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Background: Cadmium (Cd) is a highly toxic and widespread environmental oxidative stressor that causes a myriad of health problems, including osteoporosis and bone damage. Although nuclear factor erythroid 2-related factor 2 (NRF2) and its Cap 'n' Collar and basic region Leucine Zipper (CNC-bZIP) family member nuclear factor erythroid 2-related factor 1 (NRF1) coordinate various stress responses by regulating the transcription of a variety of antioxidant and cytoprotective genes, they play distinct roles in bone metabolism and remodeling. However, the precise roles of both transcription factors in bone loss induced by prolonged Cd exposure remain unclear., Objectives: We aimed to understand the molecular mechanisms underlying Cd-induced bone loss, focusing mainly on the roles of NRF2 and NRF1 in osteoclastogenesis provoked by Cd., Methods: Male wild-type (WT), global Nrf2 -knockout ( N r f 2 - / - ) and myeloid-specific Nrf2 knockout [ Nrf2 (M)-KO] mice were administered Cd (50 or 100  ppm ) via drinking water for 8 or 16 wk, followed by micro-computed tomography, histological analyses, and plasma biochemical testing. Osteoclastogenesis was evaluated using bone marrow-derived osteoclast progenitor cells (BM-OPCs) and RAW 264.7 cells in the presence of Cd (10 or 20  nM ) with a combination of genetic and chemical modulations targeting NRF2 and NRF1., Results: Compared with relevant control mice, global N r f 2 - / - or Nrf2 (M)-KO mice showed exacerbated bone loss and augmented osteoclast activity following exposure to 100  ppm Cd in drinking water for up to 16 wk. In vitro osteoclastogenic analyses suggested that Nrf2 -deficient BM-OPCs and RAW 264.7 cells responded more robustly to low levels of Cd (up to 20  nM ) with regard to osteoclast differentiation compared with WT cells. Further mechanistic studies supported a compensatory up-regulation of long isoform of NRF1 (L-NRF1) and subsequent induction of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1 (NFATc1) as the key molecular events in the Nrf2 deficiency-worsened and Cd-provoked osteoclastogenesis. L- Nrf1 silenced (via lentiviral means) Nrf2 -knockdown (KD) RAW cells exposed to Cd showed dramatically different NFATc1 and subsequent osteoclastogenesis outcomes compared with the cells of Nrf2 -KD alone exposed to Cd, suggesting a mitigating effect of the Nrf1 silencing. In addition, suppression of reactive oxygen species by exogenous antioxidants N -acetyl-l-cysteine ( 2  mM ) and mitoquinone mesylate (MitoQ; 0.2 μ M ) mitigated the L-NRF1-associated effects on NFATc1-driven osteoclastogenesis outcomes in Cd-exposed Nrf2 -KD cells., Conclusions: This in vivo and in vitro study supported the authors' hypothesis that Cd exposure caused bone loss, in which NRF2 and L-NRF1 responded to Cd and osteoclastogenic stimuli in a cooperative, but contradictive, manner to coordinate Nfatc1 expression, osteoclastogenesis and thus bone homeostasis. Our study suggests a novel strategy targeting NRF2 and L-NRF1 to prevent and treat the bone toxicity of Cd. https://doi.org/10.1289/EHP13849.

Environmental cadmium (Cd) exposure is a serious public health concern, as the kidney is the primary target for Cd exposure. The present study aimed to investigate the role and underlying mechanisms of nuclear factor erythroid-derived 2-like 2 (Nrf2) in renal fibrosis induced by chronic Cd exposure. Nrf2 knockout (Nrf2-KO) mice and their wild-type littermates (Nrf2-WT) were exposed to 100 or 200 ppm Cd in drinking water for up to 16 or 24 weeks. Following the Cd exposures, Nrf2-KO mice showed elevated urinary neutrophil gelatinase-associated lipocalin (NGAL) and BUN levels compared to Nrf2-WT mice. Masson's trichrome staining and expression of fibrosis-associated proteins revealed that more severe renal fibrosis occurred in Nrf2-KO than that in Nrf2-WT mice. Renal Cd content in the Nrf2-KO mice exposed to 200 ppm Cd was lower than that in Nrf2-WT mice, which might be a consequence of the severe renal fibrosis in the Nrf2-KO mice. Mechanistic studies showed that Nrf2-KO mice exhibited higher levels of oxidative damage, lower antioxidant levels, and more regulated cell death, apoptosis in particular, than those in Nrf2-WT mice caused by Cd exposure. In conclusion, Nrf2-KO mice were more prone to develop renal fibrosis induced by chronic Cd exposure, partially due to a weakened antioxidant, detoxification capacity and increased oxidative damage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The human body is continuously exposed to xenobiotics and internal or external oxidants. The health risk assessment of exogenous chemicals remains a complex and challenging issue. Alternative toxicological test methods have become an essential strategy for health risk assessment. As a core regulator of constitutive and inducible expression of antioxidant response element (ARE)-dependent