Peroxide Release Research Articles

Melatonin inhibits peroxide production in plant mitochondria

The effect of melatonin on respiration and production (release) of hydrogen peroxide during succinate oxidation in mitochondria isolated from lupine cotyledons and epicotyls of pea seedlings was studied. It has been shown for the first time that melatonin (10-7-10-3 M) had a significant inhibitory effect on the production of peroxide by plant mitochondria, which was characterized by concentration dependence and species specificity. At the same time, melatonin (at a concentration of up to 100 microns) had virtually no effect on mitochondrial respiration rate and respiratory control coefficient. The results confirm the antioxidant function of melatonin and indicate that it is involved in the regulation of ROS levels and maintenance of redox balance in plant mitochondria.

Quercetin, But Not Dihydroquercetin (taxifolin), Attenuates the Release of Hydrogen Peroxide from Isolated Rat Heart and Brain Mitochondria

Quercetin, But Not Dihydroquercetin (taxifolin), Attenuates the Release of Hydrogen Peroxide from Isolated Rat Heart and Brain Mitochondria

Melatonin Inhibits Peroxide Production in Plant Mitochondria.

The effect of melatonin on respiration and production (release) of hydrogen peroxide during succinate oxidation in mitochondria isolated from lupine cotyledons and epicotyls of pea seedlings was studied. It was shown for the first time that melatonin (10-7-10-3 M) had a significant inhibitory effect on the production of peroxide by plant mitochondria, which was characterized by concentration dependence and species specificity. At the same time, melatonin (at a concentration of up to 100 μM) had virtually no effect on mitochondrial respiration rate and respiratory control coefficient. The results confirm the antioxidant function of melatonin and indicate that it is involved in the regulation of ROS levels and maintenance of redox balance in plant mitochondria.

The use of site-specific suppressors to measure the relative contributions of different mitochondrial sites to skeletal muscle superoxide and hydrogen peroxide production

Reactive oxygen species are important signaling molecules crucial for muscle differentiation and adaptation to exercise. However, their uncontrolled generation is associated with an array of pathological conditions. To identify and quantify the sources of superoxide and hydrogen peroxide in skeletal muscle we used site-specific suppressors (S1QELs, S3QELs and NADPH oxidase inhibitors). We measured the rates of hydrogen peroxide release from isolated rat muscle mitochondria incubated in media mimicking the cytosol of intact muscle. By measuring the extent of inhibition caused by the addition of different site-specific suppressors of mitochondrial superoxide/hydrogen peroxide production (S1QELs for site IQ and S3QELs for site IIIQo), we determined the contributions of these sites to the total signal. In media mimicking resting muscle, their contributions were each 12–18%, consistent with a previous method. In C2C12 myoblasts, site IQ contributed 12% of cellular hydrogen peroxide production and site IIIQo contributed about 30%. When C2C12 myoblasts were differentiated to myotubes, hydrogen peroxide release increased five-fold, and the proportional contribution of site IQ doubled. The use of S1QELs and S3QELs is a powerful new way to measure the relative contributions of different mitochondrial sites to muscle hydrogen peroxide production under different conditions. Our results show that mitochondrial sites IQ and IIIQo make a substantial contribution to superoxide/hydrogen peroxide production in muscle mitochondria and C2C12 myoblasts. The total hydrogen peroxide release rate and the relative contribution of site IQ both increase substantially upon differentiation to myotubes.

Development and Characterization of an Antimicrobial Polydopamine Coating for Conservation of Humpback Whales.

