H202 Production Research Articles

The air-breathing Alaska blackfish (Dallia pectoralis) suppresses brain mitochondrial reactive oxygen species to survive cold hypoxic winters

The Alaska blackfish (Dallia pectoralis) is the only air-breathing fish in the Arctic. In the summer, a modified esophagus allows the fish to extract oxygen from the air, but this behavior is not possible in the winter because of ice and snow cover. The lack of oxygen (hypoxia) and near freezing temperatures in winter is expected to severely compromise metabolism, and yet remarkably, overwintering Alaska blackfish remain active. To maintain energy balance in the brain and limit the accumulation of reactive oxygen species (ROS), we hypothesized that cold hypoxic conditions would trigger brain mitochondrial remodeling in the Alaska blackfish. To address this hypothesis, fish were acclimated to warm (15 °C) normoxia, cold (5 °C) normoxia or cold hypoxia (5 °C, 2.1–4.2 kPa; no air access) for 5–8 weeks. Mitochondrial respiration, ADP affinity and H202 production were measured at 10 °C in isolated brain homogenates with an Oroboros respirometer. Cold acclimation and chronic hypoxia had no effects on mitochondrial aerobic capacity or ADP affinity. However, cold acclimation in normoxia led to a suppression of brain mitochondrial H202 production, which persisted and became more pronounced in the cold hypoxic fish. Overall, our study suggests cold acclimation supresses ROS production in Alaska blackfish, which may protect the fish from oxidative stress when oxygen becomes limited during winter.

Sex-dependent effects of developmental hypoxia on cardiac mitochondria from adult murine offspring

Insufficient oxygen supply (hypoxia) during fetal and embryonic development can lead to latent phenotypical changes in the adult cardiovascular system, including altered cardiac function and increased susceptibility to ischemia reperfusion injury. While the cellular mechanisms underlying this phenomenon are largely unknown, several studies have pointed towards metabolic disturbances in the heart of offspring from hypoxic pregnancies. To this end, we investigated mitochondrial function in the offspring of a mouse model of prenatal hypoxia. Pregnant C57 mice were subjected to either normoxia (21%) or hypoxia (14%) during gestational days 6–18. Offspring were reared in normoxia for up to 8 months and mitochondrial biology was assessed with electron microscopy (ultrastructure), spectrophotometry (enzymatic activity of electron transport chain complexes), microrespirometry (oxidative phosphorylation and H202 production) and Western Blot (protein expression). Our data showed that male adult offspring from hypoxic pregnancies possessed mitochondria with increased H202 production and lower respiratory capacity that was associated with reduced protein expression of complex I, II and IV. In contrast, females from hypoxic pregnancies had a higher respiratory capacity and lower H202 production that was associated with increased enzymatic activity of complex IV. From these results, we speculate that early exposure to hypoxia has long term, sex-dependent effects on cardiac metabolic function, which may have implications for cardiovascular health and disease in adulthood.

Percutaneous transluminal renal angioplasty attenuates poststenotic kidney mitochondrial damage in pigs with renal artery stenosis and metabolic syndrome.

Percutaneous transluminal renal angioplasty (PTRA) has been used to treat renovascular disease (RVD), a chronic condition characterized by renal ischemia and metabolic abnormalities. Mitochondrial injury has been implicated as a central pathogenic mechanism in RVD, but whether it can be reversed by PTRA remains uncertain. We hypothesized that PTRA attenuates mitochondrial damage, renal injury, and dysfunction in pigs with coexisting renal artery stenosis (RAS) and metabolic syndrome (MetS). Four groups of pigs (n = 6 each) were studied after 16 weeks of diet-induced MetS and RAS (MetS + RAS), MetS + RAS treated 4 weeks earlier with PTRA, and Lean and MetS Sham controls. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multidetector computed tomography, and renal tubular mitochondrial structure and function and renal injury ex vivo. PTRA successfully restored renal artery patency, but mean arterial pressure remained unchanged. Stenotic kidney RBF and GFR, which fell in MetS + RAS compared to MetS, rose after PTRA. PTRA attenuated MetS + RAS-induced mitochondrial structural abnormalities in tubular cells and peritubular capillary endothelial cells, decreased mitochondrial H2 02 production, and increased renal cytochrome-c oxidase-IV activity and ATP production. PTRA also improved cortical microvascular and peritubular capillary density and ameliorated tubular injury and tubulointerstitial fibrosis in the poststenotic kidney. Importantly, renal mitochondrial damage correlated with poststenotic injury and dysfunction. Renal revascularization attenuated mitochondrial injury and improved renal hemodynamics and function in swine poststenotic kidneys. This study suggests a novel mechanism by which PTRA might be relatively effective in ameliorating mitochondrial damage and improving renal function in coexisting MetS and RAS.

