Depletion of reactive oxygen species induced by chlorogenic acid triggers apoptosis-like death in Escherichia coli

Chlorogenic acid (CGA) is a phenolic compound with various health-promoting properties, including antioxidant effects and a wide range of antibacterial activities. However, the antibacterial mechanism remains unclear. We investigated the underlying mode of action of CGA against Escherichia coli, which shows bacterial apoptosis-like death. Cells treated with CGA showed apoptotic features such as membrane depolarisation, caspase-like protein expression, increased intracellular Ca²⁺ levels, phosphatidylserine externalisation, and DNA fragmentation. In contrast to common bacterial apoptosis-like death, which is caused by reactive oxygen species (ROS) accumulation, CGA depleted intracellular ROS. Because ROS are important intracellular signalling molecules, and ROS depletion may affect bacterial intracellular signalling pathways, leading to cell death. To determine whether deficiencies in intracellular ROS cause apoptosis-like death, the cells were treated with H₂O₂ after CGA treatment. H₂O₂ restored depleted intracellular ROS levels to similar levels as in untreated cells, and cell viability was increased compared to CGA-treated cells. Moreover, apoptotic features were attenuated in H₂O₂ post-treated cells. These results demonstrate that CGA induces bacterial apoptosis in E. coli and intracellular ROS depletion is a core regulator in the progression of bacterial apoptosis-like death.     

Antioxidant effect of homogenetisic acid on hydrogen peroxide induced oxidative stress in human lung fibroblast cells

Homogentisic acid was found to scavenge intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and thus prevented lipid peroxidation in human fibroblast (WI 38) cells. The radical scavenging activity of homogentisic acid was found to protect WI 38 cells against hydrogen peroxide (H₂O₂) induced oxidative stress, via the activation of extracellular signal regulated kinase (ERK) protein. Homogentisic acid increased the activity of catalase. Hence, from the present study, it is suggested that homogentisic acid protects WI 38 cells against H₂O₂ damage by enhancing the intracellular antioxidative activity.     

Mitochondrial transmembrane potential is diminished in phorbol myristate acetate-stimulated peritoneal resident macrophages isolated from wild-type mice,but not in those from gp91-phox-deficient mice

Macrophages produce superoxide (O₂^–) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O₂^– is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H₂DCFDA), O₂^– was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H₂DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O₂^–-producing cells, but not in O₂^–-deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H₂O₂ dismutated from O₂^–, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA.     

The effect of reactive oxygen species on ethylene production induced by osmotic stress in etiolated mungbean seedling

After 10 h osmotic stress in 25% polyethylene glycol (PEG6000) solution (–1.8 MPa) at 25 °C in darkness, the etiolated mungbean seedlings were transferred to pure water for recovery. The ethylene release rate and the level of reactive oxygen species (ROS), including superoxide radical (O₂^–) and hydrogen peroxide (H₂O₂), were investigated during the recovery process. The results showed that ethylene production rate and amount of ROS increased dramatically after osmotic stress, and a close correlation was observed between ethylene release rate and concentrations of ROS. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) or aminooxyacetic acid (AOA), could reduce the ethylene release rate, but had no significant influence to the content of O₂^– and H₂O₂. As well as, silver thiosulfate (STS), an inhibitor of ethylene action, exhibited no obvious effect to the concentration of ROS, showing stress-inducible ethylene was not the cause for the increase of stress-inducible ROS. On the other hand, exogenous generator of superoxide radical (methylviologen, MV, or sodium dithionite, Na₂S₂O₄) could enhance the ethylene production evidently, which could be inhibited by exogenous scavenger of superoxide radical (superoxide dismutase, SOD, or 1, 4-diazabicyclo (2,2,2) octane, DABCO). However, either exogenous H₂O₂ or catalase (CAT) had no significant influence on ethylene production. The results suggested that it was superoxide radical but not H₂O₂which was involved directly in osmotic stress-inducible ethylene biosynthesis. The dual-role of superoxide radical on stress ethylene biosynthesis was also discussed.     

