The effect of electromagnetic field on reactive oxygen species production in human neutrophils in vitro

The present study was undertaken in order to determine the effect of low frequency electromagnetic field (EMF) on reactive oxygen species (ROS) production in human neutrophils in peripheral blood in vitro. We investigated how differently generated EMF and several levels of magnetic induction affect ROS production. To evaluate the level of ROS production, two fluorescent dyes were used: 2′7′-dichlorofluorscein-diacetate and dihydrorhodamine. Phorbol 12-myristate 13-acetate (PMA), known as strong stimulator of the respiratory burst, was also used. Alternating magnetic field was generated by means of Viofor JPS apparatus. Three different levels of magnetic induction have been analyzed (10, 40 and 60 μT). Fluorescence of dichlorofluorescein and 123 rhodamine was measured by flow cytometry. The experiments demonstrated that only EMF tuned to the calcium ion cyclotron resonance frequency was able to affect ROS production in neutrophils. Statistical analysis showed that this effect depended on magnetic induction value of applied EMF. Incubation in EMF inhibited cell activity slightly in unstimulated neutrophils, whereas the activity of PMA-stimulated neutrophils has increased after incubation in EMF.     

Oxidative stress as a potential biomarker for determining disease activity in patients with Rheumatoid Arthritis

Rheumatoid arthritis is an inflammatory, autoimmune disease where oxidative stress has been proposed to contribute to the joint tissue damage. To establish whether measurement of the redox status in blood mirrors the oxidant status at sites of inflammation in patients with rheumatoid arthritis, we concomitantly examined their oxidant status by spectrophotometry and/or flow cytometry. The basal levels of total reactive oxygen species (ROS), superoxide and hydroxyl radicals were significantly raised in neutrophils sourced from peripheral blood and synovial infiltrate, as also showed a strong positive correlation; however, there was no major increase in the reactive nitrogen species RNS generated in monocytes from both sources. Furthermore, raised levels of superoxide in neutrophils of synovial infiltrate showed a positive correlation with NADPH oxidase activity in synovial fluid. Additionally, as ROS generated in both peripheral blood and synovial infiltrate correlated positively with both DAS 28 and CRP/anti-CCP levels, its measurement can serve as an indirect measure of the degree of inflammation in patients with RA.     

Changes in neutrophil functions during a 10‐month soccer season and their effects on the physical condition of professional Japanese soccer players

We examined changes in neutrophil function of soccer players over a 10‐month period and determined its effectiveness as an index for athlete physical condition. Subjects included 21 male professional Japanese soccer players. Data on body composition, myogenic enzymes and neutrophil function were obtained before and after 2 h of training at 3 investigation points: one week before opening season, at season mid‐point, and one week before the last game of the season. As a result, change ratios of myogenic enzyme levels before and after the 2‐hr training session at the third investigation point were significantly higher compared to the two other points. Reactive oxygen species production and phagocytic activity significantly increased after 2‐hr training session at point 1, although the extent of the increase became smaller over time and ROS production capability decreased significantly by point 3 assessment. Fatigue, especially muscle fatigue, chronically accumulated along with a gradual decrease in neutrophil immune function over the 10‐month season. Therefore, determination of neutrophil function can be used as a useful index to assess and understand an athlete's physical condition. Copyright © 2012 John Wiley & Sons, Ltd.     

