Increase In H2O2 Levels Research Articles

Caveolin‐1 is a critical determinant of autophagy and oxidative stress

Reactive oxygen species (ROS) are implicated in the pathogenesis of many disease states. Caveolin‐1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Caveolin‐1null mice have marked cardiovascular abnormalities, yet the molecular mechanisms are incompletely understood. We performed unbiased metabolomic analyses of lysates prepared from endothelial cells following siRNA‐mediated caveolin‐1 knockdown, and discovered striking (up to 30‐fold) increases in levels of cellular dipeptides, consistent with an increase in autophagy. We found that plasma 8‐isoprostanes levels were significantly higher in caveolin‐1null mice compared to wild‐type control mice (454 ± 44 vs. 319 ± 3 pg/ml, p<0.05). We injected caveolin‐1null and wild‐type mice with a recombinant lentivirus expressing the hydrogen peroxide (H2O2) biosensor HyPer2; quantitative fluorescence imaging studies revealed significant increases in H2O2 in the endothelium of arterial preparations isolated from caveolin‐1null mice (p<0.05 compared to wild type). Caveolin‐1 knockdown in endothelial cells led to significant (p<0.05) increases in H2O2 levels (measured using both the HyPer2 biosensor and the Amplex Red assay), associated with a marked decrease in the GSH/GSSG ratio. The mitochondrial inhibitor rotenone attenuated the increase in H2O2 levels seen after caveolin‐1 knockdown, whereas the NADPH oxidase inhibitor VAS‐7820 had no effect. Cellular imaging of mitochondrial ROS production (assayed with MitoSox Red) and of mitochondrial membrane potential revealed marked increases after caveolin‐1 knockdown, with no change in mitochondrial abundance. These findings establish that caveolin‐1 plays a key role in the regulation of cellular oxidative stress, and suggest that caveolin‐1‐modulated pathways may represent novel targets for the amelioration of oxidative stress in cardiovascular disease states.

Thioredoxin Reductase Deficiency Potentiates Oxidative Stress, Mitochondrial Dysfunction and Cell Death in Dopaminergic Cells

Mitochondria are considered major generators of cellular reactive oxygen species (ROS) which are implicated in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease (PD). We have recently shown that isolated mitochondria consume hydrogen peroxide (H2O2) in a substrate- and respiration-dependent manner predominantly via the thioredoxin/peroxiredoxin (Trx/Prx) system. The goal of this study was to determine the role of Trx/Prx system in dopaminergic cell death. We asked if pharmacological and lentiviral inhibition of the Trx/Prx system sensitized dopaminergic cells to mitochondrial dysfunction, increased steady-state H2O2 levels and death in response to toxicants implicated in PD. Incubation of N27 dopaminergic cells or primary rat mesencephalic cultures with the Trx reductase (TrxR) inhibitor auranofin in the presence of sub-toxic concentrations of parkinsonian toxicants paraquat; PQ or 6-hydroxydopamine; 6OHDA (for N27 cells) resulted in a synergistic increase in H2O2 levels and subsequent cell death. shRNA targeting the mitochondrial thioredoxin reductase (TrxR2) in N27 cells confirmed the effects of pharmacological inhibition. A synergistic decrease in maximal and reserve respiratory capacity was observed in auranofin treated cells and TrxR2 deficient cells following incubation with PQ or 6OHDA. Additionally, TrxR2 deficient cells showed decreased basal mitochondrial oxygen consumption rates. These data demonstrate that inhibition of the mitochondrial Trx/Prx system sensitizes dopaminergic cells to mitochondrial dysfunction, increased steady-state H2O2, and cell death. Therefore, in addition to their role in the production of cellular H2O2 the mitochondrial Trx/Prx system serve as a major sink for cellular H2O2 and its disruption may contribute to dopaminergic pathology associated with PD.

