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1.
Toxicol Appl Pharmacol ; 483: 116837, 2024 02.
Article in English | MEDLINE | ID: mdl-38278496

ABSTRACT

FLT3L-Fc is a cytokine-Fc fusion agonizing receptor-type tyrosine-protein kinase FLT3 (fms-related tyrosine kinase 3; CD135). FLT3 is expressed on dendritic cells (DCs) as well as myeloid and lymphoid progenitors. Nonclinical pharmacokinetics, pharmacodynamics and safety of FLT3L-Fc were investigated in rats and cynomolgus monkeys. FLT3L-Fc induced robust pharmacodynamic responses, evidenced by marked expansion of peripheral blood cDC1s, cDC2s, and pDCs (up to 301-fold in rats and 378-fold in monkeys), peaking at 8-10 days after the first dose. FLT3L-Fc was well tolerated with no adverse findings at doses up to 10 mg/kg administered intravenously twice three weeks apart. In both species, major clinical pathology findings consisted of expansion of white blood cell (WBC) populations including lymphocytes, monocytes, neutrophils, basophils, and large unstained cells, which were pronounced after the first dose. The WBC findings were associated microscopically with histiocytic and mononuclear cell infiltrates in multiple organs. Tissue immunohistochemistry in monkeys showed that the leukocyte infiltrates consisted of hematopoietic progenitor cells and histiocytes with a reactive morphology and were associated with a slight stimulation of regional T and B cell populations. Additional FLT3L-Fc-associated changes included decreases in red blood cell (RBC) mass, increases in RBC distribution width, variable changes in reticulocytes, and transient alterations in platelet counts (rats only). The RBC and WBC findings were associated microscopically with increased hematopoietic cellularity of the bone marrow in both species and increased splenic megakaryocytic extramedullary hematopoiesis in rats. The totality of nonclinical safety data support the clinical development of FLT3L-Fc.


Subject(s)
Membrane Proteins , Neoplasms , Rats , Animals , Dendritic Cells , Hematopoietic Stem Cells , Immunotherapy
2.
Am J Pathol ; 192(4): 687-700, 2022 04.
Article in English | MEDLINE | ID: mdl-35063406

ABSTRACT

Skin toxicity is a common safety concern associated with drugs that inhibit epidermal growth factor receptors as well as other targets involved in epidermal growth and differentiation. Recently, the use of a three-dimensional reconstructed human epidermis model enabled large-scale drug screening and showed potential for predicting skin toxicity. Although a decrease in epidermal thickness was often observed when the three-dimensional reconstructed tissues were exposed to drugs causing skin toxicity, the thickness evaluation of epidermal layers from a pathologist was subjective and not easily reproducible or scalable. In addition, the subtle differences in thickness among tissues, as well as the large number of samples tested, made cross-study comparison difficult when a manual evaluation strategy was used. The current study used deep learning and image-processing algorithms to measure the viable epidermal thickness from multiple studies and found that the measured thickness was not only significantly correlated with a pathologist's semi-quantitative evaluation but was also in close agreement with the quantitative measurement performed by pathologists. Moreover, a sensitivity of 0.8 and a specificity of 0.75 were achieved when predicting the toxicity of 18 compounds with clinical observations with these epidermal thickness algorithms. This approach is fully automated, reproducible, and highly scalable. It not only shows reasonable accuracy in predicting skin toxicity but also enables cross-study comparison and high-throughput compound screening.


Subject(s)
Deep Learning , Skin Diseases , Algorithms , Epidermis , Humans , Image Processing, Computer-Assisted , Skin
3.
Toxicol Appl Pharmacol ; 418: 115494, 2021 05 01.
Article in English | MEDLINE | ID: mdl-33722668