Oxidative pathways in response to polyunsaturated aldehydes in the marine diatom Skeletonema marinoi (Bacillariophyceae)

Polyunsaturated aldehydes (PUA) have recently been shown to induce reactive oxygen species (ROS) and possibly reactive nitrogen species (RNS, e.g., peroxynitrite) in the diatom Skeletonema marinoi (S. marinoi), which produces high amounts of PUA. We now are attempting to acquire better understanding of which reactive molecular species are involved in the oxidative response of S. marinoi to PUA. We used flow cytometry, the dye dihydrorhodamine 123 (DHR) as the main indicator of ROS (but which is also known to partially detect RNS), and different scavengers and inhibitors of both nitric oxide (NO) synthesis and superoxide dismutase activity (SOD). Both the scavengers Tempol (for ROS) and uric acid (UA, for peroxynitrite) induced a lower DHR‐derived green fluorescence in S. marinoi cells exposed to the PUA, suggesting that both reactive species were produced. When PUA‐exposed S. marinoi cells were treated with the NO scavenger 2‐4‐carboxyphenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), an opposite response was observed, with an increase in DHR‐derived green fluorescence. A higher DHR‐derived green fluorescence was also observed in the presence of sodium tungstate (ST), an inhibitor of NO production via nitrate reductase. In addition, two different SOD inhibitors, 2‐methoxyestradiol (2ME) and sodium diethyldithiocarbamate trihydrate (DETC), had an effect, with DETC inducing the strongest inhibition after 20 min. These results indicate the involvement of O₂^? generation and SOD activity in H₂O₂ formation (with downstream ROS generation dependent from H₂O₂) in response to PUA exposure. This is relevant as it refines the biological impact of PUA and identifies the specific molecules involved in the response. It is speculated that in PUA‐exposed S. marinoi cells, beyond a certain threshold of PUA, the intracellular antioxidant system is no longer able to cope with the excess of ROS, thus resulting in the observed accumulation of both O₂^?? and H₂O₂. This might be particularly relevant for population dynamics at sea, during blooms, when cell lysis increases and PUA are released. It can be envisioned that in the final stages of blooms, higher local PUA concentrations accumulate, which in turn induces intracellular ROS generation that ultimately leads to cell death and bloom decay.     

Index

Hydrogen peroxide (H₂O₂) can induce cell damage by generating reactive oxygen species (ROS), resulting in DNA damage and cell death. The aim of this study is to elucidate the protective effects of fisetin (3,7,3′,4′,-tetrahydroxy flavone) against H₂O₂-induced cell damage. Fisetin reduced the level of superoxide anion, hydroxyl radical in cell free system, and intracellular ROS generated by H₂O₂. Moreover, fisetin protected against H₂O₂-induced membrane lipid peroxidation, cellular DNA damage, and protein carbonylation, which are the primary cellular outcomes of H₂O₂ treatment. Furthermore, fisetin increased the level of reduced glutathione (GSH) and expression of glutamate-cysteine ligase catalytic subunit, which is decreased by H₂O₂. Conversely, a GSH inhibitor abolished the cytoprotective effect of fisetin against H₂O₂-induced cells damage. Taken together, our results suggest that fisetin protects against H₂O₂-induced cell damage by inhibiting ROS generation, thereby maintaining the protective role of the cellular GSH system.     

Effects of SOD/catalase mimetic platinum nanoparticles on radiation-induced apoptosis in human lymphoma U937 cells

Since polyacrylic acid capped platinum nano-particles (nano-Pts) are known to have a unique ability to quench superoxide (O₂ ^?) and hydrogen peroxide (H₂O₂), the anti-oxidant activity of nano-Pts against apoptosis induced by x-irradiation in human lymphoma U937 cells was investigated. DNA fragmentation assay, Annexin V-FITC/PI by flow cytometry and Giemsa staining revealed a significant decrease in apoptosis induced by 10 Gy, when cells were pre-treated with nano-Pts in a dose-dependent manner. Pre-treatment with nano-Pts significantly decreased radiation-induced reactive oxygen species (ROS) production, Fas expression and loss of mitochondrial membrane potential as determined by flow-cytometry. Furthermore, western blot analysis also showed that the expression of cleaved caspase-3, Bid and cytosolic cytochrome-c were significantly reduced in nano-Pts pretreated cells. Due to the catalase mimetic activity of nano-Pts, these results indicate that pre-treatment of U937 cells with nano-Pts significantly protect radiation-induced apoptosis by inhibiting intracellular ROS (mainly H₂O₂), which plays a key role in the induction of apoptosis, because of no practical observation of intracellular O₂ ^? formation.     