genes, nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in maintaining cellular redox homeostasis. Consistent with the properties of Nrf2-mediated antioxidant response, Nrf2-ARE activity is a direct indicator of oxidative stress and thus has been used to identify and characterize oxidative stressors and redox modulators. To screen and distinguish chemicals or environmental insults that affect the cellular antioxidant activity and/or induce oxidative stress, various in vitro cell models expressing distinct ARE reporters with high-throughput and high-content properties have been developed. These ARE-reporter systems are currently widely applied in drug discovery and safety assessment. In the present review, we provide an overview of the basic structures and applications of various ARE-reporter systems employed for discovering Nrf2-ARE modulators and characterizing oxidative stressors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Humans are exposed to disinfection by-products through oral, inhalation, and dermal routes, during bathing and swimming, potentially causing skin lesions, asthma, and bladder cancer. Nuclear factor E2-related factor 2 (NRF2) is a master regulator of the adaptive antioxidant response via the antioxidant reaction elements (ARE) orchestrating the transcription of a large group of antioxidant and detoxification genes. Here we used an immortalized human keratinocyte model HaCaT cells to investigate NRF2-ARE as a responder and protector in the acute cytotoxicity of seven haloacetonitriles (HANs), including chloroacetonitrile (CAN), bromoacetonitrile (BAN), iodoacetonitrile (IAN), bromochloroacetonitrile (BCAN), dichloroacetonitrile (DCAN), dibromoacetonitrile (DBAN), and trichloroacetonitrile (TCAN) found in drinking water and swimming pools. The rank order of cytotoxicity among the HANs tested was IAN ≈ BAN ˃ DBAN ˃ BCAN ˃ CAN ˃ TCAN ˃ DCAN based on their LC 50 . The HANs induced intracellular reactive oxygen species accumulation and activated cellular antioxidant responses in concentration- and time-dependent fashions, showing elevated NRF2 protein levels and ARE activity, induction of antioxidant genes, and increased glutathione levels. Additionally, knockdown of NRF2 by lentiviral shRNAs sensitized the HaCaT cells to HANs-induced cytotoxicity, emphasizing a protective role of NRF2 against the cytotoxicity of HANs. These results indicate that HANs cause oxidative stress and activate NRF2-ARE-mediated antioxidant response, which in turn protects the cells from HANs-induced cytotoxicity, highlighting that NRF2-ARE activity could be a sensitive indicator to identify and characterize the oxidative stress induced by HANs and other environmental pollutants., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Developmental arsenic exposure increases cancer risk in later life with the mechanism elusive. Oxidative stress is a dominant determinant in arsenic toxicity. However, the role of Nrf2, a key regulator in antioxidative response, in tumor-augmenting effects by developmental arsenic exposure is unclear. In the present study, wild-type C57BL/6J and Nrf2-konckout (Nrf2-KO) were developmentally exposed to inorganic arsenic via drinking water. For hepatic tumorigenesis analysis, mice were intraperitoneally injected with diethylnitrosamine (DEN) at two weeks of age. Developmental arsenic exposure aggravated tumor multiplicity and burden, and expression of PCNA and AFP in hepatic tumors induced by DEN. Nrf2 activation as indicated by over-expression of Nrf2 and its downstream genes, including Gss, Gsr, p62, Gclc and Gclm, was found in liver tumors, as well as in the livers in developmentally arsenic-exposed pups at weaning. Notably, Nrf2 deficiency attenuated tumor-augmenting effects and over-expression of Nrf2 downstream genes due to developmental arsenic exposure. Furthermore, the levels of urinary DEN metabolite (acetaldehyde) and hepatic DNA damage markers (O6-ethyl-2-deoxyguanosine adducts and γ-histone H2AX) after DEN treatment were elevated by Nrf2 agonist, 2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide. Collectively, our data suggest that augmentation of DEN-induced hepatic tumorigenesis by developmental arsenic exposure is dependent on Nrf2 activation, which may be related to the role of Nrf2 in DEN metabolic activation. Our findings reveal, at least in part, the mechanism underlying increased susceptibility to developing cancer due to developmental arsenic exposure., Competing Interests: Declaration of competing interest No potential competing financial interest was disclosed., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Aims: Keap1-Nrf2 signaling pathway is one of the most important antioxidant signaling pathways, and its abnormal activation is related to cancer metastasis and drug resistance. Many studies have shown Keap1 and Nrf2 mutations are closely associated with cancer occurrence. However, few