Gamma-glutamylcysteine Ligase Research Articles

Tempol, a Superoxide Dismutase Mimetic, Inhibits Wallerian Degeneration Following Spinal Cord Injury by Preventing Glutathione Depletion and Aldose Reductase Activation.

Spinal cord contusion injury results in Wallerian degeneration of spinal cord axonal tracts, which are necessary for locomotor function. Axonal swelling and loss of axonal density at the contusion site, characteristic of Wallerian degeneration, commence within hours of injury. Tempol, a superoxide dismutase mimetic, was previously shown to reduce the loss of spinal cord white matter and improve locomotor function in an experimental model of spinal cord contusion, suggesting that tempol treatment might inhibit Wallerian degeneration of spinal cord axons. Here, we report that tempol partially inhibits Wallerian degeneration, resulting in improved locomotor recovery. We previously reported that Wallerian degeneration is reduced by inhibitors of aldose reductase (AR), which converts glucose to sorbitol in the polyol pathway. We observed that tempol inhibited sorbitol production in the injured spinal cord to the same extent as the AR inhibitor, sorbinil. Tempol also prevented post-contusion upregulation of AR (AKR1B10) protein expression within degenerating axons, as previously observed for AR inhibitors. Additionally, we hypothesized that tempol inhibits axonal degeneration by preventing loss of the glutathione pool due to polyol pathway activity. Consistent with our hypothesis, tempol treatment resulted in greater glutathione content in the injured spinal cord, which was correlated with increased expression and activity of gamma glutamyl cysteine ligase (γGCL; EC 6.3.2.2), the rate-limiting enzyme for glutathione synthesis. Administration of the γGCL inhibitor buthionine sulfoximine abolished all observed effects of tempol administration. Together, these results support a pathological role for polyol pathway activation in glutathione depletion, resulting in Wallerian degeneration after spinal cord injury (SCI). Interestingly, methylprednisolone, oxandrolone, and clenbuterol, which are known to spare axonal tracts after SCI, were equally effective in inhibiting polyol pathway activation. These results suggest that prevention of AR activation is a common target of many disparate post-SCI interventions.

Inhibition of NOX2 or NLRP3 inflammasome prevents cardiac remote ischemic preconditioning.

Introduction: Short episodes of ischemia-reperfusion (IR) in the heart (classical ischemic preconditioning, IPC) or in a limb (remote ischemic preconditioning, RIPC) before a prolonged ischemic episode, reduce the size of the infarct. It is unknown whether IPC and RIPC share common mechanisms of protection. Animals KO for NOX2, a superoxide-producing enzyme, or KO for NLRP3, a protein component of inflammasome, are not protected by IPC. The aim of this study was to investigate if NOX2 or NLRP3 inflammasome are involved in the protection induced by RIPC. Methods: We preconditioned rats using 4 × 5min periods of IR in the limb with or without a NOX2 inhibitor (apocynin) or an NLRP3 inhibitor (Bay117082). In isolated hearts, we measured the infarct size after 30min of ischemia and 60min of reperfusion. In hearts from preconditioned rats we measured the activity of NOX2; the mRNA of Nrf2, gamma-glutamylcysteine ligase, glutathione dehydrogenase, thioredoxin reductase and sulfiredoxin by RT-qPCR; the content of glutathione; the activation of the NLRP3 inflammasome and the content of IL-1β and IL-10 in cardiac tissue. In exosomes isolated from plasma, we quantified NOX2 activity. Results: The infarct size after IR decreased from 40% in controls to 9% of the heart volume after RIPC. This protective effect was lost in the presence of both inhibitors. RIPC increased NOX2 activity in the heart and exosomes, as indicated by the increased association of p47phox to the membrane and by the increased oxidation rate of NADPH. RIPC also increased the mRNA of Nrf2 and antioxidant enzymes. Also, RIPC increased the content of glutathione and the GSH/GSSG ratio. The inflammasome proteins NLRP3, procaspase-1, and caspase-1 were all increased in the hearts of RIPC rats. At the end of RIPC protocol, IL-1β increased in plasma but decreased in cardiac tissue. At the same time, IL-10 did not change in cardiac tissue but increased by 70% during the next 50min of perfusion. Conclusion: RIPC activates NOX2 which upregulates the heart's antioxidant defenses and activates the NLRP3 inflammasome which stimulates a cardiac anti-inflammatory response. These changes may underlie the decrease in the infarct size induced by RIPC.

Diselenide-derivative of 3-pyridinol targets redox enzymes leading to cell cycle deregulation and apoptosis in A549cells.

