Formation Of Hypochlorous Acid Research Articles

Electrolyzed Water Treatment for the Control of the Zoonotic Pathogen Vibrio vulnificus in Aquaculture: A One Health Perspective.

Vibrio vulnificus (Vv) is a bacterial pathogen native to warm and brackish water ecosystems that can cause fatal septicemia (Vv-vibriosis) in humans and various farmed fish species. From a One Health perspective, controlling Vv-vibriosis outbreaks on farms is essential not only for animal but also for human health, as it reduces the risk of Vv transmission to humans. Electrolyzed water (EW) is a sustainable control method, exhibiting transient disinfectant properties due to the formation of hypochlorous acid (HOCl). We hypothesized that EW could effectively reduce Vv populations in aquaculture facilities, preventing outbreak emergence. To test this hypothesis, survival assays in EW were conducted under varying conditions of salinity, pH, and free available chlorine (FAC). The results indicated that an intermediate concentration of FAC had a significant bactericidal effect on Vv populations regardless of the condition and tested strain. Consequently, the strategic use of EW could serve as an eco-friendly preventive and control measure against Vv-vibriosis by significantly decreasing the bacterial load in farm water.

Antibacterial Efficacy of Irrigants with Varying Osmolarity on E. faecalis Biofilm: An In Vitro Study.

To evaluate the role of the addition of different concentrations of sodium chloride salt to conventional intracanal irrigants to vary their osmotic values and thereby compare their antibacterial efficacy. In an active attachment biofilm model, Enterococcus faecalis (ATCC 29212) biofilms were grown. Sodium chloride salts were added to 100 mL of distilled water to make 6M (hyperosmotic), 0.5M, and 0.25M (hypoosmotic) sodium chloride solutions, respectively. The experimental groups were divided into three groups: Group I: 5.25% sodium hypochlorite, group II: 2% chlorhexidine, and group III: 2% povidone iodine, and four subgroups within these three groups, such as subgroup A (without salt solution), subgroup B (with 6M of hyperosmotic salt solution), subgroup C (with 0.5M of hypoosmotic salt solution), and subgroup D (with 0.25M of hypoosmotic salt solution), respectively. Biofilms were treated with all the subgroups for a contact time of 15 min. A crystal violet assay was done to estimate the bacterial cell biomass. The results revealed that subgroups IIIB, IB, and IID, ID had a statistical reduction in bacterial biomass at p < 0.05. There were no significant differences between subgroups IC, IIC, and IIIC and subgroups IA, IIA, and IIIA. The antibacterial efficacy of all three irrigants was significantly affected by varying the osmolarities. The results prove that the hyperosmotic and hypoosmotic salt solutions, along with irrigants, have enhanced antibacterial efficacy on E. faecalis biofilm due to its ability to vary the turgor pressure of cell wall, as well as the inherent properties of the irrigants such as hypochlorous acid formation, ionic interaction, and free radical interactions.

Mechanism of Action of Flavin-Dependent Halogenases.

To rationally engineer the substrate scope and selectivity of flavin-dependent halogenases (FDHs), it is essential to first understand the reaction mechanism and substrate interactions in the active site. FDHs have long been known to achieve regioselectivity through an electrophilic aromatic substitution at C7 of the natural substrate Trp, but the precise role of a key active-site Lys residue remains ambiguous. Formation of hypochlorous acid (HOCl) at the cofactor-binding site is achieved by the direct reaction of molecular oxygen and a single chloride ion with reduced FAD and flavin hydroxide, respectively. HOCl is then guided 10 Å into the halogenation active site. Lys79, located in this site, has been proposed to direct HOCl toward Trp C7 through hydrogen bonding or a direct reaction with HOCl to form an -NH2Cl+ intermediate. Here, we present the most likely mechanism for halogenation based on molecular dynamics (MD) simulations and active-site density functional theory "cluster" models of FDH PrnA in complex with its native substrate l-tryptophan, hypochlorous acid, and the FAD cofactor. MD simulations with different protonation states for key active-site residues suggest that Lys79 directs HOCl through hydrogen bonding, which is confirmed by calculations of the reaction profiles for both proposed mechanisms.

