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Background: Parkinson's disease is the most common neurodegenerative disorder, characterized by loss of dopaminergic neurons in substantia nigra and depletion of dopamine in striatum due to excitotoxicity, oxidative stress and many other factors may contribute to MPTP- and PD-related neurodegeneration. The present study deals with the neuroprotective effect of Naringenin (NGN), a bioflavonoid against MPTP-induced Parkinson's disease in the mouse model., Methods: Healthy male C57BL/6J mice (18-22 g b wt) were pretreated with NGN [25, 50, 100 mg/kg/b.wt, p.o] once daily for 5 days. Thereafter, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) (80 mg/kg b.wt, i.p) was given in two divided doses (2 × 40 mg/kg at 16 h interval). The animals were observed for motor functions 48 h after the first MPTP injection. After completion of behaviour tasks, all animals were euthanized to dissect out the brain and used for biochemical, molecular and histopathological investigations., Results: Pretreatment of NGN significantly reversed the toxic effects of MPTP by reducing LPO levels and increasing the activities of glutathione reductase and catalase along with improved behavioural performance. Interestingly, pre-treatment with NGN down-regulated iNOS expression level in MPTP intoxicated mice brain. In addition, the histopathological evaluation revealed that NGN decreased the nuclear pigmentation and cytoplasmic vacuolation in the substantia nigra and striatal regions when compared to MPTP-intoxicated mice brain., Discussion: The present study showed that NGN exerts neuroprotection by suppressing oxidative stress via antioxidant mechanisms. The above finding suggests that NGN may act as a potential target in the management of PD.

Glutathione (GSH) and GSH-related enzymes constitute the most important defense system that protects cells from free radical, radiotherapy, and chemotherapy attacks. In this study, we aim to explore the potential role and regulatory mechanism of the GSH redox cycle in drug resistance in glioblastoma multiforme (GBM) cells. We found that temozolomide (TMZ)-resistant glioma cells displayed lower levels of endogenous reactive oxygen species and higher levels of total antioxidant capacity and GSH than sensitive cells. Moreover, the expression of glutathione reductase (GSR), the key enzyme of the GSH redox cycle, was higher in TMZ-resistant cells than in sensitive cells. Furthermore, silencing GSR in drug-resistant cells improved the sensitivity of cells to TMZ or cisplatin. Conversely, the over-expression of GSR in sensitive cells resulted in resistance to chemotherapy. In addition, the GSR enzyme partially prevented the oxidative stress caused by pro-oxidant L-buthionine -sulfoximine. The modulation of redox state by GSH or L-buthionine -sulfoximine regulated GSR-mediated drug resistance, suggesting that the action of GSR in drug resistance is associated with the modulation of redox homeostasis. Intriguingly, a trend toward shorter progress-free survival was observed among GBM patients with high GSR expression. These results indicated that GSR is involved in mediating drug resistance and is a potential target for improving GBM treatment., (© 2017 International Society for Neurochemistry.)

The combination of trimethoprim and sulfamethoxazole (TMP-SMX) is the most effective regimen for therapy of Pneumocystis pneumonia (PCP). As many patients with PCP are allergic or do not respond to it, efforts have been devoted to develop alternative therapies for PCP. We have found that the combination of vitamin D 3 (VitD3) (300 IU/kg/day) and primaquine (PMQ) (5 mg/kg/day) was as effective as TMP-SMX for therapy of PCP. In this study, we investigated the mechanisms by which vitamin D enhances the efficacy of PMQ. C57BL/6 mice were immunosuppressed by CD4 + cell depletion, infected with Pneumocystis murina for 8 weeks, and then treated for 9 days with the combination of VitD3 and PMQ (VitD3-PMQ) or with TMP-SMX or PMQ to serve as controls. The results showed that vitamin D supplementation increased the number of CD11c + cells, suppressed the production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], and interleukin-6 [IL-6]) and inducible nitric oxide synthase (iNOS), and enhanced the expression of genes related to antioxidation (glutathione reductase and glutamate-cysteine ligase modifier subunit), antimicrobial peptides (cathelicidin), and autophagy (ATG5 and beclin-1). These results suggest that the main action of vitamin D is enhancing the ability of the host to defend against Pneumocystis infection., (Copyright © 2017 American Society for Microbiology.)

Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate (DMF) is an effective oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. DMF activates the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) leading to increased synthesis of the major cellular antioxidant glutathione (GSH) and prominent neuroprotection in vitro . We previously demonstrated that DMF is capable of raising GSH levels even when glutathione synthesis is inhibited, suggesting enhanced GSH recycling. Here, we found that DMF indeed induces glutathione reductase (GSR), a homodimeric flavoprotein that catalyzes GSSG reduction to GSH by using NADPH as a reducing cofactor. Knockdown of GSR using a pool of E. coli RNase III-digested siRNAs or pharmacological inhibition of GSR, however, also induced the antioxidant response rendering it impossible to verify the suspected attenuation of DMF-mediated neuroprotection. However, in cystine-free medium, where GSH synthesis is abolished, pharmacological inhibition of GSR drastically reduced the effect of DMF on glutathione recycling. We conclude that DMF increases glutathione recycling through induction of glutathione reductase., Competing Interests: The authors declare that they have no competing interests.

There is a growing interest in applying tobacco agroinfiltration for recombinant protein production in a plant based system. However, in such a system, the action of proteases might compromise recombinant protein production. Protease sensitivity of model recombinant foot-and-mouth disease (FMD) virus P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely degraded when treated with the serine protease trypsin than when treated with the cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1 was degraded when incubated with such a protease-containing tobacco extract. In silico analysis revealed potential protease cleavage sites within the P1, VP1 and GOR sequences. The interaction modeling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modeling with the entire P1-polyprotein fusion complex. Several plant transcripts with differential expression were detected 24 hr post-agroinfiltration when the RNA-seq technology was applied to identify changed protease transcripts using the recently available tobacco draft genome. Three candidate genes were identified coding for proteases which included the Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested recombinant proteins are sensitive to protease action and agroinfiltration induces the expression of potential proteases that can compromise recombinant protein production.

Oxidative stress plays an important role in the development of beta-cell dysfunction and insulin resistance, two major pathophysiological abnormalities of type 2 diabetes. Expression levels of antioxidant enzymes in beta cells are very low, rendering them more susceptible to damage caused by reactive oxygen species (ROS). Although the antioxidant effects of glucagon-like peptide-1 (GLP-1) and its analogs have been previously reported, the exact mechanisms involved are still unclear. In this study, we demonstrated that GLP-1 was able to effectively inhibit oxidative stress and cell death of INS-1E beta cells induced by the pro-oxidant tert-butyl hydroperoxide (tert-BOOH). Incubation with GLP-1 enhanced cellular levels of glutathione and the activity of its related enzymes, glutathione-peroxidase (GPx) and -reductase (GR) in beta cells. However, inhibition of ERK, but not of the PI3K/AKT pathway abolished, at least in part, the antioxidant effect of GLP-1. Moreover, ERK activation seems to be protein kinase A (PKA)-dependent because inhibition of PKA with H-89 was sufficient to block the GLP-1-derived protective effect on beta cells. GLP-1 likewise increased the synthesis of GR and favored the translocation of the nuclear transcription factor erythroid 2p45-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Glucose-stimulated insulin secretion was also preserved in beta-cells challenged with tert-BOOH but pre-treated with GLP-1, probably through the down-regulation of the mitochondrial uncoupling-protein2 (UCP2). Thus, our results provide additional mechanisms of action of GLP-1 to prevent oxidative damage in beta cells through the modulation of signaling pathways involved in antioxidant enzyme regulation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea, a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k cat and k cat/K m at lower temperatures (4 °C) compared to mesophilic Baker's yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems.