Migration patterns of humpback whales have been monitored using 316L stainless steel (SS) satellite telemetry tags. The potential for tissue infection and necrosis is increased if the bacteria, naturally a part of the diverse microbiome on the skin of humpback whales, can adhere to and colonize the surface of the tags. Polydopamine (pDA) has the potential to prevent the adhesion of one of the most prevalent bacterial strains on the surface of the skin of cetaceans (Psychrobacter) through the release of hydrogen peroxide. The release of hydrogen peroxide from the pDA coatings (40–100 μM) has the ability to induce a bacteriostatic response in E. coli, a commonly used bacteria strain in antimicrobial studies and potentially P. cryohalolentis, a common humpback associated bacteria. The H2O2 dose required to induce bacteriostatic conditions in E. coli is approximately 60 μM and in P. cryohalolentis is 100 μM. Bacterial adhesion on the surface of pDA coated SS coupons was measured first using E. coli. The coating successfully prevented adhesion of E. coli on the surface of SS coupons under certain conditions (60% reduction, p < 0.05) but the same was not seen with P. cryohalolentis. When coating conditions were altered (an increase in pH and temperature) the adhesion of P. cryohalolentis was reduced (80% reduction, p < 0.001). Overall, the pDA coatings have the capacity to generate varying amounts of hydrogen peroxide by altering the coating conditions and have the ability to reduce bacterial adhesion on the surface of satellite telemetry tags, and therefore the potential to increase tag functional service lifetime.

Redox responses in skeletal muscle following denervation

Previous studies have shown a significant increase in the mitochondrial generation of hydrogen peroxide (H2O2) and other peroxides in recently denervated muscle fibers. The mechanisms for generation of these peroxides and how the muscle responds to these peroxides are not fully established. The aim of this work was to determine the effect of denervation on the muscle content of proteins that may contribute to mitochondrial peroxide release and the muscle responses to this generation. Denervation of the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles in mice was achieved by surgical removal of a small section of the peroneal nerve prior to its entry into the muscle. An increase in mitochondrial peroxide generation has been observed from 7 days and sustained up to 21 days following denervation in the TA muscle fibers. This increased peroxide generation was reduced by incubation of skinned fibers with inhibitors of monoamine oxidases, NADPH oxidases or phospholipase A2 enzymes and the muscle content of these enzymes together with peroxiredoxin 6 were increased following denervation. Denervated muscle also showed significant adaptations in the content of several enzymes involved in the protection of cells against oxidative damage. Morphological analyses indicated a progressive significant loss of muscle mass in the TA muscle from 7 days up to 21 days following denervation due to fiber atrophy but without fiber loss. These results support the possibility that, at least initially, the increase in peroxide production may stimulate adaptations in an attempt to protect the muscle fibers, but that these processes are insufficient and the increased peroxide generation over the longer term may activate degenerative and atrophic processes in the denervated muscle fibers.

Hydrogen peroxide release by bacteria suppresses inflammasome-dependent innate immunity

Hydrogen peroxide (H2O2) has a major function in host-microbial interactions. Although most studies have focused on the endogenous H2O2 produced by immune cells to kill microbes, bacteria can also produce H2O2. How microbial H2O2 influences the dynamics of host-microbial interactions is unclear. Here we show that H2O2 released by Streptococcus pneumoniae inhibits inflammasomes, key components of the innate immune system, contributing to the pathogen colonization of the host. We also show that the oral commensal H2O2-producing bacteria Streptococcus oralis can block inflammasome activation. This study uncovers an unexpected role of H2O2 in immune suppression and demonstrates how, through this mechanism, bacteria might restrain the immune system to co-exist with the host.

Peroxide impact on the fate of veterinary drugs in fertilizers

The presence of veterinary medicines in organic manure causes soil contamination which contributes to increasing resistance of indigenous microflora to drugs and results in greater susceptibility of people to allergies. The main aim of the study was to assess the efficiency of inorganic peroxide mixtures (PM) with calcium peroxide content (CaO2) in the stabilization process of manure contaminated with antiparasitic agents: albendazole (ALB) and levamisole (LEV). As a solid, CaO2 is relatively stable against decomposition. In contact with water, however, it hydrolyzes with release of oxygen. The hydrolyzation of CaO2 proceeds very slowly in soil, which guarantees the constant release of hydrogen peroxide that subsequently becomes the source of free radicals (chemical oxidation) and oxygen (aerobic conditions for the microbes). It may contribute to continuous elimination of drugs from manure. The study has demonstrated that there were significant differences in ALB and LEV conversion stimulated by the PM addition. PM supplementation increased the drug availability (on average 15% and 25% increase in the initial concentration for ALB and for LEV, respectively), thereby increasing the initial rate of reaction. Elimination of ALB and LEV from the manure sorption complex is followed by Ca2+ saturation. The initial degradation rate was affected by PM for both drugs, but the mechanisms of decomposition have been modified only for ALB. The loss of ALB in the peroxide supplemented samples was 92%, and in the samples, without the PM, it did not exceed 61%. Loss of LEV was over 90% irrespective of PM supplementation.Graphic abstract

