Genetic Evidence of an Evolutionarily Conserved Role for Nrf2 in the Protection against Oxidative Stress

Abstract

bTranscription 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). Oxidative stress causes damage to multiple cellular components, such as DNA, proteins, and lipids, and is implicated in various human pathological conditions, including cancer, neurodegeneration, and inflammatory diseases (37). Several proteins such as superoxide dismutase (SOD), catalase, glutathione peroxidase (Gpx), peroxiredoxin (Prdx), and the small thiol molecules glutathione (GSH) and thioredoxin (Txn), are directly involved in the removal of oxidative stress. Recent discoveries in the cellular antioxidant system gave rise to the novel concept of "indirect antioxidants," which act through the augmentation of cellular antioxidant capacity by enhancing the gene expression driven by the transcription factor Nrf2 (21, 22). Nrf2 is a basic-region leucine zipper (bZIP) transcription factor that heterodimerizes with small Maf proteins and binds to the antioxidant response element (ARE) within the regulatory region of its target genes (20,27). A variety of cytoprotective genes that encode phase 2 detoxifying enzymes and antioxidant proteins, such as glutathione S-transferases (GST), NAD(P)H:quinone oxidoreductase, and glutamate-cysteine ligase, are induced by Nrf2 via ARE sequences (14). Under basal conditions, Nrf2 is constantly degraded through the ubiquitin-proteasome pathway in a Keap1-dependent manner (28, 50). Upon exposure to electrophiles or oxidative stress, Nrf2 escapes from proteasomal degradation, accumulates in the nucleus, and transcriptionally activates its target genes.We previously isolated zebrafish homologs of Nrf2 and its regulator Keap1 genes (nrf2, keap1a, and keap1b) and demonstrated that ...

Nrf2-dependent protection against acute sodium arsenite toxicity in zebrafish

“…4 zebrafish Nrf2 gene (nrf2a/nfe2l2a) (Mukaigasa et al 2012). Though zebrafish has another Nrf2-related gene, nrf2b, we consider that analysis using nrf2a mutants is sufficient to study the Nrf2 activity in zebrafish, since nrf2b has been demonstrated to neither transactivate typical Nrf2 target genes nor protect larvae against oxidative stress (Timme-Laragy et al 2012).…”

Analysis of Oxidative Stress in Zebrafish Embryos

“…The dtp mutant carries a deficiency for the S-adenosylhomocysteine hydrolase (ahcy) gene and its characterization revealed that the loss of Ahcy activity causes liver degeneration by influencing ROS levels 49 . Equivalently, the zebrafish mutant nrf2 fh318 , carrying a loss-of-function mutation of the transcriptional factor nrf2 gene, has been regarded with considerable interest to investigate the protective role of this antioxidant gene in lower vertebrates 50 .…”

Role of Cytochrome P450 Monooxygenase in Carcinogen and Chemotherapeutic Drug Metabolism

“…Many are members of the steroid hormone nuclear receptor family (Conney, 2003;Evans & Mangelsdorf, 2014), while others are members of the basic helix-loop-helix transcription family (AHR) (Denison & Nagy, 2003) or basic leucine zipper (bZIP) protein family (nuclear factor (erythroid-derived 2)-like 2, Nrf2) (Mukaigasa et al, 2012;Taguchi, Motohashi, & Yamamoto, 2011). Table 3 summarizes these receptors involved in the induction of CYPs, including a discussion of their target genes.…”