Resveratrol protects adult cardiomyocytes against oxidative stress mediated cell injury

Recent studies from our laboratory have showed that resveratrol, a polyphenol found predominantly in grapes rendered strong cardioprotection in animal models of heart disease. The cardioprotection which was observed was primarily associated with the ability of resveratrol to reduce oxidative stress in these models. The aim of the current study was to corroborate the role of resveratrol as an inhibitor of oxidative stress and explore the underlying mechanisms of its action in heart disease. For this purpose, we used a cell model of oxidative stress, the hydrogen peroxide (H2O2) exposed adult rat cardiomyocytes, which was treated with and without resveratrol (30μM); cardiomyocytes which were not exposed to resveratrol served as controls. Cell injury, cell death and oxidative stress measurements as well as the activities of the major endogenous antioxidants superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were carried out in control and H2O2 exposed cardiomyocytes, treated with and without resveratrol. Pharmacological blockade using specific blockers of the antioxidant enzymes were used to confirm their role in mediating resveratrol action in H2O2 exposed cardiomyocytes. The status of H2O2 and antioxidant enzymes in serum samples from spontaneously hypertensive rats (SHR) treated with and without resveratrol (2.5mg/kg body weight) was also examined.Our results showed significant cell injury and death in H2O2 exposed cardiomyocytes which was prevented upon resveratrol treatment. SOD and CAT activities were decreased in H2O2 exposed adult rat cardiomyocytes; treatment with resveratrol significantly prevented this reduction. However, GPx activity was not altered in the H2O2 exposed cardiomyocytes in comparison to controls. Pharmacological blockade of SOD and/or CAT prevented the beneficial effect of resveratrol. In SHR, H2O2 levels were increased, but CAT activity was decreased, while SOD remained unchanged, when compared to WKY rats; resveratrol treatment significantly prevented the increase in H2O2 levels and the decrease in CAT activities in SHR.Based on our results, we conclude that treatment with resveratrol prevents oxidative stress induced cardiomyocyte injury mainly by preserving the activities of critical antioxidant enzymes. This may be a crucial mechanism by which resveratrol confers cardioprotection.

Long-term exposure to nonylphenol affects insulin signaling in the liver of adult male rats

In the present study, we sought to investigate the long-term effects of nonylphenol (NP) on insulin signaling and glucose metabolism in liver. Furthermore, reactive oxygen species (ROS) in liver was evaluated as it is known to induce insulin resistance. Rats were administered NP by oral gavage at the doses of 15, 150 and 1500 μg/ kg body weight per day for 45 days. Hydrogen peroxide (H(2)O(2)) generation and lipid peroxidation were increased, and the activities of antioxidant enzymes were decreased in the liver of NP-treated rats. NP increased the plasma glucose and insulin levels and altered the enzymes of carbohydrate metabolism. Decrease in the protein levels of insulin signaling molecules insulin receptor (IR), IR substrate (IRS)-1, IRS-2 and phosphatidylinositol-3-kinase were observed with parallel increase in H(2)O(2) levels in the liver of NP-treated rats. These results suggest that NP downregulates insulin signaling in liver, which could be due to ROS production and oxidative damage.

Inhibitors of Catalase-Amyloid Interactions Protect Cells from β-Amyloid-Induced Oxidative Stress and Toxicity

Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H(2)O(2)). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H(2)O(2). Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H(2)O(2), and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.

Chronic Hypoxia Potentiates Age-Related Oxidative Imbalance in Brain Vessels and Synaptosomes