Dissolved black carbon induced elimination of bisphenol a by peroxymonosulfate activation through HClO mediated oxidation process

Dissolved black carbon (DBC) is released into environmental media following the wide adoption of biochar-catalyzed peroxymonosulfate (PMS) advanced oxidation processes (AOPs) in wastewater treatment. However, the possible reactivity between DBC and PMS in the process has not yet been reported. In this study, the role of DBC in the biochar-initiated PMS activation process was comprehensively explored. For the first time, we discovered that DBC could significantly improve the oxidation of bisphenol A (BPA) by PMS activation with a pseudo-first-order kinetic rate constant (kapp) of 0.040 min−1. Different biomass feedstocks led to distinguishable kapp values, revealing the reactive species were largely depended on the intrinsic properties of biomass. The activation mechanism was comprehensively elucidated through in-situ and ex-situ techniques. Radical species (i.e., •OH and SO4•−) as well as singlet oxygen (1O2) were not the dominant contributors, while the Cl- induced formation of hypochlorous acid (HClO) was confirmed to be the primary oxidative species responsible for BPA oxidation. On the other hand, dissolved organic components in DBC preferentially consumed HClO and thus inhibited the oxidation of BPA. Typical chlorinated derivatives (i.e., 3-chlorobisphenol A, 3,3′-dichlorobisphenol A, 3,5-dichlorobisphenol A and 3,3′,5- trichlorobisphenol A) were identified after the oxidation process. These findings benefited further understanding of the role of biochar derived DBC and revealed the potential environmental risk of biochar-activated PMS oxidation processes.

Hypochlorous acid facilitates inducible nitric oxide synthase subunit dissociation: The link between heme destruction, disturbance of the zinc-tetrathiolate center, and the prevention by melatonin

Inducible nitric oxide synthase (iNOS) is a zinc-containing hemoprotein composed of two identical subunits, each containing a reductase and an oxygenase domain. The reductase domain contains binding sites for NADPH, FAD, FMN, and tightly bound calmodulin and the oxygenase domain contains binding sites for heme, tetrahydrobiopterin (H4B), and l-arginine. The enzyme converts l-arginine into nitric oxide (NO) and citrulline in the presence of O2. It has previously been demonstrated that myeloperoxidase (MPO), which catalyzes formation of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride (Cl−), is enhanced in inflammatory diseases and could be a potent scavenger of NO. Using absorbance spectroscopy and gel filtration chromatography, we investigated the role of increasing concentrations of HOCl in mediating iNOS heme destruction and subsequent subunit dissociation and unfolding. The results showed that dimer iNOS dissociation between 15 and 100 μM HOCl was accompanied by loss of heme content and NO synthesis activity. The dissociated subunits-maintained cytochrome c and ferricyanide reductase activities. There was partial unfolding of the subunits at 300 μM HOCl and above, and the subunit unfolding transition was accompanied by loss of reductase activities. These events can be prevented when the enzyme is preincubated with melatonin prior to HOCl addition. Melatonin supplementation to patients experiencing low NO levels due to inflammatory diseases may be helpful to restore physiological NO functions.

Freezing-induced activation of the binary chloride-Oxone system to free chlorine and its application in water treatment

