Yamashita Akihito, Deguchi Jiro, Honda Yayoi, Yamada Toru, Miyawaki Izuru, Nishimura Yuhei, Tanaka Toshio
Department of Systems Pharmacology, Mie University Graduate School of Medicine, Mie, Japan; Preclinical Research Unit, Sumitomo Dainippon Pharma Co., Ltd., Osaka, Japan.
Preclinical Research Unit, Sumitomo Dainippon Pharma Co., Ltd., Osaka, Japan.
J Pharmacol Toxicol Methods. 2019 Mar-Apr;96:34-45. doi: 10.1016/j.vascn.2018.12.006. Epub 2018 Dec 27.
Oxidative stress plays an important role in drug-induced toxicity. Oxidative stress-mediated toxicities can be detected using conventional animal models but their sensitivity is insufficient, and novel models to improve susceptibility to oxidative stress have been researched. In recent years, gene targeting methods in zebrafish have been developed, making it possible to generate homozygous null mutants. In this study, we established zebrafish deficient in the nuclear factor erythroid 2-related factor 2a (nrf2a), a key antioxidant-responsive gene, and its potential to detect oxidative stress-mediated toxicity was examined.
Nrf2a-deficient zebrafish were generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 technique. The loss of nrf2a function was confirmed by the tolerability to hydrogen peroxide and hydrogen peroxide-induced gene expression profiles being related to antioxidant response element (ARE)-dependent signaling. Subsequently, vulnerability of nrf2a-deficient zebrafish to acetaminophen (APAP)- or doxorubicin (DOX)-induced toxicity was investigated.
Nrf2a-deficient zebrafish showed higher mortality than wild type accompanied by less induction of ARE-dependent genes with hydrogen peroxide treatment. Subsequently, this model showed increased severity and incidence of APAP-induced hepatotoxicity or DOX-induced cardiotoxicity than wild type.
Our results demonstrated that anti-oxidative response might not fully function in this model, and resulted in higher sensitivity to drug-induced oxidative stress. Our data support the usefulness of nrf2a-deficient model as a tool for evaluation of oxidative stress-related toxicity in drug discovery research.
氧化应激在药物诱导的毒性中起重要作用。氧化应激介导的毒性可以使用传统动物模型检测，但它们的敏感性不足，因此人们研究了提高对氧化应激易感性的新型模型。近年来，斑马鱼中的基因靶向方法得到了发展，使得产生纯合缺失突变体成为可能。在本研究中，我们建立了核因子红细胞2相关因子2a（nrf2a）缺陷的斑马鱼，nrf2a是一种关键的抗氧化反应基因，并检测了其检测氧化应激介导毒性的潜力。
使用成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9技术生成nrf2a缺陷的斑马鱼。通过对过氧化氢的耐受性以及过氧化氢诱导的与抗氧化反应元件（ARE）依赖性信号相关的基因表达谱，证实了nrf2a功能的丧失。随后，研究了nrf2a缺陷的斑马鱼对乙酰氨基酚（APAP）或阿霉素（DOX）诱导的毒性的易感性。
nrf2a缺陷的斑马鱼比野生型显示出更高的死亡率，同时过氧化氢处理后ARE依赖性基因的诱导较少。随后，该模型显示出比野生型更高的APAP诱导的肝毒性或DOX诱导的心脏毒性的严重程度和发生率。
我们的结果表明，抗氧化反应在该模型中可能无法充分发挥作用，并导致对药物诱导的氧化应激具有更高的敏感性。我们的数据支持nrf2a缺陷模型作为药物发现研究中评估氧化应激相关毒性的工具的有用性。