This study was aimed to evaluate and compare the effect of chronic hypoxia and aging in the oxidative status of brain vessels and synaptosomes. For this purpose we isolated brain vessels and synaptosomes from 3- and 12-month-old rats subjected to chronic hypoxia (10% O₂ for 7 days) or normoxia (21% O₂). Several parameters were evaluated: mitochondrial aconitase activity, hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) levels and enzymatic [superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR)] and non-enzymatic [glutathione (GSH), glutathione disulfide (GSSG) and vitamin E] antioxidant defences. Concerning brain vessels, we observed an age-dependent increase in MDA levels and SOD, catalase, GR and GPx activities. In vessels isolated from young animals, chronic hypoxia induced an increase in H₂O₂, GSSG and vitamin E levels and CuZnSOD and catalase activities and a decrease in GSH levels. In mature animals, hypoxia induced a decrease in GSH/GSSG ratio, vitamin E levels and mitochondrial aconitase, MnSOD and GR activities and an increase in H₂O₂ levels and CuZnSOD and catalase activities. Concerning synaptosomes we observed an age-dependent increase in MDA levels, CuZnSOD and GPx activities and a decrease in MnSOD activity. In synaptosomes from young animals, chronic hypoxia induced a decrease in mitochondrial aconitase activity and GSH levels and an increase in CuZnSOD activity and GSSG levels. In synaptosomes from mature animals, hypoxia induced a decrease in mitochondrial aconitase activity, GSH/GSSG ratio, GSH and vitamin E levels and an increase in GSSG levels. Our results show that chronic hypoxia promotes and potentiates age-dependent oxidative imbalance predisposing to neurodegeneration. Further, synaptosomes and brain vessels are differently affected by aging and chronic hypoxia supporting the idea of the existence of tissue-specific susceptibilities.

PGC-1α Serine 570 Phosphorylation and GCN5-mediated Acetylation by Angiotensin II Drive Catalase Down-regulation and Vascular Hypertrophy

Angiotensin II (Ang II) is a pleuripotential hormone that is important in the pathophysiology of multiple conditions including aging, cardiovascular and renal diseases, and insulin resistance. Reactive oxygen species (ROS) are important mediators of Ang II-induced signaling generally and have a well defined role in vascular hypertrophy, which is inhibited by overexpression of catalase, inferring a specific role of H(2)O(2). The molecular mechanisms are understood incompletely. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) is a key regulator of energy metabolism and ROS-scavenging enzymes including catalase. We show that Ang II stimulates Akt-dependent PGC-1 alpha serine 570 phosphorylation, which is required for the binding of the histone acetyltransferase GCN5 (general control nonderepressible 5) to PGC-1 alpha and for its lysine acetylation. These sequential post-translational modifications suppress PGC-1 alpha activity and prevent its binding to the catalase promoter through the forkhead box O1 transcription factor, thus decreasing catalase expression. We demonstrate that overexpression of the phosphorylation-defective mutant PGC-1 alpha (S570A) prevents Ang II-induced increases in H(2)O(2) levels and hypertrophy ([(3)H]leucine incorporation). Knockdown of PGC-1 alpha by small interfering RNA promotes basal and Ang II-stimulated ROS and hypertrophy, which is reversed by polyethylene glycol-conjugated catalase. Thus, endogenous PGC-1 alpha is a negative regulator of vascular hypertrophy by up-regulating catalase expression and thus reducing ROS levels. We provide novel mechanistic insights by which Ang II may mediate its ROS-dependent pathophysiologic effects on multiple cardiometabolic diseases.

Cadmium-induced antioxidant status and sister-chromatid exchanges in Vicia faba L.

The effects of cadmium (Cd) on antioxidant responses and sister chromatid exchanges (SCE) were studied in Vicia faba L. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR), and the levels of ascorbate (ASC), glutathione (GSH), hydrogen peroxide, and malondialdehyde (MDA, indicator of lipid peroxidation) were measured. Genotoxicity was evaluated by measuring the changes in the mitotic index (MI) and sister chromatid exchanges (SCE). Increased activities of SOD and CAT were observed in plants treated with 50 and 100 µM Cd. It was found that APX activity increased remarkably with 50 µM Cd, but no significant changes were found in GR activity in any of the treatment groups. These changes in antioxidant enzyme activities were negatively correlated with lipid peroxidation. However, a positive correlation between the increase of H2O2 levels and Cd concentration occurred with all levels of Cd. A significant increase in the malondialdehyde (MDA) level was only observed at the highest (200 µM) Cd concentration. This finding suggests that membrane damage did not occur in moderate Cd frequency. SCE increased significantly in high Cd concentration (200 µM), while MI decreased. The results suggest that exposure of high Cd concentrations is cytotoxic as well as genotoxic for Vicia faba L. and also suggest that the 2 phenomena are related.