ABSTRACT

Tumor progression locus 2 (Tpl2, gene name MAP3K8), a mitogen-activated protein kinase, is widely expressed in immune and non-immune cells to integrate tumor necrosis factor (TNF), toll-like receptors (TLRs), and interleukin-1 (IL1) receptor signaling to regulate inflammatory response. Given its central role in inflammatory response, Tpl2 is an attractive small molecule drug target. However, the role of Tpl2 as an oncogene or tumor suppressor gene remains controversial, and its function outside immune cells is not understood. We therefore utilized a Tpl2 kinase dead (Tpl2-KD) mouse model in an 18-month aging study to further elucidate Tpl2 effects on lifespan and chronic disease. Histopathological studies revealed the incidence and severity of spontaneous tumors and non-neoplastic lesions were comparable between wild type and Tpl2-KD mice. The only finding was that male Tpl2-KD mice had higher bodyweight and an increased incidence of liver steatosis, suggesting a sex-specific role for Tpl2 in hepatic lipid metabolism. In conclusion, loss of Tpl2 kinase activity did not lead to increased tumorigenesis over aging in mice but affected likely alterations in lipid metabolism in male animals.


Subject(s)
Fatty Liver/enzymology , Inflammation/enzymology , Liver/enzymology , MAP Kinase Kinase Kinases/metabolism , Neoplasms/metabolism , Proto-Oncogene Proteins/metabolism , Age Factors , Animals , Fatty Liver/genetics , Fatty Liver/pathology , Female , Genotype , Inflammation/genetics , Lipid Metabolism , Liver/pathology , MAP Kinase Kinase Kinases/deficiency , MAP Kinase Kinase Kinases/genetics , Male , Mice, Inbred C57BL , Mice, Knockout , Neoplasms/genetics , Neoplasms/pathology , Phenotype , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Sex Factors
4.
Free Radic Res ; 54(7): 525-534, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32873097

ABSTRACT

Slc7a11 (xCT) and Slc3a1 (rBAT) are cystine uptake transporters that maintain intracellular concentrations of cysteine, the rate-limiting amino acid in glutathione synthesis. This study was conducted to first determine the tissue distribution of the two transporters in male and female mice. Because Slc3a1 was the primary cystine transporter in liver, its sex-divergent expression, ontogeny, diurnal rhythm and whether its mRNA expression is altered by transcription factors (AhR, CAR, PXR, PPARα, and Nrf2) was also investigated. Slc7a11 was expressed highest in brain and gonads. Slc3a1 was expressed highest in kidney and intestine, followed by liver. Duodenal and hepatic Slc3a1 was higher in females than males. Hepatic Slc3a1 was high during darkness and low during daytime. Hepatic Scl3a1 was lowest pre-birth, increased to near maximal levels at birth, decreased back to pre-birth levels between Days 3-10, and then returned to peak levels by Day 45. Except for CAR, activation of transcription factors did not increase hepatic mRNA expression of Slc3a1. Chemical activation of CAR significantly induced Slc3a1 1.4-fold in wild-type but not CAR-null mice. Slc3a1 mRNA was higher in livers of AhR- and Nrf2-null mice compared to wild-type mice. High doses of diquat but not acetaminophen induced Slc3a1, suggesting Slc3a1 may respond to oxidative stress but not necessarily to GSH depletion. Overall, Slc7a11 is mainly expressed in brain and gonads, whereas Slc3a1 is mainly expressed in kidney, small intestine and liver, and its hepatic expression is regulated by diurnal rhythm and certain xenobiotic treatments.


Subject(s)
Amino Acid Transport Systems, Basic/metabolism , Amino Acid Transport Systems, Neutral/metabolism , Cystine/metabolism , Amino Acid Transport Systems, Basic/biosynthesis , Amino Acid Transport Systems, Basic/genetics , Amino Acid Transport Systems, Neutral/biosynthesis , Amino Acid Transport Systems, Neutral/genetics , Animals , Female , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Oxidative Stress/physiology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tissue Distribution
5.
Toxicol Sci ; 170(2): 260-272, 2019 08 01.
Article in English | MEDLINE | ID: mdl-31106824

ABSTRACT

Use of genetically engineered rodents is often considered a valuable exercise to assess potential safety concerns associated with the inhibition of a target pathway. When there are potential immunomodulatory risks associated with the target, these genetically modified animals are often challenged with various pathogens in an acute setting to determine the risk to humans. However, the applicability of the results from infection models is seldom assessed when significant retrospective human data become available. Thus, the purpose of the current review is to compare the outcomes of infectious pathogen challenge in mice with genetic deficiencies in TNF-α, IL17, IL23, or Janus kinase pathways with infectious outcomes caused by inhibitors of these pathways in humans. In general, mouse infection challenge models had modest utility for hazard identification and were generally only able to predict overall trends in infection risk. These models did not demonstrate significant value in evaluating specific types of pathogens that are either prevalent (ie rhinoviruses) or of significant concern (ie herpes zoster). Similarly, outcomes in mouse models tended to overestimate the severity of infection risk in human patients. Thus, there is an emerging need for more human-relevant models that have better predictive value. Large meta-analyses of multiple clinical trials or post-marketing evaluations remains the gold-standard for characterizing the true infection risk to patients.


