Genetic evidence of an evolutionarily conserved role for Nrf2 in the protection against oxidative stress

Abstract

Transcription factor Nrf2 is considered a master regulator of antioxidant defense in mammals. However, it is unclear whether this concept is applicable to nonmammalian vertebrates, because no animal model other than Nrf2 knockout mice has been generated to examine the effects of Nrf2 deficiency. Here, we characterized a recessive loss-of-function mutant of Nrf2 (nrf2(fh318)) in a lower vertebrate, the zebrafish (Danio rerio). In keeping with the findings in the mouse model, nrf2(fh318) mutants exhibited reduced induction of the Nrf2 target genes in response to oxidative stress and electrophiles but were viable and fertile, and their embryos developed normally. The nrf2(fh318) larvae displayed enhanced sensitivity to oxidative stress and electrophiles, especially peroxides, and pretreatment with an Nrf2-activating compound, sulforaphane, decreased peroxide-induced lethality in the wild type but not nrf2(fh318) mutants, indicating that resistance to oxidative stress is highly dependent on Nrf2 functions. These results reveal an evolutionarily conserved role of vertebrate Nrf2 in protection against oxidative stress. Interestingly, there were no significant differences between wild-type and nrf2(fh318) larvae with regard to their sensitivity to superoxide and singlet oxygen generators, suggesting that the importance of Nrf2 in oxidative stress protection varies based on the type of reactive oxygen species (ROS).

Fig 1

gstp1 expression in nrf2^fh318 mutant larvae. At 5 dpf, larvae were treated with 100 μM DEM or 1 mM H₂O₂ for 6 h, and their gstp1 expression was analyzed by whole-mount in situ hybridization. Lateral views, with the anterior side on the left, are shown. Black and white arrowheads indicate positive and negative expression in gills, respectively.

Fig 2

Survival rate of nrf2^fh318 mutant larvae after exposure to oxidative stress-inducing agents, electrophiles, or their metabolic precursors. At 4 dpf, larvae were exposed to 1 mM H₂O₂ (A), 0.8 mM tBHP (B), 0.6 mM paraquat (C), 2.4 μM menadione (D), 0.1 μM rose bengal (E), 7.5 mM acetaminophen (F), or 80 μM sulforaphane (G) for 120 h or to 0.1 μM MeHg for 24 h, followed by a change to fresh medium without MeHg for an additional 96 h (H), and the survival rates were calculated using the Kaplan-Meier method. The data from at least three experiments were combined.

Fig 3

H₂O₂-dependent induction of ROS-scavenging enzyme genes. A semiquantitative RT-PCR (A) and quantitative real-time RT-PCR (B) analyses of indicated genes were performed using total RNA mixtures from whole bodies of 20 nrf2^fh318/fh318 or wild-type larvae treated with 1 mM H₂O₂ for 6 h at 5 dpf or not treated. The expression of ef1α was used to standardize the amount of cDNA. Error bars indicate standard deviations from three independent experiments.

Fig 4

Effects of sulforaphane pretreatment on the survival rate of H₂O₂-treated larvae. (A) Experimental scheme. After pretreatment with 40 μM sulforaphane for 12 h, 4-dpf larvae were treated with 1.5 mM H₂O₂ for 120 h. (B) Survival curve of H₂O₂-treated larvae with or without sulforaphane pretreatment. The data were combined from four experiments. (C) Survival rates of wild-type, nrf2^fh318/+, and nrf2^fh318/fh318 larvae at 24 h postexposure. Data are means ± standard deviations from four independent experiments. (D) Experimental scheme of BSO pretreatment. Wild-type AB larvae were pretreated with 1 mM BSO for 24 h before 1.5 mM H₂O₂ treatment at 4 dpf. (E) Survival curve of H₂O₂-treated larvae with or without BSO and sulforaphane (SF) pretreatment.