Role of extracellular peroxidase in the superoxide production by wheat root cells

Summary Extracellular peroxidase has been shown to contribute to superoxide production in wounded wheat (Triticum aestivum L. cv. Ljuba) root cells. The superoxide-synthesizing system of root cells was considerably inhibited by KCN and NaN₃ and activated by MnCl₂ and H₂O₂. Treatment of roots with salicylic acid and a range of di- and tri-carbonic acids (malic, citric, malonic, fumaric, and succinic acids) stimulated superoxide production in both root cells and extracellular solution. The H₂O₂-stimulated superoxide production in the extracellular solution was much higher when roots were preincubated with salicylic or succinic acid. Exogenous acids enhanced peroxidase activity in the extracellular solution. Pretreatment of root cells with the detergents trypsin and sodium dodecyl sulfate had similar effects on the peroxidase activity. Significant inhibition of both superoxide production and peroxidase activity by diphenylene iodonium suggests that the specificity of the latter as an inhibitor of NADPH oxidase is doubtful. Results obtained indicate that extracellular peroxidase is involved in the superoxide production in wheat root cells. The mobile form of peroxidase can be readily secreted to the apoplastic solution and serve as an emergency enzyme involved in plant wound response.Abbreviations DPI diphenylene iodonium - ECS extracellular solution - ROS reactive oxygen species - SA salicylic acid     

Production of nerve growth factor enhanced in cultured mouse astrocytes by glycerophospholipids, sphingolipids, and their related compounds

The presence of cysteine and methionine groups together with an ability to bind long-chain fatty acid (LCFA) oxidation products makes liver fatty acid binding protein (L-FABP) an attractive candidate against hepatocellular oxidative stress. In this report, we show that pharmacological treatment directed at modulating L-FABP level affected hepatocellular oxidant status. L-FABP expressing 1548-hepatoma cells, treated with dexamethasone or clofibrate, decreased and increased intracellular L-FABP levels, respectively. Oxidative stress was induced by H₂O₂ incubation or hypoxia–reoxygenation. The fluorescent marker, dichlorofluorescein (DCF), was employed to measure intracellular reactive oxygen species (ROS). Hepatocellular damage was assessed by lactate dehydrogenase (LDH) level. Dexamethasone treatment resulted in a significant increase in DCF fluorescence with higher LDH release compared to control cells. Clofibrate treatment, however, resulted in a significant decrease in both parameters (p < 0.05). Drug treatments did not affect cytosolic activites of glutathione peroxidase (GPx), superoxide dismutase (SOD), or catalase suggesting that the differences between treated and control cells may likely be associated with varying L-FABP levels. We conclude that L-FABP may act as an effective endogenous cytoprotectant against hepatocellular oxidative stress.     

Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli

Fatty acids are a promising raw material for substance production because of their highly reduced and anhydrous nature, which can provide higher fermentation yields than sugars. However, they are insoluble in water and are poorly utilized by microbes in industrial fermentation production. We used fatty acids as raw materials for l-lysine fermentation by emulsification and improved the limited fatty acid-utilization ability of Escherichia coli. We obtained a fatty acid-utilizing mutant strain by laboratory evolution and demonstrated that it expressed lower levels of an oxidative-stress marker than wild type. The intracellular hydrogen peroxide (H₂O₂) concentration of a fatty acid-utilizing wild-type E. coli strain was higher than that of a glucose-utilizing wild-type E. coli strain. The novel mutation rpsA ^D210Y identified in our fatty acid-utilizing mutant strain enabled us to promote cell growth, fatty-acid utilization, and l-lysine production from fatty acid. Introduction of this rpsA ^D210Y mutation into a wild-type strain resulted in lower H₂O₂ concentrations. The overexpression of superoxide dismutase (sodA) increased intracellular H₂O₂ concentrations and inhibited E. coli fatty-acid utilization, whereas overexpression of an oxidative-stress regulator (oxyS) decreased intracellular H₂O₂ concentrations and promoted E. coli fatty acid utilization and l-lysine production. Addition of the reactive oxygen species (ROS) scavenger thiourea promoted l-lysine production from fatty acids and decreased intracellular H₂O₂ concentrations. Among the ROS generated by fatty-acid β-oxidation, H₂O₂ critically affected E. coli growth and l-lysine production. This indicates that the regression of ROS stress promotes fatty acid utilization, which is beneficial for fatty acids used as raw materials in industrial production.     

Effects of Free Radicals on the Fluidity of Myocardial Membranes

Free radicals, including superoxide anions (O₂^?_?), hydroxyl radical (HO'), and hypohalite radical (OCl'), as well as oxidants such as hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl), have been indicated in the pathogenesis of myocardial ischemic and reperfusion injury. In this report, we compared the integrity of the myocardial membrane when exposed to these free radicals/oxidants. Isolated rat heart membrane preparations were exposed to chemically generated free radicals with or without their respective scavengers. Membrane fluidity was monitored by fluorescence polarization using the diphenylhexatriene probe, as well as by electron spin resonance (ESR) spectroscopy using 2,2,6,6-tetramethyl piperidine-n-oxyl as the spin labeling agent. HO', H₂O₂, and OCl' + HOCl increased the fluorescence polarization (FP) and microvis-cosity significantly by 1.7-fold, 1.8-fold, and 1.7-fold, respectively, as compared to an only 1.2– fold increase in FP by O₂^?_? O₂^?_? did not alter the fatty acid profiles of the membrane phospholipids. However, HO' and H₂O₂ reduced the arachidonic acid contents in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). These radicals also stimulated the lipid peroxidation by several-fold, while that by O₂^?_? was only insignificant. These results suggest that HO' and H₂O₂ decreased the membrane fluidity and induced lipid peroxidation by releasing the arachidonic acid from PC, PE. and PI.     

A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses

The generation of reactive oxygen species (ROS) in a live-cell system is routinely measured using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF). However, it is difficult to simultaneously monitor cellular oxidative responses and ROS generation in cells, and analyses of cellular oxidative responses are typically performed after ROS generation has been evaluated. In this study, we developed a modified fixed staining method that allows the simultaneous analysis of ROS generation and oxidative responses using standard immunostaining techniques. A microplate reader-based assay showed that of the fixatives tested, only methanol did not alter the hydrogen peroxide (H₂O₂)-mediated oxidation of the responsive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), a chloromethyl derivative of H₂DCFDA, or the fluorescence of oxidized DCF in vitro. Further in vivo assays using flow cytometry showed that both methanol and acetic acid maintained the fluorescence of oxidized DCF in H₂O₂-, antimycin A-, and serum starvation-treated human lung adenocarcinoma A549 cells and human microvascular endothelial HMEC-1 cells. Following acetic acid-based fixation, the ROS generation in starved HMEC-1 cells could be evaluated by flow cytometric analysis while simultaneously monitoring the phosphorylation status of p38 mitogen-activated protein kinase. Immunostaining also revealed the synchronization of ROS generation and the H₂O₂-induced phosphorylation of Src homology-2 domain-containing phosphatase2. This study describes a modified method that may be used in future biomedical investigations to simultaneously measure intracellular ROS production and cellular oxidative responses.     