Subject(s)
Disease Susceptibility/immunology , Immune System/immunology , Infections/immunology , Models, Animal , Animals , Animals, Genetically Modified , Humans , Meta-Analysis as Topic , Randomized Controlled Trials as Topic
6.
Free Radic Biol Med ; 134: 335-342, 2019 04.
Article in English | MEDLINE | ID: mdl-30659941

ABSTRACT

Mammals have developed a variety of antioxidant systems to protect them from the oxygen environment and toxic stimuli. Little is known about the mRNA abundance of antioxidant components during postnatal development of the liver. Therefore, the purpose of this study was to compare the mRNA abundance of antioxidant components during liver development. Livers from male C57BL/6J mice were collected at 12 ages from prenatal to adulthood. The transcriptome was determined by RNA-Seq with transcript abundance estimated by Cufflinks. RNA-Seq provided a complete, more accurate, and unbiased quantification of the transcriptome. Among 33 known antioxidant components examined, three ontogeny patterns of liver antioxidant components were observed: (1) Prenatal-enriched, in which the mRNAs decreased from fetal livers to adulthood, such as metallothionein and heme oxygenase-1; (2) adolescent-rich and relatively stable expression, such as peroxiredoxins; and (3) adult-rich, in which the mRNA increased with age, such as catalase and superoxide dismutase. Alternative splicing of several antioxidant genes, such as Keap1, Glrx2, Gpx3, and Txnrd1, were also detected by RNA-Seq. In summary, RNA-Seq revealed the relative abundance of hepatic antioxidant enzymes, which are important in protecting against the deleterious effects of oxidative stress.


Subject(s)
Antioxidants/metabolism , Gene Expression Regulation, Developmental , Liver/metabolism , RNA, Messenger/genetics , RNA-Seq/methods , Transcriptome , Animals , Female , Gene Expression Profiling , Liver/growth & development , Male , Mice , Mice, Inbred C57BL
7.
Cell Biosci ; 8: 45, 2018.
Article in English | MEDLINE | ID: mdl-30140426

ABSTRACT

BACKGROUND: Diabetes mellitus is associated with an increased risk in diabetic cardiomyopathy (DCM) that is distinctly not attributed to co-morbidities with other vasculature diseases. To date, while dysregulation of calcium handling is a key hallmark in cardiomyopathy, studies have been inconsistent in the types of alterations involved. In this study human cardiomyocytes were exposed to an environmental nutritional perturbation of high glucose, fatty acids, and l-carnitine to model DCM and iTRAQ-coupled LC-MS/MS proteomic analysis was used to capture proteins affected by the perturbation. The proteins captured were then compared to proteins currently annotated in the cardiovascular disease (CVD) gene ontology (GO) database to identify proteins not previously described as being related to CVD. Subsequently, GO analysis for calcium regulating proteins and endoplasmic/sarcoplasmic reticulum (ER/SR) associated proteins was carried out. RESULTS: Here, we identified CCDC47 (calumin) as a unique calcium regulating protein altered in our in vitro nutritional perturbation model. The cellular and functional role of CCDC47 was then assessed in rat cardiomyocytes. In rat H9C2 myocytes, overexpression of CCDC47 resulted in increase in ionomycin-induced calcium release and reuptake. Of interest, in a diet-induced obese (DIO) rat model of DCM, CCDC47 mRNA expression was increased in the atrium and ventricle of the heart, but CCDC47 protein expression was significantly increased only in the atrium of DIO rats compared to lean control rats. Notably, no changes in ANP, BNP, or ß-MHC were observed between DIO rats and lean control rats. CONCLUSIONS: Together, our in vitro and in vivo studies demonstrate that CCDC47 is a unique calcium regulating protein that is associated with early onset hypertrophic cardiomyopathy.