Curcumin is widely used as a traditional medicine. This work was aimed to investigate its possible protective effect against chemically induced hepatocellular carcinoma (HCC) in rats. Fifty male albino rats were divided into five groups (n=10, each). The control group received a single dose of normal saline, the diethylnitrosamine (DENA) group received a single intra-peritoneal dose at 200mg/kg body weight, and the 3rd, 4th and 5th groups were given DENA and daily administrated curcunine (CUR) via intra-gastric intubation in doses of 300,200 and 100 mg/kg b.wt. respectively for 20 weeks. Serum, and liver samples were used for determination of alpha feto-protein (AFP), interleukin-2 (IL-2), interleukine-6 (IL-6), serum liver enzymes (AST, ALT, ALP and GGT) levels as well the activities and gene expression of glutathione peroxidise (GPx), glutathione reductase (GR), catalase (CAT) and super oxide dismutase (SOD). Curcumin significantly lowered the serum levels of AFP, IL-2 and IL-6, ALT, ALT, and malondialdehyde (MDA) as well gene expression of IL-2 and IL-6. In contrast it increased the gene expression and activities of Gpx, GRD, CAT and SOD. The protective effect of CUR against DEN-induced hepatocarcinogenesis in albino rats was proven.

Multiwall carbon nanotubes (MWCNTs) have many attractive properties with potential applications in various fields. Despite their usefulness, however, the associated waste can be hazardous to the environment. To examine adverse effects in aquatic environments, Oryzias latipes were exposed to MWCNTs dispersed in water for 14 days and apoptosis and antioxidant gene expression were observed. This work showed that in gills exposed to 100 mg/L MWCNTs for 4 days, there was significant p53, caspase-3 (Cas3), caspase-8 (Cas8), and caspase-9 (Cas9) gene expression relative to the controls, while catalase (CAT) and glutathione-S-transferase (GST) expression were reduced. At 14 days, CAT, GST, and metallothionein (MT) were induced significantly in the gills and Cas3, Cas8, and Cas9 were induced in the liver. No significant gene induction was seen in intestine. Intracellular reactive oxygen species (ROS) were increased significantly only at 14 days. Histologically, no apoptosis was observed with exposure to 100 mg/L MWCNTs for 21 days. The gills were more sensitive to MWCNT toxicity than the other organs. Males had higher apoptosis gene induction than females. These results demonstrated that MWCNTs could cause apoptosis in a manner influenced by tissue and gender in aqueous environments.

Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared with A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes, and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and Western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes pyruvate kinase, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase, lactate dehydrogenase, and phosphoglycerate kinase 1 were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, whereas vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes, and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step toward a comprehensive understanding of drug resistance in ovarian cancer., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Hyperammonemia is a common finding in children with methylmalonic acidemia and propionic acidemia, but its contribution to the development of the neurological symptoms in the affected patients is poorly known. Considering that methylmalonic acid (MMA) and propionic acid (PA) predominantly accumulate in these disorders, we investigated the effects of hyperammonemia induced by urease treatment in 30-day-old rats receiving an intracerebroventricular (ICV) injection of MMA or PA on important parameters of redox homeostasis in cerebral cortex and striatum. We evaluated glutathione (GSH) concentrations, sulfhydryl content, nitrate and nitrite concentrations, 2',7'-dichlorofluorescein (DCFH) oxidation, and the activity of antioxidant enzymes. MMA decreased GSH concentrations and sulfhydryl content and increased nitrate and nitrite concentrations in cerebral cortex and striatum from hyperammonemic rats, whereas MMA or ammonia per se did not alter these parameters. MMA plus hyperammonemia also decreased glutathione reductase activity in rat cerebral cortex, but did not affect catalase, superoxide dismutase and glutathione peroxidase activities, neither DCFH oxidation. Furthermore, ICV PA administration alone or combined with hyperammonemia did not alter any of the evaluated parameters. We also found that pre-treatment with antioxidants prevented GSH reduction and sulfhydryl oxidation, whereas N(ω)-nitro-L-arginine methyl ester (L-NAME) prevented the increased nitrate and nitrite concentrations provoked by MMA plus ammonia treatments. Histological alterations, including vacuolization, ischemic neurons, and pericellular edema, were observed in brain of hyperammonemic rats injected with MMA. The data indicate a synergistic effect of MMA and ammonia disturbing redox homeostasis and causing morphological brain abnormalities in rat brain.

Alcohol abuse suppresses the immune responses of alveolar macrophages (AMs) and increases the risk of a respiratory infection via chronic oxidative stress and depletion of critical antioxidants within alveolar cells and the alveolar lining fluid. Although alcohol-induced mitochondrial oxidative stress has been demonstrated, the oxidation of the mitochondrial thioredoxin redox circuit in response to alcohol has not been examined. In vitro ethanol exposure of a mouse AM cell line and AMs from ethanol-fed mice demonstrated NADPH depletion concomitant with oxidation of mitochondrial glutathione and oxidation of the thioredoxin redox circuit system including thioredoxin 2 (Trx2) and thioredoxin 2 reductase (Trx2R). Mitochondrial peroxiredoxins (Prdx's), which are critical for the reduction of the thioredoxin circuit, were irreversibly hyperoxidized to an inactive form. Ethanol also decreased the mRNAs for Trx2, Trx2R, Prdx3, and Prdx5 plus the mitochondrial thiol-disulfide proteins glutaredoxin 2, glutathione reductase, and glutathione peroxidase 2. Thus, the mitochondrial thioredoxin circuit was highly oxidized by ethanol, thereby compromising the mitochondrial antioxidant capacity and ability to detoxify mitochondrial reactive oxygen species. Oxidation of the mitochondrial thioredoxin redox circuit would further compromise the transient oxidation of thiol groups within specific proteins, the basis of redox signaling, and the processes by which cells respond to oxidants. Impaired mitochondria can then jeopardize cellular function of AMs, such as phagocytosis, which may explain the increased risk of respiratory infection in subjects with an alcohol use disorder., (© 2013 Elsevier Inc. All rights reserved.)