Shear stress stimulates nitric oxide signaling in pulmonary arterial endothelial cells via a reduction in catalase activity: role of protein kinase Cδ

Previous studies have indicated that acute increases in shear stress can stimulate endothelial nitric oxide synthase (eNOS) activity through increased PI3 kinase/Akt signaling and phosphorylation of Ser1177. However, the mechanism by which shear stress activates this pathway has not been adequately resolved nor has the potential role of reactive oxygen species (ROS) been evaluated. Thus, the purpose of this study was to determine if shear-mediated increases in ROS play a role in stimulating Ser1177 phosphorylation and NO signaling in pulmonary arterial endothelial cells (PAEC) exposed to acute increases in shear stress. Our initial studies demonstrated that although shear stress did not increase superoxide levels in PAEC, there was an increase in H2O2 levels. The increases in H2O2 were associated with a decrease in catalase activity but not protein levels. In addition, we found that acute shear stress caused an increase in eNOS phosphorylation at Ser1177 phosphorylation and a decrease in phosphorylation at Thr495. We also found that the overexpression of catalase significantly attenuated the shear-mediated increases in H2O2, phospho-Ser1177 eNOS, and NO generation. Further investigation identified a decrease in PKCdelta activity in response to shear stress, and the overexpression of PKCdelta attenuated the shear-mediated decrease in Thr495 phosphorylation and the increase in NO generation, and this led to increased eNOS uncoupling. PKCdelta overexpression also attenuated Ser1177 phosphorylation through a posttranslational increase in catalase activity, mediated via a serine phosphorylation event, reducing shear-mediated increases in H2O2. Together, our data indicate that shear stress decreases PKCdelta activity, altering the phosphorylation pattern catalase, leading to decreased catalase activity and increased H2O2 signaling, and this in turn leads to increases in phosphorylation of eNOS at Ser1177 and NO generation.

Conjugated linoleic acid (CLA) prevents age associated skeletal muscle loss in mice by maintaining redox balance during aging

Age related skeletal muscle sarcopenia is a major health problem. Currently, there is no safe and effective therapies that selectively increase skeletal muscle mass. Previously, we have shown that CLA maintains higher skeletal muscle mass in mice. This study was designed to elucidate the molecular mechanisms of muscle loss prevention by CLA using 12 mo old C57BL/6 mice fed 10% corn oil (CO) or a diet supplemented with 0.5% c9t11 CLA (CLA 1), t10c12 CLA (CLA 2) or c9t11 CLA+t10c12 CLA (CLA mix) for 6 months. Dual energy X‐ray absorptiometry (DXA) scan was performed at baseline, and end of dietary intervention. CLA 2 and CLA mix groups showed significantly higher muscle mass as compared to CO and CLA 1 groups by DXA and muscle wet weight. Production of muscle mitochondrial ATP, and H2O2 as reactive oxygen species and muscle antioxidant enzymes (catalase and glutathione peroxidase) has been analyzed. Enhanced production of ATP, and antioxidant enzymes accompanied by slight increase in H2O2 level was noted in CLA 2 and CLA mix groups as compared to that of CO and CLA 1 groups. Finally, we have analyzed serum malondialdehyde (MDA) level. The lower level of serum MDA in CLA 2 and CLA mix groups indicate less oxidative stress in these groups. These data suggest that CLA 2 and CLA mix groups may preserve muscle mass by enhancing muscle mitochondrial energy production as well as by maintaining the redox balance during aging. Research support: NIH

Modulatory effect of hydrogen peroxide on tumoral lymphocytes proliferation

BW 5147 (murine lymphoma cell line). We analyzed the effect of H2O2 in cell proliferation testing nitric oxide and apoptosis. Enzymes involved in the regulation of H2O2 levels as superoxide dismutase (SOD) and peroxidase (PER) were analyzed. H2O2 exerted a biphasic effect. The inhibitory effect of H2O2 was related to the activation of the ERK and P38 pathway, NO production and apoptosis. The high proliferation was associated with a low level of H2O2 related to a low SOD and a high PER activities. Drugs capable of producing an increase in H2O2 levels could be used in cancer.