The aim of present study was to understand the mechanism of action of 2,2'-diselenobis(3-pyridinol) or DISPOL in human lung cancer (A549) cells. A549 cells were treated with 10µM (∼IC50) of DISPOL for varying time points to corelate the intracellular redox changes with its cytotoxic effect. The results indicated that DISPOL treatment led to a time dependant decrease in the basal level of reactive oxygen species (ROS). Additionally, DISPOL treatment elevated the ratio of reduced (GSH) and oxidised (GSSG) glutathione by upregulating gamma-glutamylcysteine ligase (γ-GCL) involved in GSH biosynthesis and inhibiting the activities of redox enzymes responsible for GSH utilization and recycling, such as glutathione-S-transferase (GST) and glutathione reductase (GR). Molecular docking analysis suggests putative interactions of DISPOL with GST and GR which could account for its inhibitory effect on these enzymes. Further, DISPOL induced reductive environment preceded G1 arrest and apoptosis as evidenced by decreased expression of cell cycle genes (Cyclin D1 and Cyclin E1) and elevation of p21 and apoptotic markers (cleaved caspase 3 and cleaved PARP). The combinatorial experiments involving DISPOL and redox modulatory agents such as N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) indeed confirmed the role of reductive stress in DISPOL-induced cell death. Finally, Lipinski's rule suggests attributes of drug likeness in DISPOL. Taken together, DISPOL exhibits a novel mechanism of reductive stress-mediated cell death in A549cells that warrants future exploration as anticancer agent.

Bone Marrow Mesenchymal Stem Cells Exert Protective Effects After Ischemic Stroke Through Upregulation of Glutathione.

Bone marrow mesenchymal stem cells (BMSCs) have been shown to promote stroke recovery, however, the underlying mechanisms are not well understood. In this study naïve rats were intravenously injected with syngeneic BMSCs to screen for potential differences in brain metabolite spectrum versus vehicle-treated controls by capillary electrophoresis-mass spectrometry. A total of 65 metabolites were significantly changed after BMSC treatment. Among them, 5-oxoproline, an intermediate in the biosynthesis of the endogenous glutathione (GSH), was increased. To confirm the obtained results and investigate the metabolic pathways, BMSCs were injected into rats 24h after middle cerebral artery occlusion (MCAO). Rats receiving vehicle solution and sham-operated animals served as controls. High performance liquid chromatography, reverse transcription-quantitative polymerase chain reaction, and Western blotting revealed that intravenous BMSC application increased the levels of 5-oxoproline and GSH in MCAO rats, as well as the expression of key enzymes involved in GSH synthesis including, gamma-glutamylcyclotransferase and gamma-glutamylcysteine ligase. Subsequent clinical investigation confirmed that acute ischemic stroke patients had higher plasma 5-oxoproline and GSH levels than age- and sex-matched non-stroke controls. The optimal cutoff value for 5-oxoproline diagnosing acute ischemic stroke (≤ 7d) was 3.127µg/mL (sensitivity, 63.4 %; specificity, 81.2 %) determined by receiver characteristic operator curve. The area under the curve was 0.782 (95 % confidence interval: 0.718-0.845). Our findings indicate that BMSCs play a protective role in ischemic stroke through upregulation of GSH and 5-oxoproline is a potential biomarker for acute ischemic stroke. Ischemic stroke causes oxidative stress and induction of endogenous, glutathione-dependent anti-oxidative mechanisms. 5-oxoproline, an important metabolite in glutathione biosynthesis, could serve as a biomarker of acute ischemic stroke. Moreover, intravenous bone marrow mesenchymal stem cell (BMSC) treatment after experimental stroke upregulates the expression of key enzymes involved in glutathione synthesis, which results in better antioxidative defense and improved stroke outcome.

Exposure of Rats to Multiple Oral Doses of Dichloroacetate Results in Upregulation of Hepatic Glutathione Transferases and NAD(P)H Dehydrogenase [Quinone] 1.

Dichloroacetate (DCA) is an investigational drug that is used in the treatment of various congenital and acquired disorders of energy metabolism. Although DCA is generally well tolerated, some patients experience peripheral neuropathy, a side effect more common in adults than children. Repetitive DCA dosing causes downregulation of its metabolizing enzyme, glutathione transferase zeta 1 (GSTZ1), which is also critical in the detoxification of maleylacetoacetate and maleylacetone. GSTZ1 (-/-) knockout mice show upregulation of glutathione transferases (GSTs) and antioxidant enzymes as well as an increase in the ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH), suggesting GSTZ1 deficiency causes oxidative stress. We hypothesized that DCA-mediated depletion of GSTZ1 causes oxidative stress and used the rat to examine induction of GSTs and antioxidant enzymes after repeated DCA exposure. We determined the expression of alpha, mu, pi, and omega class GSTs, NAD(P)H dehydrogenase [quinone] 1 (NQO1), gamma-glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by liquid chromatography-tandem mass spectrometry. Enzyme activity was measured in hepatic cytosol using 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and 2,6-dichloroindophenol as substrates. In comparison with acetate-treated controls, DCA dosing increased the relative expression of GSTA1/A2 irrespective of rodent age, whereas only adults displayed higher levels of GSTM1 and GSTO1. NQO1 expression and activity were higher in juveniles after DCA dosing. GSH concentrations were increased by DCA in adults, but the GSH:GSSG ratio was not changed. Levels of GCLC and GSS were higher and lower, respectively, in adults treated with DCA. We conclude that DCA-mediated depletion of GSTZ1 causes oxidative stress and promotes the induction of antioxidant enzymes that may vary between age groups. SIGNIFICANCE STATEMENT: Treatment with the investigational drug, dichloroacetate (DCA), results in loss of glutathione transferase zeta 1 (GSTZ1) and subsequent increases in body burden of the electrophilic tyrosine metabolites, maleylacetoacetate and maleylacetone. Loss of GSTZ1 in genetically modified mice is associated with induction of glutathione transferases and alteration of the ratio of oxidized to reduced glutathione. Therefore, we determined whether pharmacological depletion of GSTZ1 through repeat administration of DCA produced similar changes in the liver, which could affect responses to other drugs and toxicants.

