Hormone signaling plays an essential role during fetal life and is vital for brain development. Endocrine-disrupting chemicals can interfere with the hormonal milieu during this critical time-period, disrupting key neurodevelopmental processes. Hence, there is a need for the development of assays that evaluate developmental neurotoxicity (DNT) induced by an endocrine mode of action. Herein, we evaluated the neural progenitor C17.2 cell line as an in vitro test system to aid in the detection of endocrine disruption-induced DNT. For this, C17.2 cells were exposed during 10 days of differentiation to agonists and antagonists of the thyroid hormone (THR), glucocorticoid (GR), retinoic acid (RAR), retinoic x (RXR), oxysterol (LXR), estrogen (ER), androgen (AR), and peroxisome proliferator activated delta (PPARβ/δ) receptors, as well as to the agonist of the vitamin D (VDR) receptor. Upon exposure and differentiation, neuronal morphology (neurite outgrowth and branching) and the percentage of neurons in culture were assessed by immunofluorescence. For this, the cells were stained for βIII-tubulin (neuronal marker). C17.2 cells decreased neurite outgrowth and branching in response to RAR, RXR and PPARβ/δ agonists. Exposure to the GR agonist increased the number of cells differentiating into neurons, while exposure to the RXR agonist had the opposite effect. With this approach, we demonstrate that C17.2 cells are responsive to GR, RAR, RXR, and PPARβ/δ agonists and hence could be useful to develop a test system for hazard assessment of endocrine disruption-induced DNT.
Keywords: endocrine disruptors; in vitro assay development; neurodevelopment; peroxisome proliferator-activated receptor; retinoic acid.
Hormones play a vital role for an organism’s development, including brain development. Endocrine disrupting chemicals (EDCs) interfere with the hormone system. Exposure to EDCs while a fetus is developing can cause a toxic effect on the nervous system called developmental neurotoxicity (DNT). In Europe, the use of chemicals shown to be EDCs may be restricted. Animal tests for developmental neurotoxicity require many animals but cannot determine whether a chemical causes DNT via endocrine disruption or other mechanisms. We have developed a method to identify endocrine disruption-caused DNT using a mouse nerve cell line. In culture, these cells extend long processes called neurites that branch out, and we can measure the length and branching rate of the neurites. We show that this maturation process is dependent on hormonal signals and can therefore be used to identify chemicals that interfere with nerve cell maturation via these signals.