NaCl-induced expression of glutathione reductase in roots of rice (Oryza sativa L.) seedlings is mediated through hydrogen peroxide but not abscisic acid

Reactive oxygen species (ROS) play an important role in NaCl stress. Plants tolerant to NaCl stress may evolve certain strategies to remove these ROS, thus reducing their toxic effects. Therefore, the expression patterns of the gene family encoding glutathione reductase (GR, EC 1.6.4.2) were analyzed in roots of etiolated rice (Oryza sativa L.) seedlings in response to NaCl stress. Semi-quantitative RT-PCR was applied to quantify the mRNA levels for one cytosolic (OsGR2) and two chloroplastic (OsGR1 and OsGR3) isoforms of glutathione reductase identified in the rice genome. The expression of OsGR2 and OsGR3 but not OsGR1 was increased in rice roots treated with 150 mM NaCl. The Rab16A is an abscisic acid (ABA)-responsive rice gene. Increasing concentrations of ABA, from 1 to 12 μM, progressively increased the expression of OsRab16A in rice roots. In the present study, the ABA level was judged by the expression of OsRab16A in rice roots. Treatment with 150 mM NaCl induced the expression of OsRab16A, and the expression increased with increasing concentrations of ABA, which suggests that ABA may be involved in this response in rice roots. In fact, exogenous application of ABA enhanced the expression of OsGR2 and OsGR3 in rice roots. On inhibiting ABA accumulation with sodium tungstate (Tu), an inhibitor of ABA biosynthesis, the expression of OsGR2 and OsGR3 was still induced by NaCl; therefore, NaCl-triggered expression of OsGR2 and OsGR3 in rice roots is not mediated by accumulation of ABA. However, NaCl treatment could induce H2O2 production in rice roots, and H2O2 treatment resulted in enhanced OsGR2 and OsGR3 induction. On inhibiting the NaCl-induced accumulation of H2O2 with diphenylene iodonium, the expression of OsGR2 and OsGR3 was also suppressed. Moreover, the increase in H2O2 level was prior to the induction of OsGR2 and OsGR3 in NaCl-treated rice roots. Thus, H2O2, but not ABA, is involved in regulation of OsGR2 and OsGR3 expression in NaCl-treated rice roots.

Oxidative behaviour of fresh‐cut ‘Fuji’ apples treated with stabilising substances

AbstractBACKGROUND:Although changes in the quality of fresh‐cut products treated with chemical stabilisers have been extensively described in the literature, very little is known about the physiology of these products. This work aims to describe the physiological aspect and particularly the effects of chemical preservatives on the oxidative behaviour of fresh‐cut apples.RESULTS:Immediately after treatment, the samples treated with the anti‐browning agents (ascorbic acid + cysteine) exhibited a sharp increase in H2O2 levels (a five‐fold increase when compared to the other samples) and peroxidative damage, especially at the beginning of the storage period (two‐fold increase after 1 day). In line with this result, peroxidase (POX) activity decreased by 50% during the first day of storage, but no significant changes in superoxide dismutase (SOD) and catalase (CAT) activities were found between the different samples. H2O2 accumulation was not attributed to the action of cysteine but to a specific action of ascorbic acid, which mainly acted as a pro‐oxidant under these conditions.CONCLUSION:The results presented in this work showed that ascorbic acid cause important oxidative damage in fresh‐cut Fuji apples. Alternatives are required to prevent detrimental loss of quality resulting from this oxidative action. Copyright © 2008 Society of Chemical Industry