T165. ANTI-GLUTAMATERGIC PROPERTY OF N-ACETYLCYSTEINE DOCUMENTED IN VIVO WITH 1H MRS

BackgroundDeficits of brain glutathione (GSH), the primary antioxidant in living tissue, and associated redox imbalance are postulated to be implicated in schizophrenia. However, due to poor blood-brain barrier permeability of GSH, direct supplementation is ineffective in restoring its brain levels. Therefore, there has been great interest in investigating N-acetylcysteine (NAC), as a prodrug that can be deacetylated to supply cysteine (Cys), which crosses the BBB and is the rate-limiting substrate in brain GSH synthesis and restoration. Once inside the cell, Cys combines with glutamate (Glu) to initiate GSH synthesis in a reaction that is catalyzed by gamma-glutamyl cysteine ligase (GCL), the rate-limiting enzyme. Likely due to the in situ reaction of NAC-supplied Cys with Glu to spur GSH synthesis, NAC is believed to have anti-glutamatergic properties. However, direct in vivo evidence of NAC as an anti-glutamatergic agent is currently lacking. In this study, we used 1H MRS to monitor changes in brain levels of both GSH and Glu in response to 4 weeks of daily supplementation with NAC in healthy volunteer subjects. We postulated that 4 weeks of NAC treatment would elevate GSH and decrease Glu levels.MethodsSubjects: Participants recruited for this study consisted of 12 mentally and physically healthy human volunteer (HV) subjects.NAC Supplementation: To investigate the effects of dietary NAC supplementation on cortical GSH and Glu levels, each HV subject underwent 1H MRS scans at baseline. Then a 4-week supplement of 900mg NAC tablets was provided, to be taken 2 per day for a daily NAC dose of 1800mg. Finally, each subject was brought back after 4 weeks for the post-NAC 1H MRS scans to assess the effect of the treatment on cortical GSH and Glu levels.Brain 1H MRS: In vivo cortical GSH spectra were recorded in 15 min on a 3.0 T GE MRI system from a 3cmx3cmx2cm occipital cortex voxel, using the standard J-editing technique, with TE/TR 68/1500ms and 290 interleaved excitations (580 total). Levels of Glu, uncontaminated with glutamine, were obtained from the same occipital cortex voxel in 6 min using the CT-PRESS method, with TE/TR 139ms/1500ms, and 129 chemical shift encoding steps in increments of 0.8ms in t1 dimension. Peak areas for both GSH and Glu were derived by frequency-domain fitting of the recorded spectra. The resulting peak areas were then expressed as ratios relative to the area of the unsuppressed tissue water signal in the voxel.ResultsGSH Levels: The effect of 4 weeks of NAC supplementation on GSH levels was a numerical increase that did not reach statistical significance relative to baseline (p=0.33).Glu LevelsFollowing 4 weeks of NAC supplementation cortical Glu levels decreased significantly compared to baseline (p=0.04).DiscussionThis study has shown that following 4 weeks of taking NAC, brain Glu levels in HV decreased significantly, even though a numerical increase in GSH levels did not reach statistical significance. The observed Glu decrease at 4 weeks suggests a net increase in the consumption of intracellular Glu reserves in the GCL-catalyzed reaction that combines NAC-supplied Cys with Glu in GSH synthesis. The failure of GSH levels to increase significantly after NAC likely reflected the established “feedback inhibition” mechanism whereby GSH synthesis is shut off in a tissue with normal levels, with de novo synthesis due to NAC occurring only in a subset of subjects with “low” GSH levels (i.e., below the sample mean) at baseline. In summary, the results of this study support the role of NAC as an anti-glutamatergic agent, which seems to modulate Glu levels by increasing the availability of intracellular Cys that then combines with Glu to initiate GSH synthesis, leading to a lowering of total Glu levels as measured with MRS

Induction of Glutathione Transferases and NAD(P)H Quinone Dehydrogenase 1 in Rats Dosed with Dichloroacetate