Accelerated chemical reactions in frozen solutions can be applied in the degradation of organic pollutants in water. Herein we propose a novel freezing system that enables the degradation of various organic compounds in a frozen solution. Although the degradation of 4-chlorophenol (4-CP) by Oxone as the sole primary oxidant did not take place in aqueous solutions (25 °C) regardless of the presence of chloride ions (Cl−, micromolar levels) and only took place to a minor extent in frozen solutions (−20 °C) in the absence of Cl−, the addition of Cl− (micromolar levels) to the freezing/Oxone system significantly accelerated the degradation of 4-CP. Various analytical characterizations and pH measurements of the frozen solution suggested that the enhanced degradation of 4-CP in the freezing/Oxone/Cl− system could be because Cl−, Oxone, and protons are concentrated in the liquid brine upon freezing. This process subsequently facilitates the formation of hypochlorous acid (HOCl) as a secondary oxidant. The positive effect of Cl− was observed under widely varying conditions (i.e., [Cl−] = 25–1000 μM, pHi = 3–11, and freezing temperature = from −10 to −30 °C), and the freezing/Oxone/Cl− system described herein successfully degraded all 12 tested organic pollutants. In addition, outdoor freezing experiments carried out on winter days confirmed the successful performance of the freezing/Oxone/Cl− system without the requirement for electrical energy.

Selective Immobilization of Antimony Using Brucite-rich Precipitate Produced during In Situ Hypochlorous Acid Formation through Seawater Electrolysis in a Nuclear Power Plant

This study has investigated the selective immobilization of antimony using the brucite (magnesium hydroxide)-rich precipitate (BP) collected from a hypochlorous storage tank in a nuclear power plant of South Korea. The energy dispersive X-ray and X-ray diffraction analyses revealed that the BP mainly consisted of magnesium (72.5%) and its dominant mineral phase was brucite (Mg(OH)2). Therefore, brandholzite (Mg[Sb(OH)6]2·6H2O) was newly formed through the surface-induced precipitation during the adsorption of antimony using the BP. The adsorbed amount of antimony increased with decreasing pH values because of the increased positive surface charge of the BP (pHpzc = 9.6). The maximum adsorption capacity (Qmax) of BP, calculated by Langmuir adsorption isotherm, was 11.02 mg/g. The presence of competitive anions did not significantly affect the adsorption of antimony toward the BP due to its high selectivity. These results suggest that the facile utilization of the BP as a low-cost adsorbent seems to be a practical option for the selective removal of antimony from wastewater.

KINETICS AND MECHANISM OF TETRAHYDROFURAN OXIDATION BY CHLORAMINET IN ACIDIC MEDIA

The kinetics of tetrahydrofuran oxidation by sodium N-chloro-p-toluene sulfonamide in the hydrochloric acid medium was studied in this work at 308 K. The reaction rate shows a first-order dependence on [CAT] and fractional-order dependence each on [THF] and [H+]. The derivative rate law, which suitable for experimental results, is Equation 24. The first-order rate constant has been evaluated from the relationship of the plot of Log [CAT] versus Time. The variation of the ionic strength by the addition of sodium perchlorate (NaClO4) and chloride ion on the medium showed no significant effect on the reaction. The reaction rate raised with decreasing dielectric constant (D), while the addition of p-toluene sulfonamide retards the rate of reaction. The oxidation reaction of tetrahydrofuran have been studied at a different temperature, The equilibrium constants for the formation of hypochlorous acid, protonated hydrochlorous acid and protonated hydrochlorous acid–THF complex and its decomposition constant have been estimated. Also, the rate constant for the slow (rate-determining step) and the activation parameter have been calculated. A suitable mechanism for the oxidation reaction of tetrahydrofuran was proposed based on the experimental finding. The mechanism includes the reaction of active species (H2OCl) of the oxidizing agent with the tetrahydrofuran in a fast step to give the complex(X). This complex will then transformed into complex (X̅ ) in slow step then to γ-butyrolactone in another fast step.

Binding of lactoferrin to the surface of low-density lipoproteins modified by myeloperoxidase prevents intracellular cholesterol accumulation by human blood monocytes.