Hydrogen peroxide affects abscisic acid binding to ABAP1 in barley aleurones

Dramatic increases in H2O2 levels have been observed following abscisic acid (ABA) treatment of plant tissues. Following ABA treatment in aleurone cells, H2O2 reached transient levels of approximately 115 micromol/L H2O2. To determine whether ABA perception was modified by such changes, the effect of H2O2 on a recently characterized ABA-binding protein (ABAP1), cloned from barley aleurone layers, was examined. ABA binding to the protein was weakened by H2O2 in a concentration-dependent manner. A concentration of 75 micromol/L H2O2 gave a 50% decline in ABA binding in a reaction following first-order kinetics, indicative of binding-site susceptibility to its microenvironment. We monitored the unfolding of ABAP1 using steady-state and time-resolved tryptophan fluorescence, while following the capacity of ABAP1 to bind ABA. ABA binding decreased by 50% following ABAP1 denaturation with 1 mol/L guanidine hydrochloride or 2 mol/L urea, while the maximum emission spectra (lambda emi) red shifted from 338 to 347 nm at 3.5 mol/L guanidine hydrochloride and 5 mol/L urea. However, only a slight blue shift of lambda emi was observed following either ABAP1 incubation with H2O2 or binding to (+)-ABA (physiologically active ABA). The equilibrium ABA dissociation rate accelerated in the presence of 250 micromol/L H2O2, with the half-time dissociation reduced to 8 min. A comparison of inactivation kinetics and conformational changes shows that inactivation of ABAP1 occurs before any noticeable conformational change. This suggests that the ABA binding site is highly responsive to its microenvironment and is situated in a region that is more flexible than the protein molecule as a whole. The results demonstrate that H2O2, generated by ABA treatment of aleurone layers, is sufficient to affect the ABA-binding capacity of ABAP1, suggesting that this may be another level of control of ABA signal transduction.

Development of a neurotoxicity test-system using human post-mitotic, astrocytic and neuronal cell lines in co-culture

Astrocytes are essential for neuronal function and survival, so both cell types were included in a human neurotoxicity test-system to assess the protective effects of astrocytes on neurons, compared with a culture of neurons alone. The human NT2.D1 cell line was differentiated to form either a co-culture of post-mitotic NT2.N neuronal (TUJ1, NF68 and NSE positive) and NT2.A astrocytic (GFAP positive) cells (∼2:1 NT2.A:NT2.N), or an NT2.N mono-culture. Cultures were exposed to human toxins, for 4 h at sub-cytotoxic concentrations, in order to compare levels of compromised cell function and thus evidence of an astrocytic protective effect. Functional endpoints examined included assays for cellular energy (ATP) and glutathione (GSH) levels, generation of hydrogen peroxide (H2O2) and caspase-3 activation. Generally, the NT2.N/A co-culture was more resistant to toxicity, maintaining superior ATP and GSH levels and sustaining smaller significant increases in H2O2 levels compared with neurons alone. However, the pure neuronal culture showed a significantly lower level of caspase activation. These data suggest that besides their support for neurons through maintenance of ATP and GSH and control of H2O2 levels, following exposure to some substances, astrocytes may promote an apoptotic mode of cell death. Thus, it appears the use of astrocytes in an in vitro predictive neurotoxicity test-system may be more relevant to human CNS structure and function than neuronal cells alone. © 2007 Elsevier Ltd. All rights reserved.

