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.     

The priming effect of halogenated phospholipids on the functional responses of human neutrophils

Halogenated lipids formed in the reactions with myeloperoxidase (MPO)-derived species may contribute to the regulation of the functional activity of cells. In the present study we have investigated the effects of chloro- and bromohydrins formed in the HOCl and HOBr reactions, respectively, with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on three different functional responses of human neutrophils: H₂O₂ generation, degranulation (MPO exocytosis), and cell aggregation. It was shown that POPC chloro- and bromohydrins (POPC-Cl and POPC-Br) induced the priming of neutrophils, resulting in significant upregulation of cell responses to neutrophil stimulators such as N-formyl-Met-Leu-Phe and lectin from Solanum tuberosum. The stimulating effects of POPC-Cl and POPC-Br were observed at low micromolar concentrations (liposomal concentration of POPC, 0.5–5 μM; the content of POPC-Cl or POPC-Br, 38 ± 3% of total lipids) after a short exposure (about 5 min) of the neutrophils to POPC-Cl or POPC-Br. These results suggest that halogenated lipids formed in vivo via MPO-dependent reactions may be considered as a new class of biologically active substances that are potentially able to contribute to the priming of myeloid cells in the sites of inflammation and serve as inflammatory response modulators.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils

It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase — 4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC₅₀ = 20 μM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p < 0.01) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a “vicious circle” of neutrophil activation at the inflammatory site.     

Influences of chloride and hypochlorite on neutrophil extracellular trap formation

Background

The release by neutrophils of DNA-based extracellular traps (NETs) is a recently recognized innate immune phenomenon that contributes significantly to control of bacterial pathogens at tissue foci of infection. NETs have also been implicated in the pathogenesis of non-infectious diseases such as small vessel vasculitis, lupus and cystic fibrosis lung disease. Reactive oxygen species (ROS) are important mediators of NET generation (NETosis). Neutrophils with reduced ROS production, such as those from patients with chronic granulomatous disease or myeloperoxidase (MPO) deficiency, produce fewer NETs in response to inflammatory stimuli. To better understand the roles of various ROS in NETosis, we explore the role of MPO, its substrates chloride ion (Cl⁻) and hydrogen peroxide (H₂O₂), and its product hypochlorite (HOCl) in NETosis.

Findings

In human peripheral blood neutrophils, pharmacologic inhibition of MPO decreased NETosis. Absence of extracellular Cl⁻, a substrate for MPO, also reduced NETosis. While exogenous addition of H₂O₂ and HOCl stimulated NETosis, only exogenous HOCl could rescue NETosis in the setting of MPO inhibition. Neither pharmacological inhibition nor genetic deletion of MPO in murine neutrophils blocked NETosis, in contrast to findings in human neutrophils.

Conclusions

Our results pinpoint HOCl as the key ROS involved in human NETosis. This finding has implications for understanding innate immune function in diseases in which Cl⁻ homeostasis is disturbed, such as cystic fibrosis. Our results also reveal an example of significant species-specific differences in NET phenotypes, and the need for caution in extrapolation to humans from studies of murine NETosis.     

Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase

Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H₂O₂) prevents polyspermy in Arbacia punctulata. We found that it is not H₂O₂ but probably hypochlorous acid/hypochlorite (HOCl/OCl^?) derived from H₂O₂ that is toxic to the supernumerary sperm. The spermicidal activity of H₂O₂ is potentiated by at least one order of magnitude by cupric ions (Cu²⁺). This increased toxicity is not due to the formation of hydroxyl radicals (·OH) because ·OH scavengers did not counteract the activity of Cu²⁺. More-over, substitution of Cu²⁺ by ferrous ions (Fe²⁺), which are known to cause formation of ·OH from H₂O₂, had no effect on fertilization even at 10²?10³ times higher concentrations. In contrast, 3-amino-1,2,4-triazole (AT), an HOCl/OCl^? scavenger, totally reversed the toxic effects of Cu²⁺. Furthermore, we found that HOCl/OCl^? is generated in solutions of H₂O₂ and Cu²⁺ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl^?. HOCl/OCl^? generated by Cu²⁺ from H₂O₂ and Cl^?, a low concentration of exogenously added HOCl/OCl^?, or increased concentrations of H₂O₂ has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl^? from the Cl^? present in sea water through reaction with H₂O₂ generated by the newly fertilized egg.     