Introduction: Reactive oxygen species (ROS) are produced by a number of physiological and pathological processes which influence the function of a diverse array of cellular events. An imbalance between ROS generation and elimination was reported for different environmental xenobiotics exposure. Here, we analyzed the effect of chlorpyrifos (CPF) on the JEG-3 cell antioxidant defense in conditions where cell viability and morphology were not altered., Methods: Acetylcholinesterase (AChE) activity, reduced glutathione (GSH) content and catalase (CAT) antioxidant enzyme activity were measured by biochemical studies. ROS production was detected using the fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate. The transcript level of superoxide dismutase enzyme 1 (SOD1), glutathione reductase (GR), heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) as well as Nrf2 protein amount were analyzed by quantitative real time PCR and Western blot, respectively., Results: The results showed that CPF inhibited AChE activity, induced ROS production, upregulated CAT activity, and decreased GSH concentration. In response to CPF exposure, GR and HO-1 mRNA levels were increased with no changes in SOD1 mRNA. Furthermore, CPF significantly augmented Nrf2 at both mRNA and protein levels trigging the antioxidant status by increasing nuclear Nrf2 translocation., Discussion and Conclusion: Taken together, these data indicate that JEG-3 cells are able to attenuate the oxidative stress induced by CPF through the adaptive activation of the Nrf2/ARE pathway., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This study aimed to evaluate how excess selenium induces oxidative stress by determining antioxidant enzyme activity and changes in expression of selected selenoproteins in mice. BALB/c mice (n = 20 per group) were fed a diet containing 0.045 (Se-marginal), 0.1 (Se-adequate), 0.4 (Se-supernutrition), or 0.8 (Se-excess) mg Se/kg. Gene expression was quantified in RNA samples extracted from the liver, kidney, and testis by real-time quantitative reverse transcription-polymerase chain reaction. We found that glutathione peroxidase (GPx) and catalase activities decreased in livers of mice fed the marginal or excess dose of Se as compared to those in the Se-adequate group. Additionally, superoxide dismutase and glutathione reductase activities were significantly reduced only in mice fed the excess Se diet, compared to animals on the adequate Se diet. Se-supernutrition had no effect on hepatic mRNA levels of GPx isoforms 1 and 4 (GPx1 and GPx4), down-regulated GPx isoform 3 (GPx3), and upregulated selenoprotein W (SelW) mRNA expression. The excess Se diet led to decreased hepatic mRNA levels of GPx1, GPx3 and GPx4 but no change in testicular mRNA levels of GPx1, GPx3 or SelW. Dietary Se had no effect on testicular mRNA levels of GPx4. Thus, our results suggest that Se exposure can reduce hepatic antioxidant capacity and cause liver dysfunction. Dietary Se was found to differentially regulate mRNA levels of the GPx family or SelW, depending on exposure. Therefore, these genes may play a role in the toxicity associated with Se.

The correlation between changes in activities of glutathione peroxidase and glutathione reductase in heart of rats during development of adrenaline myocarditis and intensity of free radical processes estimated by biochemiluminesce parameters and the content of lipoperoxidation products was demonstrated. The maximal increase of glutathione peroxidase and glutathione reductase activities (in 1.8 and 1.4 times accordingly) was observed t 24 h after the development of the pathological process; this coincided with the maximum intensity of prosesses of free radical oxidation. Using combination of reverse transcriptions with real-time polymerase chain reaction the cardiac mRNA levels of glutathione peroxidase and glutathione reductase genes were determined during the development of adrenaline myocarditis in rats. Analysis of expression of glutathione peroxidase and glutathione reductase genes showed, that the level of this transcripts demonstrated 2,8- and 7,3- increase in rats with adrenaline myocarditis, respectively. Obviously, overexpression of these enzymes can increase the resistance of cardiomyocites to oxidative stress.

Oxidative stress is the main etiological factor behind the pathogenesis of various diseases including inflammation, cancer, cardiovascular and neurodegenerative disorders. Due to the spin trapping abilities and various pharmacological properties of nitrones, their application as therapeutic agent has been gaining attention. Though the antioxidant properties of the nitrones are well known, the mechanism by which they modulate the cellular defense machinery against oxidative stress is not well investigated and requires further elucidation. Here, we have investigated the mechanisms of cytoprotection of the nitrone spin traps against oxidative stress in bovine aortic endothelial cells (BAEC). Cytoprotective properties of both the cyclic nitrone 5,5-dimethyl-pyrroline N-oxide (DMPO) and linear nitrone α-phenyl N-tert-butyl nitrone (PBN) against H₂O₂-induced cytotoxicity were investigated. Preincubation of BAEC with PBN or DMPO resulted in the inhibition of H₂O₂-mediated cytotoxicity and apoptosis. Nitrone-treatment resulted in the induction and restoration of phase II antioxidant enzymes via nuclear translocation of NF-E2-related factor 2 (Nrf-2) in oxidatively-challenged cells. Furthermore, the nitrones were found to inhibit the mitochondrial depolarization and subsequent activation of caspase-3 induced by H₂O₂. Significant down-regulation of the pro-apoptotic proteins p53 and Bax, and up-regulation of the anti-apoptotic proteins Bcl-2 and p-Bad were observed when the cells were preincubated with the nitrones prior to H₂O₂-treatment. It was also observed that Nrf-2 silencing completely abolished the protective effects of nitrones. Hence, these findings suggest that nitrones confer protection to the endothelial cells against oxidative stress by modulating phase II antioxidant enzymes and subsequently inhibiting mitochondria-dependent apoptotic cascade., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Calmodulin (CaM), the predominant Ca(2+) receptors, is one of the best-characterized Ca(2+) sensors in all eukaryotes. In this study the role of CaM and the possible interrelationship between CaM and hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA) induced antioxidant defense were investigated in the seedling of Panax ginseng. Treatment of ABA (100 μM) and H(2)O(2) (10 mM) increased the expression of Panax ginseng calmodulin gene (PgCaM) and significantly enhanced the expression of the antioxidant marker genes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and the activities of chloroplastic and cytosolic antioxidant enzymes. Pretreatments with two CaM antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W7) and inhibitor or scavenger, diphenyleneiodonium chloride, and dimethylthiourea of reactive oxygen species almost completely suppressed the up-regulation of antioxidant and PgCaM gene. Moreover, H(2)O(2) production and CaM content was almost completely inhibited by pretreatments with two CaM antagonists. In addition, the expressions of PgCaM gene under different biotic stress were analyzed at different time intervals. Thus it may suggests that CaM are involved in ABA-induced increased expression of PgCaM which triggers H(2)O(2) production through activating trans-plasma membrane NADPH oxidase, resulting in up-regulation of defense related antioxidant gene and also plays a pivotal role in defense response against pathogens.

Hydrogen peroxide (H2O2) is considered a signal molecule inducing cellular stress. Both heat shock (HS) and Cd can increase H2O2 content. We investigated the involvement of H2O2 in HS- and Cd-mediated changes in the expression of ascorbate peroxidase (APX) and glutathione reductase (GR) in leaves of rice seedlings. HS treatment increased the content of H2O2 before it increased activities of APX and GR in rice leaves. Moreover, HS-induced H2O2 production and APX and GR activities could be counteracted by the NADPH oxidase inhibitors dipehenylene iodonium (DPI) and imidazole (IMD). HS-induced OsAPX2 gene expression was associated with HS-induced APX activity but was not regulated by H2O2. Cd-increased H2O2 content and APX and GR activities were lower with than without HS. Cd did not increase the expression of OsAPX and OsGR without HS treatment. Cd increased H2O2 content by Cd before it increased APX and GR activities without HS. Treatment with DPI and IMD effectively inhibited Cd-induced H2O2 production and APX and GR activities. Moreover, the effects of DPI and IMD could be rescued with H2O2 treatment. H2O2 may be involved in the regulation of HS- and Cd-increased APX and GR activities in leaves of rice seedlings., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Anticonvulsant properties of α-asarone were studied in mice at three doses with different toxicity. The 100mg/kg dose decreased both treadmill performance and locomotor activity, caused hypothermia, and potentiated pentobarbital-induced sleep. The last two effects and no toxicity were observed at 60 and 22mg/kg, respectively. In chemical (pentylenetetrazole, picrotoxin, N-methyl-D-aspartate, pilocarpine) and electrical (maximal electroshock) seizure tests, neither seizures nor death were prevented by 60 mg/kg α-asarone which, however, exhibited protective-like effects (delay in the onset of clonic and/or tonic seizures and/or in the death of mice). Magnesium deficiency-dependent audiogenic seizures responded to non-toxic doses of α-asarone (60 mg/kg and less): 22 mg/kg protecting 50% of tested animals. Because these seizures respond to both anti-seizure and antioxidant compounds, antioxidant properties of α-asarone were studied, indicating 5 Units of superoxide dismutase-like activity per mg α-asarone. Treatment of mice by α-asarone (daily dose of 100mg/kg during 7 days) induced brain antioxidant enzymes (superoxide dismutase, glutathione peroxidase and reductase) in striatum and hippocampus and to a lesser extent in cortex. In view of recent findings about deleterious roles of chronic inflammatory/oxidant stresses in human epilepsy outcome, antioxidant and inductive properties of α-asarone are proposed to be coherent bases for traditional clinical efficacy., (Copyright © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Astrocytes possess important roles in maintaining normal brain function and providing trophic support to the neurons. They also suffer a range of toxic insults, being a chief target of prooxidants such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP(+)), 6-hydroxydopamine (6-OHDA), 4-hydroxy-2-nonenal (HNE), and acrolein. Recently, we have observed that the cellular antioxidants and phase 2 enzymes can be upregulated by 3H-1,2-dithiole-3-thione (D3T), a nutraceutical found in cruciferous vegetables, against many prooxidants in human neuroblastoma cell lines (SH-SY5Y). However, the regulation of the above cellular factors by D3T in astrocytes and their role in ameliorating the neurotoxic effects of the above neurotoxins have not been investigated. In this study, we show that incubation of human primary astrocytes with micromolar concentrations (5-100 microM) of D3T for 24 h resulted in significant increases in the levels of reduced glutathione (GSH), glutathione reductase (GR), and the phase 2 enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1). D3T treatment also caused time-dependent increases in mRNA expression of the gamma-glutamylcysteine ligase catalytic subunit (GCLC), GR, and of NQO1 in these cells. Pretreatment of astrocytes with D3T was found to afford remarkable protection against the neurocytotoxicity elicited by MPTP, MPP(+), 6-OHDA, HNE and acrolein. Taken together, this study demonstrates for the first time that in human astrocytes, the cruciferous nutraceutical D3T potently induces the cellular GSH system and the phase 2 enzyme NQO1, which is accompanied by dramatically increased resistance of these cells to the damage induced by various neurotoxicants. The results of this study may have important implications for the development of novel neuroprotective strategies.