studies focus on Keap1-Nrf2 binding characteristics of cancer-associated mutations. The study investigated the molecular mechanism between Keap1/Nrf2 mutations and cancer., Main Methods: We have determined the crystal structure of the Keap1-Kelch domain with Nrf2 25-mer peptide. What's more, we clarified the molecular effects of Nrf2 Thr80 and Nrf2 Pro85 on the binding of Keap1 by the method isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF) and electrophoretic mobility shift assay (EMSA). Especially, we confirmed the effect of Thr80 and Pro85 mutations on Keap1/Nrf2 signaling pathway in HEK293T cells by RT-PCR and western blot (WB). Finally, we verified the effect of six cancer-related high-frequency somatic mutations Keap1 G364C , Keap1 D422N , Keap1 R470C , Keap1 G480W , Keap1 E493Q and Keap1 R601L on binding with Nrf2 through ITC experiments., Key Findings: Nrf2 Thr80 and Nrf2 Pro85 play a vital role in the Keap1-Nrf2 interaction. Mutant or modification at position Thr80 will disrupt the interaction. Especially, Nrf2 Thr80 and Nrf2 Pro85 mutations activate the expression of cytoprotective genes in HEK293T cells. As for Keap1, except G364C, the binding affinity of other cancer-related mutants to Nrf2 hardly changed, which means that Keap1 mutants can activate Nrf2 without disrupting the binding to Nrf2., Significance: The study provides new insight into Keap1/Nrf2 signaling pathway and cancer., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Alcoholic liver disease (ALD) is a major cause of morbidity and mortality from liver disorders. Various mechanisms, including oxidative stress and impaired lipid metabolism, have been implicated in the pathogenesis of ALD. Our previous studies showed that nuclear factor erythroid-derived 2-like 2 (Nrf2) is a master regulator of adaptive antioxidant response and lipid metabolism by using a liver-specific Nrf2 knockout (Nrf2(L)-KO) mouse model. In the current study, an ALD model was developed by a Lieber-DeCarli liquid-based ethanol diet given to this Nrf2(L)-KO mouse strain. We found that Nrf2(L)-KO mice were quite sensitive to lethality from 6.3% ethanol diet. We thus decreased the ethanol concentration to 4.2% to obtain tissues to analyze the role of hepatic Nrf2 in the development of ALD. We found that mild hepatic steatosis occurred with both liquid control and 4.2% ethanol diet feeding, which contain 35% fat. Both the fatty acid β-oxidation marker peroxisome proliferators-activated receptor α (PPARα), and lipogenesis regulator PPARγ were reduced with ethanol feeding in Nrf2(L)-KO mice, compared to Nrf2 floxed control mice (Nrf2-LoxP). However, Nrf2(L)-KO livers showed more cell injury than the livers of Nrf2-LoxP mice. Consistent with these data, there was increased proportion of apoptotic cells in the liver of ethanol-fed Nrf2(L)-KO mice comparing Nrf2-LoxP controls. Mechanistically, Nrf2 mediated expression of ethanol detoxification enzymes, such as alcohol dehydrogenase 1 and aldehyde dehydrogenase1a1, likely contributed to the sensitivity to ethanol toxicity. In conclusion, hepatic Nrf2 is critical to the development of ALD, particularly the morbidity and liver injury., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cadmium (Cd) is a heavy metal pollutant that adversely effects the kidney. Oxidative stress and inflammation are likely major mechanisms of Cd-induced kidney injury. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is crucial in regulating antioxidant and inflammatory responses. To investigate the role of Nrf2 in the development of subacute Cd-induced renal injury, we utilized Nrf2 knockout (Nrf2-KO) and control mice (Nrf2-WT) which were given cadmium chloride (CdCl 2 , 1 or 2 mg/kg i.p.) once daily for 7 days. While subacute CdCl 2 exposure induced kidney injury in a dose-dependent manner, after the higher Cd dosage exposure, Nrf2-KO mice showed elevated blood urea nitrogen (BUN) and urinary neutrophil gelatinase-associated lipocalin (NGAL) levels compared to control. In line with the findings, the renal tubule injury caused by 2 mg Cd/kg, but not lower dosage, in Nrf2-KO mice determined by Periodic acid-Schiff staining was more serious than that in control mice. Further mechanistic studies showed that Nrf2-KO mice had more apoptotic cells and severe oxidative stress and inflammation in the renal tubules in response to Cd exposures. Although there were no significant differences in Cd contents of tissues between Cd-exposed Nrf2-WT and Nrf2-KO mice, the mRNA expression of Nrf2 downstream genes, including heme oxygenase 1 and metallothionein 1, were significantly less induced by Cd exposures in the kidney of Nrf2-KO compared with Nrf2-WT mice. In conclusion, Nrf2-deficient mice are more sensitive to kidney injury induced by subacute Cd exposure due to a muted antioxidant response, as well as a likely diminished production of specific Cd detoxification metallothioneins.