Oral Phenelzine Treatment Mitigates Metabolic Disturbances in Mice Fed a High-Fat Diet.

Novel mechanisms and health benefits have been recently suggested for the antidepressant drug phenelzine (PHE), known as a nonselective monoamine oxidase inhibitor. They include an antilipogenic action that could have an impact on excessive fat accumulation and obesity-related metabolic alterations. We evaluated the metabolic effects of an oral PHE treatment on mice fed a high-fat diet (HFD). Eleven-week-old male C57BL/6 mice were fed a HFD and either a 0.028% PHE solution (HFD + PHE) or water to drink for 11 weeks. PHE attenuated the increase in body weight and adiposity without affecting food consumption. Energy efficiency was lower in HFD + PHE mice. Lipid content was reduced in subcutaneous fat pads, liver, and skeletal muscle. In white adipose tissue (WAT), PHE reduced sterol regulatory element-binding protein-1c and phosphoenolpyruvate carboxykinase mRNA levels, inhibited amine-induced lipogenesis, and did not increase lipolysis. Moreover, HFD + PHE mice presented diminished levels of hydrogen peroxide release in subcutaneous WAT and reduced expression of leukocyte transmigration markers and proinflammatory cytokines in visceral WAT and liver. PHE reduced the circulating levels of glycerol, triacylglycerols, high-density lipoprotein cholesterol, and insulin. Insulin resistance was reduced, without affecting glucose levels and glucose tolerance. In contrast, PHE increased rectal temperature and slightly increased energy expenditure. The mitigation of HFD-induced metabolic disturbances points toward a promising role for PHE in obesity treatment and encourages further research on its mechanisms of action. SIGNIFICANCE STATEMENT: Phenelzine reduces body fat, markers of oxidative stress, inflammation, and insulin resistance in high-fat diet mice. Semicarbazide-sensitive amine oxidase, monoamine oxidase, phosphoenolpyruvate carboxykinase, and sterol regulatory element-binding protein-1c are involved in the metabolic effects of phenelzine. Phenelzine could be potentially used for the treatment of obesity-related complications.

Rituximab Treatment Modulates the Release of Hydrogen Peroxide and the Production of Proinflammatory Cytokines by Monocyte at the Onset of Type 1 Diabetes.

Monocytes are the main blood innate mononuclear phagocyte and one of the most important effector cells expressing Fcγ receptor, which is critical for the interaction with Fc domain of antibodies. To evaluate the effect of Rituximab (RTX, a chimeric human anti-CD20 monoclonal antibody) on the functional activities of Monocytes (MOs) at the onset of human Type 1 Diabetes (T1D). MOs were isolated from peripheral blood mononuclear cells (PBMCs) obtained from volunteer patients with recent-onset T1D and healthy control donors. The levels of the production of Interleukin 1β (IL-1β) and IL-6 were significantly increased in MOs from patients with T1D when compared to MOs from healthy controls (respectively, p < 0.01 and p < 0.05). Similarly, Interferon γ (IFN-γ), and intracellular free Calcium Ion (ifCa2+) levels were increased in T1D MOs than in control MOs, but the difference did not reach a significant level. Conversely, the production levels of IL-4 and catalase activity, as well as of both phagocytosis and killing capacities were decreased in MOs of T1D patients compared to MOs from healthy controls, but the difference was not significant for catalase activity and killing capacity (respectively, p < 0.01, p > 0.05, p < 0.01, and p > 0.05). Additionally, treatment with RTX significantly upregulated phagocytosis (p < 0.05), markedly downregulated the release of IL-1β (p < 0.01), ifCa2+, hydrogen peroxide (H2O2), and slightly downregulated the Nitric Oxide Synthase (NOS) activity, NOS activity-to-arginase activity ratio, the levels of Lactate Dehydrogenase (LDH)-based cytotoxicity, and the production of IL-6 and IFN-γ. Moreover, RTX treatment significantly upregulated the production of IL-4 (p < 0.05), IL-10 (p < 0.01) and the catalase activity (p < 0.05). Our study has shown for the first time that RTX can reverse the abnormal functional activities of MOs as well as their production of proinflammatory cytokines at the onset of T1D. From a therapeutic point of view, RTX may potentially be suggested at the beginning of T1D to immunomodulate innate immunity and inflammatory conditions.