8.
Toxicol Lett ; 232(1): 326-32, 2015 Jan 05.
Article in English | MEDLINE | ID: mdl-25280775

ABSTRACT

This study utilized pharmacological activation of Nrf2 with oleanolic acid (OA, 22.5mg/kg, sc for 4 days) and the genetic alteration of Nrf2 (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.


Subject(s)
Chemical and Drug Induced Liver Injury/prevention & control , Liver/drug effects , NF-E2-Related Factor 2/metabolism , Oleanolic Acid/pharmacology , Organic Anion Transporters, Sodium-Independent/metabolism , Phalloidine , Animals , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Cytoprotection , Disease Models, Animal , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/drug effects , Gene Expression Regulation/drug effects , Inflammation Mediators/metabolism , Kelch-Like ECH-Associated Protein 1/genetics , Kelch-Like ECH-Associated Protein 1/metabolism , Lipid Peroxidation/drug effects , Liver/metabolism , Liver/pathology , Mice, Inbred C57BL , Mice, Knockout , NF-E2-Related Factor 2/deficiency , NF-E2-Related Factor 2/genetics , Organic Anion Transporters, Sodium-Independent/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effects , Solute Carrier Organic Anion Transporter Family Member 1B3
9.
PLoS One ; 9(3): e93013, 2014.
Article in English | MEDLINE | ID: mdl-24667526

ABSTRACT

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 µg/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-1ß 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 Gstα1, Gstα4, Gstµ, 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.


Subject(s)
Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/prevention & control , Liver/drug effects , Liver/metabolism , Microcystins/toxicity , NF-E2-Related Factor 2/metabolism , Animals , Biological Transport/drug effects , Chemical and Drug Induced Liver Injury/metabolism , Chemokines/genetics , Glutathione/metabolism , Lipid Peroxidation/drug effects , Liver/cytology , Mice , Microcystins/metabolism , NF-E2-Related Factor 2/deficiency , NF-E2-Related Factor 2/genetics , Oxidative Stress/drug effects , Thiobarbituric Acid Reactive Substances/metabolism
10.
Toxicol Appl Pharmacol ; 272(3): 816-24, 2013 Nov 01.
Article in English | MEDLINE | ID: mdl-23948738

ABSTRACT

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 (Ostß). OA increased the expression of Nrf2 and its target gene, Nqo1, but decreased the expression of AhR, CAR and PPARα 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.


Subject(s)
Bile Acids and Salts/metabolism , Chemical and Drug Induced Liver Injury/metabolism , Cholestasis/chemically induced , Cholestasis/metabolism , Oleanolic Acid/toxicity , Animals , Dose-Response Relationship, Drug , Female , Mice , Mice, Inbred C57BL
11.
Oxid Med Cell Longev ; 2013: 305861, 2013.
Article in English | MEDLINE | ID: mdl-23766851

ABSTRACT

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 ß , IL-6, and TNF α), 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.


Subject(s)
Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/prevention & control , NF-E2-Related Factor 2/metabolism , Animals , Cell Death/genetics , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/pathology , DNA Damage/genetics , Disease Models, Animal , Endoplasmic Reticulum Stress/genetics , Gene Expression Regulation , Inflammation/genetics , Inflammation/pathology , Liver/metabolism , Liver/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Stress, Physiological/genetics
12.
PLoS One ; 8(3): e59122, 2013.
Article in English | MEDLINE | ID: mdl-23527105

ABSTRACT

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.


Subject(s)
Fasting , Liver/metabolism , NF-E2-Related Factor 2/genetics , Oxidative Stress , Transcriptional Activation , Animals , Body Weight , Gene Expression Regulation , Glucose/metabolism , Lipid Metabolism , Liver/anatomy & histology , Male , Mice , Mice, Knockout , Mitochondria/metabolism , NF-E2-Related Factor 2/metabolism , Organ Size , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Signal Transduction , Trans-Activators/metabolism , Transcription Factors
13.
PLoS One ; 7(10): e44686, 2012.
Article in English | MEDLINE | ID: mdl-23056183

ABSTRACT

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.


