Oxidative stress inducers potentiate 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated pre-cardiac edema in larval zebrafish.

We reported the involvement of oxidative stress and prostaglandins including thromboxane and prostacyclin in pre-cardiac edema (early edema) caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While the involvement of oxidative stress in TCDD-induced toxicity has been frequently reported, the mechanism of its action is still unclear. In the present study, oxidative stress inducers including paraquat, hydrogen peroxide (H2O2) and rotenone augmented early edema (edema) induced by a low concentration of TCDD (0.1 ppb) at 55 hr post fertilization (hpf), while each of them alone did not cause edema. Edema caused by TCDD plus oxidative stress inducers was almost abolished by antioxidants, an antagonist for thromboxane receptor (ICI-192,605) and an agonist for prostacyclin receptor (beraprost), suggesting that the site of action of these inducers was in the regular signaling pathway after activation of aryl hydrocarbon receptor type 2 (AHR2) by TCDD. Oxidative stress inducers also enhanced edema caused by an agonist for the thromboxane receptor (U46619), and the enhancement was also inhibited by antioxidants. Sulforaphane and auranofin, activators of Nrf2 that is a master regulator of anti-oxidative response, did not affect U46619-evoked edema but almost abolished TCDD-induced edema and potentiation by paraquat in both TCDD- and U46619-induced edema. Taken together, the results suggest that oxidative stress augments pre-cardiac edema caused by TCDD via activation of thromboxane receptor-mediated signaling in developing zebrafish. As paraquat and other oxidative stress inducers used also are environmental pollutants, interaction between dioxin-like compounds and exogenous source of oxidative stress should also be considered.

Evaluation of Antioxidant Activity of Spice-Derived Phytochemicals Using Zebrafish.

Various dietary phytochemicals seem to display antioxidant activity through the NF-E2-related factor 2 (Nrf2) pathway. However, few studies have demonstrated its antioxidant effect and Nrf2 dependency at the animal level. We constructed a zebrafish-based assay system to analyze the in vivo antioxidant activity of phytochemicals and examined the activity of 10 phytochemicals derived from spices, using this system as a pilot study. Hydrogen peroxide and arsenite were used as oxidative stressors, and Nrf2 dependency was genetically analyzed using an Nrf2-mutant zebrafish line. The activities of curcumin, diallyl trisulfide and quercetin were involved in the reduction of hydrogen peroxide toxicity, while those of cinnamaldehyde, isoeugenol and 6-(methylsulfinyl)hexyl isothiocyanate were involved in the reduction of arsenite toxicity. The antioxidant activities of these phytochemicals were all Nrf2 dependent, with the exception of cinnamaldehyde, which showed strong antioxidant effects even in Nrf2-mutant zebrafish. In summary, we succeeded in constructing an assay system to evaluate the in vivo antioxidant activity of various phytochemicals using zebrafish larvae. Using this system, we found that each spice-derived phytochemical has its own specific property and mechanism of antioxidant action.

Nrf2 activation attenuates genetic endoplasmic reticulum stress induced by a mutation in the phosphomannomutase 2 gene in zebrafish.

Nrf2 plays critical roles in animals' defense against electrophiles and oxidative stress by orchestrating the induction of cytoprotective genes. We previously isolated the zebrafish mutant it768, which displays up-regulated expression of Nrf2 target genes in an uninduced state. In this paper, we determine that the gene responsible for it768 was the zebrafish homolog of phosphomannomutase 2 (Pmm2), which is a key enzyme in the initial steps of N-glycosylation, and its mutation in humans leads to PMM2-CDG (congenital disorders of glycosylation), the most frequent type of CDG. The pmm2it768 larvae exhibited mild defects in N-glycosylation, indicating that the pmm2it768 mutation is a hypomorph, as in human PMM2-CDG patients. A gene expression analysis showed that pmm2it768 larvae display up-regulation of endoplasmic reticulum (ER) stress, suggesting that the activation of Nrf2 was induced by the ER stress. Indeed, the treatment with the ER stress-inducing compounds up-regulated the gstp1 expression in an Nrf2-dependent manner. Furthermore, the up-regulation of gstp1 by the pmm2 inactivation was diminished by knocking down or out double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), one of the main ER stress sensors, suggesting that Nrf2 was activated in response to the ER stress via the PERK pathway. ER stress-induced activation of Nrf2 was reported previously, but the results have been controversial. Our present study clearly demonstrated that ER stress can indeed activate Nrf2 and this regulation is evolutionarily conserved among vertebrates. Moreover, ER stress induced in pmm2it768 mutants was ameliorated by the treatment of the Nrf2-activator sulforaphane, indicating that Nrf2 plays significant roles in the reduction of ER stress.

