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

Periodic Reporting for period 2 - EDCMET (Metabolic effects of Endocrine Disrupting Chemicals: novel testing METhods and adverse outcome pathways)

Reporting period: 2020-07-01 to 2021-12-31

Thus far, endocrine disruptor (ED) research has mainly focused on reproductive endocrinology and related hormones, which is reflected in the regulatory test methods assessing only endocrine effects of xenobiotics. Accumulating evidence links ED exposure to increased incidence of metabolic syndrome and further, incidence of fatty liver and type 2 diabetes. EDs may exert their adverse metabolic effects via several mechanisms, such as modulation of nuclear receptors, impairment of mitochondrial respiration or excessive cellular accumulation of lipids. 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. 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. 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. The interdisciplinary approach and complementary expertise of project participants will aid in the identification of novel mechanisms of action and the development of novel or improved validated test methods for regulatory purposes.
Interactions of EDs with NRs as well as the potential mechanisms and molecular triggers behind NR structure stabilization and activation have been explored using optimized in silico approaches. A database of omics level data for the action of EDs in hepatic cell models and in vivo (rodents) has been established. New transcriptomics data from human hepatic cell models and mice, as well as plasma and liver metabolomics data from mice, have been produced. Metabolomics data is already available from selected cohort samples and possibilities for further analyses are explored. To analyze omics data, a predictive classification pipeline has been developed and published. This pipeline is further expanded and validated by interrogation of de novo omics data generated in the project. A systems toxicology tool to predict emergent metabolic phenotype from ED exposure is under development.

NR-coregulator interaction assays are available for ten NRs. Reporter gene assays for predicting the activation of NRs involved in the regulation of metabolic pathways have been developed and evaluated by following the NCATS and OECD Framework to ensure robustness, reproducibility, and transferability of the assays. Mitochondrial respiration assays have been developed to enable assessment of functional effects of EDs on cellular level in hepatic cell models. High-throughput, fluorescence-based AdipoRed assay is available to evaluate the steatotic effects of EDs. A set of ED chemicals has been screened across the assay panel and further compound testing is ongoing. Steps towards regulatory implementation of the developed assays have been initiated in collaboration with EURION and OECD expert group.

Standard Operating Procedures (SOPs) have been developed for insulin tolerance and glucose tolerance tests as well as for inducing obesity, insulin resistance, and non-alcoholic fatty liver disease using high-fat diet in mice. Studies on ED effects on vulnerable individuals (obesity, in utero) are ongoing. Liver transcriptomics and biochemical analyses following pregnane X receptor activation have revealed widespread effects and mechanistic details on effects of xenobiotics on cholesterol synthesis. The biological knowledge gained from in vitro and in vivo studies, alongside further interrogation of legacy data, will also support AOP development later in the project.

Levels of selected ED compounds have been analysed from cohort samples and analyses on potential associations of exposure levels and systemic metabolism as well as health outcomes are underway. Several scientific papers on the epidemiological findings have been published and a workshop is planned for 2022 to discuss results and further plans.

Information on developed assays, ED test compounds, experimental protocols and cohorts have been shared and discussed in EURION meetings and working groups.
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 to increase human relevancy in testing and risk prediction and further reduce the use of laboratory animals. The developed methods undergo preliminary experimental validation and can contribute to the current OECD test systems. The methods and models will be coupled with human exposure data, linking the levels of EDs with metabolic endpoints and health outcomes. 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.

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 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.
EDCMET staff at the general assembly meeting in Berlin, November 2021
EDCMET staff at the project kick-off meeting in Brussels, February 2019