Nuclear hormone receptors control fundamental processes of human fetal neurodevelopment: Basis for endocrine disruption assessment.

Despite growing awareness of endocrine disrupting chemicals (EDCs), knowledge gaps remain regarding their effects on human brain development. EDC risk assessment focuses primarily on EATS modalities (estrogens, androgens, thyroid hormones, and steroidogenesis), overlooking the broader range of hormone receptors expressed in the developing brain. This limits the evaluation of chemicals for their potential to cause endocrine disruption-mediated developmental neurotoxicity (ED-DNT). The Neurosphere Assay, an in vitro test method for developmental neurotoxicity (DNT) evaluation, is an integral component of the DNT in vitro testing battery, which has been used to screen a broad domain of environmental chemicals. Here, we define the endocrine-related applicability domain of the Neurosphere Assay by assessing the impact and specificity of 14 hormone receptors on seven key neurodevelopmental processes (KNDPs), neural progenitor cell (NPC) proliferation, migration of radial glia, neurons, and oligodendrocytes, neurite outgrowth, and differentiation of neurons and oligodendrocytes. Comparative analyses in human and rat NPCs of both sexes revealed species- and sex-specific responses. Mechanistic insights were obtained through RNA sequencing and agonist/antagonist co-exposures. Most receptor agonists modulated KNDPs at concentrations in the range of physiologically relevant hormone concentrations. Phenotypic effects induced by glucocorticoid receptor (GR), liver X receptor (LXR), peroxisome proliferator-activated receptor beta/delta (PPARβδ), retinoic acid receptor (RAR) and retinoid X receptor (RXR) activation were counteracted by receptor antagonists, confirming specificity. Transcriptomics highlighted receptor crosstalk and the involvement of conserved developmental pathways (e.g. Notch and Wnt). Species comparisons identified limited concordance in hormone receptor-regulated KNDPs between human and rat NPCs. This study presents novel findings on cellular and molecular hormone actions in human fetal NPCs, highlights major species differences, and illustrates the Neurosphere Assay's relevance for detecting endocrine MoAs, supporting its application in human-based ED-DNT risk assessment.