Regulation of Staphylococcus aureus-induced CXCR1 expression via inhibition of receptor mobilization and receptor shedding during dual receptor (TNFR1 and IL-1R) neutralization.

Our earlier studies proposed a radically new idea suggesting interdependency between TNF-α/TNFR1 and IL-1β/IL-1R pathways in modulation of Staphylococcus aureus-induced CXCL8/CXCR1 axis. However, the effects of inhibition of cytokine receptor mobilization at intracellular level and surface TNFR1 and IL-1R shedding on S. aureus-induced CXCR1 expression have not been studied so far in peritoneal macrophages. This study aimed to investigate the role of inhibition of receptor mobilization from the intracellular pool (using brefeldin A) and surface receptor shedding (using TAPI-1) on CXCR1 expression during dual receptor (TNFR1 plus IL-1R) neutralization in peritoneal macrophages isolated from wild-type Swiss Albino mice. Release of superoxide anion, nitric oxide, and hydrogen peroxide was measured and cytokine production was done by ELISA. Expression of surface receptors (TNFR1, IL-1R, and CXCR1) and inflammatory mediators was studied by Western blot. It was observed that S. aureus-infected macrophages showed elevated ROS production, secretion of TNF-α, IL-1β, and CXCL8, along with increased expression of surface receptors (TNFR1, IL-1R, and CXCR1), and inflammatory markers (iNOS and COX-2) compared with control or treated groups (p < 0.05). However, prior treatment of macrophages with BFA or TAPI-1 in the presence of anti-TNFR1 antibody and IRAP during S. aureus infection showed significant reduction of all these parameters (p < 0.05). We can conclude that targeting of TNFR1 and IL-1R (with major focus on surface expression study) either through blockage of intracellular receptor trafficking pathway or via surface receptor shedding diminishes TNFR1/IL-1R interaction and consequently downregulates CXCR1 expression along with inflammatory signalling pathways during bacterial infections.

Sevoflurane Promotes Regeneration of the Endothelial Glycocalyx by Upregulating Sialyltransferase

BackgroundDuring ischemia-reperfusion injury, the endothelial glycocalyx is damaged by oxidative stress-induced release of hydrogen peroxide, leading to decreased endothelium-dependent vasodilation. The regenerative effects of sevoflurane on the endothelial glycocalyx and endothelium-dependent vasodilation in oxidative stress remain unclear. Sialic acid, which is a component of the glycocalyx, plays a key role in antioxidant activity and is catalyzed by the sialyltransferase, ST6Gal-I. We hypothesized that ST6Gal-I is involved in the sevoflurane-induced promotion of endothelial glycocalyx restoration and endothelium-dependent vasodilation after oxidative stress. Materials and methodsTo assess vasodilation, isometric tension in the rat aorta was measured. Aortic rings were treated with 0.5 mM hydrogen peroxide pre-exposure or post exposure to sevoflurane, with or without an ST6Gal-I inhibitor. The rings were then used for glycocalyx imaging using fluorescein isothiocyanate-labeled lectin staining and for immunohistochemistry for ST6Gal-I. ResultsVasodilation was significantly decreased by treatment with hydrogen peroxide compared to controls. Application of sevoflurane after treatment with hydrogen peroxide revived endothelium-dependent vasodilatation, whereas pretreatment with sevoflurane had no such effect. Sevoflurane after-treatment revived the intensity of fluorescence of the endothelial glycocalyx compared to the hydrogen peroxide group. However, pretreatment with sevoflurane had no such effect. Sevoflurane significantly upregulated the reduced expression of ST6Gal-I induced by hydrogen peroxide treatment. ConclusionsSevoflurane exerts regenerative effects on endothelium-dependent vasodilation and the endothelial glycocalyx following oxidative stress in the rat aorta.