Small CAB-like proteins prevent formation of singlet oxygen in the damaged photosystem II complex of the cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterial small CAB-like proteins (SCPs) are single-helix membrane proteins mostly associated with the photosystem II (PSII) complex that accumulate under stress conditions. Their function is still ambiguous although they are assumed to regulate chlorophyll (Chl) biosynthesis and/or to protect PSII against oxidative damage. In this study, the effect of SCPs on the PSII-specific light-induced damage and generation of singlet oxygen ((1)O(2)) was assessed in the strains of the cyanobacterium Synechocystis sp. PCC 6803 lacking PSI (PSI-less strain) or lacking PSI together with all SCPs (PSI-less/scpABCDE(-) strain). The light-induced oxidative modifications of the PSII D1 protein reflected by a mobility shift of the D1 protein and by generation of a D1-cytochrome b-559 adduct were more pronounced in the PSI-less/scpABCDE(-) strain. This increased protein oxidation correlated with a faster formation of (1)O(2) as detected by the green fluorescence of Singlet Oxygen Sensor Green assessed by a laser confocal scanning microscopy and by electron paramagnetic resonance spin-trapping technique using 2, 2, 6, 6-tetramethyl-4-piperidone (TEMPD) as a spin trap. In contrast, the formation of hydroxyl radicals was similar in both strains. Our results show that SCPs prevent (1)O(2) formation during PSII damage, most probably by the binding of free Chl released from the damaged PSII complexes.     

Seasonal changes in antioxidant level of scots pine (Pinus sylvestris L.) needles exposed to industrial pollution. I. ascorbate and thiol content

Current and previous year needles from three 16 years-old populations of Scots pine (Pinus sylvestris L.) trees were seasonally collected at the three experimental areas: Luboń- close to the phosphate fertiliser factory, Głogów — close to the copper foundry and Kórnik — control site. Głogów is the most polluted site, where at 1998 monthly mean daily concentrations of different pollutants were: SO₂ - 17 μg·m⁻³, NO_x - 12 μg·m⁻³ and dust containing heavy metals as Cu, Pb, Cd - 29 μg·m⁻³. Trees growing in Luboń were influenced for many years by high concentration of SO₂ and fluor compounds. A few years ago emissions were markedly reduced, but low pH of soil and high concentration of aluminium ions still influence the growth of trees. Seasonal changes of ascorbate and thiol content were observed in each needle class and population, with the maximum in the winter and minimum in the summer. In needles from trees growing on polluted sites higher level of ascorbic acid and thiols comparing to control site was observed. Significant differences appeared in each population of Scots pine growing under higher pollution stress in the Głogów site. In needles from trees growing in Luboń significant differences in ascorbic acid and thiols content were evidently less numerous. Needles from polluted sites in some seasons contained significantly more malondialdehyde (MDA) and those was more frequent in Głogów than in Luboń. The results indicated that in the Głogów site trees are more influenced by pollution stress than in Luboń and the defense reaction measured as an increase of the antioxidant level is more evident.     

Effects of a bout of traditional and original sumo training on neutrophil immune function in amateur university sumo wrestlers

In order to examine in detail the influence on the neutrophil immune function in sumo wrestlers of performing traditional and original training we examined changes in the neutrophil immune function in 17 male amateur university sumo wrestlers (aged 20.2 ± 1.5 years), before (‘Pre’) and after the training (‘Post’) for 2.5 h under fasting conditions. Assays included blood leukocyte and neutrophil counts, serum concentration of immunoglobulins, complements, myogenic enzymes and neutrophil oxidative burst activity (OBA) and phagocytic activity (PA). Myogenic enzymes, neutrophil counts, the ratio of neutrophil counts:leukocyte counts significantly increased and immunoglobulins and complements decreased in Post compared with Pre. There was a positive correlation between the change of neutrophil counts before and after the training and the change of creatine kinase (r = 0.667, p < 0.01). The Post OBA significantly increased and PA significantly decreased compared with Pre. It was concluded that sumo training causes muscular damage and an increase in the neutrophil count as a response. In this time, although OBA increased, PA decreased after training. Compensation between PA and reactive oxygen species production may exist to maintain the overall integrity of the neutrophil immune function. Copyright © 2008 John Wiley & Sons, Ltd.     