Formation of protein S-nitrosylation by reactive oxygen species

Abstract

In the present study, the formation of whole cellular S-nitrosylated proteins (protein-SNOs) by the reactive oxygen species (ROS), hydrogen peroxide (H₂O₂), and superoxide (O₂^??) is demonstrated. A spectrum of protein cysteine oxidative modifications was detected upon incubation of serum-starved mouse embryonic fibroblasts with increasing concentrations of exogenous H₂O₂, ranging from exclusive protein-SNOs at low concentrations to a mixture of protein-SNOs and other protein oxidation at higher concentrations to exclusively non-SNO protein oxidation at the highest concentrations of the oxidant used. Furthermore, formation of protein-SNOs was also detected upon inhibition of the antioxidant protein Cu/Zn superoxide dismutase that results in an increase in intracellular concentration of O₂^??. These results were further validated using the phosphatase and tensin homologue, PTEN, as a model of a protein sensitive to oxidative modifications. The formation of protein-SNOs by H₂O₂ and O₂^?? was prevented by the NO scavenger, c-PTIO, as well as the peroxinitrite decomposition catalyst, FETPPS, and correlated with the production or the consumption of nitric oxide (NO), respectively. These data suggest that the formation of protein-SNOs by H₂O₂ or O₂^?? requires the presence or the production of NO and involves the formation of the nitrosylating intermediate, peroxinitrite.     

Pycnogenol enhances endothelial cell antioxidant defenses

Summary

Reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide anion (O₂^?), and hydroxyl radical (OH^?) have been implicated in mediating various pathological events such as cancer, atherosclerosis, diabetes, ischemia, inflammatory diseases, and the aging process. The glutathione (GSH) redox cycle and antioxidant enzymes—superoxide dismutase (SOD) and catalase (CAT)—play an important role in scavenging ROS and preventing cell injury. Pycnogenol has been shown to protect endothelial cells against oxidant-induced injury. The present study determined the effects of pycnogenol on cellular metabolism of H₂O₂ and O₂^? and on glutathione-dependent and -independent antioxidant enzymes in bovine pulmonary artery endothelial cells (PAEC). Confluent monolayers of PAEC were incubated with pycnogenol, and oxidative stress was triggered by hypoxanthine and xanthine oxidase or H₂O₂. Pycnogenol caused a concentration-dependent enhancement of H₂O₂ and O₂^? clearance. It increased the intracellular GSH content and the activities of GSH peroxidase and GSH disulfide reductase. It also increased the activities of SOD and CAT. The results suggest that pycnogenol promotes a protective antioxidant state by upregulating important enzymatic and nonenzymatic oxidant scavenging systems.     

Cadmium and zinc-mediated oxidative burst in tobacco BY-2 cell suspension cultures

Generation of reactive oxygen species (ROS) constitutes an important first reaction under many stress conditions in plants. We demonstrate that Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells in suspension cultures, generate superoxide radical and hydrogen peroxide upon treatment with cadmium and zinc. Addition of catalase and N,N-diethyldithiocarbamate (DDC) decreased the level of H₂O₂, whereas superoxide dismutase (SOD) induced a slight increase of the H₂O₂ production. The effects of catalase, DDC and SOD on the heavy metal-induced ROS production indicate that it occurs outside of the cells, and that at least part of the hydrogen peroxide is produced by dismutation of the superoxide radical (O₂ ^·−). The effect of pretreatment of the cell cultures with commonly used mammalian NADPH oxidase inhibitors was also tested. Strong inhibitions of cadmium and zinc-mediated ROS production were obtained with the flavoprotein inhibitors—diphenylene iodonium (DPI) and quinacrine and with an inhibitor of b-type cytochromes—imidazol. Membrane permeable-N-ethyl maleimide (NEM) and iodoacetate, and membrane non-permeable thiol reagents—para-chloromercuribenzoic acid (pCMBS) also inhibited the ROS production. These results suggested that the enzyme responsible for cadmium and zinc-induced ROS production in tobacco cells contains a flavocytochrome. They also show the importance of intra- and extracellular thiol groups in the observed stress reaction. The induction of ROS production with heavy metals showed properties comparable to the elicitor-induced oxidative burst in other plant cells.     