Nuclear factor-E2-related factor 2 (Nrf2) is a master transcription factor in antioxidant response, protecting against oxidative damage and various diseases. Previous studies suggest that Nrf2 is suppressed in fibrotic skin and Nrf2 agonists represent a therapeutic strategy, which is mainly attributed to Nrf2 function in fibroblasts. However, constitutive activation of Nrf2 may endow cells with proliferation and survival advantage, facilitating skin tumorigenesis. Non-invasive and mild modulation of Nrf2 via topical application may be helpful. Keratinocytes, which are essential for epidermal formation and function maintenance, have been shown to modulate differentiation of fibroblasts in different stages of fibrosis. In this respect, the role of Nrf2 in keratinocytes in skin fibrosis remains elusive. In the present study, bleomycin (BLM)-induced skin fibrosis model was applied to keratinocyte-specific Nrf2 knockout (Nrf2(K)-KO) mice generated with Keratin 14-Cre/loxp system. BLM treatment significantly suppressed Nrf2 expression in the epidermis. Nrf2 deficiency in keratinocytes exacerbated BLM-induced skin fibrosis according to dermal thickness, and immunostaining of collagen and α-SMA. One-dose BLM treatment led to the emergence of apoptotic cells in the epidermis and an elevated number of macrophages and neutrophils in the dermis, which was aggravated by Nrf2 deficiency, as indicated by TUNEL staining, and expression of F4/80 and Ly6G. In line with in vivo evidence, NRF2 silencing in HaCaT cells significantly decreased cell survival rate in response to BLM due to suppressed expression of antioxidative genes and increased intracellular levels of reactive oxygen species (ROS). The mRNA levels of chemokines and cytokines that are capable of recruiting macrophages and neutrophils, including Mcp-1, Il-6 and Il-8, were increased by Nrf2 deficiency in primary mouse keratinocytes. Moreover, bardoxolone methyl (CDDO-Me), a potent Nrf2 activator, ameliorated BLM-induced skin fibrosis after topical administration. These findings indicate that Nrf2 in keratinocytes protects against skin fibrosis via regulating cell resistance to apoptosis and expression of cytokines and chemokines. The restoration of Nrf2 through topical application might be a potential pharmacologic approach to combat skin fibrosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) is an emerging global disease with increasing prevalence. However, the mechanism of NAFLD development is not fully understood. To elucidate the cell-specific role of nuclear factor erythroid-derived 2-like 2 (NRF2) in the pathogenesis of NAFLD, we utilized hepatocyte- and macrophage-specific Nrf2-knockout [Nrf2(L)-KO and Nrf2(Mϕ)-KO] mice to examine the progress of NAFLD induced by high-fat diet (HFD). Compared to Nrf2-LoxP littermates, Nrf2(L)-KO mice showed less liver enlargement, milder inflammation and less hepatic steatosis after HFD feeding. In contrast, Nrf2(Mϕ)-KO mice displayed no significant difference in HFD-induced hepatic steatosis from Nrf2-LoxP control mice. Mechanistic investigations revealed that Nrf2 deficiency in hepatocytes dampens the expression of peroxisome proliferator-activated receptor γ (PPARγ) and its downstream lipogenic genes in the liver and/or primary hepatocytes induced by HFD and palmitate exposure, respectively. While PPARγ agonists augmented PPARγ expression and its transcriptional activity in primary hepatocytes in a NRF2-dependent manner, forced overexpression of PPARγ1 or γ2 distinctively reversed the decreased expression of their downstream genes fatty acid binding protein 4, lipoprotein lipase and/or fatty acid synthase caused by Nrf2 deficiency. We conclude that NRF2-dependent expression of PPARγ in hepatocytes is a critical initiating process in the development of NAFLD, suggesting that inhibition of NRF2 specifically in hepatocytes may be a valuable approach to prevent the disease., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Chronic exposure to arsenic induces a variety of cancers, particularly in the skin. Autophagy is a highly conserved process which plays a dual role in tumorigenesis. In the present study, we found that chronic exposure to an environmentally relevant dose of arsenite induced malignant transformation of human keratinocytes (HaCaT) with dysregulated autophagy as indicated by an increased number of autophagosomes, activation of mTORC1 pathway, and elevated protein levels of p62 and LC3II. Meanwhile, arsenite-transformed cells showed lower intracellular levels of reactive oxygen species compared with control. Silencing p62 ameliorated elevation in mRNA levels of NRF2 downstream genes ( AKR1C1 and NQO1 ) and malignant phenotypes (acquired invasiveness and anchor-independent growth) induced by chronic arsenite exposure. On the other hand, silencing NRF2 abrogated the increase in mRNA and protein levels of p62 and malignant phenotypes induced by arsenite. In response to acute arsenite exposure, impaired autophagic flux with an increase in p62 protein level and interrupted autophagosome-lysosome fusion was observed. The increase in p62 protein levels in response to arsenite was not completely dependent on NRF2 activation and at least partially attributed to protein degradation. Our data indicate that accumulation of p62 by impaired autophagic flux is involved in the activation of NRF2 and contributes to skin tumorigenesis due to chronic arsenite exposure., Competing Interests: No potential conflicts of interest were disclosed., (Copyright © 2019 Xiafang Wu et al.)