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress-elicited changes in plants at the proteome level. Hydroponically grown 2-wk-old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H(2)O(2) contents in roots. A total of 23 As-regulated proteins including predicted and novel ones were identified using 2-DE coupled with MS analyses. The expression levels of S-adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST-tau, and tyrosine-specific protein phosphatase proteins (TSPP) were markedly up-regulated in response to arsenate, whereas treatment by H(2)O(2) also regulated the levels of CS suggesting that its expression was certainly regulated by As or As-induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose-dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.

The current investigation has been conducted to investigate the influence of ginger on hepatic antioxidant enzymes system in ethanol treated rats. Ethanol significantly decreased the superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione content while an increase of malondialdehyde (MDA) levels were estimated in the hepatic tissue. This effect was reversed by a treatment with 1% dietary ginger for 4 weeks in rats by improved antioxidant status which suggest that treatment of ginger may have protective role against the ethanol induced hepatotoxicity.

We describe the gene expression profile of third leaves of rice (cv. Nipponbare) seedlings subjected to salt stress (130 mM NaCl). Transcripts of Mn-SOD, Cu/Zn-SOD,cytosolic and stromal APX, GR and CatB were regulated, whereas expression of thylakoid-bound APX and CatA were down-regulated. The levels of the compatible solute proline and of transcripts of its biosynthetic gene, Delta1-pyrroline-5-carboxylate synthetase (P5CS), were strongly increased by salt stress. Interestingly, a potential compatible solute, gamma-aminobutyric acid (GABA), was also found to be strongly induced by salt stress along with marked up-regulation of transcripts of GABA-transaminase. A dye-swap rice DNA microarray analysis identified a large number of genes whose expression in third leaves was altered by salt stress. Among 149 genes whose expression was altered at all the times assayed (3, 4 and 6 days) during salt stress, there were 47 annotated novel genes and 76 unknown genes. These results provide new insight into the effect of salt stress on the expression of genes related to antioxidant enzymes, proline and GABA as well as of genes in several functional categories.

Objective: Thiol oxidative stress leads to macrophage dysfunction and cell injury, and has been implicated in the development of atherosclerotic lesions. We investigated if strengthening the glutathione-dependent antioxidant system in macrophages by overexpressing glutathione reductase (GR) decreases the severity of atherosclerosis., Methods and Results: Bone marrow cells infected with retroviral vectors expressing either enhanced green fluorescent protein (EGFP) or an EGFP-fusion protein of cytosolic GR (GR(cyto)-EGFP) or mitochondrial GR (GR(mito)-EGFP) were transplanted into low-density lipoprotein receptor-deficient mice. Five weeks after bone marrow transplantation, animals were challenged with a Western diet for 10 weeks. No differences in either plasma cholesterol and triglyceride levels or peritoneal macrophage content were observed. However, mice reconstituted with either GR(cyto)-EGFP or GR(mito)-EGFP-expressing bone marrow had lesion areas (P<0.009) that were 32% smaller than recipients of EGFP-expressing bone marrow. In cultured macrophages, adenovirus-mediated overexpression of GR(cyto)-EGFP or GR(mito)-EGFP protected cells from mitochondrial hyperpolarization induced by oxidized low-density lipoprotein., Conclusion: This study provides direct evidence that the glutathione-dependent antioxidant system in macrophages plays a critical role in atherogenesis, and suggests that thiol oxidative stress-induced mitochondrial dysfunction contributes to macrophage injury in atherosclerotic lesions.

NADPH is an essential electron donor in numerous biosynthetic and detoxification reactions. In animal, yeast and bacteria, the NADP-dependent isocitrate dehydrogenase (NADP-ICDH), which catalyzes the production of NADPH, is being recognized as an essential component of the antioxidative defence mechanisms. In plant cells, there is little information on the antioxidant properties of NADP-ICDH. Using a pea cDNA lambdagt11 library, the full-length cDNA of a NADP-ICDH was obtained. In pea leaves, the analyses of activity, protein and transcript expression of NADP-ICDH under six different abiotic stress conditions (CL, continuous light, HLI, high light intensity, D, continuous dark, LT, low-temperature HT, high-temperature and W, mechanical wounding) revealed a differential regulation at transcriptional and post-translational level depending on the abiotic stress. The activity and protein expression of NADP-ICDH and catalase increased only under HLI but the NADP-ICDH transcripts were up-regulated by cold stress (70%) and W (40%). Under the same conditions, the transcript analysis of glutathione reductase (GR), monodehydroascorbate reductase (MDAR) and ascorbate peroxidase (APX), key components of the antioxidative ascorbate-glutathione cycle, showed similar inductions. These data indicate that in pea plants the cytosolic NADP-ICDH shows a differential response, at mRNA and activity level, depending on the type of abiotic stress and suggests that this dehydrogenase could have a protective antioxidant role against certain environmental stresses in plants.

This study was undertaken to investigate the inducibility of glutathione (GSH), glutathione reductase (GR) and glutathione peroxidase (GPx) by 3H-1,2-dithiole-3-thione (D3T) in beta-cells, and the resultant cytoprotection against oxidant injury. Incubation of the insulin-secreting RINm5F cells with D3T led to significant induction of GSH, GR and GPx. D3T-mediated induction of GSH was abolished by buthionine sulfoximine (BSO), suggesting a critical involvement of gamma-glutamylcysteine ligase (gammaGCL). Consistently, incubation of RINm5F cells with D3T resulted in increased expression of gammaGCL protein and mRNA. Pretreatment of RINm5F cells with D3T provided remarkable protection against oxidant-elicited cytotoxicity. On the other hand, depletion of cellular GSH by BSO sensitized RINm5F cells to oxidant injury. Furthermore, cotreatment of RINm5F cells with BSO to reverse D3T-mediated GSH induction abolished the cytoprotective effects of D3T on oxidant injury. Taken together, this study demonstrates that upregulation of glutathione system by D3T is effective for protecting against oxidative beta-cell injury.

