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Metabolic effects of Endocrine Disrupting Chemicals: novel testing METhods and adverse outcome pathways

Periodic Reporting for period 1 - EDCMET (Metabolic effects of Endocrine Disrupting Chemicals: novel testing METhods and adverse outcome pathways)

Reporting period: 2019-01-01 to 2020-06-30

Endocrine disruptors (EDs) are defined as exogenous chemicals that alter functions of the endocrine system, thereby causing adverse health effects in an organism or its progeny. Historically, the field of ED research has focused on reproductive endocrinology and related hormones, which is reflected in the regulatory test methods assessing endocrine effects of xenobiotics. Recent evidence links increased incidence of metabolic syndrome - a cluster of metabolic risk factors including abdominal obesity, dyslipidaemia, elevated blood pressure, and elevated fasting glucose - to ED exposure, further increasing the incidence of atherosclerosis and type 2 diabetes. However, the current testing tools do not appropriately identify effects related to less-studied endocrine-mediated pathways and health outcomes, such as disruption of lipid and glucose metabolism, in humans. Activation of nuclear receptors (NRs) has been linked to the development of several metabolic and liver-related diseases. While public databases, such as ToxCast, contain numerous assays for the assessment of NR modulation, important metabolism-related NRs have not been included in the analyses or the information available is misleading due to lack of performance information of the assays. In addition to the NRs, EDs may alter metabolic functions by other mechanisms, such as impairment of mitochondrial respiration, which has been linked to the development of insulin resistance and non-alcoholic fatty liver disease. New and improved approaches are needed to increase the quality, efficiency and effectiveness of existing methods to evaluate the effects of EDs and to meet the demanding and evolving regulatory requirements worldwide.

The Metabolic effects of Endocrine Disrupting Chemicals: novel testing METhods and adverse outcome pathways (EDCMET) project focuses on developing novel or improved computational and in vitro methods, such as non-cellular assays and cell culture systems, as well as standardized in vivo animal models to assess the metabolic effects of EDs, with a focus on NRs regulating these processes. Further, unbiased omics techniques will be used to investigate tissue and plasma markers in vivo. Epidemiological and field monitoring data is used to gain information regarding the human exposure to EDs and related metabolic effects. EDCMET will also apply the adverse outcome pathway (AOP) paradigm to identify molecular initiating events (MIEs) and predict the emerging adverse biological phenotype. EDCMET comprises of academic and non-academic partners in various research fields, including systems toxicologists, experimental biologists with a thorough understanding of the molecular mechanisms of metabolic disease and comprehensive methodological skills and ultimately, epidemiologists linking environmental exposure to adverse metabolic outcomes. The interdisciplinary approach and complementary expertise of the participants will aid in the identification of novel mechanisms of action and the development of novel or improved validated test methods for regulatory purposes.
EDCMET has been exploring and collecting publicly available resources and data, including NR proteins and ligands, ED compounds and omics data for the action of EDs in hepatic cell models and in vivo. An initial analysis pipeline for prediction of chemicals with ED potential has been generated. Liver transcriptomics data from mice has been produced and plasma and liver metabolomics from wildtype and NR knockout mice have been planned. Metabolomics data is already available from selected cohort samples and possibilities for further metabolomics analyses are explored. The de novo omics data will be used to further develop and validate the omics analysis pipelines.

To evaluate a battery of NR-cofactor interaction assays, a small set of ED chemicals has been screened across a panel of relevant NRs and further assays are under development. Reporter gene assays for predicting the activation of NRs involved in the regulation of metabolic pathways have been largely optimized and detailed evaluation of assay performance is on-going. The NCATS and OECD Framework guidance on standardized in vitro test systems and scientific assay validation have been followed and implemented in the development and scientific pre-validation of the NR assays to ensure robustness and reproducibility of results as well as transferability of the assays. These, together with established standard operating procedures (SOPs), will ensure the applicability of the protocols also in the international regulatory context. Mitochondrial respiration assays are under development to enable assessment of functional effects of EDs on cellular level in hepatic cell models in high-throughput format. High-throughput, fluorescence-based AdipoRed assay is available to evaluate the steatotic effects of EDs.

SOPs have been developed for performing insulin tolerance and glucose tolerance tests and for inducing obesity, insulin resistance, and non-alcoholic fatty liver disease using high-fat diet in mice. Standard protocols for glucose and insulin tolerance tests and liver steatosis have been developed in humanized mice. Experiments with ED reference compounds are on-going. Multiple approaches, including traditional risk assessment, guideline values and toxicological cut-off points, toxicokinetic modelling and WHO risk assessment methodology and health effects based on epidemiological studies, have been considered. Analyses of EDs from cohort samples have been started and further analysis methods are under development.
EDCMET will provide an array of new or improved testing tools for risk assessment of metabolism disrupting EDs. These tools are expected to identify novel disrupted pathways in human liver and adipose tissues and to increase human relevancy in testing and risk prediction and further reduce the use of laboratory animals. The methods and models will be coupled with human exposure data, linking the levels of EDs with metabolic endpoints and health outcomes. The developed methods undergo preliminary experimental validation and can contribute to the current OECD test systems. All information concerning the EDs and protocols, will be provided for the regulatory use and the regulatory implementation will be done in collaboration with contacts in national and EU-level. EDCMET will lay the groundwork for future testing and ED toxicity assessment and contribute to a better understanding of human exposure to chemicals and the associated burden of metabolic diseases. EDCMET will also deliver methods to be commercialised by the SMEs involved in the project.

Endocrine-related diseases represent a high cost for healthcare systems in the EU countries and metabolic effects of EDs are poorly understood. EDCMET will increase the knowledge on metabolic diseases by characterization of the affected critical pathways in liver and adipose tissues using the AOP approach. EDCMET will also aid in the development of novel biomarkers for metabolic diseases and provide a relevant base for assessing adverse human health effects of environmental chemicals. The availability of fast and cheaper test systems can stimulate research around environmental chemicals and contribute to safer marketed products and thereby have a positive impact on the environment.