Arsenic exposure increases the risk of various bone disorders. For instance, chronic exposure to low level arsenic can cause bone resorption by promoting osteoclast differentiation. Osteoclast precursor cells produce hydrogen peroxide after low level arsenic exposure and then undergo differentiation, producing cells which break down bone matrix. Nuclear factor E2-related factor 2 (Nrf2) regulates receptor activator of nuclear factor-κB dependent osteoclastogenesis by modulating intracellular reactive oxygen species (ROS) signaling via expression of cytoprotective enzymes. Here we tested the hypothesis that loss of Nrf2 will increase arsenic-induced bone loss. We treated 40 week-old Nrf2 +/+ and Nrf2 -/- mice with 5 ppm arsenic in the drinking water, which produces a blood arsenic level similar to humans living in areas where arsenic exposure is endemic. After 4 months, Micro-CT and dual-energy x-ray analysis revealed a drastic overall decrease in the bone volume with arsenic treatment in mice lacking Nrf2. Deficiency of Nrf2 in RAW 264.7 cells or bone marrow-derived macrophages (BMMs) promoted arsenic-induced osteoclast differentiation. Lack of Nrf2 increases arsenic-induced ROS levels and phosphorylation of p38. N-Acetyl-cysteine and SB203580 pretreatment essentially abolished arsenic-induced phosphorylation of p38 and reversed arsenic-induced increased osteoclast differentiation in Nrf2 deficiency. Taken together, our data suggest that loss of Nrf2 causes increased oxidative stress and enhanced susceptibility to arsenic-induced bone loss., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Non-small cell lung cancer (NSCLC) has a high mortality rate worldwide. Various treatments strategies have been used against NSCLC including individualized chemotherapies, but innate or acquired cancer cell drug resistance remains a major obstacle. Recent studies revealed that the Kelch-like ECH associated protein 1/Nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) pathway is intimately involved in cancer progression and chemoresistance. Thus, antagonizing Nrf2 would seem to be a viable strategy in cancer therapy. In the present study a traditional Chinese medicine, triptolide, was identified that markedly inhibited expression and transcriptional activity of Nrf2 in various cancer cells, including NSCLC and liver cancer cells. Consequently, triptolide made cancer cells more chemosensitivity toward antitumor drugs both in vitro and in a xenograft tumor model system using lung carcinoma cells. These results suggest that triptolide blocks chemoresistance in cancer cells by targeting the Nrf2 pathway. Triptolide should be further investigated in clinical cancer trials., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Binge alcohol drinking is an important health concern and well-known risk factor for the development of numerous disorders. Oxidative stress plays a critical role in the pathogenesis of acute alcoholism. Nuclear factor erythroid 2 like 2 (NRF2) is a master regulator of cellular adaptive response to oxidative insults. However, the role of NRF2 in acute alcoholism and associated pathologies remains unclear. We found that Nrf2-knockout (Nrf2-KO) mice had exaggerated hypoglycemia and hypothermia and increased mortality compared to wildtype mice after binge ethanol exposure. This phenotype was partially rescued by providing warm environment and/or glucose administration. Acute high dose of alcohol exposure resulted in substantially worsened liver and pancreatic injuries in Nrf2-KO mice. Importantly, deficiency of Nrf2 allowed severe pancreatitis and pancreatic β-cell injury with increased insulin secretion and/or leaking during binge ethanol exposure, which contributed to hypoglycemia. In contrast, a clinically used NRF2 activator dimethyl fumarate (DMF) protected against hypoglycemia and lethality induced by acute ethanol exposure. Furthermore, Nrf2-KO mice likely had defective hepatic acetaldehyde metabolism. Taken together, NRF2 plays an important protective role against acute binge alcohol-induced hepatic and pancreatic damage, which may be partially attributable to its primary regulating role in antioxidant response and impact on ethanol metabolism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Alcoholic liver disease (ALD) is a leading cause of morbidity and mortality of liver disorders and a major health issue globally. ALD refers to a spectrum of liver pathologies ranging from steatosis, steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma. Various mechanisms, including oxidative stress, protein and DNA modification, inflammation and impaired lipid metabolism, have been implicated in the pathogenesis of ALD. Further, reactive oxygen species (ROS) in particular, have been identified as a key component in the initiation and progression of ALD. Nuclear factor erythroid 2 like 2 (Nrf2) is a master regulator of the intracellular adaptive antioxidant response to oxidative stress, and aids in the detoxification of a variety of toxicants. Given its cytoprotective role, Nrf2 has been extensively studied as a therapeutic target for ALD. Paradoxically, however, emerging evidence have revealed that Nrf2 may be implicated in the progression of ALD. In this review, we summarize