Properties of a cryptic lysyl oxidase from haloarchaeon Haloterrigena turkmenica.

BackgroundLysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic.MethodsLOX open reading frame was cloned from Haloterrigena turkmenica in an E. coli expression vector. Recombinant Haloterrigena turkmenica lysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding. Amine oxidase activity has been measured fluorometrically as hydrogen peroxide release coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting.ResultsCultured H. turkmenica has no detectable amine oxidase activity. HTU-LOX may be expressed in E. coli with a high protein yield. The full-length protein gives no catalytic activity. For this reason, we hypothesized that the hydrophobic N-terminal region may interfere with proper folding and its removal may be beneficial. Indeed, truncated His-tagged HTU-LOX lacking the N-terminal hydrophobic signal peptide purified under denaturing conditions can be successfully refolded into an active enzyme, and a larger N-terminal truncation further increases the amine oxidase activity. Refolding is optimal in the presence of Cu2+ at pH 6.2 and is not sensitive to salt. HTU-LOX is sensitive to LOX inhibitor 3-aminopropionitrile. HTU-LOX deaminates usual substrates of mammalian LOX such as lysine-containing polypeptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed substrate specificity of the former. HTU-LOX readily oxidizes various primary amines including such compounds as taurine and glycine, benzylamine being a poor substrate. Of note, HTU-LOX is also active towards several aminoglycoside antibiotics and polymyxin. Western blotting indicates that epitopes for the anti-HTU-LOX polyclonal antibodies coincide with a high molecular weight protein in H. turkmenica cells.ConclusionH. turkmenica contains a lysyl oxidase gene that was heterologously expressed yielding an active recombinant enzyme with important biochemical features conserved between all known LOXes, for example, the sensitivity to 3-aminopropionitrile. However, the native function in the host appears to be cryptic.SignificanceThis is the first report on some properties of a lysyl oxidase from Archaea and an interesting example of evolution of enzymatic properties after hypothetical horizontal transfers between distant taxa.

Development of a Novel In Situ Activity Assay for Lysyl Oxidase Like‐2 (LOXL2)

Lysyl oxidase like‐2 (LOXL2) is a member of the lysyl oxidase (LOX) super family, which are copper‐dependent amine oxidases that initialize collagen and elastin cross‐linking to form a stable and organized matrix. Augmented LOXL2 expression is highly associated with diverse disease processes with a known element of matrix accumulation including fibrosis, pulmonary hypertension, and cancer metastasis. To date, lack of sensitive and specific assays that can detect LOXL2 catalytic function in situ in the extracellular matrix (ECM) of cells and tissue has limited the ability to fully elucidate the role of LOXL2 in pathobiological mechanisms.Current methods to detect LOXL2 enzymatic activity are inefficient and limited in scope. The most widely used assay is hydrogen peroxide based: oxidation of the LOX active site occurs when substrate is added to samples, and releases hydrogen peroxide. The level of hydrogen peroxide is detected using an HRP‐coupled system, that utilizes the conversion of Amplex Red to the fluorescent end product, resorufin. While the results are semi‐quantitative, this assay has limited dynamic range and cannot distinguish hydrogen peroxide arising from other pathways. Moreover, the assay requires LOXL2 in solution. The harsh conditions required to obtain a solubilized ECM sample, where the pertinent LOXL2 activity occurs, cause LOXL2 unfolding and loss of activity, rendering this assay unfeasible. Thus, there is a need to develop a sensitive, specific, and practicable in situ activity assay that overcomes these limitations.This study provides a proof of concept for a novel in situ activity assay for LOXL2. Here, we developed an assay to fluorescently label the products of LOXL2 catalysis. Specifically, LOXL2 catalyzes an oxidative de‐amination of lysine and hydroxylysine residues (found on collagen and elastin) to highly reactive semi‐aldehydes called allysines. In this assay, we labeled the newly generated allysine residues with biotin hydrazide using 1‐ethyl‐3‐(3‐dimethyl‐aminopropyl) carbodiimide hydrochloride (EDC) crosslinker. This biotinylation allows for a robust method of measuring LOXL2 activity through western blotting, immunoprecipitation and immunofluorescence imaging using biotin‐streptavidin interactions.Support or Funding InformationFunded by MedImmune research funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Mitochondrial Peroxide Release in the Mechanisms Underlying Age‐Related Sarcopenia