Glutathione reductase (GR) plays an essential role in cell defense against reactive oxygen metabolites by sustaining the reduced status of an important antioxidant, glutathione. To address the effect of oxidative stresses on the intertidal copepod Tigriopus japonicus, we exposed specimens to hydrogen peroxide, heavy metals and different salinity levels, cloned and sequenced the oxidative stress-related GR gene. T. japonicus GR gene (Tigriopus GR) cDNA contained 1526 bp including an open reading frame (ORF) encoding 458 amino acids with a theoretical pI of 6.58 and a calculated molecular weight of 49.6 kDa. Tigriopus GR showed a high similarity to frog Xenopus laevis GR (identity 57%) and the filarial parasite, Onchocerca volvulus GR (identity 57%). Specific motifs such as flavin adenine dinucleotide-binding site (LVLGGGSGGIASARRAAEF), pyridine nucleotide-disulphide oxidoreductases class-I active site (GGTCVNVGCVP), and NADPH binding motif (GxGYIAx18Rx5R) were highly conserved in the deduced amino acid sequence of Tigriopus GR. Interestingly, its expression and enzyme characteristics were different from GR homologue of filarial parasite O. volvulus. To investigate the biochemical and enzymatic characteristics of Tigriopus GR protein, we constructed the expression vector, pCRT7/TOPO NT containing Tigriopus GR. Tigriopus pCRT7/TOPO NT/GR was expressed in Escherichia coli, and the soluble protein was purified by 6x His-tag chromatography. The recombinant Tigriopus GR enzyme was found to make homodimer complexes of approximately 108 kDa on 12% native gel electrophoresis and showed enzymatic activity with NADPH and oxidized glutathione (GSSG) as substrates. To analyze the gene expression of Tigriopus GR against different environmental stresses (hydrogen peroxide, salinity, and heavy metals), we performed real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR). Slight down-regulation in the expression of Tigriopus GR at the initial stage was observed upon exposure to hydrogen peroxide. The expression recovered in 2h, while there were significant changes upon heavy metal (Cu and Mn) exposures in a time-dependent manner. Also, Tigriopus GR expression was significantly increased with moderately high salt stress (24 and 40 ppt). In the case of low salt stress (0 and 12 ppt) the expression was found to be down-regulated. These findings provide a better understanding of cellular protection mechanisms in the intertidal copepod T. japonicus against the environmental stressors caused by non-optimal salt levels.

Objective: To study the effects of deltamethrin (DM) on the mRNA expression of copper-zinc dependent SOD (CuZn-SOD), glutathione reductase (GR) and gamma glutamylcysteine synthetase (gamma-GCS) light subunit (GCSl), as well as on expression of both mRNA and protein of gamma-GCS heavy subunit (GCSh) and NFE2 related factor 2 (Nrf2) in cerebral cortex and hippocampus of rats., Methods: Eighteen Wistar male rats were randomizedly divided into three groups, six for each group. The low dosage and high dosage DM treated groups were administrated intraperitoneally with DM (the daily dosage was 3.125, 12.500 mg/kg BWT respectively) for five consecutive days while the control group was administered intraperitoneally with olive oil. The relative amount of mRNA expression of these genes was measured by the method of reverse transcription polymerase chain reaction (RT-PCR) (n = 6). The protein level was detected by the method of immunohistochemistry and image analysis system (n = 4)., Results: There was no change in mRNA expression level of CuZn-SOD, GR, GCSh and Nrf2 gene in both cerebral cortex and hippocampus tissue in rats administrated with DM. However, the mRNA level of GCSl gene in cerebral cortex of high dosage group as well as in both cerebral cortex and hippocampus of the low dosage group was significantly lower than that in corresponding tissue in the control group, and was decreased to 71.1%, 63.6% and 75.2% of mRNA level of corresponding tissue in the control group (P < 0.01). There was no obvious effect on protein level of both GCSh and Nrf2 in CA1, CA2, CA3 and dentate gyrus (DG) of hippocampus as well as on that in cerebral cortex in rats treated with DM., Conclusion: Under the experimental conditions, there is no obvious effect in the mRNA expression level of CuZn-SOD, GR gene, as well as on expression of both mRNA and protein of Nrf2 gene in both cerebral cortex and hippocampus tissue in rats administered with DM. DM depresses the mRNA expression of GCSl gene, but does not affect the mRNA expression of GCSh gene.

Aim: To define the role of enzymes involved in glutathione metabolism in 6-hydroxydopamine (6-OHDA)-induced glutathione alteration in primary cultured astrocytes., Methods: Total glutathione (GSx) levels were determined using the modified enzymatic microtiter plate assay. The mRNA levels of gamma-glutamylcysteine synthetase (gammaGCS), gamma-glutamyltransferase (gammaGT), glutathione peroxidase (GPx), GR(glutathione reductase), and glutathione transferases (GST) were determined using RT-PCR. gammaGT activity was determined using gammaGT assay kits., Results: In primary cultured astrocytes, 6-OHDA induced a significant elevation of cellular GSx levels after treatment for 24 h. However, the GSx levels decreased after 24 h and the values were even lower than the value in the control group without 6-OHDA at 48 h. RT-PCR data showed that the mRNA levels of gammaGCS, the rate-limiting enzyme of gamma-L-glutamyl-L-cysteinylglycine (GSH) synthesis, were increased by 6-OHDA after treatment for 24 h and 48 h; the mRNA levels of GPx, GR, and GST did not alter in 6-OHDA-treated astrocytes after treatment for 24 h and 48 h; and 6-OHDA increased the mRNA levels and the activity of gammaGT after treatment for 48 h, which induced a decrease in GSx levels, despite the up-regulation of gammaGCS after exposure to 6-OHDA for 48 h., Conclusion: The change in gammaGCS correlated with the increase in GSH levels induced by 6-OHDA after treatment for 24 h. GSx levels decreased because of increased gammaGT mRNA levels and gammaGT activity induced by 6-OHDA after treatment for 48 h.

Polychaetes, Eurythoe complanata, from the Gulf of Cariaco,Venezuela, were exposed to 0.3, 1.6, and 3.3% water-soluble fraction (WSF) of used crankcase oil during 15 and 21 days. The antioxidant enzymes glutathione peroxidase (GPX), glutathione reductase (GR), catalase (CAT), and glutathione-S-transferase (GST) were assayed in the body wall tissue. Furthermore, after chemical exposure, the polychaetes were cut into equal halves; then wound healing and the number of regenerated body segments were recorded periodically. GST activity was affected by all the experimental treatments, with activity increasing with WSF concentrations. GPx activity was altered for the contamination period. GR and CAT activities rose in response to increasing WSF concentrations, and were higher for long-term than for short-term exposures. The wound healing of the transected body regions was retarded by WSF exposure. WSF affected the tissue regeneration, which was almost abolished at 3.3% WSF. The exposure period did not affect the tissue-repairing responses. Alteration of GST in contaminated organisms suggested equivalent changes in detoxication of bioaccumulated organic contaminants. The variation of GR and CAT suggests induction of oxidative stress that could reduce the ability of WSF-exposed worms to repair damaged tissue.

