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Protection against phalloidin-induced liver injury by oleanolic acid involves Nrf2 activation and suppression of Oatp1b2.
Toxicol. Lett.
PUBLISHED: 08-26-2014
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This study utilized pharmacological activation of Nrf2 with oleanolic acid (OA, 22.5mg/kg, sc for 4 days) and the genetic Nrf2 activation (Nrf2-null, wild-type, and Keap1-HKO mice) to examine the role of Nrf2 in protection against phalloidin hepatotoxicity. Mice were given phalloidin (1.5mg/kg, ip for 8h) to examine liver injury and the expression of toxicity-related genes. Phalloidin increased serum enzyme activities and caused extensive hepatic hemorrhage and necrosis in Nrf2-null and wild-type mice, but less injury was seen in Keap1-HKO mice and OA-pretreated mice. Phalloidin increased the expression of neutrophil-specific chemokine mKC and MIP-2 in Nrf2-null and WT mice, but such increases were attenuated in Keap1-HKO and OA-pretreated mice. Phalloidin increased, while Nrf2 activation attenuated, the expression of genes involved in acute-phase response (Ho-1) and DNA-damage response genes (Gadd45 and Chop10). Phalloidin is taken up by hepatocytes through Oatp1b2, but there was no difference in basal and phalloidin-induced Oatp1b2 expression among Nrf2-null, wild-type, and Keap1-HKO mice. In contrast, OA decreased phalloidin-induced Oatp1b2. Phalloidin activated MAPK signaling (p-JNK), which was attenuated by activation of Nrf2. In conclusion, this study demonstrates that protection against phalloidin hepatotoxicity by OA involves activation of Nrf2 and suppression of Oatp1b2.
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Overexpression of Nrf2 protects against microcystin-induced hepatotoxicity in mice.
PLoS ONE
PUBLISHED: 01-01-2014
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Oxidative stress and glutathione (GSH) depletion are implicated in mycocystin hepatotoxicity. To investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in microcystin-induced liver injury, Nrf2-null, wild-type, and Keap1-hepatocyte knockout (Keap1-HKO) mice were treated with microcystin (50 ?g/kg, i.p.). Blood and liver samples were collected 8 h thereafter. Microcystin increased serum alanine aminotransferase and aspartate aminotransferase activities, and caused extensive inflammation and necrosis in Nrf2-null and wild-type mice, but not in Keap1-HKO mice. Oxidative stress and inflammation are implicated in microcystin-induced hepatotoxicity, as evidenced by increased lipid peroxidation and increased expression of pro-inflammatory genes, such as neutrophil-specific chemokines mKC and MIP-2, and pro-inflammatory cytokines IL-1? and IL-6. The increased expression of these pro-inflammatory genes was attenuated in Keap1-HKO mice. Nrf2 and Nqo1 mRNA and protein were higher in Keap1-HKO mice at constitutive levels and after microcystin. To further investigate the mechanism of the protection, hepatic GSH and the mRNA of GSH-related enzymes were determined. Microcystin markedly depleted liver GSH by 60-70% in Nrf2 and WT mice but only 35% in Keap1-HKO mice. The mRNAs of GSH conjugation and peroxide reduction enzymes, such as Gst?1, Gst?4, Gst?, and Gpx2 were higher in livers of Keap1-HKO mice, together with higher expression of the rate-limiting enzyme for GSH synthesis (Gclc). Organic anion transport polypeptides were increased by microcystin with the most increase in Keap1-HKO mice. In conclusion, this study demonstrates that higher basal levels of Nrf2 and GSH-related genes in Keap1-HKO mice prevented microcystin-induced oxidative stress and liver injury.
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Oleanolic acid alters bile acid metabolism and produces cholestatic liver injury in mice.
Toxicol. Appl. Pharmacol.