Dichloroacetate (DCA) is an investigational drug for treatment of children and adults with genetic or acquired mitochondrial diseases and cancer. The pharmacokinetics and dynamics of DCA vary by subject age. For example, the chronic use of DCA in adults is associated with the development of reversible peripheral neuropathy, a side effect that is seldom observed in children, who metabolize DCA more quickly. The dechlorination of DCA to its inactive metabolite, glyoxylate, is catalyzed by maleylacetoacetate isomerase/glutathione transferase zeta 1 (MAAI/GSTZ1). GSTZ1 is also critical in tyrosine catabolism through trans‐isomerization of maleylacetoacetate (MAA) and maleylacetone (MA). However, DCA causes mechanism‐based inactivation of GSTZ1, resulting in inhibition of both DCA metabolism and isomerization of MAA and MA. The resulting tissue accumulation of DCA, MAA, and MA may be a mechanism of toxicity. Studies in GSTZ1(−/−) mice showed induction of antioxidant enzymes and GSTs A1/A2, M1 and M5, as well as an increase in the GSSG:GSH ratio, suggesting that GSTZ1 deficiency causes oxidative stress. In this study, we determined if loss of the expression and activity of GSTZ1 in rat tissues following multiple DCA dosages that mimicked therapy resulted in similar effects. Adult (52 wk) female Sprague Dawley rats (8 per group) and young (4 wk) male and female Sprague Dawley rats (4 per group) were dosed with sodium DCA (100 mg/kg) or sodium acetate (100 mg/kg) by oral gavage daily for 8 consecutive days. On the 9th day, rats were euthanized, livers were removed and cytosolic and mitochondrial subcellular fractions were prepared. Protein expression was assessed by Western blot using antibodies to A, M, P, T and O‐class GSTs and to NAD(P)H quinone dehydrogenase 1 (NQO1), gamma‐glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by LC/MS/MS. Enzyme activity was measured in hepatic cytosol using 1‐chloro‐2,4‐dinitrobenzene (CDNB), 1,2‐dichloro‐4‐nitrobenzene (DCNB), and 2,6‐dichloroindophenol (DCPIP) as substrates. In comparison to acetate‐treated controls, GST activities with CDNB and DCNB were higher in DCA‐treated rats irrespective of age (p < 0.05), as was the relative expression of GSTA1/A2 (p < 0.01), whereas DCA‐treated adults displayed higher levels of GSTM1 (p < 0.05) and GSTO1 (p < 0.05). Changes in the expression of P and T‐class GSTs following multiple doses of DCA were not observed for either group. NQO1 expression and activity were higher in juveniles after DCA dosing (p < 0.05). GSH concentrations were increased by DCA in adults but the GSH:GSSG ratio was not changed. Levels of GCLC and GSS were higher and lower, respectively, in adults treated with DCA. We conclude that DCA‐mediated depletion of GSTZ1 causes oxidative stress and promotes the induction of antioxidant enzymes that may vary between age groups. These findings will be important when interpreting outcomes of ongoing clinical trials of DCA in children and adults.Support or Funding InformationSupported in part by NIH RO1‐GM099871.

Effects of dietary supplementation with enzymatically treated Artemisia annua on growth performance, intestinal morphology, digestive enzyme activities, immunity, and antioxidant capacity of heat-stressed broilers

The gastrointestinal tract is considered as one of the main target organs affected by heat stress. Phytogenic feed additives containing phenolics and flavonoids can improve the resistance of broilers to heat stress. This study was conducted to investigate the effects of dietary supplementation with enzymatically treated Artemisia annua (EA) on growth performance, intestinal morphology, digestive enzyme activities, immunity and antioxidant capacity of broilers challenged with heat stress. One hundred and forty-four 21-day-old male Arbor Acres broilers were randomly distributed into 3 treatments: 1) non-challenged control (CON); 2) heat-stress-challenged control (HS); and 3) heat-stress-challenged group + 1 g EA/kg diet (HS-EA). From 22 to 41 d, broilers in the CON group were housed at 22 ± 1°C, the HS and HS-EA groups, in which broilers were raised at 34 ± 1°C for 8 h (0900–1700 h) and the temperature for the rest time was the same as that of the CON group. The EA supplementation alleviated the compromised body weight gain and intestinal morphology impairment caused by heat stress challenge (P < 0.05). The EA attenuated heat-stress-induced decreased intestinal lipase, trypsin and total superoxide dismutase activities, and reduced intestinal secretory immunoglobulin A (SIgA) and IgG concentrations (P < 0.05). The EA inclusion prevented the elevation of intestinal malondialdehyde content and reduction of intestinal glutathione concentration induced by heat stress challenge (P < 0.05). The intestinal mRNA abundances of nuclear factor erythroid 2-related factor 2, heme oxygenase 1, glutathione peroxidase, gamma-glutamyl cysteine ligase larger catalytic subunit and gamma-glutamyl cysteine ligase smaller modulator subunit in heat-stressed broilers were increased in response to dietary EA treatment (P < 0.05). In conclusion, dietary supplementation of 1 g/kg EA could alleviate heat-stress-induced compromised growth performance and intestinal damage of broilers.

