为了道德,经济,和科学原因,动物实验,用于评估化学品在市场上发布之前的潜在神经毒性,需要用新的方法来取代。为了说明新方法方法的使用,人类诱导的多能干细胞衍生的3D模型BrainSpheres急性(48小时)或反复(7天)暴露于胺碘酮(0.625-15µM),一种据报道对神经系统有有害影响的亲脂性抗心律失常药物。使用转录组学评估神经毒性,细胞类型特异性标记的免疫组织化学,以及与脂质代谢有关的各种基因的实时逆转录聚合酶链反应。通过将分布动力学模型与神经毒性读数相结合,我们表明,胺碘酮的神经毒性作用的时间和浓度依赖性增加是由重复给药后胺碘酮的细胞积累驱动的.室性体外分布动力学模型的发展使我们能够预测脑球中细胞相关浓度随时间和不同暴露情况的变化。结果表明,人类细胞本质上对胺碘酮比啮齿动物细胞更敏感。首次在脑细胞中观察到胺碘酮诱导的脂质代谢基因调节。星形胶质细胞似乎是体外最敏感的人脑细胞类型。总之,在重复给药人类诱导多能干细胞衍生的BrainSppheres后,结合体外动力学的隔室模型评估不同分子水平的读数,提供了一种评估神经毒性途径和完善人类化学安全性评估的机制手段。
For ethical, economical, and scientific reasons, animal experimentation, used to evaluate the potential neurotoxicity of chemicals before their release in the market, needs to be replaced by new approach methodologies. To illustrate the use of new approach methodologies, the human induced pluripotent stem cell-derived 3D model BrainSpheres was acutely (48 h) or repeatedly (7 days) exposed to amiodarone (0.625-15 µM), a lipophilic antiarrhythmic drug reported to have deleterious effects on the nervous system. Neurotoxicity was assessed using transcriptomics, the immunohistochemistry of cell type-specific markers, and real-time reverse transcription-polymerase chain reaction for various genes involved in the lipid metabolism. By integrating distribution kinetics modeling with neurotoxicity readouts, we show that the observed time- and concentration-dependent increase in the neurotoxic effects of amiodarone is driven by the cellular accumulation of amiodarone after repeated dosing. The development of a compartmental in vitro distribution kinetics model allowed us to predict the change in cell-associated concentrations in BrainSpheres with time and for different exposure scenarios. The results suggest that human cells are intrinsically more sensitive to amiodarone than rodent cells. Amiodarone-induced regulation of lipid metabolism genes was observed in brain cells for the first time. Astrocytes appeared to be the most sensitive human brain cell type in vitro. In conclusion, assessing readouts at different molecular levels after the repeat dosing of human induced pluripotent stem cell-derived BrainSpheres in combination with the compartmental modeling of in vitro kinetics provides a mechanistic means to assess neurotoxicity pathways and refine chemical safety assessment for humans.