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

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.