Aquaporin transport of hydrogen peroxide in skeletal muscle

In addition to the established intracellular production of H202 by skeletal muscle it is also produced extracellularly and studies have shown that H202 levels contribute to the pathophysiology of the age‐related loss of skeletal muscle mass and function [1, 2]. Furthermore, externally applied H2O2, inhibits skeletal neurotransmission by an undefined intracellular mechanism [3]. Despite considerable advancement of our understanding of the sources, sinks and functions of H202 within skeletal muscle fibres, there have been no previous reports of how H202 is transported into skeletal muscle fibres. Recent evidence in other systems have implicated aquaporin (AQP) channels in this role. These are a family of integral membrane proteins that are often described as “diffusion facilitators” and have been shown to aid the movement of H202 across membranes in yeast, plants and some mammalian cells [3]. In this study we investigated whether AQP are also a conduit for H2O2 in muscle.To measure unbiased AQP transcript abundance we used RNAseq (150bp‐read depth >280 M clusters per lane, n=20 subjects) on muscles from young and old mice (approx. 6 and 26 months) both control and peroneal nerve crushed TA muscle. To measure H2O2 permeability, we used an AAV6‐HyPer2 transfection method that allows measurement of intracellular H202 in response to extracellular H2O2 exposure. We also used the well‐established swell assay to assess H20 permeability. The peroneal crush model was used to induce motor unit turnover as seen in ageing. RNAseq studies used TA muscle, but for in vitro assays FDB preparations were used as they are more amenable to isolation of single fibres. Primary muscle fibres were isolated from FDB of young adult mice and once dissociated, plated onto matrigel coated dishes to enable permeability assays to be carried out.RNA deep seq studies identified significant expression of 8 AQP mRNA transcripts present within murine skeletal muscle. A decrease of AQP4 and AQP1, but a significant increase in AQP3 was detected with ageing. To verify functional expression of AQP's within the muscles we measured AQP permeability with a standard swell assay [4, 5]. Challenging FDB fibres with a low osmolality solution (80mOSm) led to volume increase that was inhibited by 5 mins pre‐treatment with the established AQP blocker HgCl2 and the selective AQP1 blocker AQP1b. Similarly, extracellular H2O2, applied at physiological levels (10uM), led to an increase in intracellular H2O2 levels, recorded in terms of AAV6‐Hyper2 signal intensity. This was significantly inhibited by 5 mins pre‐treatment with AQP blockers HgCl2 (30μM), TEA (3mM) and Bumetanide (5μM).These data clearly demonstrate, for the first time, that AQPs contribute to H2O2 membrane transport within skeletal muscle. Future work aims to examine changes of these transport mechanisms with age and injury using specific AQP shRNA and the role of AQP isoforms in neuromuscular transmission.Support or Funding InformationWork funded by British MRCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial targeted peptides preserve mitochondrial organization and decrease reversible myocardial changes in early swine metabolic syndrome.