Cardiac ischemia reperfusion leads to oxidative stress and poor physiological recovery. Selenium deficiency down-regulates thioredoxin reductase (Txnrd) and glutathione peroxidase (Gpx) activity, impairing recovery from ischemia-reperfusion. Furthermore, selenium supplementation has been shown to be cardioprotective and lessens oxidative stress in reperfused rat hearts. In this study we have investigated the role of selenium in the mRNA expression of these, and related antioxidant proteins, post ischemia-reperfusion. Male rats were fed varying doses of selenium for five weeks. Hearts were isolated and perfused using the Langendorff method with 22.5 min of global ischemia and 45 min reperfusion. RNA was extracted for quantitative real-time PCR analysis of glutathione peroxidase (Gpx)-1 and 4, glutathione reductase (Gsr), thioredoxin peroxidase-2 (Prdx2), thioredoxin (Txn) and thioredoxin reductase (Txnrd)-1 and 2 gene expression. Selenium deficiency produced significant reductions in Gpx-1, Gpx-4, Prdx2, Txnrd-1 and Txnrd-2 expression. Conversely, selenium supplementation of 1000 microg/kg significantly up-regulated Gpx-1, Gpx-4, Txn, Txnrd-1 and Txnrd-2 transcription. Our results show selenium modulates the cardiac mRNA expression of thioredoxin and glutathione related enzymes post ischemia-reperfusion, and impacts on tolerance to ischemia-reperfusion.

A strain of mice (Gr1a1Neu) that exhibited tissue glutathione reductase (GR) activities that were substantially lower (less than 10% in liver) than the corresponding activities in control mice has been reported. The present report describes characterization of the mutation(s) in the GR gene of these mice. RT-PCR of mRNA from the Neu mice indicated a substantial deletion in the normal GR coding sequence. Southern blots revealed that the deletion involved a region spanning from intron 1 through intron 5. The exact breakpoints of the deletion were characterized by PCR and sequencing through the region encompassing the deletion. The deletion involves nucleotides 10840 through 23627 of the genomic GR gene and functionally deletes exons 2 through 5. In addition, the deletion produces a frame shift in exon 6 and introduces a stop codon in exon 7 that would prevent translation of the remainder of the protein. Consequently, the Neu mice are incapable of producing a functional GR protein and appear to be genetic knockouts for GR. The Neu mice offer live animal models with which to test hypotheses regarding oxidant mechanisms of tissue injury in vivo.

A population of killifish (Fundulus heteroclitus) inhabiting a creosote-polluted inlet of the Elizabeth River demonstrates tolerance to the acute toxic effects exerted by contaminated sediments on reference site killifish. Previous data have suggested that upregulated antioxidant defenses contribute to short-term tolerance in killifish exposed to Elizabeth River sediments. This study investigated population differences in antioxidant defenses from wild caught Elizabeth River and reference population killifish in different seasons, and after being held in the laboratory. Parameters measured in the killifish were total glutathione concentrations (GSH(T)), activities of glutathione reductase (GR), glutathione peroxidase (GPx), and lipid peroxidation (LPO), all in adult hepatic tissues. The Elizabeth River population exhibited greater GSH(T), higher GPx activities, and increased LPO as compared to the reference population. Sex specific population differences were also observed in GSH(T) and GPx. Both populations displayed decreased GSH(T) and increased GR from early to late summer, as well as after being held in the laboratory. This study indicates that there are many factors that may contribute to differences in levels of antioxidant defenses in addition to exposure to contaminants, including reproductive status and environmental conditions.

The role of endosymbiotic diatoms as pro-oxidant stressors in porifera has been investigated in the Antarctic sponge Haliclona dancoi in which the presence of diatoms is influenced by marked seasonal variations during the austral summer. Both chlorophaeopigments and frustules were absent in sponge tissues sampled in early November at the beginning of the summer and increased from the mid of December with slightly shifted temporal trends. The efficiency of antioxidant defenses in the sponge showed a marked response to symbionts with clearly enhanced values corresponding to the peak of diatoms.

Offshore oil production releases large amounts of lipophilic compounds in produced water and into the ocean. The discharge of produced water from the Norwegian petroleum sector has increased from 26 million m3 in 1993 to 120 million m3 in 2001, and it continues to increase. Produced water contains significant amounts of alkylphenols, which have been reported to be estrogenic, causing endocrine disruption in fish. In year 2000, approximately 44 tons of alkylphenols were released on the Norwegian continental shelf in connection with discharge of produced water. Except from being estrogenic, relatively little is known about the effects of alkylphenols when released in the marine environment. Our objective was to study how alkylphenols affect the redox status in first spawning Atlantic cod (Gadus morhua) of both sexes. Model compounds tested included 4-tert-butylphenol (C4), 4-n-pentylphenol (C5), 4-n-hexylphenol (C6) and 4-n-heptylphenol (C7), all found in produced water. First spawning Atlantic cod were force-fed a mixture of these four alkylphenols, ranging between 0.02 and 80 ppm or 5 ppm 17beta-estradiol (E2), for 1 or 4 weeks. Increased hepatic total glutathione concentration in response to alkylphenol exposure was detected in female fish compared to control group after 1-week exposure, an effect not seen after 4 weeks. Furthermore, hepatic total glutathione concentration was sex dependent, where male fish sampled after 4 weeks had higher levels of glutathione than female fish. Increased glutathione reductase catalytic activities in both male and female fish were seen after exposure to 0.02 ppm alkylphenol mixture in 4 weeks. The glutathione S-transferase activity was only affected in male fish exposed to 0.02 ppm alkylphenols, and glucose-6-phosphate dehydrogenase activity increased in female fish exposed to 0.02 ppm alkylphenol mixture for 1 week. The increase of hepatic total glutathione content as well as the effects on glutathione reductase activities suggests that alkylphenol exposure affects the redox status in Atlantic cod., (Copyright 2004 Elsevier B.V.)

Harbours can be considered as model environments for developing and validating field monitoring procedures and to investigate mechanistic relationships between different biological responses. In this study, several biomarkers were investigated in marine mussels caged for 4 weeks into an industrialised harbour of north-west Italy. Organisms were collected at different time intervals to better characterise the sensitivity, temporal variations and interactions of analysed responses. Besides single antioxidants (catalase, glutathione S-transferases, glutathione reductase, total glutathione), the total oxyradical scavenging capacity (TOSC) assay was used to analyse the capability of the whole antioxidant system to neutralise specific forms of radicals: these data were further integrated by measurement of DNA integrity, oxidised bases and the impairment of lysosomal membrane stability in haemocytes. Results showed a biphasic trend for single antioxidants and TOSC, with no variation or increase during the first 2 weeks of exposure to the polluted site followed by a progressive decrease up to a severe depletion in the final part of the experiment. These findings suggest an initial counteractive response of mussels toward the enhanced prooxidant challenge, while antioxidants appeared overwhelmed at longer exposure periods. The hypothesis of reactive oxygen species (ROS) mediated toxicity is supported by the appearance of cell damages (DNA integrity and lysosome membrane stability), which exhibited a progressive enhancement during the course of the experiment with a maximum impairment after 30 days of exposure.