Myeloperoxidase deficiency induces MIP-2 production via ERK activation in zymosan-stimulated mouse neutrophils

Abstract

Myeloperoxidase (MPO), a major constituent of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide (H₂O₂) and chloride anion. We have previously reported that MPO-deficient (MPO^?/?) neutrophils produce greater amount of macrophage inflammatory protein-2 (MIP-2) in vitro than do wild type when stimulated with zymosan. In this study, we investigated the molecular mechanisms governing the up-regulation of MIP-2 production in the mutant neutrophils. Interestingly, we found that zymosan-induced production of MIP-2 was blocked by pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK), and with BAY11-7082, an inhibitor of nuclear factor (NF)-κB. Western blot analysis indicated that U0126 also inhibited the phosphorylation of p65 subunit of NF-κB (p65), indicating that MIP-2 was produced via the ERK/NF-κB pathway. Intriguingly, we found that ERK1/2, p65, and alpha subunit of inhibitor of κB (IκBα) in the MPO^?/? neutrophils were phosphorylated more strongly than in the wild type when stimulated with zymosan. Exogenous H₂O₂ treatment in addition to zymosan stimulation enhanced the phosphorylation of ERK1/2 without affecting the zymosan-induced MIP-2 production. In contrast, exogenous HOCl inhibited the production of MIP-2 as well as IκBα phosphorylation without affecting ERK activity. The zymosan-induced production of MIP-2 in the wild-type neutrophils was enhanced by pre-treatment of the MPO inhibitor 4-aminobenzoic acid hydrazide. Collectively, these results strongly suggest that both lack of HOCl and accumulation of H₂O₂ due to MPO deficiency contribute to the up-regulation of MIP-2 production in mouse neutrophils stimulated with zymosan.     

The relationship between exhaled carbon monoxide and human neutrophil function in the Japanese general population

We have evaluated the relationship between exhaled carbon monoxide (CO) level and neutrophil‐related functions such as reactive oxygen species (ROS) production capability, phagocytic activity and serum opsonic activity in the general population. Serum opsonic activity was determined by measuring the effects of serum on neutrophil ROS production capability using lucigenin‐ and luminol‐dependent chemiluminescence (LgCL, LmCL). LgCL is associated with the detection of O₂^?, whereas LmCL mainly detects H₂O₂ and HOCl, which are higher reactive oxygen radicals. In females, exhaled CO level was found to have positive associations with ROS production capability and LgCL. However, the opposite tendency was seen between exhaled CO level and LmCL in both genders. This result suggests that neutrophil ROS production in females may have contributed to oxidative stress, which led to the increases in intrinsic CO and exhaled CO consequently. Such changes then may have inhibited the process of changing reactive oxygen radicals into higher oxidizing potential levels. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of hydrogen peroxide on neutrophil ability to generate reactive oxygen and chlorine species and to secrete myeloperoxidase in vitro

In this work, the effects of H₂O₂ at concentrations of 10^?8–10^?2 mol/l on the neutrophil ability to generate reactive oxygen and chlorine species (ROCS) and to secrete myeloperoxidase (MPO) were studied, as well as the H₂O₂ damaging action on neutrophils. It was found that H₂O₂ at concentrations of 2 × 10^?3–10^?2 mol/l led to disturbances of neutrophil membrane barrier properties and to a lactate dehydrogenase release. Incubation of neutrophils with an addition of 10^?4–10^?7 mol/l H₂O₂ was accompanied by an increase of the cell ability to generate ROCS during phagocytosis and a decrease of neutrophil ability to secrete MPO and ROCS into the extracellular medium during adhesion. Mechanisms of the H₂O₂ action are coupled with arachidonic acid metabolism. Inhibition of the 5-lipoxygenase or cyclooxygenase metabolism pathways produced an enhancement of the H₂O₂ destructive effect. Block of 5-lipoxygenase pathway led to elimination of the H₂O₂ action on MPO and ROCS secretion and to an enhancement of the H₂O₂ effect on the neutrophil ability to generate ROCS during phagocytosis. The obtained data indicate a high blood neutrophil resistance to the H₂O₂ destructive action and confirm the H₂O₂ regulatory role with respect to the neutrophil functions.     

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100

Oxidation of LDL by the myeloperoxidase (MPO)-H₂O₂-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H₂O₂-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H₂O₂-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.     