The mechanisms responsible for cardiac damage in the early stages of metabolic syndrome (MetS) remain unknown. Mitochondria are intimately associated with cellular myofibrils, with the cytoskeleton functioning as a linkage coordinator, and closely associated to the calcium release sites of the sarcoplasmic reticulum (SR). We hypothesized that early MetS is characterized by mitochondria-related myocardial damage, associated with altered cytoskeletal-mitochondria-SR interaction. Domestic pigs were studied after 16 weeks of diet-induced MetS, MetS treated for the last 4 weeks with the mitochondrial-targeted peptide elamipretide (ELAM; 0.1 mg/kg SC q.d), or Lean controls (n = 6/group). Cardiac remodeling and function were assessed by fast comuted tomography. Myocardial mitochondrial structure, SR-mitochondria interaction, calcium handling, cytoskeletal proteins, oxidative stress, and apoptosis were studied ex-vivo. MetS pigs developed hyperlipidemia, hypertension, and insulin resistance, yet cardiac function was preserved. MetS-induced mitochondrial disorganization, decreased (C18:2)4 cardiolipin, disrupted ATP/ADP balance, and decreased cytochrome-c oxidase (COX)-IV activity. MetS also increased mitochondrial hydrogen peroxide (H2O2) production, decreased nicotinamide adenine dinucleotide phosphate (NADPH)/NADP and GSH/GSSG, and decreased myocardial desmin and β2 tubulin immunoreactivity, and impaired SR-mitochondrial interaction and mitochondrial calcium handling, eliciting myocardial oxidative stress and apoptosis. ELAM improved mitochondrial organization and cardiolipin species profile, restored ATP/ADP ratio and COX-IV activity, decreased H202 production, and improved generation of NADPH and GSH. ELAM also improved cytoskeletal-mitochondria-SR interaction and mitochondrial calcium handling, attenuating oxidative stress, and apoptosis. Disorganization of cardiomyocyte cytoskeletal-mitochondria-SR network is associated with cardiac reversible changes in early MetS, preceding overt cardiac dysfunction. These findings may introduce novel therapeutic targets for blunting cardiac damage in early MetS.

The effect of antibiotics on the mitochondrial function in rat skeletal muscle

BackgroundMitochondria are the main energy producing organelle of the cell through oxidative phosphorylation (OXPHOS) and the electron transport system, and major sources of reactive oxygen species (ROS) result from superoxide (O2−) and hydrogen peroxide (H2O2). Accumulation of ROS has the potential to harm macromolecules such as DNA and RNA. How antibiotics contribute to oxidative damage in cells have not been sufficiently investigated, although Kohanski et al. (1) have proposed a mechanism of how bactericidal antibiotics increases O2− production causing an excessive production of hydroxyl radical (OH−) via the Fenton reaction. This study has therefore investigated the effect of clarithromycin, trimethoprim and penicillin on the mitochondrial respiration and H202 production in rat skeletal muscle to see if our findings correlate with Kohanski's proposal.MethodAn approach combining high‐resolution respirometry and fluorometric measurement of H2O2 production by the Oroboros Oxygraph 2K instrument was used. Skeletal muscle tissue from mice (C57BL/6JRJ) was prepared and permeabilized using saponin stock solution. A stepwise multiple substrate‐uncoupler‐inhibitor titration (SUIT) protocol was used to analyze the oxidative phosphorylation, the mitochondrial respiratory control, and H2O2 production. The drug concentrations of antibiotics were equivalent to those from clinical use. 6 muscle samples were exposed to each antibiotic and 6 were used as control. The software Datlab was used to calculate mitochondrial oxygen flux per mass [pmol/(s*mg)] and the H2O2 production using a fluorescent probe.ResultsOur findings showed that clinical doses of clarithromycin increased the mitochondrial respiration by 3.62 pmol, SD= ±4.71 compared to control which showed a decrease in respiration by −0.84 pmol, SD= ±1.16 (p‐value = 0.063). There were not observed any significant alterations in H202 production with the addition of clarithromycin as the H2O2 production increased by 0.52 pmol, SD= ±0.75 compared to control 0.011 pmol, SD= ±0.037 (p‐value = 0.164). Trimethoprim and penicillin on the other hand caused a decrease in mitochondrial respiration by −1.11 pmol SD= ±3.01 and −4.65 pmol SD= ±25.09 compared to their respective control −0.46 pmol SD= ±1.27 and 3.46 pmol SD= ±5.38, (p‐value = 0.706). No significant findings were observed with the H2O2 production.ConclusionOur findings suggest that clarithromycin possibly increases the mitochondrial respiration and leads to an increased production of O2−. Clarithromycin did increase the H202 production slightly. This study does not finally elucidate that antibiotics contribute to the formation of ROS, but can on the other hand assist the further understanding of drugs and mitochondrial dysfunction.Support or Funding InformationEdon Morina1, Vanja Cejvanovic1, Laura Kofoed Kjær1, Henrik Enghusen Poulsen1, Søren Nielsen21Q7642, Department of Clinical Pharmacology, Bispebjerg Hospital; 2Department of Health Science and Technology Aalborg University