Reduced Glutathione Level Promotes Epithelial-Mesenchymal Transition in Lens Epithelial Cells via a Wnt/β-Catenin–Mediated Pathway: Relevance for Cataract Therapy

The epithelial-mesenchymal transition (EMT) process plays a pivotal role in the pathogenesis of posterior capsular opacification because of remnant lens epithelial cell proliferation, migration, and transformation after cataract surgery. The latter, we hypothesize, may result in posterior capsule wrinkling and opacification because of a profound change in the lens growth environment via a 1000-fold reduction of extracellular glutathione (GSH) levels. To test this hypothesis, we investigated the EMT process in cell culture and GSH biosynthesis deficiency mouse models. Our data indicate a dramatic increase of pro-EMT markers, such as type I collagen, α-smooth muscle actin, vimentin, and fibronectin, under conditions of lens GSH depletion. Further study suggests that decreased GSH triggers the Wnt/β-catenin signal transduction pathway, independent of transforming growth factor-β. Equally important, the antioxidants N-acetyl cysteine and GSH ethyl ester could significantly attenuate the EMT signaling stimulated by decreased GSH levels. These findings were further confirmed by mock cataract surgery in both gamma glutamyl-cysteine ligase, catalytic subunit, and gamma glutamyl-cysteine ligase, modifier subunit, knockout mouse models. Remarkably, increased EMT marker expression, β-catenin activation, and translocation into the nucleus were found in both knockout mice compared with the wild type, and such increased expression could be significantly attenuated by N-acetyl cysteine or GSH ethyl ester treatment. This study, for the first time we believe, links oxidative stress to lens fibrosis and posterior capsular opacification formation via EMT-mediated mechanisms.

Apocynin protects against neurological damage induced by quinolinic acid by an increase in glutathione synthesis and Nrf2 levels

Apocynin (APO) is a well-known NADPH oxidase (NOX) inhibitor. However, several studies have reported its ability to increase glutathione (GSH) levels. Due to GSH is a major non-enzymatic antioxidant in brain, the aim of this study was to evaluate, in the striatum of control and quinolinic acid (QUIN) injected rats, the effect of APO administration on: (1) GSH levels, (2) activity of some enzymes involved in the GSH metabolism, and (3) nuclear factor erythroid-2-related factor 2 (Nrf2) mRNA levels. Animals received QUIN 240nmol in right striatum and APO (5mg/kg, i.p.), 30min before and 60min after intrastriatal injection. APO treatment prevented the QUIN-induced histological damage to the striatum. In control rats, APO treatment increased GSH and Nrf2 mRNA levels and the activities of gamma-glutamylcysteine ligase (γ-GCL), glutathione-S-transferase (GST) and glutathione peroxidase (GPx). On the other hand, APO treatment prevented the QUIN-induced decrease in GSH and Nrf2 levels, and in γ-GCL and GPx activities. These data indicate that APO is able to increase GSH levels and the activity of proteins involved in its metabolism, which could be associated with its ability to increase the Nrf2 mRNA levels.

Gclc deficiency in mouse CNS causes mitochondrial damage and neurodegeneration.

Gamma glutamyl cysteine ligase (GCL) is the rate-limiting enzyme for intracellular glutathione (GSH) synthesis. The GSH concentration and GCL activity are declining with age in the central nervous system (CNS), and is accompanied by elevated reactive oxygen species (ROS). To study the biological effects of low GSH levels, we disrupted its synthesis both at birth by breeding a Gclc loxP mouse with a thy1-cre mouse (NEGSKO mouse) and at a later age by breeding with a CaMKII-ERT2-Cre (FIGSKO mouse). NEGSKO mice with deficiency of the Gclc in their entire CNS neuronal cells develop at 4 weeks: progressive motor neuron loss, gait problems, muscle denervation and atrophy, paralysis, and have diminished life expectancy. The observed neurodegeneration in Gclc deficiency is of more chronic rather than acute nature as demonstrated by Gclc targeted single-neuron labeling from the inducible Cre-mediated knockout (SLICK) mice. FIGSKO mice with inducible Gclc deficiency in the forebrain at 23 weeks after tamoxifen induction demonstrate profound brain atrophy, elevated astrogliosis and neurodegeneration, particularly in the hippocampus region. FIGSKO mice also develop cognitive abnormalities, i.e. learning impairment and nesting behaviors based on passive avoidance, T-Maze, and nesting behavior tests. Mechanistic studies show that impaired mitochondrial glutathione homeostasis and subsequent mitochondrial dysfunction are responsible for neuronal cell loss. This was confirmed by mitochondrial electron transporter chain activity analysis and transmission electron microscopy that demonstrate remarkable impairment of state 3 respiratory activity, impaired complex IV function, and mitochondrial swollen morphology in the hippocampus and cerebral cortex. These mouse genetic tools of oxidative stress open new insights into potential pharmacological control of apoptotic signaling pathways triggered by mitochondrial dysfunction.