Changes in the antigenic properties of proteins of laboratory mice during oxidative stress

Changes in the level of oxidative damage to proteins in CD1 outbred mice γ irradiated with a dose of 3 Gy have been studied. The changes were estimated from the amount of carbonyl groups (CG) in the proteins. It was found that two hours after exposure to γ radiation, the amount of CG in the cytoplasmic and nuclear fractions of the liver, heart, brain, and spleen sharply increased. Two months after irradiation, the level of CG in the cytoplasmic and nuclear subcellular fractions of the liver and brain decreased to the level of CG in the control animals, which were not exposed to radiation. In the subcellular fractions of the heart and spleen, the increase in the degree of damage was more significant and a high level of damage was observed even two months after irradiation. An enhancement of the antigenic properties of proteins from the liver, heart, and spleen in the postirradiation period was found. Spleen proteins were most immunogenic. A comparison of the antigenic properties of proteins isolated from the tissues 60 days after irradiation revealed a correlation between the level of oxidative damage and the immunogenicity of the total protein fraction.     

Reduction of mitochondrial H2O2 by overexpressing peroxiredoxin 3 improves glucose tolerance in mice

H(2)O(2) is a major reactive oxygen species produced by mitochondria that is implicated to be important in aging and pathogenesis of diseases such as diabetes; however, the cellular and physiological roles of mitochondrial H(2)O(2) remain poorly understood. Peroxiredoxin 3 (Prdx3/Prx3) is a thioredoxin peroxidase localized in mitochondria. To understand the cellular and physiological roles of mitochondrial H(2)O(2) in aging and pathogenesis of age-associated diseases, we generated transgenic mice overexpressing Prdx3 (Tg(PRDX3) mice). Tg(PRDX3) mice overexpress Prdx3 in a broad range of tissues, and the Prdx3 overexpression occurs exclusively in the mitochondria. As a result of increased Prdx3 expression, mitochondria from Tg(PRDX3) mice produce significantly reduced amount of H(2)O(2), and cells from Tg(PRDX3) mice have increased resistance to stress-induced cell death and apoptosis. Interestingly, Tg(PRDX3) mice show improved glucose homeostasis, as evidenced by their reduced levels of blood glucose and increased glucose clearance. Tg(PRDX3) mice are also protected against hyperglycemia and glucose intolerance induced by high-fat diet feeding. Our results further show that the inhibition of GSK3 may play a role in mediating the improved glucose tolerance phenotype in Tg(PRDX3) mice. Thus, our results indicate that reduction of mitochondrial H(2)O(2) by overexpressing Prdx3 improves glucose tolerance.     

Carnosol protects against spinal cord injury through Nrf-2 upregulation

Background: Carnosol is an ortho-diphenolic diterpene with excellent antioxidant potential. The present study was designed to identify the protective role of carnosol against spinal cord injury (SCI)-induced oxidative stress and inflammation in Wistar rats. Methods: In the present study, oxidative stress status was determined through estimating total antioxidant capacity, total oxidant status, lipid peroxide content, protein carbonyl and sulfhydryl levels, reactive oxygen species (ROS), antioxidant status (superoxide-dismutase, catalase, glutathione, glutathione peroxidase, glutathione-S-transferase). Inflammatory effects were determined by analyzing the expression of NF-κB and COX-2 through Western blot analysis. Further, carnosol-mediated redox homeostasis was analyzed by determining p-AKT and Nrf-2 levels. Results: SCI resulted in a significant increase in oxidative stress status through increased ROS generation, total oxidant levels, lipid peroxide content, protein carbonyl and sulfhydryl levels. The antioxidant status in SCI rats was significantly reduced, indicating imbalance in redox status. In addition, the expression of NF-κB and COX-2 was significantly upregulated, while p-AKT and Nrf-2 levels were downregulated in SCI rats. However, treatment with carnosol showed a significant enhancement in the antioxidant status with concomitant decline in oxidative stress parameters. Further, carnosol treatment regulated the key proteins in inflammation and redox status through significant downregulation of NF-κB and COX-2 levels and upregulation of p-AKT and Nrf-2 expression. Conclusion: Thus, the present study shows for the first time on the protective role of carnosol against SCI-induced oxidative stress and inflammation through modulating NF-κB, COX-2 and Nrf-2 levels in Wistar rats.     