Subject(s)
Antioxidant Response Elements/genetics , Intracellular Signaling Peptides and Proteins/metabolism , NF-E2-Related Factor 2/metabolism , Organic Chemicals/pharmacology , Signal Transduction/drug effects , Animals , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , Gene Expression Regulation, Neoplastic/drug effects , Humans , Kelch-Like ECH-Associated Protein 1 , Luciferases/genetics , Luciferases/metabolism , Luminescent Measurements/methods , Mice , Molecular Structure , NAD(P)H Dehydrogenase (Quinone)/genetics , Organic Chemicals/chemistry , Protein Binding/drug effects , Pyridones/chemistry , Pyridones/pharmacology , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Small Molecule Libraries
14.
Toxicol Appl Pharmacol ; 264(3): 305-14, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-23017736

ABSTRACT

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.


Subject(s)
Dietary Fats/administration & dosage , Dietary Fats/adverse effects , Glucose Intolerance/metabolism , NF-E2-Related Factor 2/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animal Feed/analysis , Animals , Blood Glucose , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Diet , Fatty Acids/metabolism , Gene Expression Regulation/physiology , Glucose Intolerance/genetics , Homeostasis , Hormones , Insulin/metabolism , Kelch-Like ECH-Associated Protein 1 , Liver/metabolism , Mice , Obesity , RNA
15.
Free Radic Res ; 46(10): 1220-9, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22762311

ABSTRACT

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.


Subject(s)
Chemical and Drug Induced Liver Injury/metabolism , Diquat/toxicity , Lung Injury/metabolism , NF-E2-Related Factor 2/metabolism , Alanine Transaminase/blood , Animals , Antioxidants/metabolism , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/pathology , Glutamate-Cysteine Ligase/biosynthesis , Glutathione/metabolism , Glutathione Disulfide/metabolism , Herbicides/toxicity , Inbreeding , Lung Injury/chemically induced , Lung Injury/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , NF-E2-Related Factor 2/deficiency , Oxidative Stress/drug effects , Oxidative Stress/physiology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reactive Oxygen Species/metabolism , Signal Transduction , Survival Analysis , Thiobarbituric Acid Reactive Substances/metabolism
16.
PLoS One ; 7(7): e39006, 2012.
Article in English | MEDLINE | ID: mdl-22808024

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cytoskeletal Proteins/genetics , Hepatocytes/metabolism , Liver/metabolism , Metabolic Detoxication, Phase II , Metabolic Detoxication, Phase I , NF-E2-Related Factor 2/genetics , RNA, Messenger/genetics , Adaptor Proteins, Signal Transducing/deficiency , Animals , Biological Transport , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Cytoskeletal Proteins/deficiency , Gene Expression Profiling , Gene Expression Regulation , Kelch-Like ECH-Associated Protein 1 , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mice , Mice, Knockout , NF-E2-Related Factor 2/deficiency , Oligonucleotide Array Sequence Analysis , Oxidative Stress , RNA, Messenger/metabolism
17.
Toxicol Appl Pharmacol ; 262(3): 321-9, 2012 Aug 01.
Article in English | MEDLINE | ID: mdl-22627062

ABSTRACT

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.


Subject(s)
Ethanol/pharmacology , Liver/drug effects , NF-E2-Related Factor 2/physiology , Oxidative Stress/drug effects , Adaptor Proteins, Signal Transducing/physiology , Alanine Transaminase/metabolism , Animals , Cytoskeletal Proteins/physiology , Ethanol/antagonists & inhibitors , Fatty Acids, Nonesterified/analysis , Kelch-Like ECH-Associated Protein 1 , L-Lactate Dehydrogenase/metabolism , Lipid Metabolism/drug effects , Lipid Peroxidation/drug effects , Lipids/analysis , Lipids/biosynthesis , Liver/chemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction
18.
Toxicol Sci ; 123(2): 590-600, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21775727

ABSTRACT

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.


Subject(s)
Liver/metabolism , NADP/metabolism , NF-E2-Related Factor 2/metabolism , Animals , Cell Nucleus/metabolism , Gene Dosage , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NADP/genetics , NF-E2-Related Factor 2/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Transcription, Genetic
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