Antimicrobial Activity of Myeloperoxidase from Neutrophilic Peroxisome

Myeloperoxidase plays the key role in antimicrobial oxygen-dependent activity of neutrophils. This heme-containing enzyme catalyzes HOCl formation from H₂O₂ and Cl^–. HOCl is a strong oxidation agent produced at the significant level by neutrophils. Myeloperoxidase easily oxidizes thiocyanate to hypothiocyanate and Br^– to HOBr, which are involved in protective reactions. Myeloperoxidase reacts quickly with nitric oxide and peroxynitrite in inflammation foci. All these reactions affect neutrophil-induced oxidative stress.     

Leukotriene B4 and its action with a free-radical-generating system

The concentration of Leukotriene B₄ (LTB₄) demonstrated in early inflammation has been shown to induce leukocyte aggregation, chemotaxis and degranulation of polymorphonuclear leukocytes (PMN) $\text{in vitro}$. N-f-Met. Leu-Phe, a potent chemotactic factor, has been shown to activate neutrophils to produce chemiluminescence and produce superoxide radicals. The characteristics of the LTB₄-induced degranulation of rabbit neutrophils are strikingly similar to those of the chemotactic factors. Thiols, and in partiicular glutathione, have been shown to have a marked inhibitory effect in clinical assays of superoxide dismutase (SOD) activity, using reactions which are supposedly specific for the superoxide ion. SOD is most frequently assessed by coupling a generator of O₂^? with an indicating scavenger for the radical. The enzyme then competes with the scavenger for the available O₂^? and inhibits the processes being observed, thus, the inhibition serves as a basis for estimation of SOD activity. A method proposed by Misra and Fridovich for the estimation of SOD activity is based on the photo-oxidation of dianisidine sensitised by riboflavin.This assay can be used to classify compounds as either SOD-like or glutathione-like. With a small quantity if LTB₄ and LTD₄, we obtained preliminary results for their effect on the assay (Table 1). They appear to be glutathione-like, i.e., reactive with the free-radical-generating system in preference to a specific reaction with O₂^? and are only slightly less effective than glutathione.Although our results are preliminary it is clear that the leukotrienes are effective as radical scavengers in this reaction. Further studies with two prostaglandis (products of the cycloxygenase pathway) will also be presented.     

H2O2-Induced Cell Death in Human Glioma Cells: Role of Lipid Peroxidation and PARP Activation

underlying mechanism of ROS-induced cell injury remains to be defined. This study was undertaken to examine the role of lipid peroxidation and poly (ADP-ribose) polymerase (PARP) activation in H₂O₂-induced cell death in A172 cells, a human glioma cell line. H₂O₂ induced a dose- and time-dependent cell death. The cell death was prevented by thiols (dithiothreitol and glutathione), iron chelators (deferoxamine and phenanthroline), H₂O₂ scavengers (catalase and pyruvate), and a hydroxyl radical scavenger (dimethylthiourea). Antioxidants N,N-diphenyl-p-phenylenediamine (DPPD) and Trolox had no effect on the H₂O₂-induced cell death. Lipid peroxidation did not increase in human glioma cells exposed to H₂O₂. The PARP inhibitor 3-aminobenzamide prevented the cell death induced by H₂O₂. The PARP activity was increased by H₂O₂ and the H₂O₂ effect was prevented by 3-aminobenzamide, dithiothreitol, and phenanthroline. The ATP depletion induced by H₂O₂ was prevented by catalase, dithiothreitol, phenanthroline, and 3-aminobenzamide, but not by DPPD. These results indicate that the H₂O₂-induced cell death is mediated by PARP activation but not by lipid peroxidation in human glioma cells.     

β3 Integrin Promotes TGF-β1/H2O2/HOCl-Mediated Induction of Metastatic Phenotype of Hepatocellular Carcinoma Cells by Enhancing TGF-β1 Signaling