The antioxidant enzyme peroxiredoxin-2 is depleted in lymphocytes seven days after ultra-endurance exercise

Abstract

Purpose. Peroxiredoxin-2 (PRDX-2) is an antioxidant and chaperone-like protein critical for cell function. This study examined whether the levels of lymphocyte PRDX-2 are altered over 1 month following ultra-endurance exercise. Methods. Nine middle-aged men undertook a single-stage, multi-day 233 km (145 mile) ultra-endurance running race. Blood was collected immediately before (Pre), upon completion/retirement (Post), and following the race at Day 1, Day 7 and Day 28. Lymphocyte lysates were examined for PRDX-2 by reducing and non-reducing SDS-PAGE with western blotting. In a sub-group of men who completed the race (n = 4), PRDX-2 oligomeric state (indicative of redox status) was investigated. Results. Ultra-endurance exercise caused significant changes in lymphocyte PRDX-2 (F_(4,32) 3.409, p = 0.020, η² = 0.299): 7 days after the race, PRDX-2 levels in lymphocytes had fallen to 30% of pre-race values (p = 0.013) and returned to near-normal levels at Day 28. Non-reducing gels demonstrated that dimeric PRDX-2 (intracellular reduced PRDX-2 monomers) was increased in three of four race completers immediately post-race, indicative of an ‘antioxidant response’. Moreover, monomeric PRDX-2 was also increased immediately post-race in two of four race-completing subjects, indicative of oxidative damage, which was not detectable by Day 7. Conclusions. Lymphocyte PRDX-2 was decreased below normal levels 7 days after ultra-endurance exercise. Excessive accumulation of reactive oxygen species induced by ultra-endurance exercise may underlie depletion of lymphocyte PRDX-2 by triggering its turnover after oxidation. Low levels of lymphocyte PRDX-2 could influence cell function and might, in part, explain reports of dysregulated immunity following ultra-endurance exercise.     

Redox treatment ameliorates diabetes mellitus-induced skin flap necrosis via inhibiting apoptosis and promoting neoangiogenesis

Intractable wound healing is the habitual problem of diabetes mellitus. High blood glucose limits wound healing by interrupting inflammatory responses and inhibiting neoangiogenesis. Oxidative stress is commonly thought to be a major pathogenic cause of diabetic complications. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, EDV) is a free radical scavenger which suppress oxidative stress. This study investigates whether EDV can reduce oxidative stress in wound healing HaCaT/human dermal fibroblasts cells (HDFs) in vitro and in vivo animal model. Cell viability and wound healing assays, FACS flow cytometry, and Hoechst 33342 staining were performed to confirm apoptosis and cytotoxicity in H₂O₂ and EDV-treated HaCaT and HDFs. A streptozotocin-induced hyperglycemic animal model was made in adult C57BL6 mice. Full-thickness skin flap was made on dorsomedial back and re-sutured to evaluate the wound healing process. EDV was delivered slowly in the skin flap with degradable fibrin glue. The flap was monitored and analyzed on postoperative days 1, 3, and 5. CD31/DAPI staining was done to detect newly formed blood vessels. The expression levels of NF-κB, bcl-2, NOX3, and STAT3 proteins in C57BL6 mouse tissues were also examined. The wound healing process in hyper- and normoglycemic mice showed a difference in protein expression, especially in oxidative stress management and angiogenesis. Exogenous H₂O₂ reduced cell viability in a proportion to the concentration via apoptosis. EDV protected HaCaT cells and HDFs from H₂O₂ induced reactive oxygen species cell damage and apoptosis. In the mouse model, EDV with fibrin resulted in less necrotic areas and increased angiogenesis on postoperative day 5, compared to sham-treated mice. Our results indicate that EDV could protect H₂O₂-induced cellular injury via inhibiting early apoptosis and inflammation and also increasing angiogenesis. EDV might be valuable in the treatment of diabetic wounds that oxidative stress has been implicated.     