Upregulation of cellular glutathione levels in human ABCB5- and murine Abcb5-transfected cells.

BackgroundPreviously, we have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCB5-transfected cells showed resistance to taxanes and anthracyclines. Herein, we further screened ABCB5 substrates, and explored the mechanism of resistance.MethodsSensitivity of the cells to test compounds was evaluated using cell growth inhibition assay. Cellular levels of buthionine sulfoximine (BSO), glutathione and amino acids were measured using HPLC and an enzyme-based assay. Cellular and vesicular transport of glutathione was evaluated by a radiolabeled substrate. Expression levels of glutathione-metabolizing enzymes were assessed by RT-PCR.ResultsHuman ABCB5-transfected 293/B5-11 cells and murine Abcb5-transfected 293/mb5-8 cells showed 6.5- and 14-fold higher resistance to BSO than the mock-transfected 293/mock cells, respectively. BSO is an inhibitor of gamma-glutamylcysteine ligase (GCL), which is a key enzyme of glutathione synthesis. 293/B5-11 and 293/mb5-8 cells also showed resistance to methionine sulfoximine, another GCL inhibitor. A cellular uptake experiment revealed that BSO accumulation in 293/B5-11 and 293/mb5-8 cells was similar to that in 293/mock cells, suggesting that BSO is not an ABCB5 substrate. The cellular glutathione content in 293/B5-11 and 293/mb5-8 cells was significantly higher than that in 293/mock cells. Evaluation of the BSO effect on the cellular glutathione content showed that compared with 293/mock cells the BSO concentration required for a 50 % reduction in glutathione content in 293/B5-11 and 293/mb5-8 cells was approximately 2- to 3-fold higher. This result suggests that the BSO resistance of the ABCB5- and Abcb5-transfected cells can be attributed to the reduced effect of BSO on the transfectants. Cellular and vesicular transport assays showed that the transport of radiolabeled glutathione in 293/B5-11 cells was similar to that in 293/mock cells. The mRNA expression of genes encoding glutathione-metabolizing enzymes in 293/B5-11 cells was similar to that in 293/mock cells. The cellular content of Glu, a precursor of glutathione, in 293/B5-11 and 293/mb5-8 cells was higher than that in 293/mock cells.ConclusionsABCB5/Abcb5-transfected cells showed resistance to BSO, which is not a substrate of ABCB5. Our results suggest that ABCB5/Abcb5 upregulates cellular glutathione levels to protect cells from various poisons.

Sulfur amino acid metabolism in Zucker diabetic fatty rats

The present study was aimed to investigate the metabolomics of sulfur amino acids in Zucker diabetic fatty (ZDF) rats, an obese type 2 diabetic animal model. Plasma levels of total cysteine, homocysteine and methionine, but not glutathione (GSH) were markedly decreased in ZDF rats. Hepatic methionine, homocysteine, cysteine, betaine, taurine, spermidine and spermine were also decreased. There are no significant difference in hepatic S-adenosylmethionine, S-adenosylhomocysteine, GSH, GSH disulfide, hypotaurine and putrescine between control and ZDF rats. Hepatic SAH hydrolase, betaine-homocysteine methyltransferase and methylene tetrahydrofolate reductase were up-regulated while activities of gamma-glutamylcysteine ligase and methionine synthase were decreased. The area under the curve (AUC) of methionine and methionine-d4 was not significantly different in control and ZDF rats treated with a mixture of methionine (60mg/kg) and methionine-d4 (20mg/kg). Moreover, the AUC of the increase in plasma total homocysteine was comparable between two groups, although the homocysteine concentration curve was shifted leftward in ZDF rats, suggesting that the plasma total homocysteine after the methionine loading was rapidly increased and normalized in ZDF rats. These results show that the AUC of plasma homocysteine is not responsive to the up-regulation of hepatic BHMT in ZDF rats. The present study suggests that the decrease in hepatic methionine may be responsible for the decreases in its metabolites, such as homocysteine, cysteine, and taurine in liver and consequently decreased plasma homocysteine levels.

Oxidative stress and mitochondrial dysfunction in Kindler syndrome.

