CVRM Safety, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Gothenburg, Sweden.
Functional and Mechanistic Safety, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Gothenburg, Sweden.
Arch Toxicol. 2020 Apr;94(4):1251-1263. doi: 10.1007/s00204-020-02682-w. Epub 2020 Feb 28.
In addition to hepatocytes, the liver comprises a host of specialised non-parenchymal cells which are important to consider in the development of in vitro models which are both physiologically and toxicologically relevant. We have characterized a 3D co-culture system comprising primary human hepatocytes (PHH) and non-parenchymal cells (NPC) and applied it to the investigation of acetaminophen-induced toxicity. Firstly, we titrated ratios of PHH:NPC and confirmed the presence of functional NPCs via both immunohistochemistry and activation with both LPS and TGF-β. Based on these data we selected a ratio of 2:1 PHH:NPC for further studies. We observed that spheroids supplemented with NPCs were protected against acetaminophen (APAP) toxicity as determined by ATP (up to threefold difference in EC at day 14 compared to hepatocytes alone) and glutathione depletion, as well as miR-122 release. APAP metabolism was also altered in the presence of NPCs, with significantly lower levels of APAP-GSH detected. Expression of several CYP450 enzymes involved in the bioactivation of APAP was also lower in NPC-containing spheroids. Spheroids containing NPCs also expressed higher levels of miRNAs which have been implicated in APAP-induced hepatotoxicity, including miR-382 and miR-155 which have potential roles in liver regeneration and inflammation, respectively. These data indicate that the interaction between hepatocytes and NPCs can have significant metabolic and toxicological consequences important for the correct elucidation of hepatic safety mechanisms.
除了肝细胞，肝脏还包含许多特化的非实质细胞，在开发具有生理和毒理学相关性的体外模型时，这些细胞是需要考虑的。我们已经对包含原代人肝细胞（PHH）和非实质细胞（NPC）的 3D 共培养系统进行了特征描述，并将其应用于对乙酰氨基酚诱导的毒性的研究。首先，我们滴定了 PHH：NPC 的比例，并通过免疫组织化学和 LPS 和 TGF-β的激活确认了功能性 NPC 的存在。基于这些数据，我们选择了 2：1 PHH：NPC 的比例进行进一步研究。我们观察到，补充 NPC 的球体能够抵抗乙酰氨基酚（APAP）毒性，这可以通过 ATP（与仅肝细胞相比，第 14 天的 EC 差异高达三倍）和谷胱甘肽耗竭以及 miR-122 释放来确定。在 NPC 的存在下，APAP 代谢也发生了改变，检测到的 APAP-GSH 水平明显降低。参与 APAP 生物活化的几种 CYP450 酶的表达在含有 NPC 的球体中也较低。含有 NPC 的球体还表达了更高水平的 miRNA，这些 miRNA 与 APAP 诱导的肝毒性有关，包括 miR-382 和 miR-155，它们分别在肝再生和炎症中具有潜在作用。这些数据表明，肝细胞和 NPC 之间的相互作用可能会产生重要的代谢和毒理学后果，这对于正确阐明肝脏安全机制非常重要。