Myeloperoxidase (MPO) is a unique heme-containing peroxidase that can catalyze the formation of hypochlorous acid (HOCl). The strong interaction of MPO with low-density lipoproteins (LDL) promotes proatherogenic modification of LDL by HOCl. The MPO-modified LDL (Mox-LDL) accumulate in macrophages, resulting in the formation of foam cells, which is the pathognomonic symptom of atherosclerosis. A promising approach to prophylaxis and atherosclerosis therapy is searching for remedies that prevent the modification or accumulation of LDL in macrophages. Lactoferrin (LF) has several application points in obesity pathogenesis. We aimed to study LF binding to Mox-LDL and their accumulation in monocytes transformed into macrophages. Using surface plasmon resonance and ELISA techniques, we observed no LF interaction with intact LDL, whereas Mox-LDL strongly interacted with LF. The affinity of Mox-LDL to LF increased with the degree of oxidative modification of LDL. Moreover, an excess of MPO did not prevent interaction of Mox-LDL with LF. LF inhibits accumulation of cholesterol in macrophages exposed to Mox-LDL. The results obtained reinforce the notion of LF potency as a remedy against atherosclerosis.

The Transcription Factor ArcA Modulates Salmonella's Metabolism in Response to Neutrophil Hypochlorous Acid-Mediated Stress.

Salmonella Typhimurium, a bacterial pathogen with high metabolic plasticity, can adapt to different environmental conditions; these traits enhance its virulence by enabling bacterial survival. Neutrophils play important roles in the innate immune response, including the production of microbicidal reactive oxygen species (ROS). In addition, the myeloperoxidase in neutrophils catalyzes the formation of hypochlorous acid (HOCl), a highly toxic molecule that reacts with essential biomolecules, causing oxidative damage including lipid peroxidation and protein carbonylation. The bacterial response regulator ArcA regulates adaptive responses to oxygen levels and influences the survival of Salmonella inside phagocytic cells. Here, we demonstrate by whole transcriptomic analyses that ArcA regulates genes related to various metabolic pathways, enabling bacterial survival during HOCl-stress in vitro. Also, inside neutrophils, ArcA controls the transcription of several metabolic pathways by downregulating the expression of genes related to fatty acid degradation, lysine degradation, and arginine, proline, pyruvate, and propanoate metabolism. ArcA also upregulates genes encoding components of the oxidative pathway. These results underscore the importance of ArcA in ATP generation inside the neutrophil phagosome and its participation in bacterial metabolic adaptations during HOCl stress.

Myeloperoxidase-mediated oxidation of edaravone produces an apparent non-toxic free radical metabolite and modulates hydrogen peroxide-mediated cytotoxicity in HL-60 cells

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H2O2) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 μM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV–Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 μM H2O2 or a flux of H2O2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H2O2-induced toxicity was ameliorated at high edaravone concentrations (100–200 μM). In contrast, low concentrations of edaravone (1–10 μM) exacerbated the H2O2-induced toxicity. However, the effect of edaravone at low concentration (0–10 μM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug.

Oxidation of cysteine by ceruloplasmin leads to formation of hydrogen peroxide, which can be utilized by myeloperoxidase

Myeloperoxidase (MPO) is the enzyme of azurophilic granules of neutrophils, which catalyzes two electron oxidation of either chloride or bromide in the so-called “halogenating cycle”. Interaction of hydrogen peroxide with MPO in the presence of chloride ions leads to formation of hypochlorous acid (HOCl). Ceruloplasmin (CP) is known to be an effective physiological inhibitor of the MPO activity. However, despite the large excess of CP in blood plasma, MPO-dependently modified biomolecules were found in variety of inflammation loci, including vessel walls. This study shows that CP, which is supposed to inhibit MPO, can provide its action in physiological conditions due to hydrogen peroxide formation during oxidation of free cysteine. The key role of labile copper ions in said process is also demonstrated.

Chlorine Dioxide-Pollutant Transformation and Formation of Hypochlorous Acid as a Secondary Oxidant.