BackgroundKindler Syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering, photosensitivity, premature aging, and propensity to skin cancer. In spite of the knowledge underlying cause of this disease involving mutations of FERMT1 (fermitin family member 1), and efforts to characterize genotype-phenotype correlations, the clinical variability of this genodermatosis is still poorly understood. In addition, several pathognomonic features of KS, not related to skin fragility such as aging, inflammation and cancer predisposition have been strongly associated with oxidative stress. Alterations of the cellular redox status have not been previously studied in KS. Here we explored the role of oxidative stress in the pathogenesis of this rare cutaneous disease.MethodsPatient-derived keratinocytes and their respective controls were cultured and classified according to their different mutations by PCR and western blot, the oxidative stress biomarkers were analyzed by spectrophotometry and qPCR and additionally redox biosensors experiments were also performed. The mitochondrial structure and functionality were analyzed by confocal microscopy and electron microscopy.ResultsPatient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits. Electron microscopy analysis of both, KS skin biopsies and keratinocytes showed marked morphological mitochondrial abnormalities. Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement. Imbalance of oxidative stress biomarkers together with abnormalities in the mitochondrial network and function are consistent with a pro-oxidant state.ConclusionsThis is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0211-8) contains supplementary material, which is available to authorized users.

Treadmill exercise activates Nrf2 antioxidant system to protect the nigrostriatal dopaminergic neurons from MPP+ toxicity

Exercise induces oxidative stress, which may activate adaptive antioxidant responses. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays an important role in the defense of oxidative stress by regulating the expression of antioxidant enzymes, gamma-glutamylcysteine ligase (γGCL) and heme oxygenase-1 (HO-1). We investigated whether treadmill exercise protects dopaminergic neurons by regulating the Nrf2 antioxidant system in a 1-methyl-4-phenylpyridine (MPP+)-induced parkinsonian rat model. We found that MPP+ induced early decreases in total glutathione level and Nrf2/γGCLC (catalytic subunit of γGCL) expression, but late upregulation of HO-1 expression in association with loss of nigral dopaminergic neurons and downregulation of tyrosine hydroxylase and dopamine transporter expression in the striatum. Treadmill exercise for 4weeks induced upregulation of Nrf2 and γGCLC expression, and also prevented the MPP+-induced downregulation of Nrf2/γGCLC/glutathione, HO-1 upregulation, and nigrostriatal dopaminergic neurodegeneration. Moreover, the protective effect of exercise was blocked by the knockdown of Nrf2 using a lentivirus-carried shNrf2 delivery system. These results demonstrate an essential role of Nrf2 in the exercise-mediated protective effect that exercise enhances the nigrostriatal Nrf2 antioxidant defense capacity to protect dopaminergic neurons against the MPP+-induced toxicity.

Review of Nrf2-regulated genes induced in response to antioxidants

Nuclear factor (erythroid-derived 2) -like 2 (Nrf2) is a transcription factor that plays an important role in the cellular protection against free radical damage and reduce the incidence of radical derived degenerative diseases such as cancer. Nrf2 is referred to as the "master regulator" of the antioxidant response due to the fact that it modulates the expression of several genes including phase-2 and antioxidant enzymes playing a crucial role in detoxification of electrophiles and reactive oxygen species (ROS), including glutathione-S-transferase (GST), gamma-glutamyl cysteine ligase (γ-GCL), glutathione-S-reductase (GSR), NAD(P)H:quinoneoxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), etc. Following dissociation from it obligatory partner Kelch like ECHassociated protein 1 (Keap1), Nrf2 translocates to the nucleus and transactivates the antioxidant response element (ARE) in the promoter region of several antioxidant genes. In this review, we discuss the role of the Nrf2 system, with particular focus on Nrf2-controlled target genes.

Expression of hepatic antioxidant enzymes in non-obese type-2 diabetic Goto–Kakizaki rats

Diabetes mellitus and its complications have been attributed in part to oxidative stress, against which antioxidant enzymes constitute a major protective mechanism. The present study was performed to investigate the effects of early stage type 2 diabetes in the absence of obesity and liver damage on hepatic antioxidant enzyme expression and oxidative stress using 9-week-old Goto-Kakizaki (GK) rats. Hepatic total antioxidant capacity determined by total oxygen radical scavenging capacity and lipid peroxidation determined by malondialdehyde in plasma and liver were not significantly different between normal Wistar rats and GK rats. These results indicated that oxidative stress is not evident in these type 2 diabetic rats. Hepatic expression levels of antioxidant enzymes, including superoxide dismutase-1, catalase, glutathione peroxidase and reductase, thioredoxin-1, mu- and pi-class glutathione S-transferase (GST), and the gamma-glutamylcysteine ligase catalytic subunit, were not different between normal rats and GK rats. But, hepatic level and activity of alpha-class GST were decreased and peroxiredoxin-1 level was increased in GK rats, suggesting that upregulation of peroxiredoxin-1 compensates for downregulation of alpha-class GST. These results suggest that alpha-class GST and peroxiredoxin-1 in liver can be altered during the early stages of type 2 diabetes in the absence of obesity and severe oxidative stress.

