Selection of appropriate fit-for-purpose in vitro and in silico models is critical for non-animal safety assessment of chemical-induced hepatoxicity. The present study evaluated the feasibility of integrating in vitro data from three-dimensionally (3D)-cultured HepaRG cells and physiologically based pharmacokinetic (PBPK) modeling to predict chemical-induced liver toxicity. A 3D organoid culture system was established using an ultralow attachment method. HepaRG cells cultured in a two-dimensional (2D) monolayer and under 3D conditions were exposed to acetaminophen (APAP) at concentrations of 0.16-20 mM. The results showed that the viability of both 3D- and 2D cultured cells was significantly decreased by APAP in a concentration-dependent manner. Furthermore, 3D cultures were more sensitive to APAP-induced mitochondrial damage than 2D cultures were, based on measurements of mitochondrial superoxide accumulation and mitochondrial membrane potential loss. PBPK simulations using nominal in vitro concentrations showed that the APAP concentration eliciting mitochondrial damage was closer to the predicted peak liver concentration in humans in 3D cultures than it was in 2D cultures. In summary, our results suggest that combining in vitro data from 3D HepaRG cultures and PBPK modeling provides a promising tool for assessment of liver injury.

目的 利用人肝癌HepaRG细胞系建立3D肝细胞模型,并应用该模型对抗糖尿病药物曲格列酮所诱导的肝毒性进行评价.方法 采用低吸附法构建3D HepaRG细胞/组织模型.3D HepaRG细胞给予曲格列酮(3.125、6.25、12.5、25和50 μmol/L)处理不同时间后,应用Alamar blue法测定细胞存活率,高内涵成像分析检测线粒体活性氧自由基、线粒体膜电位和线粒体丰度,以评价曲格列酮引起的肝细胞损伤.结果 以1000或3 000个细胞/孔密度接种细胞,细胞在第3天自发聚集形成致密的球形,第7天后可形成稳定球体.曲格列酮可剂量依赖性地降低3D HepaRG细胞的存活率,诱导3D细胞线粒体损伤,表现为线粒体活性氧自由基生成增加,线粒体膜电位和线粒体丰度下降.结论 采用低吸附法成功构建了3D HepaRG模型并应用该模型阐明曲格列酮诱导的肝毒性特征,提示3D HepaRG模型在评估药物性肝损伤等肝毒性测试中具有潜在的重要应用价值.

肝脏是机体代谢外源性化学物的主要场所,也是化学物及其代谢产物毒作用的重要靶器官.为了更加快速、准确地对化学物引起的肝损伤进行评估,选择贴近人体的细胞模型和培养方法至关重要.近年来研究发展了多种人源体外肝细胞模型,其中新兴的三维(3D)肝细胞体外模型具有类似体内肝脏表型、代谢能力,并适于长期体外培养,为药物等化学物的肝毒性测试提供了有力的体外评价工具.本文主要介绍目前常用的肝细胞模型的特点,以及球体模型、生物反应器、3D打印和肝脏芯片等3D培养系统,概述了这些模型在化学性肝损伤评估中的应用进展.

Background: Zinc oxide nanoparticles (ZnO NPs) are one of the most widely used nanomaterials in a variety of fields such as industrial, pharmaceutical, and household applications. Increasing evidence suggests that ZnO NPs could elicit unignorable harmful effect to the cardiovascular system, but the potential deleterious effects to human cardiomyocytes remain to be elucidated. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been increasingly used as a promising in vitro model of cardiomyocyte in various fields such as drug cardiac safety evaluation. Herein, the present study was designed to elucidate the cardiac adverse effects of ZnO NPs and explore the possible underlying mechanism using hiPSC-CMs. Methods: ZnO NPs were characterized by transmission electron microscopy and dynamic light scattering. The cytotoxicity induced by ZnO NPs in hiPSC-CMs was evaluated by determination of cell viability and lactate dehydrogenase release. Cellular reactive oxygen species (ROS) and mitochondrial membrane potential were measured by high-content analysis (HCA). Mitochondrial biogenesis was assayed by detection of mtDNA copy number and PGC-1 alpha pathway. Moreover, microelectrode array techniques were used to investigate cardiac electrophysiological alterations. Results: We demonstrated that ZnO NPs concentration- and time-dependently elicited cytotoxicity in hiPSC-CMs. The results from HCA revealed that ZnO NPs exposure at low-cytotoxic concentrations significantly promoted ROS generation and induced mitochondrial dysfunction. We further demonstrated that ZnO NPs could impair mitochondrial biogenesis and inhibit PGC-1 alpha pathway. In addition, ZnO NPs at insignificantly cytotoxic concentrations were found to trigger cardiac electrophysiological alterations as evidenced by decreases of beat rate and spike amplitude. Conclusion: Our findings unveiled the potential harmful effects of ZnO NPs to human cardiomyocytes that involve mitochondrial biogenesis and the PGC-1 alpha pathway that could affect cardiac electrophysiological function.

In vitro to in vivo extrapolation (IVIVE) for next-generation risk assessment (NGRA) of chemicals requires computational modeling and faces unique challenges. Using mitochondria-related toxicity data of troglitazone (TGZ), a prototype drug known for liver toxicity, from HepaRG, HepG2, HC-04, and primary human hepatocytes, we explored inherent uncertainties in IVIVE, including cell models, cellular response endpoints, and dose metrics. A human population physiologically-based pharmacokinetic (PBPK) model for TGZ was developed to predict in vivo doses from in vitro point-of-departure (POD) concentrations. Compared to the 200-800 mg/d dose range of TGZ where liver injury was observed clinically, the predicted POD doses for the mean and top one percentile of the PBPK population were 28-372 and 15-178 mg/d respectively based on C-max, dosimetry, and 185-2552 and 83-1010 mg/d respectively based on AUC. In conclusion, although with many uncertainties, integrating in vitro assays and PBPK modeling is promising in informing liver toxicity-inducing TGZ doses.