In addition to being an important mediator of migration and invasion of tumor cells, β3 integrin can also enhance TGF-β1 signaling. However, it is not known whether β3 might influence the induction of metastatic phenotype of tumor cells, especially non-metastatic tumor cells which express low level of β3. Here we report that H₂O₂ and HOCl, the reactive oxygen species produced by neutrophils, could cooperate with TGF-β1 to induce metastatic phenotype of non-metastatic hepatocellular carcinoma (HCC) cells. TGF-β1/H₂O₂/HOCl, but not TGF-β1 or H₂O₂/HOCl, induced β3 expression by triggering the enhanced activation of p38 MAPK. Intriguingly, β3 in turn promoted TGF-β1/H₂O₂/HOCl-mediated induction of metastatic phenotype of HCC cells by enhancing TGF-β1 signaling. β3 promoted TGF-β1/H₂O₂/HOCl-induced expression of itself via positive feed-back effect on p38 MAPK activation, and also promoted TGF-β1/H₂O₂/HOCl-induced expression of α3 and SNAI2 by enhancing the activation of ERK pathway, thus resulting in higher invasive capacity of HCC cells. By enhancing MAPK activation, β3 enabled TGF-β1 to augment the promoting effect of H₂O₂/HOCl on anoikis-resistance of HCC cells. TGF-β1/H₂O₂/HOCl-induced metastatic phenotype was sufficient for HCC cells to extravasate from circulation and form metastatic foci in an experimental metastasis model in nude mice. Inhibiting the function of β3 could suppress or abrogate the promoting effects of TGF-β1/H₂O₂/HOCl on invasive capacity, anoikis-resistance, and extravasation of HCC cells. These results suggest that β3 could function as a modulator to promote TGF-β1/H₂O₂/HOCl-mediated induction of metastatic phenotype of non-metastatic tumor cells, and that targeting β3 might be a potential approach in preventing the induction of metastatic phenotype of non-metastatic tumor cells.     

Carbamoylated free amino acids in uremia: HOCl generates volatile protein modifying and cytotoxic oxidant species from N-carbamoyl-threonine but not threonine

N-carbamoylation is the non-enzymatic reaction of cyanate with amino groups. Due to urea-formed cyanate in uremic patients beside carbamoylated proteins also free amino acid carbamoylation has been detected, a modification which has been linked to disturbed protein synthesis as NH₂-derivatisation interferes with peptide bond formation. HOCl the product of the activated MPO/H₂O₂/Cl⁻ system is known to react with the NH₂-group of free amino acids to form chloramines which could exert some protective effect against protein modification and cytotoxicity induced by HOCl. As N-carbamoylation may inhibit formation of chloramines we have used N-carbamoyl-threonine as a model amino acid to study its ability to limit the reactivity of HOCl with proteins (LDL and human serum albumin) and cells (THP-1 monocytes and coronary artery endothelial cells). The data indicate that N-carbamoylation completely abolished the protein- and cell-protective effect of threonine against HOCl attack. In contrast to threonine the reaction of HOCl with carbamoyl-threonine resulted in the formation of volatile oxidant species with protein modifying and cytotoxic potential. The volatile lipophilic inorganic monochloramine (NH₂Cl) was identified as a breakdown product of this reaction.     

Dissociation of DNA double strand by hypohalous acids

Abstract

Calf thymus DNA was treated with authentic HOCl, and hypohalous acid-generating systems. This caused a decrease in fluorescence of ethidium-DNA complexes when ethidium bromide was subsequently added to the DNA. The fluorescence continued to decrease up to 30 min after adding HOCl. Loss in fluorescence was proportional to the concentration of HOCl and was complete when a 3-fold excess of HOCl was added to the DNA. No significant decrease in the fluorescence was observed when the chlorination was carried out in the presence of a concentration of monochlorodimedone (MCD) equivalent to that of HOCl. MCD is known to react stoichiometrically with HOCl. The decrease in fluorescence was completely inhibited by H₂O₂, ascorbate and glutathione (GSH). We have estimated the rate constant for the reaction of HOCl with H₂O₂ to be 1–2×10⁵ M^-1s^-1. When compared with authentic HOCl, HOCl-generating systems (Cl^-+H₂O₂+MPO or chloroperoxidase) were found to be inefficient in damaging DNA. This result most likely arises because the rate constant for reaction of HOCl with H₂O₂ is about 1000-fold faster than that for the reaction with DNA. HOBr and HOI generating systems also had a limited ability to damage DNA. We conclude that good chlorine acceptors and antioxidants protect DNA from hypohalous acid-induced oxidative damage.     

Formation of cholesterol ozonolysis products through an ozone-free mechanism mediated by the myeloperoxidase-H2O2-chloride system

The presence of the cholesterol ozonolysis products, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (atheronal-A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (atheronal-B) in human atherosclerotic tissues was recently reported as evidence for the generation of ozone by activated human neutrophils. However, the mechanism for the formation of atheronals in atherosclerotic tissues is unknown. In this study, we found that atheronals were formed by the reaction of cholesterol with human myeloperoxidase (MPO) in the presence of its substrates H₂O₂ and Cl⁻. The omission of either H₂O₂ or Cl⁻ from the MPO-H₂O₂-Cl⁻ system resulted in a significant reduction in yields. The formation of atheronals by the MPO-H₂O₂-Cl⁻ system was inhibited by an inhibitor of MPO and scavengers of reactive oxygen species such as sodium azide, methionine, β-carotene, and vinylbenzoic acid. Our results suggest that MPO produces atheronals at least partly through an ozone-free mechanism, via the reaction of cholesterol with singlet oxygen generated from HOCl and H₂O₂.     

