Transcriptional activation by NRs is controlled by the ligand-binding domain with different binding modes for receptor ligands, such as EDs. Existing NR crystal structures often fail to capture the dynamic conformational changes triggered by ligands and protein partners, which poses a challenge to understand these interactions. To address the challenge, EDCMET developed an in silico molecular modeling approach to identify and map interactions between NRs and EDs and applied the method to study the NR-ED interactions of metabolically relevant NRs. The developed tool is able to reliably predict ED interactions with multiple NRs with both agonist (activating) and antagonist (repressing) modes, and thus, can identify the initial MIEs to predict ED-protein interactions potentially leading to metabolic dysfunction. The EDTox tool was developed to prioritize chemicals based on their ED potential and to study the mode of action of EDs within toxicogenomic-driven gene networks. A combination of approaches, ranging from simple statistical analyses to more complex machine learning algorithms was used to identify potential exposure and effect biomarkers based on publicly available and de novo EDCMET data. A model predicting NR regulation of liver metabolism and emerging phenotypes was constructed.
To assess the metabolic adverse effects of EDs mediated by NRs, EDCMET developed several in vitro assays to assess NR-coregulator interactions, as well as cell-based assays to study NR activation in response to ED exposure. Further, high throughput mitochondrial respiration and triglyceride accumulation assays in hepatic cells have been developed. The assays have been used to screen a set of over 30 ED compounds as well as assay-specific controls to scientifically validate the developed assays for use in addressing the metabolic effects of EDs. In addition, changes in HepG2 and HepaRG human liver cell transcriptomes upon exposure to EDs were profiled. The in vitro assays were evaluated using the Test readiness criteria implemented in the EURION Cluster. The triglyceride accumulation assay, achieving the highest readiness score, was selected for further validation by the PEPPER initiative. Further validation and regulatory review are needed to ensure that the EDCMET in vitro assays meet the scientific and regulatory standards for use in toxicology assessments.
Standardized in vivo protocols have been established for several metabolic endpoint measuring disturbances in lipid and glucose homeostasis. EDCMET showed that application of a repeated dose 28-day oral toxicity test is insensitive in detecting metabolic disruption by EDs and therefore is not suitable for screening purposes. Instead, combination of nutritional challenge with high-fat diet (HFD) and treatment with EDCs was found to be a useful method of detecting EDC-induced metabolic effects and a protocol utilizing 20-week ED exposure under HFD-feeding was established. Another protocol investigating effects of in utero exposure on later metabolic health was developed to represent the effects in developing individuals. The EDCMET in vivo studies have also provided a significant amount of new mechanistic information on metabolic disruption through major pathways such as the NRs PXR and PPARα as MIEs. An AOP concept was developed and published for PXR-induced hypercholesterolemia predominantly based on data provided by the EDCMET project.
The most important findings related to human health risks in EDCMET are i) the importance of considering sex differences, as health biomarkers and outcomes were found to be different in males and females, ii) the difference in effects of exposure found also in internal exposome on metabolic effects, iii) the physiological status of children and pregnant women is affecting the outcomes of exposure and iv) the levels of older, partially banned, pesticides and other environmental contaminants in serum are decreasing, while levels of newer EDCs are increasing.