Chlorine dioxide (ClO2) has been used as a disinfectant in water treatment for a long time, and its use for micropollutant abatement in wastewater has recently been suggested. Surprisingly, a mechanistic understanding of ClO2 reactions in (waste)water matrices is largely lacking. The present study contributes to this mechanistic understanding by performing a detailed investigation of ClO2 reactions with organic matter using phenol as a surrogate for reactive phenolic moieties. A concept for indirectly determining HOCl using 2- and 4-bromophenol was developed. The reaction of phenol with ClO2 formed chlorite (62 ± 4% per ClO2 consumed) and hypochlorous acid (HOCl) (42 ± 3% per ClO2 consumed). The addition of ClO2 to wastewater (5 × 10-5 M ClO2) resulted in 40% atenolol and 47% metoprolol transformation. The presence of the selective HOCl scavenger glycine largely diminished their transformation, indicating that atenolol and metoprolol were transformed by a fast reaction with HOCl (e.g., k (atenolol + HOCl) = 3.5 × 104 M-1 s-1) that formed in ClO2 reactions with the wastewater matrix. The formation of HOCl may thus increase the number of transformable micropollutants in ClO2 applications. However, chlorine related byproducts may also be formed.

Aging resistant TiO2/silicone rubber composites

We have recently shown [1,2] that one component room temperature vulcanized (RTV-1) silicone rubbers (SIR) based on polydimethylsiloxane (PDMS) can be rapidly degraded by low voltage (LV) energized aqueous salt solutions by previously unreported aging mechanisms related to the formation of hypochlorous acid in high voltage (HV) transmission line applications. In this study, we are showing how to improve the resistance of the rubbers to extreme environmental aging by embedding TiO2 micro-particles. Molecular dynamics (MD) simulations were conducted to determine the combined effect of TiO2 and different concentrations of hydrophobic PDMS methyl groups on surface hydrophobicity of a TiO2/PDMS composite. In addition, the effects of both TiO2 and silica on the diffusivities of LV aqueous salt components in the PDMS were predicted and related to unique interfacial interactions between the particles and the methyl groups of the PDMS. Rutile TiO2 reoriented methyl groups away from the particles reducing the diffusivities of water and hypochlorous acid. This effect shielded the PDMS network against environmental chain scissions. On the other hand, silica attracted the groups accelerating acid and water migrations and thus enhancing damage to the network. In the experimental part, TiO2/RTV was subjected independently to hypochlorous acid and electrolyzed LV aqueous salt. As expected, TiO2 greatly increased the contact angle, reduced the surface energy and improved the hydrophobicity of the composite, mitigating the negative effect of the reduced concentrations of methyl groups. As a result, aging damage to the rubber was dramatically reduced by about 50% in highly oxidative environments.

Pathways of myeloperoxidase-induced cellular damage in atherosclerosis

The formation of hypochlorous acid (HOCl) by myeloperoxidase (MPO) during chronic inflammation plays a key role in the development of atherosclerosis. HOCl is a powerful oxidant that causes extensive tissue damage. In the presence of thiocyanate (SCN-), the production of HOCl by MPO is decreased by the formation of hypothiocyanous acid (HOSCN), which is a milder, thiol-specific, oxidant. It has been proposed that SCN- may reduce the extent of MPO-induced damage during inflammation, but SCN- is elevated in the plasma of smokers, who are at greater risk of atherosclerosis. In this study, we compared the reactivity of HOCl and HOSCN with macrophages, endothelial cells, and smooth muscle cells, which are involved in the progression of atherosclerosis. The sensitivity and responses to each oxidant vary with cell and treatment conditions, but culminate in altered calcium fluxes, mitochondrial dysfunction, release of cytokines and cell death by apoptosis and necrosis. We show that HOSCN can have a dual role, acting in both a pro-inflammatory and protective capacity, with repair of thiols and altered metabolic flux promoting cell survival and adaption to stress. Our data provide new insight into how SCN- could modulate MPO-induced damage and influence the development of atherosclerosis.