The possible repositioning of an oral anti-arthritic drug, auranofin, for Nrf2-activating therapy: The demonstration of Nrf2-dependent anti-oxidative action using a zebrafish model.

The Nrf2 pathway is a biological defense system against oxidative stress. The pharmacological activation of the Nrf2 pathway is a promising therapy for oxidative stress-related diseases, but it has been challenging to find an Nrf2 activator with acceptable toxicity. To circumvent this problem, we focused on an already approved oral anti-arthritic drug, auranofin that has been reported to have the potential to activate Nrf2. We used a zebrafish model to investigate whether auranofin has protective action against oxidative stress in vivo. Auranofin pre-treatment considerably improved the survival of zebrafish larvae that were challenged with a lethal dose of hydrogen peroxide. This protective effect was not observed in an Nrf2 mutant zebrafish strain, suggesting that the activation of the biological defense against oxidative stress was Nrf2-dependent. Auranofin-induced protection was further tested by challenges with redox-active heavy metals. A clear protective effect was observed against arsenite, a highly redox-reactive toxicant. In addition, this effect was also demonstrated to be Nrf2-dependent based on the analysis of an Nrf2 mutant strain. These results clearly demonstrate the anti-oxidative action of auranofin and encourage the repositioning of auranofin as a drug that improves oxidative stress-related pathology.

Conservation of the Keap1-Nrf2 System: An Evolutionary Journey through Stressful Space and Time.

The Keap1-Nrf2 system is an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress. Its regulatory mechanisms, e.g., stress-sensing mechanism, proteasome-based regulation of Nrf2 activity and selection of target genes, have been elucidated mainly in mammals. In addition, emerging model animals, such as zebrafish, fruit fly and Caenorhabditis elegans, have been shown to have similar anti-stress systems to mammals, suggesting that analogous defense systems are widely conserved throughout the animal kingdom. Experimental evidence in lower animals provides important information beyond mere laboratory-confined utility, such as regarding how these systems transformed during evolution, which may help characterize the mammalian system in greater detail. Recent advances in genome projects of both model and non-model animals have provided a great deal of useful information toward this end. We herein review the research on Keap1-Nrf2 and its analogous systems in both mammals and lower model animals. In addition, by comparing the amino acid sequences of Nrf2 and Keap1 proteins from various species, we can deduce the evolutionary history of the anti-stress system. This combinatorial approach using both experimental and genetic data will suggest perspectives of approach for researchers studying the stress response.

Nrf2-dependent protection against acute sodium arsenite toxicity in zebrafish.

Transcription factor Nrf2 induces a number of detoxifying enzymes and antioxidant proteins to confer protection against the toxic effects of a diverse range of chemicals including inorganic arsenicals. Although a number of studies using cultured cells have demonstrated that Nrf2 has a cell-protective function against acute and high-dose arsenic toxicity, there is no clear in vivo evidence of this effect. In the present study, we genetically investigated the protective role of Nrf2 against acute sodium arsenite toxicity using the zebrafish Nrf2 mutant, nrf2a(fh318). After treatment with 1mM sodium arsenite, the survival of nrf2a(fh318) larvae was significantly shorter than that of wild-type siblings, suggesting that Nrf2 protected the zebrafish larvae against high-dose arsenite exposure. To understand the molecular basis of the Nrf2-dependent protection, we analyzed the gene expression profiles after arsenite exposure, and found that the genes involved in the antioxidative function (prdx1 and gclc), arsenic metabolism (gstp1) and xenobiotic elimination (abcc2) were induced in an Nrf2-dependent manner. Furthermore, pre-treatment with sulforaphane, a well-known Nrf2 activator improved the survival of zebrafish larvae after arsenic exposure. Based on these results, we concluded that Nrf2 plays a fundamental and conserved role in protection against acute sodium arsenite toxicity.