Nitric oxide increases toxicity of hydrogen peroxide against rat liver endothelial cells and hepatocytes by inhibition of hydrogen peroxide degradation

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) show cooperativity in their cytotoxic action. The present study was performed to decipher the mechanisms underlying this phenomenon. In cultured liver endothelial cells and in cultured, glutathione-depleted hepatocytes, the combined exposure to NO (released by spermine NONOate, 1 mM) and H(2)O(2) (released by glucose oxidase) induced cell injury that was far higher than the injury elicited by NO or H(2)O(2) alone. In both cell types, the addition of the NO donor increased H(2)O(2) steady-state levels, although with different kinetics: in hepatocytes, the increase in H(2)O(2) levels was already evident at early time points while in liver endothelial cells it became evident after > or =2 h of incubation. NO exposure inhibited H(2)O(2) degradation, assessed after addition of 50 microM, 200 microM, or 4 mM authentic H(2)O(2), significantly in both cell types. However, again, early and delayed inhibition was observed. The late inhibition of H(2)O(2) degradation in endothelial cells was paralleled by a decrease in glutathione peroxidase activity. Glutathione peroxidase inactivation was prevented by hypoxia or by ascorbate, suggesting inactivation by reactive nitrogen oxide species (NO(x)). Early inhibition of H(2)O(2) degradation by NO, in contrast, could be mimicked by the catalase inhibitor azide. Together, these results suggest that the cooperative effect of NO and H(2)O(2) is due to inhibition of H(2)O(2) degradation by NO, namely to inhibition of catalase by NO itself (predominant in hepatocytes) and/or to inhibition of glutathione peroxidase by NO(x) (prevailing in endothelial cells).

Causes of root growth retardation induced by ultraviolet-B irradiation of shoots in Barley seedlings

In barley seedlings (Hordeum vulgare L.) during two days after irradiation of shoots with UV-B (0.5 W/m2, 6 h), the rate of elongation of primary roots decreased 2–3 times compared to that in control plants. The modulus of elasticity of roots (e) increased at most twofold in 12 h after the onset of irradiation; the hydraulic conductivity (Lp) diminished by a factor of two in 12 h, and the root osmotic pressure gradually decreased by 0.08 MPa in 24 h. Changes in e and Lp were shown to be related to oxidative stress in growing roots, which was evidenced from the increase in H2O2 level up to 15-fold increase in 6 h and in activity of guaiacol peroxidase (3.5-fold in 12 h). After 48 h, the characteristics of oxidative metabolism and root characteristics e and Lp became identical in untreated and treated plants. On the third day, the rate of root growth in treated plants reached its initial value. It is concluded that the main causes of retardation of root growth under these conditions were as follows: the increase in cell wall rigidity related to formation of oxidative cross-links in the apoplast and the decrease in root osmotic pressure due to limited transport of assimilates from irradiated leaves. After the intensity of UV-B irradiation applied to shoots was enhanced (1.6 W/m2, 4 h), another physiological status of roots was observed on the 2nd day characterized by twofold increase in Lp, tenfold decreased root elongation rate, and by a progressing increase of root diameter in growing roots. The comparison of root responses induced by irradiation of shoots with the root responses to sodium salicylate and ABA suggests that both agents might participate in the transmission of signals from irradiated leaves to roots.

High glucose-induced oxidative stress inhibits Na+/glucose cotransporter activity in renal proximal tubule cells