Age related loss of muscle mass and function (sarcopenia) is a major contributor to reduced quality of life in the elderly. Previous studies from our laboratory have shown that denervation induced alterations in muscle mitochondrial reactive oxygen species (ROS) generation is an integral player in this process. To specifically determine the role of mitochondrial function, generation of ROS and lipid peroxides in denervation atrophy, we utilized two transgenic mouse models with increased expression of the hydrogen peroxide scavengers catalase or peroxiredoxin 3, specifically in skeletal muscle. First, we asked whether these mitochondrial antioxidants can reduce denervation induced changes in muscle mitochondrial ROS production and reduce muscle atrophy in a surgical denervation model. Gastrocnemius mass is significantly reduced (~20%) at 7 days post‐sciatic nerve transection in wildtype, skmPrdx3 and skmMCAT mice, i.e., there was no protective effect of hydrogen peroxide scavengers on denervation atrophy. ROS production measured using the fluorescent probe Amplex Red was elevated in permeabilized muscle fibers and isolated mitochondria from denervated hindlimb 7 days post transection and neither Prdx3 or MCAT expression inhibits the signal. This data supports that the Amplex Red signal does not represent hydrogen peroxide alone, but includes the response from other lipid peroxide species and suggests that hydrogen peroxide scavenging is not sufficient to reduce denervation induced atrophy. Next, we asked whether Prdx3 muscle specific expression can modulate the sarcopenia phenotype in an established mouse model of accelerated sarcopenia, mice lacking CuZnSOD (Sod1KO/skmPrdx3Tg mice). ROS generation is significantly elevated in isolated muscle mitochondria from Sod1KO mice, and normalized by Prdx3 overexpression in the Sod1KO/skmPrdx3Tg mice. The oxygen consumption rate is reduced in skeletal muscle mitochondria isolated from Sod1KO muscle and restored by Prdx3 overexpression. We also found that in vitro contractile force generation was decreased by ~20% in Sod1KO mice, and the force reduction wwas rescued in Sod1KO/skmPrdx3Tg mice. Importantly, the muscle atrophy that occurs in the Sod1KO mice is fully restored by Prdx3 overexpression. This finding demonstrates a direct role of skeletal muscle‐specific mitochondrial H2O2 in promoting contractile abnormalities and muscle atrophy. Together these results implicate mitochondrial H2O2 generation as a novel therapeutic target to ameliorate skeletal muscle pathologies and support further studies to elucidate the role of lipid peroxide mediators released by mitochondria in denervation induced atrophy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Platelet Serotonin Aggravates Myocardial Ischemia/Reperfusion Injury via Neutrophil Degranulation.

