原代人肝细胞球体中揭示的慢性 Fialuridine 肝毒性机制。,Toxicological Sciences

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原代人肝细胞球体中揭示的慢性 Fialuridine 肝毒性机制。
Toxicological Sciences ( IF 3.4 ) Pub Date : 2019-10-01 , DOI: 10.1093/toxsci/kfz195
Delilah F G Hendriks ₁ , Tracey Hurrell ₁ , Julia Riede ₁ , Muriëlle van der Horst ₁ , Sarianna Tuovinen ₁ , Magnus Ingelman-Sundberg ₁

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药物肝毒性通常延迟发作。一个典型案例是氟尿嘧啶的慢性肝毒性，由于临床前研究未能确定这种人类特异性肝毒性，导致临床试验中数名患者死亡。常规的临床前体外模型主要用于评估急性药物毒性的风险。在这里，我们评估了原代人肝细胞 (PHH) 的 3D 球体培养物在评估慢性药物肝毒性事件中的效用，以氟尿苷为例。氟尿嘧啶的毒性只有在反复接触 7 天后才能检测到。很容易确定临床表现，包括活性氧的形成、脂质积累和细胞凋亡的诱导。沉默人平衡核苷转运蛋白 1 (ENT1) 的表达或活性，参与 fialuridine 的线粒体转运，适度保护 PHH 球体免受 fialuridine 毒性。通过沉默胸苷激酶 2 (TK2) 干扰法尿苷磷酸化为活性三磷酸代谢物提供了实质性保护，而同时沉默 ENT1 和 TK2 提供了近乎完全的保护。线粒体 DNA 编码基因表达的减少提示了氟尿嘧啶诱导的线粒体功能障碍，这与毒性的发生相关，并且在 ENT1 和 TK2 的同时沉默下被阻止。此外，干扰对脱氧核糖核苷三磷酸 (dNTP) 库稳态至关重要的核糖核苷酸还原酶 (RNR) 的表达或活性会导致选择性增强氟尿嘧啶毒性。

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Mechanisms of Chronic Fialuridine Hepatotoxicity as Revealed in Primary Human Hepatocyte Spheroids.

Drug hepatotoxicity is often delayed in onset. An exemplar case is the chronic nature of fialuridine hepatotoxicity, which resulted in the deaths of several patients in clinical trials as preclinical studies failed to identify this human-specific hepatotoxicity. Conventional preclinical in vitro models are mainly designed to evaluate the risk of acute drug toxicity. Here, we evaluated the utility of 3D spheroid cultures of primary human hepatocytes (PHHs) to assess chronic drug hepatotoxicity events using fialuridine as an example. Fialuridine toxicity was only detectable after 7 days of repeated exposure. Clinical manifestations, including reactive oxygen species formation, lipid accumulation, and induction of apoptosis, were readily identified. Silencing the expression or activity of the human equilibrative nucleoside transporter 1 (ENT1), implicated in the mitochondrial transport of fialuridine, modestly protected PHH spheroids from fialuridine toxicity. Interference with the phosphorylation of fialuridine into the active triphosphate metabolites by silencing of thymidine kinase 2 (TK2) provided substantial protection, whereas simultaneous silencing of ENT1 and TK2 provided near-complete protection. Fialuridine-induced mitochondrial dysfunction was suggested by a decrease in the expression of mtDNA-encoded genes, which correlated with the onset of toxicity and was prevented under the simultaneous silencing of ENT1 and TK2. Furthermore, interference with the expression or activity of ribonucleotide reductase (RNR), which is critical to deoxyribonucleoside triphosphate (dNTP) pool homeostasis, resulted in selective potentiation of fialuridine toxicity. Our findings demonstrate the translational applicability of the PHH 3D spheroid model for assessing drug hepatotoxicity events which manifest only under chronic exposure conditions.

更新日期：2019-11-01

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