0420: Acute exercise preconditioning induced mitochondrial protection against skeletal muscle ischemia–reperfusion injury

Ischemia-reperfusion (I-R) induces mitochondrial function impairments and oxidative stress in skeletal muscle. In cardiac muscle, one-to-several days of exercise attenuate I-R injury. The objective of the present study was to show that a single exercise (EXO) prior to the I-R period protects the hind limb mitochondrial function. Twenty mice were randomly divided into 2 groups: The control group (CTL I-R, n=10) underwent I-R without exercise and the exercise group (EXO I-R, n=10) ran before I-R. Mice underwent 2 hours of ischemia induced by a rubber band tourniquet applied on the right root of the hind limb (the left leg was used as control leg), followed by a reperfusion period of 2 h. The EXO was performed on a treadmill with 10% incline 1h before I-R. The mice ran at a speed of 30cm until 40cm/sec during 30min. Blood lactate obtained from the tip of the tail at the end of EXO was above 9 mmol/L. In skinned gastrocnemius fibers, I-R significantly decreased the maximal mitochondrial respiration (Vmax) of the ischemic leg in comparison to the control leg in the CTL group (23.6 vs 28.6 pmol/(sec*mg wet weight); p<0.05). EXO I-R mice increased Vmax in comparison to CTL I-R mice (39.5 vs 31.8 pmol/(sec*dw) in the ischemic leg and the control leg respectively; p<0.05)). After EXO, the impairment between ischemic leg and control leg was partially restored (p=0.22). Without preconditioning, I-R increased H202 production of the ischemic leg in comparison to the control leg (42.6 vs 37.8 umol/mg dw; p=0.23). EXO I-R mice significantly decreased the H202 production in comparison to CTL I-R mice (26.6 vs 42.6 umol/mg dw; p<0.01) in the ischemic leg. After EXO, the impairment between ischemic leg and control leg was completely abolished (p=0.8). These results show that a preconditioning exercise protect the maximal mitochondrial respiration in mice and reduce the mitochondrial production of ROS following a period of I-R.

Sand fly saliva: effects on host immune response and Leishmania transmission

The feeding success of sand flies (Diptera: Phlebotominae) is linked to the vast array of pharmacological substances in their saliva, which interferes with the host haemostasis and immune response. Modification of feeding site plays also an important role in Leishmania transmission. In naive hosts, co-inoculation of saliva and Leishmania parasites increases the chance of successful transmission. Disease exacerbation seems to be associated with enhanced production of type 2 cytokines and selective inhibition of some macrophage functions including the production of NO and H202. On the other hand, hosts repeatedly exposed to sand fly bites develop anti-saliva immune response that results in a protection against Leishmania infection. This led to a new interesting approach to anti-Leishmania vaccine--using salivary components to block parasite transmission. The review is therefore focused on the interactions that run between immunomodulatory molecules in sand fly saliva and host immune response, with the impact on Leishmania infection development. Recent studies revealed that saliva-based vaccine for leishmaniasis might be effective and feasible, however, several questions still require to be solved. The knowledge based on experimental mouse model cannot be fully extrapolated to dogs or humans and due to differences in salivary antigens between sand fly species the protective effect is species-specific. On the other hand, the specificity of salivary antigens enables the use of anti-saliva antibodies for monitoring the exposure of hosts to sand fly bites and might be used as a marker of risks for Leishmania transmission in endemic areas.