The recognition of the critical involvement of oxidative and electrophilic stress in cardiac disorders has led to extensive investigation of the protective effects of exogenous antioxidants on cardiac injury. On the other hand, another strategy for protecting against oxidative/electrophilic cardiac injury may be through induction of the endogenous antioxidants and phase 2 enzymes in myocardium by chemical inducers. However, our understanding of the chemical inducibility of cardiac antioxidants/phase 2 enzymes in vivo is very limited. In addition, careful studies on the basal levels of a scope of endogenous antioxidants/phase 2 enzymes in myocardium as compared with other tissues, such as liver, are lacking. Accordingly, this study was undertaken to determine the basal levels of endogenous antioxidants/phase 2 enzymes, including superoxide dismutase (SOD), catalase, reduced glutathione (GSH), GSH peroxidase (GPx), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase 1 (NQO1), and investigate the inducibility of the above antioxidants/phase 2 enzymes by the chemoprotectant, 1,2-dithiole-3-thione (D3T), in cardiac as well as hepatic tissues in C57BL/6 mice. Our results demonstrated that in C57BL/6 mice, the levels of catalase, GSH, GPx, GR, and GST were significantly lower in cardiac tissue than in hepatic tissue. The level of total SOD did not differ significantly between mouse heart and liver. Notably, heart contained a much higher NQO1 activity than liver. Immunoblotting and RT-PCR analyses further demonstrated the high expression of NQO1 protein and mRNA in myocardium. Oral administration of D3T at 0.25 and 0.5 mmol/kg body weight for 3 consecutive days resulted in a significant induction of cardiac SOD, catalase, GR, GST, and NQO1. No significant induction of cardiac GSH and GPx was observed with the above D3T treatment. Only GR, GST, and NQO1 in mouse liver were induced by the D3T treatment. Unexpectedly, we observed a significant D3T dose-dependent decrease in hepatic GPx activity. Taken together, this study demonstrates for the first time that: (1) the expression of NQO1 is remarkably high in mouse myocardium though other cardiac antioxidants/phase 2 enzymes are relatively lower as compared with liver; (2) a number of endogenous antioxidants/phase 2 enzymes in mouse cardiac tissue can be significantly induced by D3T following oral administration; and (3) the inducibility of endogenous antioxidants/phase 2 enzymes by D3T differs between mouse cardiac and hepatic tissues. This study provides a basis for future investigation of the cardioprotection of chemically induced endogenous antioxidants and phase 2 enzymes in myocardium in animal models of oxidative/electrophilic cardiac disorders.

Escherichia coli thioredoxin reductase (TR) and glutathione reductase (GR) are dimeric proteins that require a flavin adenine dinucleotide (FAD) cofactor for activity. A cell-free protein synthesis (CFPS) reaction supplemented with FAD was used to produce TR at 760 microg/ml with 89% of the protein being soluble. GR accumulated to 521 microg/ml in a cell-free reaction with 71% solubility. The TR produced was fully active with a specific activity of 1390 min(-1). The GR had a specific activity of 139 U/mg, which is significantly more active than reported for GR purified from cells. The specific activity for both TR and GR decreased without FAD supplementation. This research demonstrates that CFPS can be used to produce enzymes that are multimeric and require a cofactor.

This work provides the first absolute expression patterns of genes coding for all known components of both thioredoxin (Trx) and glutaredoxin (Grx) systems in mouse: Trx1, Trx2, Grx1, Grx2, TrxR1, TrxR2, thioredoxin/glutathione reductase, and glutathione reductase. We devised a novel assay that, combining the advantages of multiplex and real-time PCR, streamlines the quantitation of the actual mRNA copy numbers in whole-animal experiments. Quantitations reported establish differences among adult organs and embryonic stages, compare mRNA decay rates, explore the significance of alternative mRNA isoforms derived from TrxR1 and Grx2 genes, and examine the time-course expression upon superoxide stress promoted by paraquat. Collectively, these quantitations show: i) unique expression profiles for each transcript and mouse organ examined, yet with some general trends like the higher amounts of mRNA species coding for thioredoxins than those coding for the reductases that control their redox states and activities; ii) continuous expression during embryogenesis with outstanding up-regulations of Trx1 and TrxR1 mRNAs in specific temporal sequences; iii) drastic differences in mRNA stability, liver decay rates range from 2.8 h (thioredoxin/glutathione reductase) to >/= 35 h (Trx1 and Trx2), and directly correlate with mRNA steady-state values; iv) testis-specific differences in the amounts (relative to total isoforms) of transcripts yielding the mitochondrial Grx2a and 67-kDa TrxR1 variants; and v) coordinated up-regulation of TrxR1 and glutathione reductase mRNAs in response to superoxide stress in an organ-specific manner. Further insights into in vivo roles of these redox systems should be gained from more focused studies of the mechanisms underlying the vast differences reported here at the transcript level.

The influence of oxygen availability during cultivation on the biosynthetic processes and enzymatic activities in the microaerophilic bacterium Spirillum winogradskii D-427 was studied, and the roles played by different systems of the defense against oxidation stress were determined. The metabolic adjustments caused by transition from microaerobic (2% O2) aerobic conditions (21% O2 of the gas phase) were found to slow down constructive metabolism and increase synthesis of exopolysaccharides as a means of external protection of cells from excess oxygen. This resulted in a twofold decline of the growth yield coefficient. Even though the low activity of catalase is compensated for by a multifold increase in the activities of other cytoplasmic enzymes protecting from toxic forms of O2--peroxidase and enzymes of the redox system of glutathione (glutathione peroxidase and glutathione reductase)--massive lysis of cells starts in the mid-exponential phase and leads to culture death in the stationary phase because of H2O2 accumulation in the periplasm (up to 10 micrograms/mg protein). The absence in cells of cytochrome-c-peroxidase, a periplasmic enzyme eliminating H2O2, was shown. It follows that the major cause of oxidative stress in cells is that active antioxidant defenses are located in the cytoplasm, whereas H2O2 accumulates in the periplasm due to the lack of cytochrome-c-peroxidase. The addition to the medium of thiosulfate promotes elimination of H2O2, stops cell lysis under aerobic conditions, lends stability to cultures, and results in a threefold increase in the growth yield.

It has recently been discovered that glutathione-dependent formaldehyde dehydrogenase (FALDH) exhibits a strong S-nitrosoglutathione reductase activity. Plants use NO and S-nitrosothiols as signaling molecules to activate defense mechanisms. Therefore, it is interesting to investigate the regulation of FALDH by mechanical wounding and plant hormones involved in signal transduction. Our results show that the gene encoding FALDH in Arabidopsis (ADH2) is down-regulated by wounding and activated by salicylic acid (SA). In tobacco, FALDH levels and enzymatic activity decreased after jasmonate treatment, and increased in response to SA. This is the first time that regulation of FALDH in response to signals associated with plant defense has been demonstrated.

Cell-based models have been used extensively in screening novel bioactive chemical entities. In this study, seven well-established mammalian cell lines, which have different origins, were utilized to compare their responses to the treatments of three detoxifying enzyme inducers, tert-butylhydroquinone (tBHQ), beta-naphthoflavone (beta-NF), and sulforaphane (SUL), which are potential chemopreventive compounds. The enzymatic activities of glutathione s-transferase (GST), NAD(P)H:quinone oxidoreductase (QR), aldehyde reductase (AR), and glutathione reductase (GR) were measured by kinetics methods using UV-Vis spectroscopy, and analyzed statistically by Student's t-test. Among these mammalian cell lines, the mouse hepatoma Hepa1c1c7 cells were the most robust and sensitive cells, which had higher basal as well as upregulated enzymatic activities. In human cell lines, the prostate LNCaP and hepatic HepG2 cells were also very responsive to the inducers. The results suggested that different cell lines responded differently to individual detoxifying gene inducer, and the selection of appropriate cell line is important for screening potential chemopreventive agents.