the role of Nrf2 in the development of ALD and discuss the underlying mechanisms. Clearly, more comprehensive studies with proper animal and cell models and in human are needed to verify the potential therapeutic role of Nrf2 in ALD., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a central mediator of cellular responses to oxidative stress. We hypothesized that Nrf2 modulates progression from acute tubular damage to renal fibrosis. We asked whether Nrf2 deletion increases renal injury in mice following unilateral ureteral obstruction (UUO)., Methods: We explored the time course of renal injury and Nrf2 expression in Nrf2+/+ mice following UUO. We compared Nrf2+/+ and Nrf2-/- mice following UUO in tubular damage, transdifferentiation [vimentin, proliferating cell nuclear antigen (PCNA)], fibrosis [fibronectin, α-smooth muscle actin (SMA)], antioxidative and inflammatory responses. We studied Nrf2 in renal biopsies of patients with acute, subacute and chronic tubulointerstitial nephritis (TIN)., Results: In Nrf2+/+ mice, renal Nrf2 expression and Nrf2-regulated glutamate-cysteine ligase catalytic (Gclc) and heme oxygenase-1 (Ho-1) were elevated, and renal injury occurred between 2 and 14 days after UUO. On Day 2 following UUO, in Nrf2-/- mice compared with Nrf2+/+ mice, tubular damage, apoptotic cell numbers, cleaved caspase3 and cleaved-poly ADP-ribose polymerase were increased. On Day 5, protein levels of vimentin and PCNA and the co-expressed cells of both proteins were increased. On Day 14, fibronectin and α-SMA protein levels were increased. Nrf2 deletion decreased expression of antioxidative genes (Gclc and Ho-1) and increased expression of inflammatory response genes (Tgfβ, Tnf, IL-6, IL-1β and F4/80). Finally, Nrf2 expression was upregulated in renal biopsies of patients with TIN., Conclusions: Following UUO, Nrf2 deficiency increased tubular damage, transdifferentiation, fibrosis and inflammatory response while decreasing antioxidative responses. The renal protective role of Nrf2 in the development of tubulointerstitial fibrosis in UUO may be mediated by antioxidative and anti-inflammatory pathways.

Inorganic arsenic (iAs) is a known toxicant and carcinogen. Worldwide arsenic exposure has become a threat to human health. The severity of arsenic toxicity is strongly correlated with the speed of arsenic metabolism (methylation) and clearance. Furthermore, oxidative stress is recognized as a major mechanism for arsenic-induced toxicity. Nuclear factor-E2-related factor 2 (Nrf2), a key regulator in cellular adaptive antioxidant response, is clearly involved in alleviation of arsenic-induced oxidative damage. Multiple studies demonstrate that Nrf2 deficiency mice are more vulnerable to arsenic-induced intoxication. However, what effect Nrf2 deficiency might have on arsenic metabolism in mice is still unknown. In the present study, we measured the key enzymes involved in arsenic metabolism in Nrf2-WT and Nrf2-KO mice. Our results showed that basal transcript levels of glutathione S-transferase omega 2 (Gsto2) were significantly higher and GST mu 1 (Gstm1) lower in Nrf2-KO mice compared to Nrf2-WT control. Arsenic speciation and methylation rate in liver and urine was then studied in mice treated with 5mg/kg sodium arsenite for 12h. Although there were some alterations in arsenic metabolism enzymes between Nrf2-WT and Nrf2-KO mice, the Nrf2 deficiency had no significant effect on arsenic methylation. These results suggest that the Nrf2-KO mice are more sensitive to arsenic than Nrf2-WT mainly because of differences in adaptive antioxidant detoxification capacity rather than arsenic methylation capacity., (Copyright © 2017. Published by Elsevier Inc.)

Background: Resistance to chemotherapy is a major obstacle in the treatment of human hepatocellular carcinoma (HCC). Despite playing an important role in chemoprevention, nuclear factor erythroid 2-related factor 2 (NRF2) also contributes to chemo- and radio-resistance. The current study focusses on camptothecin as a novel NRF2 inhibitor to sensitise HCC to chemotherapy., Methods: The expression and transcriptional activity of NRF2 in human HCC biopsies and camptothecin-treated culture cells were determined using immunostaining, western blot, reverse-transcription quantitative real-time PCR (RT-qPCR) and luciferase reporter assay. The effect of camptothecin on chemosensitivity of cancer cells was assessed in vitro and in xenografts., Results: The expression and transcriptional activity of NRF2 were substantially elevated in HCC biopsies compared with corresponding adjacent tissues, and positively correlated with serum α-fetoprotein, a clinical indicator of pathological progression. In searching chemicals targeting NRF2 for chemotherapy, we discovered that camptothecin is a potent NRF2 inhibitor. Camptothecin markedly suppressed NRF2 expression and transcriptional activity in different types of cancer cells including HepG2, SMMC-7721 and A549. As a result, camptothecin sensitised these cells to chemotherapeutic drugs in vitro and in xenografts., Conclusions: Camptothecin is a novel NRF2 inhibitor that may be repurposed in combination with other chemotherapeutics to enhance their efficacy in treating high NRF2-expressing cancers.

