激素信号在胎儿生命中起着至关重要的作用,对大脑发育至关重要。在这个关键的时间段内,破坏内分泌的化学物质会干扰荷尔蒙环境,破坏关键的神经发育过程。因此,需要开发评估由内分泌作用模式诱导的发育神经毒性(DNT)的测定法。在这里,我们评估了神经祖细胞C17的适用性。2个细胞系,作为体外测试系统,以帮助检测内分泌干扰(ED)诱导的DNT。为此,C17.2细胞在分化的10天期间暴露于甲状腺激素(Thr)的激动剂和拮抗剂,糖皮质激素(Gr),视黄酸(Rar),视黄酸x(Rxr),氧固醇(Lxr),雌激素(Er),雄激素(Ar),和过氧化物酶体增殖物激活的δ(Pparβ/δ)受体,以及维生素D(Vdr)受体的激动剂。暴露和分化后,神经元形态(神经突生长和分支),并通过免疫荧光评估培养物中神经元的百分比。为此,将细胞与Hoechst一起孵育(核染色)并对βIII-微管蛋白(神经元标记物)进行染色。C17.2细胞对Rar有反应,Rxr和Pparβ/δ激动剂减少神经突生长和分支。此外,暴露于Gr激动剂增加了分化为神经元的细胞数量,而暴露于Rxr激动剂具有相反的效果。通过这种方法,我们已经确定C17.2细胞对Gr有反应,rar,Rxr,和Pparβ/δ激动剂,因此有助于开发ED引起的DNT的危害评估测试系统。
内分泌干扰化学物质(EDC)干扰激素信号。由于激素对生物体的发育起着至关重要的作用,EDC暴露是高度关注的问题。在欧洲法规中,如果化学物质的毒性是由激素干扰介导的,则可以限制其使用。许多EDC影响大脑发育。然而,在动物试验中,无法证明化学物质通过内分泌干扰(ED)诱导发育神经毒性(DNT)。此外,监管的DNT测试需要大量的动物。因此,迫切需要体外测试系统来鉴定ED诱导的DNT。在此,我们介绍了基于鼠神经祖细胞系C17.2的这种方法的开发,该方法可以模拟神经元分化过程。我们表明C17.2细胞的分化对类视黄醇敏感,糖皮质激素,和过氧化物酶体增殖物激活受体信号中断,从而为识别ED诱导的DNT提供了一种替代方法。
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 applicability of the neural progenitor C17. 2 cell-line, as an in vitro test system to aid in the detection of endocrine disruption (ED) 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), oxysterols (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 incubated with Hoechst (nuclear staining) and stained for βIII-tubulin (neuronal marker). The C17.2 cells were responsive to the Rar, Rxr and Pparβ/δ agonists which decreased neurite outgrowth and branching. Additionally, 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 have identified that the C17.2 cells are responsive to Gr, Rar, Rxr, and Pparβ/δ agonists, hence contributing to the development of test systems for hazard assessment of ED-induced DNT.
Endocrine disrupting chemicals (EDCs) interfere with hormonal signaling. As hormones play a vital role for an organism’s development, EDC exposure is of high concern. In European regulations, the use of a chemical can be restricted if its toxicity is mediated by hormonal interference. A number of EDCs affect brain development. However, in animal tests, it is impossible to prove that a chemical induces developmental neurotoxicity (DNT) via endocrine disruption (ED). Furthermore, the regulatory DNT tests require large amounts of animals. Thus, there is an urgent need for in vitro test systems to identify ED-induced DNT. Herein we present the development of such a method based on the murine neural progenitor cell-line C17.2 with which neuronal differentiation processes can be mimicked. We show that differentiation of C17.2 cells are sensitive to retinoid, glucocorticoid, and peroxisome proliferator activated receptor signaling disruption, thus providing an alternative method for identifying ED-induced DNT.