PUBLISHED: 06-19-2013
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Oleanolic acid (OA) is a triterpenoids that exists widely in plants. OA is effective in protecting against hepatotoxicants. Whereas a low dose of OA is hepatoprotective, higher doses and longer-term use of OA produce liver injury. This study characterized OA-induced liver injury in mice. Adult C57BL/6 mice were given OA at doses of 0, 22.5, 45, 90, and 135 mg/kg, s.c., daily for 5 days, and liver injury was observed at doses of 90 mg/kg and above, as evidenced by increases in serum activities of alanine aminotransferase and alkaline phosphatase, increases in serum total bilirubin, as well as by liver histopathology. OA-induced cholestatic liver injury was further evidenced by marked increases of both unconjugated and conjugated bile acids (BAs) in serum. Gene and protein expression analysis suggested that livers of OA-treated mice had adaptive responses to prevent BA accumulation by suppressing BA biosynthetic enzyme genes (Cyp7a1, 8b1, 27a1, and 7b1); lowering BA uptake transporters (Ntcp and Oatp1b2); and increasing a BA efflux transporter (Ost?). OA increased the expression of Nrf2 and its target gene, Nqo1, but decreased the expression of AhR, CAR and PPAR? along with their target genes, Cyp1a2, Cyp2b10 and Cyp4a10. OA had minimal effects on PXR and Cyp3a11. Taken together, the present study characterized OA-induced liver injury, which is associated with altered BA homeostasis, and alerts its toxicity potential.
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Genetic activation of Nrf2 protects against fasting-induced oxidative stress in livers of mice.
PLoS ONE
PUBLISHED: 02-11-2013
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Acute fasting causes elevated oxidative stress. The current study investigated the effects of the nuclear factor erythoid 2-related factor 2 (Nrf2), the sensor of oxidative stress in cells, on energy homeostasis and liver pathophysiology during fasting. Feed was removed from mice possessing none (Nrf2-null), normal (wild-type, WT), enhanced (Keap1-knockdown, K1-KD), and maximum (hepatocyte-specific Keap1-knockout, K1-HKO) Nrf2 activity in liver for 24 h. Body weight, blood glucose, and blood lipid profiles were similar among mice with graded Nrf2 activity under either fed or fasted conditions. Fasting reduced liver size in mice expressing Nrf2, but not in Nrf2-null mice. Nrf2-null mice accumulated more non-esterified free fatty acids and triglycerides in liver after fasting than the other genotypes of mice. Fatty acids are mainly catabolized in mitochondria, and Nrf2-null mice had lower mitochondrial content in liver under control feeding conditions, which was further reduced by fasting. In contrast, mitochondrial contents in mice with enhanced Nrf2 activity were not affected by fasting. Oxidative stress, determined by staining of free radicals and quantification of malondialdehyde equivalents, was highest in Nrf2-null and lowest in K1-HKO mice after fasting. The exacerbated oxidative stress in livers of Nrf2-null mice is predicted to lead to damages to mitochondria, and therefore diminished oxidation and increased accumulation of lipids in livers of Nrf2-null mice. In summary, the Nrf2-regulated signaling pathway is critical in protecting mitochondria from oxidative stress during feed deprivation, which ensures efficient utilization of fatty acids in livers of mice.
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Nrf2 protection against liver injury produced by various hepatotoxicants.
Oxid Med Cell Longev
PUBLISHED: 01-14-2013
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To investigate the role of Nrf2 as a master defense against the hepatotoxicity produced by various chemicals, Nrf2-null, wild-type, Keap1-knock down (Keap1-Kd) and Keap1-hepatocyte knockout (Keap1-HKO) mice were used as a "graded Nrf2 activation" model. Mice were treated with 14 hepatotoxicants at appropriate doses, and blood and liver samples were collected thereafter (6 h to 7 days depending on the hepatotoxicant). Graded activation of Nrf2 offered a Nrf2-dependent protection against the hepatotoxicity produced by carbon tetrachloride, acetaminophen, microcystin, phalloidin, furosemide, cadmium, and lithocholic acid, as evidenced by serum alanine aminotransferase (ALT) activities and by histopathology. Nrf2 activation also offered moderate protection against liver injury produced by ethanol, arsenic, bromobenzene, and allyl alcohol but had no effects on the hepatotoxicity produced by D-galactosamine/endotoxin and the Fas ligand antibody Jo-2. Graded Nrf2 activation reduced the expression of inflammatory genes (MIP-2, mKC, IL-1 ? , IL-6, and TNF ?), oxidative stress genes (Ho-1, Egr1), ER stress genes (Gadd45 and Gadd153), and genes encoding cell death (Noxa, Bax, Bad, and caspase3). Thus, this study demonstrates that Nrf2 prevents the liver from many, but not all, hepatotoxicants. The Nrf2-mediated protection is accompanied by induction of antioxidant genes, suppression of inflammatory responses, and attenuation of oxidative stress.
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Beneficial role of Nrf2 in regulating NADPH generation and consumption.
Toxicol. Sci.