Arsenic is a well-known human carcinogen. Stem cells are indicated to be involved in arsenic carcinogenesis and have a survival selection advantage during arsenic exposure with underlying mechanisms undefined. In the present study, we demonstrated that CD34 high -enriched cells derived from HaCaT human keratinocytes showed stem-like phenotypes. These cells were more resistant to arsenic toxicity and had higher arsenic efflux ability than their mature compartments. The master transcription factor in antioxidant defense, nuclear factor erythroid 2-related factor 2 (NRF2) with its downstream genes, was highly expressed in CD34 high -enriched cells. Stable knockdown of NRF2 abolished the hyperresistance to arsenic toxicity and holoclone-forming ability of CD34 high -enriched cells. Our results suggest that skin epithelial stem/progenitor cells are more resistant to arsenic toxicity than mature cells, which is associated with the high innate expression of NRF2 in skin epithelial stem/progenitor cells.

The Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor regulating a wide array of genes for antioxidant and detoxification enzymes in response to oxidative and xenobiotic stress. A large number of Nrf2-antioxidant response element (ARE) activators have been screened for use as chemopreventive agents in oxidative stress-related diseases and even cancer. However, constitutive activation of Nrf2 occurs in a variety of cancers. Aberrant activation of Nrf2 is correlated with cancer progression, chemoresistance, and radioresistance. In this review, we examine recent studies of Nrf2-ARE inhibitors in the context of cancer therapy. We enumerate the possible Nrf2-inhibiting mechanisms of these compounds, their effects sensitizing cancer cells to chemotherapeutic agents, and the prospect of applying them in clinical cancer therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response element (ARE)-dependent transcription, plays a pivotal role in chemical detoxification in normal and tumor cells. Consistent with previous findings that NRF2-ARE contributes to chemotherapeutic resistance of cancer cells, we found that stable knockdown of NRF2 by lentiviral shRNA in human acute monocytic leukemia (AML) THP-1 cells enhanced the cytotoxicity of several chemotherapeutic agents, including arsenic trioxide (As2O3), etoposide and doxorubicin. Using an ARE-luciferase reporter expressed in several human and mouse cells, we identified a set of compounds, including isonicotinic acid amides, isoniazid and ethionamide, that inhibited NRF2-ARE activity. Treatment of THP-1 cells with ethionamide, for instance, significantly reduced mRNA expression of multiple ARE-driven genes under either basal or As2O3-challenged conditions. As determined by cell viability and cell cycle, suppression of NRF2-ARE by ethionamide also significantly enhanced susceptibility of THP-1 and U937 cells to As2O3-induced cytotoxicity. In THP-1 cells, the sensitizing effect of ethionamide on As2O3-induced cytotoxicity was highly dependent on NRF2. To our knowledge, the present study is the first to demonstrate that ethionamide suppresses NRF2-ARE signaling and disrupts the transcriptional network of the antioxidant response in AML cells, leading to sensitization to chemotherapeutic agents., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Oxidative stress is implicated in the pathogenesis of pancreatic β-cell dysfunction that occurs in both type 1 and type 2 diabetes. Nuclear factor E2-related factor 2 (NRF2) is a master regulator in the cellular adaptive response to oxidative stress. The present study found that MIN6 β-cells with stable knockdown of Nrf2 (Nrf2-KD) and islets isolated from Nrf2-knockout mice expressed substantially reduced levels of antioxidant enzymes in response to a variety of stressors. In scramble MIN6 cells or wild-type islets, acute exposure to oxidative stressors, including hydrogen peroxide (H2O2) and S-nitroso-N-acetylpenicillamine, resulted in cell damage as determined by decrease in cell viability, reduced ATP content, morphology changes of islets, and/or alterations of apoptotic biomarkers in a concentration- and/or time-dependent manner. In contrast, silencing of Nrf2 sensitized MIN6 cells or islets to the damage. In addition, pretreatment of MIN6 β-cells with NRF2 activators, including CDDO-Im, dimethyl fumarate (DMF), and tert-butylhydroquinone (tBHQ), protected the cells from high levels of H2O2-induced cell damage. Given that reactive oxygen species (ROS) are involved in regulating glucose-stimulated insulin secretion (GSIS) and persistent activation of NRF2 blunts glucose-triggered ROS signaling and GSIS, the present study highlights the distinct roles that NRF2 may play in pancreatic β-cell dysfunction that occurs in different stages of diabetes.

