National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, United States of America.
PLoS One. 2011;6(6):e18540. doi: 10.1371/journal.pone.0018540. Epub 2011 Jun 7.
The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC₅₀) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation.
环境化学物质的广泛存在促使人们需要替代传统的整体动物生物测定方法来进行毒性测试。胚胎干细胞（ES 细胞）为胚胎发育提供了体外模型，也是评估发育毒性的替代方法。在这里，我们使用小鼠 ES 细胞平台评估了 ToxCast™化学文库中的 309 种环境化学物质，这些化学物质主要是食品用农药。ES 细胞在缺乏多能性因子的情况下培养，以促进自发分化，并在 DMSO 溶解的不同浓度的化学物质存在下培养，以测试暴露对分化和细胞毒性的影响。通过 In-Cell Western™分析测量心肌细胞分化（α，β肌球蛋白重链；MYH6/MYH7）和细胞毒性（DRAQ5™/Sapphire700™）。确定分化和细胞毒性终点的半数最大活性浓度（AC₅₀）值，化学文库中有 18%的化学物质在任一终点显示出显著活性。对 ToxCast 第一阶段测定（约 500 个）进行这些效应挖掘，发现了一组化学物质（26 个）的显著关联，这些化学物质扰乱了基于转录的活性并损害了 ES 细胞分化。包括 BMPR2、PAX6 和 OCT1 在内的几个关键发育基因的转录活性增加与 ES 细胞分化减少强烈相关。多个涉及活性氧信号通路的基因（NRF2、ABCG2、GSTA2、HIF1A）与 ES 细胞分化减少也密切相关。从这些数据构建的多元模型显示，ABCG2 转运蛋白的改变是 ES 细胞分化受损的一个强有力预测因子。总之，这些结果提供了一些环境化学物质可能通过破坏 ES 细胞生长和分化来作用的代谢和调节途径的初步特征。