Antioxidant enzymes, hydrogen peroxide metabolism, and respiration in rat heart during experimental hyperammonemia

The effects of toxic ammonia doses on H2O2 metabolism, energy metabolism, and antioxidant enzyme activities in rat heart were studied. Ammonium acetate administration to animals proved to increase total superoxide dismutase (SOD), catalase, and glutathione peroxidase activities in the heart cytoplasmic fraction as well as Mn-SOD, catalase, and glutathione reductase in heart mitochondria. Conversely, ammonia inhibited the same activities in the brain, liver, and erythrocytes. Hyperammonemia had no effect on the levels of ATP, ADP and total adenine nucleotides in the heart but decreased them in the brain. Ammonia impaired oxidative phosphorylation and increased the rate of H202 production in heart and brain mitochondria. The ammonia concentration inhibiting antioxidant enzymes in the liver and brain can be insufficient for such effect in the heart.

Effects of endurance training on the production of reactive oxygen species in rat muscle and liver isolated mitochondria.

Mitochondria is one of the major sites of reactive oxygen species (ROS) production but little is known about the effects of chronic exercise on this function. We measured liver and skeletal muscle mitochondrial oxygen consumption (JO2) and H2O2 production after a 6-week treadmill endurance training in rats. Substrates and inhibitors of the complexes of the respiratory chain were used to characterize the sites of alterations. Results show that endurance training increased cytochrome C oxydase activity in liver and muscle mitochondria (+18% and +20% respectively) and increased both citrate synthase and JO2 in liver and muscle. H202 production in non-phosphorylating condition by liver mitochondria increased with training when succinate (S), glutamate+malate+succinate (GMS), or octanoyl-carnitine (OC) were used as substrates (+57%, +42%, +22%, respectively) but remained unchanged with glutamate+malate (GM) or palmitoyl-carnitine (PC) as substrates. H202 production in ADP-stimulated condition by liver mitochondria was decreased with training when GM or GMS were used as substrates (−88% and −44%, respectively). H202 production by muscle mitochondria remained unchanged with training except when GMS were used as substrates (−30%). These results suggest that endurance training alters mitochondrial ROS production in liver that appear specific to changes on the complex II of the respiratory chain but has limited effects on mitochondrial ROS production in skeletal muscle.

Interleukin-1β (IL-1β) induced H2O2 production contributes to the motor dysfunction in human ulcerative colitis (UC)

We have shown that neurokmin-A (NKA) is an important excitatory neumtransmitter in normal human sig~mid colonic circular muscle and that NKA-induced contraction is mediated by release of calcium from intracelhdar stores. We have also shm~m that NKA induced contraction of sigmoid circular muscle and its intracellular Ca 2+ stores are reduced in ulcerative colitis and that H2Oa may contribute to the obsep,'ed changes in the response to NKA. In this study we examined the role of 1L-lf~ in the motor dysfunction in UC and in H202 production. IL-I~ content was significantly higher(ll.2+_4.7pgkng protein) m the circular muscle layer of UC patients than in nom~al citvular muscle (0.7 _+ O. lpg/mg protein). In circular muscle cells isolated by enzymatic digestion exposure to IL-I~ (100tdml, 23 h) significantly reduced NKA and thapsigargin-induced contraction. Because NKA and thapsigargin cause release of Ca ~ + from intracellular stores, the data suggest that 1L1 ~ may reduce intracellular Ca > stores. Tbe reduced contractions were partially reversed by the H20~ scavenger catalase (87u/ml, lhr), suggesnng that the motor dyshmction ts partially mediated by production of H~O> in fura 2 loaded cells, 20 mM caffeine, which selectively releases Ca ~ + from intracellular stores, caused a 630.+_ 62 nM increase in intracellular Ca ~ + in normal cells and 324+ 71riM in UC cells, confirming that intracellular Ca > stores are reduced in UC. 1L-II3 treatment similarly reduced the caffeine-induced Ca > signal (432 _+ 69 riM) and this reduction was partially restored by catalase (520+-78 riM), confirming that IL-l[3 reduces intraceflular Ca > stores by producing H~O_,. H20~ levels in circular smooth muscle of UC patients were higher (026 +-O.06nmd/mg protein) than in normal sigmoid (0.05-+ 0,01nmol/ng protein). In normal sigmoid circular muscle 1L-113 treatment (200u/ ml, 2hr) significantly increased the production of HaO~ (from O. 15 +0.02 to 0.3 -+ 0.05nmol/ mg protein). The increase was signihcantly mhihited by the MEK inhibitor PD98059 and by the cyIosohc phosphofipase A~ (cPLa,~) inhibitor AACOCF3, but not by' the p38 MAP kin_ase inhibitor SB203580 or by the NF-KB inhibitor D'rrolidine dithiocarbamate, suggesting that lL-I~-induced production of H~O~ depends on activation of ERK1/ERK2 MAP kinase and cPLA> We conclude that IL-l[3 can mimic the changes observed ni ulceratwe colitis and may contribute to the motor dysfunction in UC by producing H~O~. Supported by Litespan Research Funds.