Inhibitive Effects of Quercetin on Myeloperoxidase-Dependent Hypochlorous Acid Formation and Vascular Endothelial Injury

Myeloperoxidase (MPO) from activated neutrophils plays important roles in multiple human inflammatory diseases by catalyzing the formation of powerful oxidant hypochlorous acid (HOCl). As a major flavonoid in the human diet, quercetin has been suggested to act as antioxidant and anti-inflammatory agent in vitro and in vivo. In this study, we showed that quercetin inhibited MPO-mediated HOCl formation (75.0 ± 6.2% for 10 μM quercetin versus 100 ± 5.2% for control group, P < 0.01) and cytotoxicity to endothelial cells in vitro, while this flavonoid was nontoxic to endothelial cell cultures ( P > 0.05, all cases). Moreover, quercetin inhibited HOCl generation by stimulated neutrophils (a rich source of MPO) and protected endothelial cells from neutrophils-induced injury. Furthermore, quercetin could inhibit HOCl-induced endothelial dysfunction such as loss of cell viability, and decrease of nitric oxide formation in endothelial cells ( P < 0.05, all cases). Consistent with these in vitro data, quercetin attenuated lipopolysaccharide-induced endothelial dysfunction and increase of MPO activity in mouse aortas, while this flavonoid could protect against HOCl-mediated endothelial dysfunction in isolated aortas ( P < 0.05). Therefore, it was proposed that quercetin attenuated endothelial injury in inflammatory vasculature via inhibition of vascular-bound MPO-mediated HOCl formation or scavenging of HOCl. These data indicate that quercetin is a nontoxic inhibitor of MPO activity and MPO/neutrophils-induced cytotoxicity in endothelial cells and may be useful for targeting MPO-dependent vascular disease and inflammation.

Sonochemical Synthesis of Optically Tuneable Conjugated Polymer Nanoparticles

AbstractThe development of novel and simple methodologies for the obtaining of semiconductive polymer nanoparticles with fine‐tuned optical properties represents nowadays a challenging research area as it involves a simultaneous chemical modification and nanostructuration of the polymer. Here, starting from poly[2‐methoxy‐5‐(2‐ethylhexyloxy)‐1,4‐phenylenevinylene], this objective is achieved with the one‐pot synthesis of oligomers with tunable conjugation length and their nanostructuration, employing a miniemulsion method. Ultrasound irradiation of heterogeneous mixtures leads to the formation of hypochlorous acid that disrupts the electronic conjugation through polymer chain cleavage. Moreover, control over the degree of the electronic conjugation of the oligomers, and therefore of the optical properties, is achieved simply by varying the polymer concentration of the initial solution. Finally, the presence of surfactants during the sonication allows for the formation of nanoparticles with progressive spectral shift of the main absorption (from λmax = 476 to 306 nm) and emission bands (from λmax = 597 to 481 nm). The integration of conducting polymer nanoparticles into polymeric matrices yields self‐standing and flexible fluorescent films.

The Production of Reactive Oxygen and Halogen Species by Neutrophils in Response to Monomeric Forms of Myeloperoxidase

It was shown for the first time that myeloperoxidase, a homodimer that consists of two disulfidebonded identical protomers and catalyzes the formation of hypochlorous acid (HOCl), is decomposed by HOCl into monomers (MPO-Cl). Dimeric myeloperoxidase can also be converted into monomers (hemimyeloperoxidase) by reduction of the disulfide bond. In this study, the effects of two monomeric forms of myeloperoxidase, MPO-Cl and hemi-myeloperoxidase, and native dimeric myeloperoxidase on the production of reactive oxygen (•O 2 − and H2O2) and halogen (HOCl) species by neutrophils were compared. Neutrophil production of these species was monitored after addition of hemi-myeloperoxidase, MPO-Cl, or dimeric myeloperoxidase and also after the subsequent addition of activators, phorbol-12-myristate-13-acetate or N-formyl-Met-Leu-Phe. HOCl production was assessed by chemiluminescence in the presence of luminol; •O 2 − production was assessed by chemiluminescence in the presence of lucigenin and by cytochrome c reduction determined spectrophotometrically, and H2O2 production was measured using fluorimetry with scopoletin. The results indicate that MPO-Cl and hemi-myeloperoxidase, which can occur in blood under halogenative stress, do not prime neutrophil NADPH oxidase, and do not enhance the production of reactive oxygen (•O 2 − and H2O2) and halogen (HOCl) species.