Platelets store large amounts of serotonin that they release during thrombus formation or acute inflammation. This facilitates hemostasis and modulates the inflammatory response. Infarct size, heart function, and inflammatory cell composition were analyzed in mouse models of myocardial reperfusion injury with genetic and pharmacological depletion of platelet serotonin. These studies were complemented by in vitro serotonin stimulation assays of platelets and leukocytes in mice and men, and by measuring plasma serotonin levels and leukocyte activation in patients with acute coronary syndrome. Platelet-derived serotonin induced neutrophil degranulation with release of myeloperoxidase and hydrogen peroxide (H2O2) and increased expression of membrane-bound leukocyte adhesion molecule CD11b, leading to enhanced inflammation in the infarct area and reduced myocardial salvage. In patients hospitalized with acute coronary syndrome, plasmatic serotonin levels correlated with CD11b expression on neutrophils and myeloperoxidase plasma levels. Long-term serotonin reuptake inhibition-reported to protect patients with depression from cardiovascular events-resulted in the depletion of platelet serotonin stores in mice. These mice displayed a reduction in neutrophil degranulation and preserved cardiac function. In line, patients with depression using serotonin reuptake inhibition, presented with suppressed levels of CD11b surface expression on neutrophils and lower myeloperoxidase levels in blood. Taken together, we identify serotonin as a potent therapeutic target in neutrophil-dependent thromboinflammation during myocardial reperfusion injury.

Central Effects of Beta-Blockers May Be Due to Nitric Oxide and Hydrogen Peroxide Release Independently of Their Ability to Cross the Blood-Brain Barrier.

Propranolol is the first-line treatment for infants suffering from infantile hemangioma. Recently, some authors raised the question of potential neurologic side effects of propranolol due to its lipophilic nature and thus its ability to passively cross the blood-brain barrier (BBB) and accumulate into the brain. Hydrophilic beta-blockers, such as atenolol and nadolol, where therefore introduced in clinical practice. In addition to their classical mode of action in the brain, circulating factors may modulate the release of reactive oxygen/nitrogen species (ROS/RNS) from endothelial cells that compose the BBB without entering the brain. Due to their high capacity to diffuse across membranes, ROS/RNS can reach neurons and modify their activity. The aim of this study was to investigate other mechanisms of actions in which these molecules may display a central effect without actually crossing the BBB. We first performed an oral treatment in mice to measure the accumulation of propranolol, atenolol and nadolol in different brain regions in vivo. We then evaluated the ability of these molecules to induce the release of nitric oxide (NO) and hydrogen peroxide (H2O2) ex vivo in the hypothalamus. As expected, propranolol is able to cross the BBB and is found in brain tissue in higher amounts than atenolol and nadolol. However, all of these beta-blockers are able to induce the secretion of signaling molecules (i.e., NO and/or H2O2) in the hypothalamus, independently of their ability to cross the BBB, deciphering a new potential deleterious impact of hydrophilic beta-blockers in the brain.

Selectivity of Dietary Phenolics for Inhibition of Human Monoamine Oxidases A and B.

Monoamine oxidases (MAOs) regulate local levels of neurotransmitters such as dopamine, norepinephrine, and serotonin and thus have been targeted by drugs for the treatment of certain CNS disorders. However, recent studies have shown that these enzymes are upregulated with age in nervous and cardiac tissues and may be involved in degeneration of these tissues, since their metabolic mechanism releases hydrogen peroxide leading to oxidative stress. Thus, targeting these enzymes may be a potential anti-aging strategy. The purpose of this study was to compare the MAO inhibition and selectivity of selected dietary phenolic compounds, using a previously validated assay that would avoid interference from the compounds. Kynuramine metabolism by human recombinant MAO-A and MAO-B leads to formation of 4-hydroxyquinoline, with Vmax values of 10.2±0.2 and 7.35±0.69 nmol/mg/min, respectively, and Km values of 23.1±0.8 μM and 18.0±2.3 μM, respectively. For oral dosing and interactions with the gastrointestinal tract, curcumin, guaiacol, isoeugenol, pterostilbene, resveratrol, and zingerone were tested at their highest expected luminal concentrations from an oral dose. Each of these significantly inhibited both enzymes except for zingerone, which only inhibited MAO-A. The IC50 values were determined, and selectivity indices (MAO-A/MAO-B IC50 ratios) were calculated. Resveratrol and isoeugenol were selective for MAO-A, with IC50 values of 0.313±0.008 and 3.72±0.20 μM and selectivity indices of 50.5 and 27.4, respectively. Pterostilbene was selective for MAO-B, with IC50 of 0.138±0.013 μM and selectivity index of 0.0103. The inhibition of resveratrol (MAO-A) and pterostilbene (MAO-B) was consistent with competitive time-independent mechanisms. Resveratrol 4'-glucoside was the only compound which inhibited MAO-A, but itself, resveratrol 3-glucoside, and pterostilbene 4'-glucoside failed to inhibit MAO-B. Additional studies are needed to establish the effects of these compounds on MAO-A and/or MAO-B in humans.