Roles of sirtuins in the regulation of antioxidant defense and bioenergetic function of mitochondria under oxidative stress

Abstract

In addition to serving as the power house of mammalian cells, mitochondria are crucial for the maintenance of cellular homeostasis in response to physiological or environmental changes. Several lines of evidence suggest that posttranslational modification (PTM) of proteins plays a pivotal role in the regulation of the bioenergetic function of mitochondria. Among them, reversible lysine acetylation of mitochondrial proteins has been established as one of the key mechanisms in cellular response to energy demand by modulating the flux of a number of key metabolic pathways. In this article, we focus on the role of Sirt3-mediated deacetylation in: (1) flexibility of energy metabolism, (2) activation of antioxidant defense, and (3) maintenance of cellular redox status in response to dietary challenge and oxidative stress. We suggest that oxidative stress-elicited down-regulation of Sirt3 plays a role in the pathophysiology of diabetes, cardiac hypotrophy, mitochondrial diseases, and age-related diseases. Besides, the physiological role of newly identified lysine acylation mediated by Sirt5 and its biochemical effects on oxidative metabolism are also discussed. Moreover, we have integrated the regulatory function of several protein kinases that are involved in the phosphorylation of mitochondrial enzymes during oxidative stress. Finally, the functional consequence of the synergistic regulation through diverse protein modifications is emphasized on the maintenance of the bioenergetic homeostasis and metabolic adaptation of the animal and human cells. Together, we have provided an updated review of PTM in mitochondrial biology and their implications in aging and human diseases through an intricate regulation of energy metabolism under oxidative stress.     

Reactive oxygen species (ROS) production in human peripheral blood neutrophils exposed in vitro to static magnetic field

The aim of this study was to determine the effect of gradient static magnetic field (SMF) on reactive oxygen species (ROS) production in human neutrophils in peripheral blood in vitro. Blood samples collected from healthy individuals were incubated in an inhomogeneous SMF (in a south or north pole of the field) for 15, 30 or 45 minutes. The maximum value of induction (B _max) amounted to ≈ 60 mT. To determine the strength of the ROS production, dihydrorhodamine (123DHR) as fluorophore and phorbol 12-myristate 13-acetate (PMA) as respiratory burst stimulator were used. 123DHR oxidation by ROS was measured by flow cytometry. The exposure of blood samples to SMF induced statistically significant changes in ROS production in unstimulated and PMA-stimulated neutrophils. The observed effects were highly correlated with the exposure time and depended on the orientation of the field. Although intracellular mechanisms underlying such interactions are not thoroughly understood, it could be presumed that SMF affects ROS metabolic oscillations and their formation and inactivation. This study emphasizes the importance of proper adjustment of exposure time to SMF for any potential therapeutic applications.     

Induction of oxidative stress causes functional alterations in mouse urothelium via a TRPM8‐mediated mechanism: implications for aging

The incidence of bladder conditions such as overactive bladder syndrome and its associated urinary incontinence is highly prevalent in the elderly. However, the mechanisms underlying these disorders are unclear. Studies suggest that the urothelium forms a ‘sensory network’ with the underlying innervation, alterations in which, could compromise bladder function. As the accumulation of reactive oxygen species can cause functional alterations with age, the aim of this study was to investigate whether oxidative stress alters urothelial sensory signalling and whether the mechanism underlying the effect of oxidative stress on the urothelium plays a role in aging. Five‐month‐old(young) and 24‐month‐old (aged) mice were used. H₂O₂, used to induce oxidative stress, resulted in an increase in bladder afferent nerve activity and urothelial intracellular calcium in preparations from young mice. These functional changes were concurrent with upregulation of TRPM8 in the urothelium. Moreover, application of a TRPM8 antagonist significantly attenuated the H₂O₂‐induced calcium responses. Interestingly, an upregulation of TRPM8 was also found in the urothelium from aged mice, where high oxidative stress levels were observed, together with a greater calcium response to the TRPM8 agonist WS12. Furthermore, these calcium responses were attenuated by pretreatment with the antioxidant N‐acetyl‐cysteine. This study shows that oxidative stress affects urothelial function involving a TRPM8‐mediated mechanism and these effects may have important implications for aging. These data provide an insight into the possible mechanisms by which oxidative stress causes physiological alterations in the bladder, which may also occur in other organs susceptible to aging.     