Liver is one of the major target organs of arsenic toxicity and carcinogenesis. Nuclear factor (erythroid-2 related) factor 2 (Nrf2) is a redox-sensitive transcription factor, regulating critically cellular defense responses against the toxic metallic arsenic in many cell types and tissues. This study was conducted to evaluate the hepato-cellular Nrf2 and Nrf2-regulated antioxidant reactions of sodium arsenite exposure in Chang human hepatocytes. Nrf2 and heme oxygenase-1 (HO-1) protein levels were detected by Western blot, and Nrf2-regulated HO-1 mRNA expressions were determined using semiquantitative RT-PCR by 0∼50 μmol/L of sodium arsenite exposure for 2, 6, 12, and 24 h. We also observed the changes of intracellular reactive oxygen species (ROS) and total cellular glutathione (GSH) by flow cytometry and spectrophotometry, respectively. Our results showed that intracellular ROS were both dose- and time-dependent induced by inorganic arsenic; Cellular Nrf2 protein levels increased rapidly after 2 h of exposure, elevated significantly at 6 h, and reached the maximum at 12 h. The endogenous Nrf2-regulated downstream HO-1 mRNA and protein were also induced dramatically and lasted for as long as 24 h. In addition, intracellular GSH levels elevated in consistent with Nrf2 activation. Our findings here suggest that inorganic arsenic alters cellular redox balance in hepatocytes to trigger Nrf2-regulated antioxidant responses promptly, which may represent an adaptive cell defense mechanism against inorganic arsenic induced liver injuries and hepatoxicity., (Copyright © 2011 Wiley Periodicals, Inc.)

The Nrf2-Keap1 signaling pathway is a protective mechanism promoting cell survival. Activation of the Nrf2 pathway by natural compounds has been proven to be an effective strategy for chemoprevention. Interestingly, a cancer-promoting function of Nrf2 has recently been observed in many types of tumors due to deregulation of the Nrf2-Keap1 axis, which leads to constitutive activation of Nrf2. Here, we report a novel mechanism of Nrf2 activation by arsenic that is distinct from that of chemopreventive compounds. Arsenic deregulates the autophagic pathway through blockage of autophagic flux, resulting in accumulation of autophagosomes and sequestration of p62, Keap1, and LC3. Thus, arsenic activates Nrf2 through a noncanonical mechanism (p62 dependent), leading to a chronic, sustained activation of Nrf2. In contrast, activation of Nrf2 by sulforaphane (SF) and tert-butylhydroquinone (tBHQ) depends upon Keap1-C151 and not p62 (the canonical mechanism). More importantly, SF and tBHQ do not have any effect on autophagy. In fact, SF and tBHQ alleviate arsenic-mediated deregulation of autophagy. Collectively, these findings provide evidence that arsenic causes prolonged activation of Nrf2 through autophagy dysfunction, possibly providing a scenario similar to that of constitutive activation of Nrf2 found in certain human cancers. This may represent a previously unrecognized mechanism underlying arsenic toxicity and carcinogenicity in humans.

Currently, the sole treatment option for patients with heart failure is transplantation. The battle of prolonging graft survival and modulating innate and adaptive immune responses is still being waged in the clinic and in research labs. The transcription factor Nrf2 controls major cell survival pathways and is central to moderating inflammation and immune responses. In this study the effect of Nrf2 levels in host recipient C57BL/6 mice on Balb/c allogeneic graft survival was examined. Importantly, Nrf2(-/-) recipient mice could not support the graft for longer than 7.5 days on average, whereas activation of Nrf2 by sulforaphane in Nrf2(+/+) hosts prolonged graft survival to 13 days. Several immune cells in the spleen of recipient mice were unchanged; however, CD11b(+) macrophages were significantly increased in Nrf2(-/-) mice. In addition, IL-17 mRNA levels were elevated in grafts transplanted into Nrf2(-/-) mice. Although Nrf2 appears to play a crucial role in graft survival, the exact mechanism is yet to be fully understood.

In response to oxidative stress, the transcription factor Nrf2 is upregulated and controls activation of many genes that work in concert to defend cells from damages and to maintain cellular redox homeostasis. p53 has been regarded as the guardian of the genome through its pro-oxidant and antioxidant functions. Under low levels of reactive oxygen species (ROS), "normal" amounts of p53 upregulates expression of antioxidant genes, protecting macromolecules from ROS-induced damage. However, at high levels or extended exposure of ROS, p53 expression is enhanced, activating pro-oxidant genes and resulting in p53-dependent apoptosis. We observed a two-phase Nrf2 expression controlled by p53. (i) The induction phase: when p53 expression is relatively low, p53 enhances the protein level of Nrf2 and its target genes to promote cell survival in a p21-dependent manner. (ii) The repression phase: when p53 expression is high, the Nrf2-mediated survival response is inhibited by p53. Our observation leads to the hypothesis that the p53-mediated biphasic regulation of Nrf2 may be key for the tumor-suppressor function of p53 by coordinating cell survival and death pathways.