Rapid Assay Development Branch, Biomolecular and Computational Toxicology Division, Center for Computational Toxicology and Exposure, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
Oak Ridge Institute for Science and Education, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
Toxicol Sci. 2022 Feb 28;186(1):118-133. doi: 10.1093/toxsci/kfab151.
Development of in vitro new approach methodologies has been driven by the need for developmental neurotoxicity (DNT) hazard data on thousands of chemicals. The network formation assay characterizes DNT hazard based on changes in network formation but provides no mechanistic information. This study investigated nervous system signaling pathways and upstream physiological regulators underlying chemically induced neural network dysfunction. Rat primary cortical neural networks grown on microelectrode arrays were exposed for 12 days in vitro to cytosine arabinoside, 5-fluorouracil, domoic acid, cypermethrin, deltamethrin, or haloperidol as these exposures altered network formation in previous studies. RNA-seq from cells and gas chromatography/mass spectrometry analysis of media extracts collected on days in vitro 12 provided gene expression and metabolomic identification, respectively. The integration of differentially expressed genes and metabolites for each neurotoxicant was analyzed using ingenuity pathway analysis. All 6 compounds altered gene expression that linked to developmental disorders and neurological diseases. Other enriched canonical pathways overlapped among compounds of the same class; eg, genes and metabolites altered by both cytosine arabinoside and 5-fluorouracil exposures are enriched in axonal guidance pathways. Integrated analysis of upstream regulators was heterogeneous across compounds, but identified several transcriptomic regulators including CREB1, SOX2, NOTCH1, and PRODH. These results demonstrate that changes in network formation are accompanied by transcriptomic and metabolomic changes and that different classes of compounds produce differing responses. This approach can enhance information obtained from new approach methodologies and contribute to the identification and development of adverse outcome pathways associated with DNT.
体外新方法学的发展是由对数千种化学物质的发育神经毒性（DNT）危害数据的需求驱动的。网络形成测定法根据网络形成的变化来描述 DNT 危害，但不提供任何机制信息。本研究调查了化学诱导的神经网络功能障碍的神经信号通路和上游生理调节剂。在体外培养 12 天的大鼠原代皮质神经网络在微电极阵列上生长，然后暴露于胞嘧啶阿拉伯糖苷、5-氟尿嘧啶、海兔毒素、氯菊酯、溴氰菊酯或氟哌啶醇中，因为这些暴露在以前的研究中改变了网络形成。细胞的 RNA-seq 和体外第 12 天收集的培养基提取物的气相色谱/质谱分析分别提供了基因表达和代谢组学鉴定。使用 Ingenuity 通路分析分析了每种神经毒物的差异表达基因和代谢物的整合。这 6 种化合物均改变了与发育障碍和神经疾病相关的基因表达。相同类别化合物之间存在重叠的其他丰富的经典途径；例如，胞嘧啶阿拉伯糖苷和 5-氟尿嘧啶暴露改变的基因和代谢物在轴突导向途径中丰富。跨化合物的上游调节剂综合分析是异构的，但鉴定了几个转录组调节剂，包括 CREB1、SOX2、NOTCH1 和 PRODH。这些结果表明，网络形成的变化伴随着转录组和代谢组的变化，并且不同类别的化合物产生不同的反应。这种方法可以增强从新方法学中获得的信息，并有助于识别和开发与 DNT 相关的不良结局途径。