Sodium percarbonate as a novel intracoronal bleaching agent: assessment of the associated risk of cervical root resorption.

To evaluate the extraradicular peroxide release from sodium percarbonate compared to sodium perborate as an intracoronal bleaching agent. Sixty mandibular single-rooted premolars with intact CEJ were selected. After root filling, gutta-percha was removed 4mm apical to CEJ and 2mm of GIC was condensed over the root filling. Intracoronal bleaching agents were placed into six groups of teeth (n=10): sodium perborate with distilled water (SPW); sodium percarbonate with distilled water (SPCW); sodium perborate with 30% hydrogen peroxide (SPHP); sodium percarbonate with 30% hydrogen peroxide (SPCHP); 30% hydrogen peroxide as positive control (HP) and distilled water as negative control (CL). The teeth were then mounted in vials filled with distilled water, kept in an incubator and taken out at 1, 3 and 6days for spectrophotometric analysis. Extraradicular peroxide release was quantified by the ferrothiocyanate method. Statistical analysis was undertaken with one-way anova and Scheffe post hoc tests. The greatest peroxide release occurred in the HP group, followed by the SPCHP and SPHP groups, and then by the SPCW and SPW groups. Intergroup comparison revealed that there was no significant difference in peroxide release among the groups SPCW and SPW on days 1, 3 and 6 (P>0.05). Similarly, no significant difference was found between the SPCHP and SPHP treated groups on days 1, 3 and 6 (P>0.05). Extraradicular peroxide release from sodium percarbonate was comparable to that of sodium perborate, as the differences were not significant.

Pharmacologic ascorbate as a pro-drug for hydrogen peroxide release to kill mycobacteria

Background and purposeTuberculosis is one of the most highly fatal diseases worldwide, and one-third of the world's population has been infected with Mycobacterium tuberculosis (M. tuberculosis). A previous study showed that M. tuberculosis was highly susceptible to being killed by ascorbate (i.e. vitamin C, VC), but the molecular mechanisms of the bactericidal activity of VC against M. tuberculosis are still not well understood. Experimental approachWe assayed the effects of VC as an anti-tuberculosis drug against mycobacteria (i.e. M. bovis BCG or M. tuberculosis H37Rv) in macrophages (i.e. RAW 264.7 cells). Relative global protein expression changes in 5 mM VC-treated and control samples of H37Rv were investigated by Tandem mass tag (TMT)-based quantitative proteomic analysis. qRT-PCR was also used to measure the differential expression of six intracellular stress response mycobacteria genes. Key resultsQuantitative proteomic analysis showed that 11 peptide components including rip3, fdxA, Rv2028c, mtp, LH57_00670, hspX, pfkB, Rv1824, Rv1813c, LH57_08410 and Rv2030c were up-regulated and 17 peptide components were down-regulated in 5 mM VC-treated H37Rv compared with the control samples. qRT-PCR also verified that VC could induce the expression of six genes (hsp, fdxD, furA, devR, hspX, and dnaB) in BCG and H37Rv. We also found that exosomes from RAW 264.7 cells treated with pharmacologic VC could kill M. bovis BCG in vitro. Conclusion and implicationsOur results demonstrated that the bactericidal activity of VC against mycobacteria, as a pro-drug for hydrogen peroxide formation (H2O2), was dependent on reactive oxygen species production and the activated oxidative stress pathway, which suggested that pharmaceutical VC and exosomes from macrophages treated with VC could be used as potential anti-tuberculosis drugs.