Modulation of tnf-α-induced icam-1 expression, no and h202 production by alginate, allicin and ascorbic acid in human endothelial cells

Plant nutrients are believed to provide protection against various diseases including inflammation. Since interactions of the cell adhesion molecules are known to play important roles in mediating inflammation, inhibiting adhesion protein upregulation is a possible therapeutic target. In this study, the interacellular adhesion molecule-1 (ICAM-1) was induced in human umbilical endothelial cells (HUVECs) after stimulation with TNF-alpha. In addition, alginate, ascorbic acid and allicin were demonstrated to inhibit the TNF-alpha induced expression of ICAM-1 on the HUVECs in a dose-dependent manner. These compounds also inhibited the production of NO and H2O2 induced by TNF-alpha, which suggests that the inhibition of ICAM-1 expression by the three compounds may be due to the modulated production of the reactive oxygen/nitrogen components. Overall, these results indicate that these dietary components have a therapeutic potential in the treatment of various inflammatory disorders associated with an increase in endothelial leukocyte adhesion molecules.

Factors Affecting the Ascorbate- and Phenolic-dependent Generation of Hydrogen Peroxide in Dulbecco's Modified Eagles Medium

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H₂0₂), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H₂0₂ production. H₂0₂ gene ration from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% C0₂ was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% C0₂. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H₂0₂ generated by ascorbate and phenolic compounds, but there was still substantial H₂0₂ generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H₂0₂ generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H₂0₂ generation is discussed.

Differentiation and Hydrogen Peroxide Production in Sclerotium rolfsii Are Induced by the Oxidizing Growth Factors, Light and Iron

Hydrogen peroxide is produced by Sclero- tium rolfsii during sclerotial differentiation, in re- sponse to oxidative growth conditions of light and Fe. An additive effect of (? light ? Fe)-induced oxida- tive stress is observed on H202 production levels and on sclerotia numbers during fungal development. High H202 production rates are correlated with high oxidative stress and are observed in less lipid-perox- idized young mycelia and sclerotia (sclerotia initials and early developing sclerotia) and not in differen- tiated, highly lipid-peroxidized older mycelia and sclerotia (developed and mature sclerotia). H202 production by undifferentiated mycelia and their ex- posure to exogenous H202 show a rate/dose-depen- dent correlation with degree of sclerotial differenti- ation (sclerotia numbers). Hydrogen peroxide and lipid peroxide concentration gradients are observed in fungal colonies during their transition from un- differentiated (mycelial) to differentiated (sclerotial) stage. Growth-stage-dependent changes in H202 pro- duction rates during development are not due to changes in glucose concentration and pH of the growth medium. Light/Fe-induced H202 production and its effect on S. rolfsii differentiation, are dis- cussed in the context of our hypothesis of oxidative stress-induced sclerotial differentiation.

