Linking nuclear receptor antagonism with neuronal morphology improves detection of developmental neurotoxicants

Brain development is highly dependent on hormonal signaling, hence developmental exposure to endocrine disrupting chemicals (EDCs) is of high concern. However, existing EDC hazard assessment methods lack the capacity to connect developmental neurotoxicity (DNT) to an endocrine mode of action, creating an urgent need for innovative tools addressing endocrine disruption (ED)-induced DNT. We have previously shown the applicability of the immortalized C17.2 murine neural progenitor cells for addressing ED-DNT. In this test sys-tem, the key neurodevelopmental processes (KNDPs) neurite outgrowth and branching were decreased by agonism of retinoic acid receptor (RAR), retinoic X receptor (RXR) and peroxisome proliferator-activated recep-tor (PPAR) β/δ. In the current study we further validate the use, and assess the added value, of the C17.2 test system for detection of ED-induced DNT by testing the effects of 17 chemicals on neuronal morphology and comparing the results to published DNT in vitro toxicity data. Out of the tested chemicals, endosulfan and cy-permethrin decreased neurite outgrowth and branching when tested alone. However, while permethrin, bi-sphenol F (BPF), benzyl butyl phthalate (BBzP) and its metabolite MBzP, perfluorooctanesulfonic acid (PFOS), and 3-phenoxybenzoic acid (3-PBA) did not independently reduce neurite outgrowth, they counteracted RAR, RXR or PPARβ/δ agonist-induced decreases, indicating antagonistic activity. Compared to neurite morphology-related endpoints in ToxCast, C17.2 cells identified more chemicals as active, with increased sensitivity to this endpoint category. These findings confirm that the C17.2 test system is a valuable in vitro model for detecting ED-induced DNT, and that receptor agonist co-treatment enables identification of additional DNT compounds.