study of the disruption of thyroid hormone transport between mother and foetus for placenta, BBB and BCSFB. A placenta perfusion model has also been set up to test transplacental T4 transport to identify the important TH transmembrane transporters that may be inhibited by THDCs. One of these transporters is undergoing development for a nonradioactive high throughput assay to allow screening of inhibitors.

There has been some delay due to the COVID-19 pandemic where in vivo tests, screening and collaborative work had to be postponed due to national lockdowns and restrictions on travel. However, the ATHENA consortium is well positioned to continue to meet the goals of the project, albeit not without a cost-neutral extension.
Certain chemicals have been identified as thyroid system disruptors, leading to a decrease in thyroid hormone, however they are not necessarily recognised as such, due to their mechanism of action. The current status quo of regulatory test guidelines has a very limited definition of what constitutes a THDC, which means that harmful chemicals are not recognised and their negative impacts to human health cannot be prevented. The ATHENA project will provide an improved understanding of the thyroid hormone system with regards to endpoints and targets of THDCs, this will allow the development of a testing strategy to incorporate into regulatory guidelines at international level. In the first 18 months of the project, the ATHENA consortium has worked towards all the expected deliverables and milestones of the project.
Heterotopia -misplaced cells in the corpus callosum of rat