Inhibition of Myeloperoxidase- and Neutrophil-Mediated Hypochlorous Acid Formation in Vitro and Endothelial Cell Injury by (-)-Epigallocatechin Gallate.

Myeloperoxidase (MPO) plays important roles in various diseases through its unique chlorinating activity to catalyze excess hypochlorous acid (HOCl) formation. Epidemiological studies indicate an inverse correlation between plant polyphenol consumption and the incidence of cardiovascular diseases. Here we showed that (-)-epigallocatechin gallate (EGCG), the main flavonoid present in green tea, dose-dependently inhibited MPO-mediated HOCl formation in vitro (chlorinating activities of MPO: 50.2 ± 5.7% for 20 μM EGCG versus 100 ± 5.6% for control, P < 0.01). UV-vis spectral and docking studies indicated that EGCG bound to the active site (heme) of MPO and resulted in the accumulation of compound II, which was unable to produce HOCl. This flavonoid also effectively inhibited HOCl generation in activated neutrophils (HOCl formation: 65.0 ± 5.6% for 20 μM EGCG versus 100 ± 6.2% for control, P < 0.01) without influencing MPO and Nox2 release and superoxide formation, suggesting that EGCG specifically inhibited MPO but not NADPH oxidase activity in activated neutrophils. Moreover, EGCG inhibited MPO (or neutrophil)-mediated HOCl formation in human umbilical vein endothelial cells (HUVEC) culture and accordingly protected HUVEC from MPO (or neutrophil)-induced injury (P < 0.05, all cases), although it did not induce cytotoxicity to HUVEC (P > 0.05, all cases). Our results indicate that dietary EGCG is an effective and specific inhibitor of MPO activity and may participate in the regulation of immune responses at inflammatory sites.

107 - Hypothiocyanous Acid (HOSCN) Re-Directs Macrophage Glycolytic Flux Through the Pentose Phosphate Pathway - An Adaption to Oxidative Stress in Inflammation?

A host of chronic inflammatory diseases are accelerated by the formation of hypochlorous acid (HOCl) by myeloperoxidase (MPO), which is released by activated neutrophils. HOCl is a powerful oxidant that causes extensive tissue damage and cell death. In the presence of thiocyanate (SCN – ), the production of HOCl by MPO is decreased in favour of the formation of a milder oxidant, hypothiocyanous acid (HOSCN). Unlike HOCl, HOSCN reacts selectively with thiols to result in reversible modifications that can be repaired, which has led to the hypothesis that SCN – may reduce the extent of MPO-induced damage during inflammation. In this study, we examined the cellular targets and consequences of the reaction of HOSCN with macrophages, a key inflammatory cell type. We show that exposure of macrophages to HOSCN results in the reversible modification of multiple proteins associated with glycolysis and metabolism. This leads to decreased glycolysis, glycolytic reserve, and glycolytic capacity, which is associated with the reduced formation of ATP, NADH and lactate, the end-product of glycolysis. HOSCN did not influence glucose uptake in the macrophages, but was associated with reduced respiration, consistent with a decrease in the pyruvate pool available for mitochondrial oxidative phosphorylation. However, HOSCN treatment also led to an increase in the activity of glucose 6-phosphate dehydrogenase and elevated levels of NADPH, suggesting re-routing of the glycolytic flux through the pentose phosphate pathway. This glycolytic switch was not observed on exposure of macrophages to HOCl. These results suggest that HOSCN may induce an adaptive response in the macrophages, whereby the increased NADPH could increase the capacity of the cellular enzymatic antioxidant systems to deal with an oxidative insult. This provides new insight into potential pathways by which SCN – could modulate the reactivity of MPO-derived oxidants, which may have implications for the treatment of inflammatory diseases.