Gene expression of three antioxidant enzymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,ZnSOD), and glutathione reductase (GR) was investigated in stationary phase Saccharomyces cerevisiae during menadione-induced oxidative stress. Both GR and Cu,ZnSOD mRNA steady state levels increased, reaching a plateau at about 90 min exposure to menadione. GR mRNA induction was higher than that of Cu,ZnSOD (about 14-fold and 9-fold after 90 min, respectively). A different pattern of response was obtained for MnSOD mRNA, with a peak at about 15 min (about 8-fold higher) followed by a decrease to a plateau approximately 4-fold higher than the control value. However, these increased mRNA levels did not result in increased protein levels and activities of these enzymes. Furthermore, exposure to menadione decreased MnSOD activity to half its value, indicating that the enzyme is partially inactivated due to oxidative damage. Cu,ZnSOD protein levels were increased 2-fold, but MnSOD protein levels were unchanged after exposure to menadione in the presence of the proteolysis inhibitor phenylmethylsulfonyl fluoride. These results indicate that the rates of Cu,ZnSOD synthesis and proteolysis are increased, while the rates of MnSOD synthesis and proteolysis are unchanged by exposure to menadione. Also, the translational efficiency for both enzymes is probably decreased, since increases in protein levels when proteolysis is inhibited do not reflect the increases in mRNA levels. Our results indicate that oxidative stress modifies MnSOD, Cu,ZnSOD, and GR gene expression in a complex way, not only at the transcription level but also at the post-transcriptional, translational, and post-translational levels.

The chemopreventive potential of cycloartenol on benzoyl peroxide and UVB radiation-induced cutaneous tumor promotion markers and oxidative stress in murine skin is assessed. Benzoyl peroxide treatment (20 mg/animal/0.2 ml acetone) and UVB radiation (0.420 J/m(2)/s) caused a decrease in the activities of cutaneous antioxidant enzymes namely, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, phase II metabolizing enzyme such as glutathione-S-transferase and quinone reductase and depletion in the level of cutaneous glutathione. There was also enhancement in cutaneous microsomal lipid peroxidation, xanthine oxidase activity, [(14)C]-ornithine decarboxylase activity and [(3)H]-thymidine incorporation into cutaneous DNA. Cycloartenol was topically applied prior to the application of benzoyl peroxide at dose levels of 0.2 mg and 0.4 mg/kg body weight in acetone, which resulted in significant inhibition of epidermal ornithine decarboxylase activity and DNA synthesis (P < 0.001). There was also significant reduction of lipid peroxidation and xanthine oxidase activity (P < 0.001). In addition, the depleted levels of glutathione, inhibited activities of antioxidant and phase II metabolizing enzymes, were also recovered to a significant level (P < 0.001). The data indicate that cycloartenol is an effective chemopreventive agent in skin carcinogenesis.

The brain is widely responsive to gonadal hormones. The functional significance of ovarian hormones in the brain is evident from biochemical studies indicating that estradiol or progesterone treatment of testectomized rats produces changes of antioxidant enzyme activities. The effect of estradiol benzoate (EB) and progesterone (P) in the control of antioxidant (AO) enzyme activities was studied in the brain of adult male Wistar rats. The activities of catalase (CAT), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST) and glutathione reductase (GR) were measured in appropriate subcellular fractions, prepared from brains of animals belonging to various experimental groups. These groups were designed with the intention to follow changes in enzyme activities 2 h or 24 h after systemic administration of 5 microg EB or 2 mg P to testectomized (TX) animals. The obtained results show that both EB and P increase CAT activity, whereas EB decreases GSH-Px, GST and GR activities. These findings clearly show the modulatory role of EB and P in the control of enzymes responsible for the protection of rat nerve cells against oxidative damage caused by free oxygen radicals.

This study used rainbow trout (Oncorhynchus mykiss) to investigate the biological effects of long-term exposure to a total chlorine-free (TCF) pulp mill effluent. Fish tanks were set up on the premises of the pulp mill, and fresh effluent water was led through a pipe directly from the pulp mill to the tanks. The fish were exposed to effluent for up to 50 days and kept for up to 70 days afterward to study the recovery process. Two independent experiments were carried out. Ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), and glutathione reductase (GR) activity were elevated in fish exposed to a 2% concentration of pulp mill effluent, as were levels of DNA adducts. These effects could be detected during exposure and also after a period of recovery. The results demonstrate that the pulp mill effluent contains substances that affect the detoxification process and also have genotoxic potential. The continued occurrence of effects after a 70-day recovery period was demonstrated in both experiments, and may indicate that the effluent contained compounds with persistent properties. The content of free and conjugated resin acids in the bile of the fish was found to be a useful indicator of exposure to pulp mill effluent.

Free radicals are involved in the pathogenesis and/or progression of Parkinson's disease (PD). Several ergot derivative dopamine (DA) agonists have been reported to scavenge free radicals in vitro and to show a neuroprotective effect in vivo. We investigated the in vitro free radical scavenging and antioxidant activities of cabergoline, a long-acting ergot DA agonist, as well as its ability to activate glutathione (GSH), catalase (Cat) and superoxide dismutase (SOD) activating effects and its in vivo neuroprotective properties against 6-hydroxydopamine (6-OHDA) intracerebroventricularly (i.c.v.) in mice. The striatal DA turnover induced by i.c.v. injection of 6-OHDA was completely normalized by pretreatment with cabergoline. Moreover, cabergoline scavenged free radicals in vitro and significantly reduced lipid peroxidation in vitro and in vivo. Furthermore, daily administration of cabergoline to mice significantly increased striatal GSH levels by activation of RNA expressions of GSH-related enzymes, although striatal Cat and SOD activities did not change. In addition, our present results suggest that repeated administration of cabergoline attenuates both 6-OHDA-induced nigrostriatal DAergic dysfunction and DA neuronal cell death, since cabergoline also had a neuroprotective effect in the immunohistochemical experiment. In conclusion, our findings indicate that the multiple antioxidant mechanisms of cabergoline, such as activation of the GSH system and the direct free radical scavenging activity, may explain the neuroprotective effect of this ergot DA agonist.

Considerable evidence suggests that reactive oxygen species (ROS) are crucially involved in the pathogenesis of cardiovascular diseases, such as myocardial ischemia-reperfusion injury. Consistent with this notion, administration of exogenous antioxidative compounds has been shown to provide protection against oxidative cardiac injury. However, whether induction of endogenous cellular antioxidants by chemicals (drugs) also offers protection against oxidative cardiac injury has not been extensively investigated. In the present study, with rat cardiomyocyte H9C2 cells as an in vitro model, we have investigated the induction of cellular antioxidants by the unique chemoprotective agent, 3 H -1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-induced cellular antioxidants against ROS-mediated injury in cardiac cells. Incubation of H9C2 cells with micromolar concentrations of D3T for 24 h resulted in a significant induction of a battery of cellular antioxidants, including reduced glutathione (GSH), GSH peroxidase, GSSG reductase, GSH S-transferase and catalase. To further examine the protective effects of the induced endogenous antioxidants against oxidative cell injury, H9C2 cells were pre-treated with D3T and then incubated with xanthine oxidase (XO) plus xanthine, a system that generates ROS. We observed that D3T pre-treatment of H9C2 cells led to significant protection against XO/xanthine-induced cytotoxicity as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction and morphological changes. Taken together, this study demonstrates for the first time that a number of endogenous antioxidants in cardiomyocytes can be induced by exposure to D3T, and that this chemical (drug) induction of cellular antioxidants is accompanied by markedly increased resistance to ROS-mediated cardiac cell injury.