A demonstration that o2− is a crucial intermediate in the high quantum yield luminescence of luminol

The chemiluminescence of luminol, due to its reaction with alkaline H₂O₂, is inhibited by Superoxide dismutase or by hydroxyl radical scavengers. Hematin markedly enhances this H₂O₂-induced luminescence of luminol and lessens, but does not eliminate, the sensitivity towards these inhibitors. Reaction mechanisms are proposed to account for these results. Since luminol luminescence depends upon a reaction between the luminol radical and O₂⁻, and since the luminol radical can reduce dioxygen to O₂⁻, Superoxide dismutase-inhibitable luminol luminescence cannot be reliably used as a detector of O₂⁻ production.     

The free amino acid tyrosine enhances the chlorinating activity of human myeloperoxidase

A key function of neutrophil myeloperoxidase (MPO) is the synthesis of hypochlorous acid (HOCl), a potent oxidizing agent that plays a cytotoxic role against invading bacteria and viruses at inflammatory sites and in phagosomes. MPO displayed a chlorinating activity preferably at acidic pH but at neutral pH MPO catalyzes mainly reactions of the peroxidase cycle. In the present work effects of tyrosine on the chlorinating activity of MPO were studied. At pH 7.4 we detected an increased HOCl production in the presence of tyrosine not only by the MPO-H₂O₂-Cl^- system but also in suspensions of zymosan-activated neutrophils. An excess of H₂O₂ is known to cause an accumulation of compound II of MPO blocking the generation of HOCl at neutral pH. As evidenced by spectral changes, tyrosine-induced activation of MPO to synthesize HOCl was due to the ability of tyrosine to reduce compound II back to the native state, thus accelerating the enzyme turnover. MPO-induced oxidation of tyrosine is relevant to what can be in vivo; we detected MPO-catalyzed formation of dityrosine in the presence of plasma under experimental conditions when tyrosine concentration was about three magnitudes of order less than the Cl⁻ concentration. At acidic pH formation of compound II was impaired in the presence of chloride and dityrosine couldn't be detected in plasma. In conclusion, the ability of tyrosine to increase the chlorinating activity of MPO at neutral pH and enhanced values of H₂O₂ may be very effective for the specific enhancement of HOCl production under acute inflammation.     

Inhibition of myeloperoxidase- and neutrophil-mediated oxidant production by tetraethyl and tetramethyl nitroxides

The powerful oxidant HOCl (hypochlorous acid and its corresponding anion, ⁻OCl) generated by the myeloperoxidase (MPO)–H₂O₂–Cl⁻ system of activated leukocytes is strongly associated with multiple human inflammatory diseases; consequently there is considerable interest in inhibition of this enzyme. Nitroxides are established antioxidants of low toxicity that can attenuate oxidation in animal models, with this ascribed to superoxide dismutase or radical-scavenging activities. We have shown (M.D. Rees et al., Biochem. J. 421, 79–86, 2009) that nitroxides, including 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl radical), are potent inhibitors of HOCl formation by isolated MPO and activated neutrophils, with IC₅₀ values of ~1 and ~6 µM respectively. The utility of tetramethyl-substituted nitroxides is, however, limited by their rapid reduction by biological reductants. The corresponding tetraethyl-substituted nitroxides have, however, been reported to be less susceptible to reduction. In this study we show that the tetraethyl species were reduced less rapidly than the tetramethyl species by both human plasma (89–99% decreased rate of reduction) and activated human neutrophils (62–75% decreased rate). The tetraethyl-substituted nitroxides retained their ability to inhibit HOCl production by MPO and activated neutrophils with IC₅₀ values in the low-micromolar range; in some cases inhibition was enhanced compared to tetramethyl substitution. Nitroxides with rigid structures (fused oxaspiro rings) were, however, inactive. Overall, these data indicate that tetraethyl-substituted nitroxides are potent inhibitors of oxidant formation by MPO, with longer plasma and cellular half-lives compared to the tetramethyl species, potentially allowing lower doses to be employed.