PUBLISHED: 07-20-2011
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Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that promotes the transcription of cytoprotective genes in response to oxidative and electrophilic stresses. Most functions of Nrf2 were identified by studying biological models with Nrf2 deficiency, however, little is known about the effects of graded Nrf2 activation. In the present study, genomic gene expression profiles by microarray analysis were characterized with a "gene dose-response" model in livers of Nrf2-null mice, wild-type mice, Kelch-like ECH associating protein 1 (Keap1)-knockdown (Keap1-KD) mice with enhanced Nrf2 activation, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum hepatic Nrf2 activation. Hepatic nuclear Nrf2 protein, glutathione concentrations, and known Nrf2 target genes were increased in a dose-dependent manner. In total, 115 genes were identified to be constitutively induced and 80 genes suppressed with graded Nrf2 activation. Messenger RNA of genes encoding enzymes in the pentose phosphate pathway and enzyme were low with Nrf2 deficiency and high with Nrf2 activation, indicating that Nrf2 is important for NADPH production. NADPH is the major reducing resource to scavenge oxidative stress, including regenerating glutathione and thioredoxin and is also used for anabolic pathways including lipid synthesis. High performance liquid chromatography-ultraviolet absorbance analysis confirmed that hepatic NADPH concentration was lowest in Nrf2-null mice and highest in Keap1-HKO mice. In addition, genes involved in fatty acid synthesis and desaturation were downregulated with graded Nrf2 activation. In conclusion, the present study suggests that Nrf2 protects against environmental insults by promoting the generation of NADPH, which is preferentially consumed by aiding scavenging of oxidative stress rather than fatty acid synthesis and desaturation.
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Implementation of a high-throughput screen for identifying small molecules to activate the Keap1-Nrf2-ARE pathway.
PLoS ONE
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Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE) located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits) of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01-30 µM. Of these 238 compounds, 231 (96%) increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones). In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.
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Nrf2 deficiency improves glucose tolerance in mice fed a high-fat diet.
Toxicol. Appl. Pharmacol.
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Nrf2, a master regulator of intracellular redox homeostasis, is indicated to participate in fatty acid metabolism in liver. However, its role in diet-induced obesity remains controversial. In the current study, genetically engineered Nrf2-null, wild-type (WT), and Nrf2-activated, Keap1-knockdown (K1-KD) mice were fed either a control or a high-fat Western diet (HFD) for 12 weeks. The results indicate that the absence or enhancement of Nrf2 activity did not prevent diet-induced obesity, had limited effects on lipid metabolism, but affected blood glucose homeostasis. Whereas the Nrf2-null mice were resistant to HFD-induced glucose intolerance, the Nrf2-activated K1-KD mice exhibited prolonged elevation of circulating glucose during a glucose tolerance test even on the control diet. Feeding a HFD did not activate the Nrf2 signaling pathway in mouse livers. Fibroblast growth factor 21 (Fgf21) is a liver-derived anti-diabetic hormone that exerts glucose- and lipid-lowering effects. Fgf21 mRNA and protein were both elevated in livers of Nrf2-null mice, and Fgf21 protein was lower in K1-KD mice than WT mice. The inverse correlation between Nrf2 activity and hepatic expression of Fgf21 might explain the improved glucose tolerance in Nrf2-null mice. Furthermore, a more oxidative cellular environment in Nrf2-null mice could affect insulin signaling in liver. For example, mRNA of insulin-like growth factor binding protein 1, a gene repressed by insulin in hepatocytes, was markedly elevated in livers of Nrf2-null mice. In conclusion, genetic alteration of Nrf2 does not prevent diet-induced obesity in mice, but deficiency of Nrf2 improves glucose homeostasis, possibly through its effects on Fgf21 and/or insulin signaling.
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Effect of graded Nrf2 activation on phase-I and -II drug metabolizing enzymes and transporters in mouse liver.
