CRISPR-Generated Nrf2a Loss- and Gain-of-Function Mutants Facilitate Mechanistic Analysis of Chemical Oxidative Stress-Mediated Toxicity in Zebrafish.,Chemical Research in Toxicology

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CRISPR-Generated Nrf2a Loss- and Gain-of-Function Mutants Facilitate Mechanistic Analysis of Chemical Oxidative Stress-Mediated Toxicity in Zebrafish.
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2020-01-08 , DOI: 10.1021/acs.chemrestox.9b00346
Margaret G Mills ₁ , Richard Ramsden ₁ , Eva Y Ma ₁ , Jone Corrales ₂ , Lauren A Kristofco ₂ , W Baylor Steele ₂ , Gavin N Saari ₂ , Fjodor Melnikov ₃ , Jakub Kostal ₄ , Terrance J Kavanagh ₁ , Julie B Zimmerman _{3,

5} , Adelina M Voutchkova-Kostal ₄ , Bryan W Brooks ₂ , Philip Coish ₃ , Paul T Anastas _{3,

6} , Evan Gallagher ₁

Affiliation

The transcription factor Nrf2a induces a cellular antioxidant response and provides protection against chemical-induced oxidative stress, as well as playing a critical role in development and disease. Zebrafish are a powerful model to study the role of Nrf2a in these processes but have been limited by reliance on transient gene knockdown techniques or mutants with only partial functional alteration. We developed several lines of zebrafish carrying different null (loss of function, LOF) or hyperactive (gain of function, GOF) mutations to facilitate our understanding of the Nrf2a pathway in protecting against oxidative stress. The mutants confirmed Nrf2a dependence for induction of the antioxidant genes gclc, gstp, prdx1, and gpx1a and identified a role for Nrf2a in the baseline expression of these genes, as well as for sod1. Specifically, the 4-fold induction of gstp by tert-butyl hydroperoxide (tBHP) in wild type fish was abolished in LOF mutants. In addition, baseline gstp expression in GOF mutants increased by 12.6-fold and in LOF mutants was 0.8-fold relative to wild type. Nrf2a LOF mutants showed increased sensitivity to the acute toxicity of cumene hydroperoxide (CHP) and tBHP throughout the first 4 days of development. Conversely, GOF mutants were less sensitive to CHP toxicity during the first 4 days of development and were protected against the toxicity of both hydroperoxides after 4 dpf. Neither gain nor loss of Nrf2a modulated the toxicity of R-(-)-carvone (CAR), despite the ability of this compound to potently induce Nrf2a-dependent antioxidant genes. Similar to other species, GOF zebrafish mutants exhibited significant growth and survival defects. In summary, these new genetic tools can be used to facilitate the identification of downstream gene targets of Nrf2a, better define the role of Nrf2a in the toxicity of environmental chemicals, and further the study of diseases involving altered Nrf2a function.

中文翻译：

CRISPR 生成的 Nrf2a 功能缺失和获得突变体有助于对斑马鱼化学氧化应激介导的毒性进行机制分析。

转录因子 Nrf2a 诱导细胞抗氧化反应，提供针对化学诱导的氧化应激的保护，并在发育和疾病中发挥关键作用。斑马鱼是研究 Nrf2a 在这些过程中的作用的强大模型，但由于依赖瞬时基因敲低技术或仅部分功能改变的突变体而受到限制。我们开发了几种携带不同无效（功能丧失，LOF）或过度活跃（功能获得，GOF）突变的斑马鱼品系，以帮助我们了解 Nrf2a 通路在防止氧化应激方面的作用。这些突变体证实了 Nrf2a 依赖于诱导抗氧化基因 gclc、gstp、prdx1 和 gpx1a，并确定了 Nrf2a 在这些基因以及 sod1 的基线表达中的作用。具体而言，在野生型鱼中叔丁基过氧化氢（tBHP）对 gstp 的 4 倍诱导作用在 LOF 突变体中被消除。此外，相对于野生型，GOF 突变体中的基线 gstp 表达增加了 12.6 倍，LOF 突变体中的基线 gstp 表达增加了 0.8 倍。 Nrf2a LOF 突变体在发育的前 4 天对氢过氧化异丙苯 (CHP) 和 tBHP 的急性毒性表现出更高的敏感性。相反，GOF 突变体在发育的前 4 天对 CHP 毒性不太敏感，并且在 4 dpf 后受到保护，免受两种氢过氧化物的毒性。尽管 R-(-)-香芹酮 (CAR) 能够有效诱导 Nrf2a 依赖性抗氧化基因，但 Nrf2a 的增加或减少都不会调节该化合物的毒性。与其他物种类似，GOF 斑马鱼突变体表现出显着的生长和生存缺陷。总之，这些新的遗传工具可用于促进Nrf2a下游基因靶标的识别，更好地确定Nrf2a在环境化学物质毒性中的作用，并进一步研究涉及Nrf2a功能改变的疾病。

更新日期：2020-01-08

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