Control of glutamine metabolism by the tumor suppressor Rb

Retinoblastoma (Rb) protein is a tumor suppressor that is dysregulated in a majority of human cancers. Rb functions to inhibit cell cycle progression in part by directly disabling the E2F family of cell cycle-promoting transcription factors. Because the de novo synthesis of multiple glutamine-derived anabolic precursors is required for cell cycle progression, we hypothesized that Rb also may directly regulate proteins involved in glutamine metabolism. We examined glutamine metabolism in mouse embryonic fibroblasts (MEFs) isolated from mice that have triple knock-outs (TKO) of all three Rb family members (Rb-1, Rbl1, and Rbl2) and found that loss of global Rb function caused a marked increase in 13C-glutamine uptake and incorporation into glutamate and TCA cycle intermediates in part via upregulated expression of the glutamine transporter ASCT2 and the activity of glutaminase 1 (GLS1). The Rb-controlled transcription factor E2F-3 altered glutamine uptake by direct regulation of ASCT2 mRNA and protein expression, and E2F-3 was observed to associate with the ASCT2 promoter. We next examined the functional consequences of the observed increase in glutamine uptake and utilization and found that glutamine exposure potently increased oxygen consumption whereas glutamine deprivation selectively decreased ATP concentration in the Rb TKO MEFs but not the WT MEFs. In addition, TKO MEFs exhibited elevated production of glutathione from exogenous glutamine, and had increased expression of gamma-glutamylcysteine ligase relative to WT MEFs. Importantly, this metabolic shift towards glutamine utilization was required for the proliferation of Rb TKO MEFs but not for the proliferation of the WT MEFs. Last, addition of the TCA cycle intermediate α-ketoglutarate to the Rb TKO MEFs reversed the inhibitory effects of glutamine deprivation on ATP, GSH levels, and viability. Taken together, these studies demonstrate that the Rb/E2F cascade directly regulates a major energetic and anabolic pathway that is required for neoplastic growth.

Naringin modulates oxidative stress and inflammation in 3-nitropropionic acid-induced neurodegeneration through the activation of nuclear factor-erythroid 2-related factor-2 signalling pathway

Nuclear factor-erythroid 2-related factor-2 (Nrf2) mediated regulation of cellular antioxidant production and the anti-inflammatory mechanism play an important role in neuroprotection against neurodegenerative diseases. Naringin a citrus flavonone, has been reported to possess neuroprotective effect against Huntington’s disease, and other neurodegenerative disorders, however the mechanisms underlying its beneficial effects on 3-nitropropionic acid (3-NP)-induced neurodegeneration are poorly defined. The objective of the present study was to investigate the neuroprotective role of naringin and delineate the mechanism of action on 3-NP-induced neurodegeneration. Rats were injected with 3-NP (10mg/kg body weight/day, i.p.) for 2weeks to develop neurodegeneration, while naringin (80mg/kg body weight/day, orally) was administered throughout the experimental period, 1h prior to 3-NP exposure. Thereafter rats were euthanized for biochemical, histological, and molecular studies. Treatment with naringin ameliorated the reduced glutathione/oxidized glutathione ratio with concomitant decrease in the levels of hydroxyl radical, hydroperoxide and nitrite in 3-NP-induced rats. Nissl staining and transmission electron microscopic studies showed that naringin modulated 3-NP-induced histological changes. Naringin induces NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, glutathione S-transferase P1 and gamma-glutamylcysteine ligase mRNA expressions through the activation of Nrf2 and decreased the expressions of pro-inflammatory mediators like tumour necrosis factor-alpha, cyclooxygenase-2 and inducible nitric oxide synthase. These results indicate that naringin might be beneficial in mitigating 3-NP-induced neurodegeneration through the enhancement of phase II and antioxidant gene expressions via Nrf2 activation; thereby modulating the oxidative stress and inflammatory responses.

Expression of hepatic and ovarian antioxidant enzymes during estrous cycle in rats

The regulation of antioxidant enzymes has received increased attention in terms of protection from many diseases. Despite reports that administered estradiol derivatives can change antioxidant enzyme levels, comprehensive information is not available regarding the effects of the human menstrual cycle or the rat estrous cycle on the expression of the antioxidant enzyme system. The present study was performed to determine the expression levels of cytosolic antioxidant enzymes, including superoxide dismutase-1, catalase, glutathione peroxidase-1, glutathione reductase, peroxiredoxin (Prx)-1, Prx-2, thioredoxin-1, gamma-glutamylcysteine ligase catalytic subunit, alpha-class glutathione S-transferase (GST), pi-class GST, and mu-class GST, in the liver and ovaries of female rats during diestrus and proestrus. Our results indicate that hepatic expression of Prx-1 and Prx-2, and ovarian expression of alpha-class GST were increased significantly during the proestrus phase compared with the diestrus phase. These results suggest that the hepatic Prx family and ovarian alpha-class GST are sensitive to changes during the estrous cycle. Further studies are needed to determine the physiological significance of the regulation of the Prx family and alpha-class GST during the estrous cycle.