From Neurodevelopmental Models to Endocrine Disruption: Metabolomic Insights from In Vitro and In Vivo Studies

Endocrine-disrupting chemicals (EDCs) are increasingly recognized for their potential to interfere with brain development during early-life windows of vulnerability. Epidemiological and experimental studies link developmental EDC exposure to impairments in cognition, behavior, and emotional regulation. Because neurodevelopment depends on tightly coordinated endocrine, metabolic, and neurotransmitter-mediated processes, disturbances during critical stages may lead to lasting effects on brain function. From a regulatory perspective, in vivo studies remain essential for assessing endocrine disruption–induced developmental neurotoxicity (DNT), while human-relevant in vitro systems, in silico tools, and multi-omics technologies complement in vivo data by providing mechanistic insight at the pathway level. Within this context, the Horizon 2020 ENDpoiNTs project integrates in vivo and in vitro models, in silico approaches, multi-omics technologies, and epidemiological data to establish mechanistically anchored neurodevelopmental endpoints for ED-induced DNT and to support the development of Adverse Outcome Pathways for developmental neurotoxicity (DNT AOPs). This thesis contributes to these objectives through analytical method development, molecular characterization of neurodevelopmental model systems, and investigation of early-life metabolic perturbations induced by EDC exposure and their relationship with long-term behavioral outcomes. First, a high-throughput LC-MS/MS method was developed for quantifying steroid hormones in rat plasma and liver, later adapted for brain tissue and extended to neurosteroids and thyroid hormones. This platform underpins the endocrine profiling used in the in vivo studies. To enhance human relevance, the thesis incorporates an in vitro component based on human cortical brain organoids (CBOs). Although CBOs have been characterized at transcriptomic, proteomic, and structural levels, their metabolic and lipidomic maturation had not been systematically mapped. Using untargeted metabolomics and lipidomics together with targeted neurotransmitter profiling across 25–200 days of differentiation, this work provides the first integrated metabolic and lipidomic reference map of CBO development and evaluates how well CBOs recapitulate neurodevelopmental pathways, supporting their use as system models for exposure studies and DNT assessment. The central in vivo component examines early-life hippocampal metabolic and lipidomic alterations in rats perinatally exposed to six structurally diverse EDCs. Targeted endocrine and neurotransmitter profiling combined with untargeted metabolomics and lipidomics revealed both chemical-specific and shared effects across endocrine, neurotransmitter, and lipid-mediated pathways, including alterations in glucocorticoids, neurosteroids, N-acylethanolamines, and PUFA-enriched lipid classes. To assess whether early molecular alterations relate to long-term behavioral impairments, the same developmental exposure cohorts were followed into adulthood for Morris water maze testing. A biologically informed selection of metabolites, neurotransmitters, hormones, and lipid classes was evaluated using group-based comparisons, correlation analyses, and functional ratios. Distinct sex- and domain-specific associations emerged: male acquisition impairments were linked to lower corticosterone, testosterone, T3, and acetylcholine, whereas female reversal impairments were associated with elevated PREG-S, DHEA-S, and GABA. Additional findings for N-acylethanolamines and PUFA-enriched lipid classes suggested broader disruption of neurochemical coordination. Finally, the findings were interpreted in the context of developing DNT AOPs, using metabolomic and lipidomic signatures to propose plausible molecular initiating events (MIEs) and key events (KEs). The data indicated potential enzymatic MIEs, including altered steroid sulfation and desulfation and changes in N-acylethanolamine turnover consistent with FAAH regulation. Nuclear receptor pathways—including glucocorticoid receptors, PPARs, and LXRs—emerged as nodes integrating disruptions in neurosteroids, N-acylethanolamines, and PUFA-enriched lipid classes, with possible GR–PPAR cross-talk. Cholesterol changes suggested involvement of the cholesterol–oxysterol–LXR axis. The thesis concludes by situating these findings within the broader landscape of ED-induced DNT testing and highlighting the metabolically characterized CBO model as a next-generation platform for validating pathways and biomarkers identified in vivo. Future directions include mixture and dose–response testing and stage-specific exposure studies to strengthen mechanistic evidence and support predictive, regulatory-relevant DNT assessment.