Conservation of the Nrf2-Mediated Gene Regulation of Proteasome Subunits and Glucose Metabolism in Zebrafish.

The Keap1-Nrf2 system is an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress. Besides the exogenous stress response, Nrf2 has been found to regulate numerous cellular functions, including protein turnover and glucose metabolism; however, the evolutionary origins of these functions remain unknown. In the present study, we searched for novel target genes associated with the zebrafish Nrf2 to answer this question. A microarray analysis of zebrafish embryos that overexpressed Nrf2 revealed that 115 candidate genes were targets of Nrf2, including genes encoding proteasome subunits and enzymes involved in glucose metabolism. A real-time quantitative PCR suggested that the expression of 3 proteasome subunits (psma3, psma5, and psmb7) and 2 enzymes involved in glucose metabolism (pgd and fbp1a) were regulated by zebrafish Nrf2. We thus next examined the upregulation of these genes by an Nrf2 activator, diethyl maleate, using Nrf2 mutant zebrafish larvae. The results of real-time quantitative PCR and whole-mount in situ hybridization showed that all of these 5 genes were upregulated by diethyl maleate treatment in an Nrf2-dependent manner, especially in the liver. These findings implied that the Nrf2-mediated regulation of the proteasome subunits and glucose metabolism is evolutionarily conserved among vertebrates.

LSD1/KDM1A promotes hematopoietic commitment of hemangioblasts through downregulation of Etv2.

The hemangioblast is a progenitor cell with the capacity to give rise to both hematopoietic and endothelial progenitors. Currently, the regulatory mechanisms underlying hemangioblast formation are being elucidated, whereas those controllers for the selection of hematopoietic or endothelial fates still remain a mystery. To answer these questions, we screened for zebrafish mutants that have defects in the hemangioblast expression of Gata1, which is never expressed in endothelial progenitors. One of the isolated mutants, it627, showed not only down-regulation of hematopoietic genes but also up-regulation of endothelial genes. We identified the gene responsible for the it627 mutant as the zebrafish homolog of Lys-specific demethylase 1 (LSD1/KDM1A). Surprisingly, the hematopoietic defects in lsd1(it627) embryos were rescued by the gene knockdown of the Ets variant 2 gene (etv2), an essential regulator for vasculogenesis. Our results suggest that the LSD1-dependent shutdown of Etv2 gene expression may be a significant event required for hemangioblasts to initiate hematopoietic differentiation.

Heme-mediated inhibition of Bach1 regulates the liver specificity and transience of the Nrf2-dependent induction of zebrafish heme oxygenase 1.

The induction of the gene encoding heme oxygenase 1 (Hmox1, HO-1) by Nrf2 is unique compared with other Nrf2 targets. We previously showed that the Nrf2a-mediated induction of zebrafish hmox1a was liver specific and transient. We screened transcription factors that could repress the induction of hmox1a but not other Nrf2a targets and concluded that Bach1b was a prime candidate. In bach1b-knocked-down larvae, the induction of hmox1a was observed ectopically in nonliver tissues and persisted longer than normal fish, suggesting that Bach1 is the only regulator for both the liver-specific and transient induction of hmox1a. Co-knockdown of bach1b with its co-ortholog bach1a enhanced these effects. To determine why Bach1 could not repress the hmox1a induction in the liver, we analyzed the effects of a heme biosynthesis inhibitor, succinylacetone, and a heme precursor, hemin. Succinylacetone decreased the Nrf2a-mediated hmox1a induction, whereas pre-treatment with hemin caused ectopic induction of hmox1a in nonliver tissues, implying that the high heme levels in the liver may release the repressive activity of Bach1. Our results suggested that Bach1 regulates the liver specificity and transience of the Nrf2a-dependent induction of hmox1a and that heme mediates this regulation through Bach1 inhibition based on its level in each tissue.