Characterization of the Protective Effect of Tetrahydrobiopterin Against S-Nitroso-N -Acetyl-D ,L-Penicillamine Induced Endothelial Cell Injury

The purpose of this study was to characterize the protective effect of tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, against NO-induced endothelial cell injury. The addition of S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, to endothelial cells induced the release of lactate dehydrogenase (LDH), a marker for cell injury. The SNAP-induced endothelial cell injury was markedly reduced by pretreatment with sepiapterin, a precursor of BH4 synthesis. On the other hand, exogenous BH4 had little effect on the SNAP-induced endothelial cell injury. We recently found that NO-induced endothelial cell injury involves a part of H202 production, since the injury was blocked by the treatment with catalase. Although BH4 released reactive oxygen species (ROS) in cell-free conditions, the increase in intracellular BH4 by pretreatment with sepiapterin strongly reduced H202-induced intracellular oxidative stress. These findings suggest that the increase in intracellular BH4 content but not extracellular BH4, strongly attenuates NO-induced endothelial cell injury by at least one of the mechanisms by which BH4 reduces H202-induced oxidative stress. Intracellular BH4 seems mainly to play a role as an antioxidant or as a ROS-scavenger.

REACTIVE OXYGEN SPECIES DO NOT CAUSE ARSINE-INDUCED HEMOGLOBIN DAMAGE

Previous work suggested that arsine- (AsH3-) induced hemoglobin (Hb02) damage may lead to hemolysis (Hatlelid et al., 1996). The purpose of the work presented here was to determine whether reactive oxygen species are formed by AsH3 in solution, in hemoglobin solutions, or in intact red blood cells, and, if so, to determine whether these species are responsible for the observed hemoglobin damage. Hydrogen peroxide (H202) was detected in aqueous solutions containing AsH3 and Hb02 or AsH3 alone but not in intact red blood cells or lysates. Additionally, high-activity catalase (19,200 U/ml) or glutathione peroxidase (68 U/ml) added to solutions of Hb02 and AsH3 had only a minor protective effect against AsHyinduced damage. Further, the differences between the visible spectra of AsH3-treated Hb02 and H202-treated HbO2 indicate that two different degradative processes occur. The presence of superoxide anion (Of) was measured by O2 -dependent reduction of nitro blue tetrazolium (NBT). The results were negative for Of. Exogenous superoxide dismutase (100 ug/ml) did not affect AsH3-induced Hb02 spectral changes, nor did the hydroxyl radical scavengers, mannitol, and DMSO (20 mM each). The general antioxidants ascorbate (<10 mM) and glutathione (<1 mM) also had no effect. These results indicate that the superoxide anion and the hydroxyl radical COH) are not involved in the mechanism of AsH3-induced Hb02 damage. The results also indicate that although AsH3 contributes to H202 production in vitro, cellular defenses are adequate to detoxify the amount formed. An alternative mechanism by which an arsenic species is the hemolytic agent is proposed.

ミストチャンバーによる気相ヒドロペルオキシドの捕集

The new-type forest decline found in Europe and North America has been ascribed toatmospheric oxidants (Ref. 1-3). Organic hydroperoxides are important oxidants (Ref. 4), andits formations from ozone-natural hydrocarbon reactions were reported. To investigate theformation mechanism as well as to determine atmospheric concentration of hydroperoxides, a technique for efficient collection is necessary. We tested the applicability of mist chamberdeveloped by Cofer et al. (Ref. 7) to the collection of gaseous hydroperoxides. The mistchamber was found to have the advantage in following three points : 1) large flow rate, 2)collection efficiency as high as bubbling, and 3) less H202 production in the trapping solutionduring the sampling of ozone-containing air.

