State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau 999078, China.
Center of Reproduction, Development and Aging (CRDA), Faculty of Health Sciences, University of Macau, Taipa, Macau 999078, China.
Toxicol Sci. 2022 Sep 24;189(2):175-185. doi: 10.1093/toxsci/kfac082.
Larval zebrafish is emerging as a new model organism for studying drug-induced liver injury (DILI) with superiorities in visual assessment, genetic engineering as well as high throughput. Metabolic bioactivation to form reactive intermediates is a common event that triggers DILI. This study first addressed the correlation between acetaminophen metabolism and hepatotoxicity in zebrafish larvae (3-day postfertilization) and demonstrated the occurrence of cytochrome P450 enzymes-mediated acetaminophen (APAP) bioactivation at early developmental stage through characterizing the dose-effect (0-1.6 mg/ml) and the time course (0-72 h) of liver injury and metabolism in the AB strain and LiPan transgenic line Tg(lfabp10a: DsRed; elaA:egfp) expressing the liver-specific fluorescent protein. APAP caused multiorgan developmental retardation and elicited dose- and time-dependent hepatotoxicity. Liver imaging revealed significant changes earlier than histological and biochemical measurements. APAP bioactivation in larval zebrafish was first confirmed by the detection of the glutathione conjugate of the reactive intermediate NAPQI (NAPQI-GSH) and subsequent mercapturate derivatives NAPQI-cysteine and NAPQI-N-acetylcysteine after even short (0.5-h postexposure) or low (0.2 mg/ml) APAP exposure. APAP overdose impaired metabolic function, in particular sulfation, whereas facilitated GSH depletion and APAP sulfate excretion. Meanwhile, APAP displayed triphasic accumulation in the larvae, agreeing with fluctuating metabolic capabilities with sulfation dominating the early larval developmental stage. Most importantly, the dose-response effects and time course of APAP accumulation and metabolism agree well with those of the liver injury development. Overall, larval zebrafish has developed mammalian-like metabolic function, enabling it an ideal model organism for high-throughput screening hepatotoxicity and mechanistic study of bioactivation-based DILI.
幼鱼斑马鱼作为一种新的模式生物，在药物诱导的肝损伤（DILI）研究中具有明显优势，包括可视化评估、基因工程以及高通量等方面。代谢物生物活化形成活性中间产物是引发 DILI 的常见事件。本研究首先探讨了幼鱼斑马鱼（受精后 3 天）中对乙酰氨基酚（APAP）代谢与肝毒性之间的相关性，并通过表征 AB 品系和 LiPan 转基因系 Tg(lfabp10a: DsRed; elaA:egfp)中剂量效应（0-1.6mg/ml）和时间进程（0-72 小时）的肝损伤和代谢，证实了早期发育阶段细胞色素 P450 酶介导的对乙酰氨基酚（APAP）生物活化的发生，该品系表达肝特异性荧光蛋白。APAP 导致多器官发育迟缓，并引起剂量和时间依赖性肝毒性。肝成像比组织学和生化测量更早地显示出显著变化。通过检测活性中间产物 NAPQI 的谷胱甘肽缀合物（NAPQI-GSH）以及随后的半胱氨酸和 NAPQI-N-乙酰半胱氨酸的巯基化物，首次证实了幼鱼斑马鱼中 APAP 的生物活化，甚至在短时间（暴露后 0.5 小时）或低剂量（0.2mg/ml）APAP 暴露后也能检测到这些产物。APAP 过量会损害代谢功能，特别是磺化作用，同时促进 GSH 耗竭和 APAP 硫酸盐排泄。同时，APAP 在幼鱼中呈现出三相积累，与具有磺化作用主导早期幼鱼发育阶段的代谢能力波动相一致。最重要的是，APAP 积累和代谢的剂量反应效应和时间进程与肝损伤发展的情况非常吻合。总体而言，幼鱼斑马鱼已发展出类似哺乳动物的代谢功能，使其成为一种理想的高通量筛选肝毒性和基于生物活化的 DILI 机制研究的模式生物。