A simple boronic acid-based fluorescent probe for selective detection of hydrogen peroxide in solutions and living cells

An approach of high sensitivity and selectivity for hydrogen peroxide (H₂O₂) detection is highly demanded due to its important roles in regulating diverse biological process. In this work, we introduced an easily synthesized fluorescent “turn off” probe, BNBD. It is designed based on the core structure of 4-chloro-7-nitrobenzofurazan as a fluorophore and incorporated with a specific H₂O₂-reactive group, aryl boronate, for sensitive and selective detection of H₂O₂. We demonstrated its selectivity by incubating the probe with other types of ROS, and measured the limit of detection of BNBD as 1.8 nM. BNBD is also conducive to H₂O₂ detection at physiological conditions. We thus applied it to detect both exogenous and endogenous changes of H₂O₂ in living cells by confocal microscopy, supporting its future applications to selectively monitor H₂O₂ levels and identify H₂O₂-related physiological or pathological responses from live cells or tissues in the near future.     

Autophagy protects ovarian cancer-associated fibroblasts against oxidative stress

RNA-Seq and gene set enrichment anylysis revealed that ovarian cancer associated fibroblasts (CAFs) are mitotically active compared with normal fibroblasts (NFs). Cellular senescence is observed in CAFs treated with H₂O₂ as shown by elevated SA-β-gal activity and p21 (WAF1/Cip1) protein levels. Reactive oxygen species (ROS) production and p21 (WAF1/Cip1) elevation may account for H₂O₂-induced CAFs cell cycle arrest in S phase. Blockage of autophagy can increase ROS production in CAFs, leading to cell cycle arrest in S phase, cell proliferation inhibition and enhanced sensitivity to H₂O₂-induced cell death. ROS scavenger NAC can reduce ROS production and thus restore cell viability. Lactate dehydrogenase A (LDHA), monocarboxylic acid transporter 4 (MCT4) and superoxide dismutase 2 (SOD2) were up-regulated in CAFs compared with NFs. There was relatively high lactate content in CAFs than in NFs. Blockage of autophagy decreased LDHA, MCT4 and SOD2 protein levels in CAFs that might enhance ROS production. Blockage of autophagy can sensitize CAFs to chemotherapeutic drug cisplatin, implicating that autophagy might possess clinical utility as an attractive target for ovarian cancer treatment in the future.     

T cell activation induces CuZn superoxide dismutase (SOD)-1 intracellular re-localization,production and secretion

Reactive oxygen species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H₂O₂), more stable and able to freely diffuse through cell membranes. Copper–zinc superoxide dismutase (CuZn-SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H₂O₂ and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H₂O₂ generation are involved in T cell receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the “non-T” cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors.     

Specific aquaporins facilitate Nox-produced hydrogen peroxide transport through plasma membrane in leukaemia cells

In the last decade, the generation and the role of reactive oxygen species (ROS), particularly hydrogen peroxide, in cell signalling transduction pathways have been intensively studied, and it is now clear that an increase of ROS level affects cellular growth and proliferation pathways related to cancer development. Hydrogen peroxide (H₂O₂) has been long thought to permeate biological membranes by simple diffusion since recent evidence challenged this notion disclosing the role of aquaporin water channels (AQP) in mediating H₂O₂ transport across plasma membranes. We previously demonstrated that NAD(P)H oxidase (Nox)-generated ROS sustain glucose uptake and cellular proliferation in leukaemia cells. The aim of this study was to assess whether specific AQP isoforms can channel Nox-produced H₂O₂ across the plasma membrane of leukaemia cells affecting downstream pathways linked to cell proliferation. In this work, we demonstrate that AQP inhibition caused a decrease in intracellular ROS accumulation in leukaemia cells both when H₂O₂ was produced by Nox enzymes and when it was exogenously added. Furthermore, AQP8 overexpression or silencing resulted to modulate VEGF capacity of triggering an H₂O₂ intracellular level increase or decrease, respectively. Finally, we report that AQP8 is capable of increasing H₂O₂-induced phosphorylation of both PI3K and p38 MAPK and that AQP8 expression affected positively cell proliferation. Taken together, the results here reported indicate that AQP8 is able to modulate H₂O₂ transport through the plasma membrane affecting redox signalling linked to leukaemia cell proliferation.