Interactions of Granulocyte-Macrophage Colony-Stimulating Factor (CSF), Granulocyte CSF, and Tumor Necrosis Factor a in the Priming of the Neutrophil Respiratory Burst

AbstractExposure of neutrophils to a range of cytokines augments their response to subsequent agonist-induced activation of the respiratory burst. We have examined the effects of several of these factors, both singly and in combination, on the priming of f-met-leu-phe (FMLP) and complement C5a-stimulated neutrophil H2O2 production, using a whole blood flow cytometric assay designed to minimize artefactual activation. Both granulocyte-macrophage colony-stimulat-ing factor (GM-CSF) and tumor necrosis factor a (TNFα) produced a similar degree of priming of the FMLP-stimulated burst in vitro (558% ± 86%, n = 41, and 581% ± 95%, n = 21, of the response seen with FMLP alone, respectively), but with markedly different kinetics (half-maximal response 20 minutes and 7 minutes, respectively). Preincubation with granulocyte colony-stimulating factor (G-CSF) alone caused only modest priming (202% ± 39%, n = 14). Priming with cytokine combinations of the FMLP-stimulated burst showed that the combinations of G-CSF and TNFα and GM-CSF and TNFα are highly synergistic, with recruitment of neutrophils unresponsive to priming by single agents. Priming with the combination of GM-CSF and G-CSF was not significantly different to priming with GM-CSF alone. Similar results were obtained using C5a as the respiratory burst stimulus. Significant priming of the FMLP-stimulated respiratory burst was seen in vivo in patients receiving an infusion of GM-CSF (332% ± 50% of preinfusion response to FMLP, P < .005, n = 8). Priming was also seen in patients receiving G-CSF (152% ± 58%, n = 5), although this did not reach conventional significance levels (.05 < P < .1). Although GM-CSF infusion caused priming in vivo, this was 48% less than predicted by preinfusion in vitro responses. This result was not due to inadequate GM-CSF levels as addition of further GM-CSF ex vivo did not correct the response. However, these neutrophils were still able to respond appropriately to ex vivo priming with TNFα, with a doubling in H202 production.© 1992 by The American Society of Hematology. 0006-497119217903-0031$3.0010

Effect of Hydrogen Peroxide on Preventing or Contributing to Polyspermic Fertilization in Sea Urchin: Modification by Tamoxifen and Macroglobulin.

The sea urchin has been described recently as “the most facile experimental system for studying molecular mechanisms of fertilization” (1). A major contributor to successful fertilization is the induction of hydrogen peroxide (H202), which is produced, with the participation of protein kinase C, by the oxidative burst caused by the sperm’s entry into the ovum. During the oxidative burst, NADPH reduces oxygen to superoxide anion radical (. O,J in a NADPH-oxidase catalyzed reaction, and Hz02 is formed by dismutation of . 02-. H202 could prevent entry of other sperms in two ways: conversion by myeloperoxidase to hypochlorite, which would kill excess sperms (2) that are ready to enter and fertilize the ova; and by hardening the cell envelope by a cross-linking reaction catalyzed by a peroxidase that couples the tyrosyl residues between proteins (1). The oxidative burst induced by the sperm’s entry has a mechanism similar to that induced by a tumor promoter, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in human neutrophils; i.e., both involve the production of H202. In contrast, many chemopreventive agents, including protease inhibitors (that are capable of inhibiting chymotrypsin) and tamoxifen (an anti-cancer agent), suppress the formation of H202 in TPA-treated human neutrophils

Hydrogen peroxide is an intermediate in the platelet activation cascade triggered by collagen, but not by thrombin

Human blood platelets produce oxidant species when stimulated by collagen and thrombin. The oxidative burst of platelets has been studied by cytofluorimetry taking advantage of the fluorogenic dye DCFH2-DA, which is taken up and deacetylated by platelets and then oxidized to the fluorescent derivative DCF. The oxidation of DCFH2 is induced by stimulation with collagen but not with thrombin and inhibited by external catalase. Catalase also inhibited the aggregation induced by collagen, but not that induced by thrombin. Aspirin and indomethacin inhibited the formation of the fluorochrome only when platelets were stimulated by thrombin. Externally added H202 increased the cytoplasmic calcium content as probed by the fluorescence of Indo-1. The present data suggest that collagen induces the production of H202, which in turn may stimulate the aggregation of platelets through a calcium mobilization. Instead the stimulation by thrombin does not require the intermediacy of H202.