Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI.

中文翻译：

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高通量共聚焦成像的差异化3D肝样球状细胞应激反应报告基因，用于鉴定药物引起的肝损伤责任。

适应性应激反应途径在适应和逆境之间的转换中起关键作用，在药物性肝损伤中很重要。以前，我们已经建立了HepG2荧光蛋白报道平台，以监测药物治疗后的适应性应激反应激活。HepG2细胞通常用于高通量初级毒性筛查，但这些细胞中的代谢能力较低，因此反复进行剂量毒性试验本来就很困难。在这里，我们应用了基于细菌人工染色体的GFP报告基因细胞系，它们代表Nrf2激活（Srxn1-GFP和NQO1-GFP），未折叠的蛋白应答（BiP-GFP和Chop-GFP）和DNA损伤应答（p21-GFP和Btg2 -GFP）作为长期分化的3D肝样球状培养物。所有HepG2 GFP报告基因系都分化为与野生型HepG2细胞相似的3D球体。我们使用高通量共聚焦显微镜和一组DILI化合物的参照物，系统地优化了单个球体中GFP报告子活性的自动化成像和定量，该参比物在原始人类肝细胞的转录水平上激活了这三个应激反应途径。在这六个3D GFP报道分子中，在单个48小时治疗或每日6天重复治疗中进一步测试了33种具有确定的DILI责任的化合物。在重复治疗6天后，观察到最强的应激反应激活，其中BiP和Srxn1-GFP报道分子反应最灵敏，并鉴定出特定的严重DILI发作化合物。在二维（2D）单层中未显示GFP报告基因激活的化合物在3D球体中显示了GFP报告基因应激反应激活。我们的数据表明，BAC-GFP HepG2细胞应力报告基因在分化的3D球体中的应用是一种有前景的策略，用于基于机理的化合物识别具有DILI责任的化合物。