Oxidative stress plays an important role in the pathogenesis of renal diseases such as diabetic nephropathy. The metabolism of excessive intracellular glucose may involve a number of processes. One consequence of excessive intracellular glucose levels is an increased rate of oxidative phosphorylation under hyperglycemic conditions, whereas another consequence is an increase in the metabolism of glucose to sorbitol by aldose reductase. In addition, hyperglycemia may result in the activation of NADPH oxidase, the production of superoxide anion, and hydrogen peroxide (H2O2). In this report, we investigate the mechanisms responsible for the H2O2 production that occurs as the consequence of hyperglycemia and the effect of H2O2 on the activity of the Na+/glucose cotransport system (SGLT) in primary cultures of renal proximal tubule cells (PTCs). When primary PTCs were cultured in the presence of high glucose, one consequence was that the Na+/glucose cotransport system was inhibited, as indicated by uptake studies utilizing alpha-methyl-D-glucoside (alpha-MG), a nonmetabolizable analog of D-glucose. Pretreatment of the cultures with either 1) aminoguanidine or pyridoxamine [inhibitors of the accumulation of advanced glycation end products (AGEs)], 2) rotenone (an inhibitor of the mitochondrial electron transport chain), or 3) apocynin or diphenylene iodonium (DPI; inhibitors of NADPH oxidase) blocked the observed changes that occurred as a consequence of the incubation of the PTCs with high glucose. Included among these changes were the observed increase in H2O2 levels, as well as an increase in lipid peroxide production, and a decrease both in the activity of catalase and in the level of glutathione (GSH), endogenous antioxidants. The high glucose-induced decrease in the level of the Na+/glucose cotransporter was similarly prevented by either aminoguanidine, rotenone, or apocynin. Thus the inhibitory effect of high glucose on both the level of the Na+/glucose cotransport system and the activity of the Na+/glucose cotransport system can be explained, at least in part, as being due to the effects of the H2O2, the consequent formation of AGEs, the increase in mitochondrial metabolism, and in NADPH oxidase activity in the PTCs. Other related changes observed in the PTCs that could be reversed by treatment with either aminoguanidine, pyridoxamine, rotenone, apocynin, or DPI included an increase in transforming growth factor-beta1 secretion and the activation of the NF-kappaB signal transduction pathway.

Two Distinct Redox Signaling Pathways for Cytosolic APX Induction under Photooxidative Stress

The cross-talk of two redox factors, H2O2 accumulation and redox status of photosynthetic electron transport (PET) in tobacco chloroplasts, on the expression of cytosolic ascorbate peroxidase (cAPX) was studied. Transgenic tobacco plants, which expressed respectively the Escherichia coli catalase and spinach thylakoid-membrane-bound APX in chloroplasts and showed increased tolerance to photooxidative stress, were used for evaluation of the relationship between H2O2 accumulation or change of redox status of PET and cAPX induction under photooxidative stress condition. There was no difference in the increase in the transcript level of cAPX in the first 1 h after irradiation with high-intensity light between the wild-type and either of the transgenic plants. The transcript level of cAPX in the wild-type showed a steady gradual increase during the next 5 h, while that in the transgenic plants during this period was stable. The H2O2 level of wild-type plants increased after 1 h, while those of transgenic plants remained constant. The decrease in q(p) value in all types of plants occurred earlier than the increase in H2O2 level. These results indicate that the induction of cAPX expression is caused by a redox change in PET, probably through the plastquinone pool at an early stage and thereafter by an increase in the cellular H2O2 level.

Increased Nox1 and hydrogen peroxide in prostate cancer.

Reactive oxygen species (ROS) are emerging as candidate mediators of growth and angiogenesis in cancer. Increased ROS often correlates with cell growth, e.g., Ras-transformed cells and cells treated with growth factors. While non-transformed cells respond to growth factors/cytokines with the regulated production of ROS, tumor cells in culture frequently overproduce H(2)O(2). We propose that NADPH oxidases (Nox) account for increased levels of ROS in some cancers. Previously, transfection of Nox1 into a prostate cancer cell line dramatically enhanced tumor growth (Arbiser et al.: PNAS 99:715-720, 2001). Using immunohistochemistry, immunofluorescence, dihydroethidium staining, and Flow cytometry, we investigated the correlation between Nox1 and ROS in prostate cancer. Here, we demonstrate that human prostate tumors show increased H(2)O(2) levels. Furthermore, 80% of human prostate tumor samples show markedly increased Nox1 protein levels and increased mRNA levels. In addition, a series of cell lines developed from LNCaP prostate cancer cells that demonstrate increasing tumor and metastatic potential, show increased Nox1 and a parallel increase in H(2)O(2) levels. The results illustrate that human prostate cancer frequently show both increased H(2)O(2) and Nox1, and that in an animal model system increased Nox1/H(2)O(2) correlates with increased tumorigenicity.