Biomarker potential of C-peptide for screening of insulin resistance in diabetic and non-diabetic individuals

Insulin resistance is a hallmark feature of type-2 diabetes mellitus (T2DM). We determined the homeostatic model assessment insulin resistance (HOMA-IR) and evaluated its association with C-peptide, insulin, fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) in T2DM patients and non-diabetic subjects. This study comprised a total of 47 T2DM patients and 38 non-diabetic controls. Venous blood samples from all the subjects were collected and sera were analyzed for FBG, HbA1c, insulin and C-peptide using an autoanalyzer. HOMA-IR was calculated using the following equation: HOMA-IR?=?fasting insulin (µU/ml)?×?fasting glucose (mmol/L)/22.5. There was a significant increase in the levels of FBG and HbA1c in diabetic patients. Although the levels of C-peptide and insulin did not differ significantly between the two groups, a significant increase in HOMA-IR was observed in T2DM patients. Both insulin and C-peptide were significantly correlated with HOMA-IR. In conclusion, C-peptide may serve as a simple and convenient predictor of HOMA-IR.     

The role of oxidative stress in lung injury induced by cigarette smoke

Oxidative stress is a damaging process resulting from an imbalance between excessive generation of oxidant compounds and insufficient antioxidant defence mechanisms. Oxidative stress plays a crucial role in the initiation and progression of cigarette smoke-induced lung injury, deterioration in lung functions, and development of chronic obstructive pulmonary disease (COPD). In smokers and in patients with COPD, the increased oxidant burden derives from cigarette smoke per se, and from activated inflammatory cells releasing enhanced amounts of reactive oxygen and nitrogen species (ROS, RNS, respectively). Although mild oxidative stress resulting from cigarette smoking leads to the upregulation of the antioxidative enzymes synthesis in the lungs, high levels of ROS and RNS observed in patients with COPD overwhelm the antioxidant enzymes capacities, resulting in oxidant-mediated lung injury and cell death. In addition, depletion of antioxidative systems in the systemic circulation was consistently observed in such patients. The imbalance between the generation of ROS/RNS and antioxidant capacities — the state of “oxidative stress” — is one of the major pathophysiologic hallmarks in the development of COPD. Detrimental effects of oxidative stress include impairment of membrane functions, inactivation of membrane-bound receptors and enzymes, and increased tissue permeability. In addition, oxidative stress aggravates the inflammatory processes in the lungs, and contributes to the worsening of the protease-antiprotease imbalance. Several markers of oxidative stress, such as increases in lipid peroxidation products and reductions in glutathione peroxidase activity, have been shown to be related to the reductions in pulmonary functions. In the present article we review the current knowledge about the vicious cycle of cigarette smoking, oxidative stress, and inflammation in the pathogenesis of COPD.     

Plant sugars are crucial players in the oxidative challenge during abiotic stress: extending the traditional concept

Plants suffering from abiotic stress are commonly facing an enhanced accumulation of reactive oxygen species (ROS) with damaging as well as signalling effects at organellar and cellular levels. The outcome of an environmental challenge highly depends on the delicate balance between ROS production and scavenging by both enzymatic and metabolic antioxidants. However, this traditional classification is in need of renewal and reform, as it is becoming increasingly clear that soluble sugars such as disaccharides, raffinose family oligosaccharides and fructans – next to their associated metabolic enzymes – are strongly related to stress‐induced ROS accumulation in plants. Therefore, this review aims at extending the current concept of antioxidants functioning during abiotic stress, with special focus on the emanate role of sugars as true ROS scavengers. Examples are given based on their cellular location, as different organelles seem to exploit distinct mechanisms. Moreover, the vacuole comes into the picture as important player in the ROS signalling network of plants. Elucidating the interplay between the mechanisms controlling ROS signalling during abiotic stress will facilitate the development of strategies to enhance crop tolerance to stressful environmental conditions.