PLoS ONE
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Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes in response to oxidative/electrophilic stress. Kelch-like ECH associating protein 1 (Keap1) sequesters Nrf2 in the cytosol. The purpose of this study was to investigate the role of Nrf2 in regulating the mRNA of genes encoding drug metabolizing enzymes and xenobiotic transporters. Microarray analysis was performed in livers of Nrf2-null, wild-type, Keap1-knockdown mice with increased Nrf2 activation, and Keap1-hepatocyte knockout mice with maximum Nrf2 activation. In general, Nrf2 did not have a marked effect on uptake transporters, but the mRNAs of organic anion transporting polypeptide 1a1, sodium taurocholate cotransporting polypeptide, and organic anion transporter 2 were decreased with Nrf2 activation. The effect of Nrf2 on cytochrome P450 (Cyp) genes was minimal, with only Cyp2a5, Cyp2c50, Cyp2c54, and Cyp2g1 increased, and Cyp2u1 decreased with enhanced Nrf2 activation. However, Nrf2 increased mRNA of many other phase-I enzymes, such as aldo-keto reductases, carbonyl reductases, and aldehyde dehydrogenase 1. Many genes involved in phase-II drug metabolism were induced by Nrf2, including glutathione S-transferases, UDP- glucuronosyltransferases, and UDP-glucuronic acid synthesis enzymes. Efflux transporters, such as multidrug resistance-associated proteins, breast cancer resistant protein, as well as ATP-binding cassette g5 and g8 were induced by Nrf2. In conclusion, Nrf2 markedly alters hepatic mRNA of a large number of drug metabolizing enzymes and xenobiotic transporters, and thus Nrf2 plays a central role in xenobiotic metabolism and detoxification.
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Nrf2 protects against diquat-induced liver and lung injury.
Free Radic. Res.
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Diquat is an herbicide that generates superoxide anions through redox cycling. Nuclear factor erythroid-derived 2- like 2 (Nrf2) is a transcription factor that up-regulates cytoprotective genes in response to oxidative stress. To investigate the protective effect of Nrf2 against diquat-induced toxicity, wild-type, Nrf2-null and Kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2 activity were treated with diquat dibromide (125 mg/kg, i.p.). Blood and tissues were collected at 1, 2, 4 and 6 hours after treatment. Administration of diquat resulted in lipid peroxidation and lethality in wild-type mice, which were more in Nrf2-null mice and less in Keap1-KD mice. Diquat produced liver injury in Nrf2-null mice, as evidenced by increased serum ALT activity and extensive hepatic necrosis, but not in wild-type and Keap1-KD mice. Diquat produced more severe lung injury in Nrf2-null than in wild-type mice, as evidenced by increased lung weight and alveolar collapse. In contrast, Keap1-KD mice had attenuated lung edema and no histopathological alterations. To further investigate the mechanism of the protective effects of Nrf2, lung and liver glutathione (GSH) concentrations were quantified. Diquat decreased GSH in lung and liver in wild-type mice, and the decrease was more in Nrf2-null mice, and less in Keap1-KD mice. After diquat treatment, the mRNA of the GSH synthesis enzyme Gclc was increased in Keap1-KD, but not in Nrf2-null mice. Collectively, Nrf2 plays an important role in preventing diquat-induced liver and lung injury, and this protective effect results from Nrf2-regulated elevation of cellular GSH and expression of GSH synthetic genes.
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Role of Nrf2 in preventing ethanol-induced oxidative stress and lipid accumulation.
Toxicol. Appl. Pharmacol.
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Oxidative stress and lipid accumulation play important roles in alcohol-induced liver injury. Previous reports showed that, in livers of nuclear factor erythroid 2-related factor 2 (Nrf2)-activated mice, genes involved in antioxidant defense are induced, whereas genes involved in lipid biosynthesis are suppressed. To investigate the role of Nrf2 in ethanol-induced hepatic alterations, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation, were treated with ethanol (5 g/kg, po). Blood and liver samples were collected 6h thereafter. Ethanol increased alanine aminotransferase and lactate dehydrogenase activities as well as thiobarbituric acid reactive substances in serum of Nrf2-null and wild-type mice, but not in Nrf2-enhanced mice. After ethanol administration, mitochondrial glutathione concentrations decreased markedly in Nrf2-null mice but not in Nrf2-enhanced mice. H(2)DCFDA staining of primary hepatocytes isolated from the four genotypes of mice indicates that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. Ethanol increased serum triglycerides and hepatic free fatty acids in Nrf2-null mice, and these increases were blunted in Nrf2-enhanced mice. In addition, the basal mRNA and nuclear protein levels of sterol regulatory element-binding protein 1(Srebp-1) were decreased with graded Nrf2 activation. Ethanol further induced Srebp-1 mRNA in Nrf2-null mice but not in Nrf2-enhanced mice. In conclusion, Nrf2 activation prevented alcohol-induced oxidative stress and accumulation of free fatty acids in liver by increasing genes involved in antioxidant defense and decreasing genes involved in lipogenesis.
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