Center for Alternative to Animal Testing (CAAT), Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205, USA.
European Commission, Joint Research Centre, European Reference Laboratory - European Centre for the Validation of Alternative Methods (EURL ECVAM), Via Enrico Fermi 2749, Ispra, VA 21027, Italy.
Toxicol Appl Pharmacol. 2018 Sep 1;354:101-114. doi: 10.1016/j.taap.2018.02.003. Epub 2018 Feb 8.
Growing concern suggests that some chemicals exert (developmental) neurotoxicity (DNT and NT) and are linked to the increase in incidence of autism, attention deficit and hyperactivity disorders. The high cost of routine tests for DNT and NT assessment make it difficult to test the high numbers of existing chemicals. Thus, more cost effective neurodevelopmental models are needed. The use of induced pluripotent stem cells (iPSC) in combination with the emerging human 3D tissue culture platforms, present a novel tool to predict and study human toxicity. By combining these technologies, we generated multicellular brain spheroids (BrainSpheres) from human iPSC. The model has previously shown to be reproducible and recapitulates several neurodevelopmental features. Our results indicate, rotenone's toxic potency varies depending on the differentiation status of the cells, showing higher reactive oxygen species (ROS) and higher mitochondrial dysfunction during early than later differentiation stages. Immuno-fluorescence morphology analysis after rotenone exposure indicated dopaminergic-neuron selective toxicity at non-cytotoxic concentrations (1 μM), while astrocytes and other neuronal cell types were affected at (general) cytotoxic concentrations (25 μM). Omics analysis showed changes in key pathways necessary for brain development, indicating rotenone as a developmental neurotoxicant and show a possible link between previously shown effects on neurite outgrowth and presently observed effects on Ca2+ reabsorption, synaptogenesis and PPAR pathway disruption. In conclusion, our BrainSpheres model has shown to be a reproducible and novel tool to study neurotoxicity and developmental neurotoxicity. Results presented here support the idea that rotenone can potentially be a developmental neurotoxicant.
人们越来越担心，某些化学物质会产生（发育）神经毒性（DNT 和 NT），并且与自闭症、注意力缺陷多动障碍发病率的上升有关。DNT 和 NT 评估的常规测试费用高昂，使得很难对大量现有的化学物质进行测试。因此，需要更具成本效益的神经发育模型。诱导多能干细胞（iPSC）与新兴的人类 3D 组织培养平台的结合，为预测和研究人类毒性提供了一种新工具。通过结合这些技术，我们从人类 iPSC 中生成了多细胞脑球体（BrainSpheres）。该模型以前已经显示出可重复性，并再现了几个神经发育特征。我们的结果表明，鱼藤酮的毒性效力取决于细胞的分化状态，在早期分化阶段比晚期分化阶段表现出更高的活性氧（ROS）和更高的线粒体功能障碍。鱼藤酮暴露后的免疫荧光形态分析表明，在非细胞毒性浓度（1 μM）下，多巴胺能神经元具有选择性毒性，而在（一般）细胞毒性浓度（25 μM）下，星形胶质细胞和其他神经元细胞类型受到影响。组学分析显示，大脑发育所必需的关键途径发生变化，表明鱼藤酮是一种发育性神经毒物，并显示出先前观察到的对神经突生长的影响与目前观察到的对 Ca2+重吸收、突触发生和 PPAR 途径破坏的影响之间可能存在联系。总之，我们的 BrainSpheres 模型已被证明是研究神经毒性和发育性神经毒性的一种可靠的新型工具。这里呈现的结果支持鱼藤酮可能是一种发育性神经毒物的观点。
