Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.     

Enzymatic production of hydrogen peroxide in a non-aqueous environment

Enzymatic production of hydrogen peroxide in organic solvents has been demonstrated using immobilizedPichia pastoris alcohol oxidase. Enzyme life was shown to be independent of the solvent used; increasing the solvent polarity resulted in higher levels of hydrogen peroxide production.     

In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice

In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria‐targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro‐apoptotic and pro‐inflammatory redox signaling pathways.     

Superoxide and hydrogen peroxide production by macrophages of New Zealand black mice

Resident peritoneal macrophages from New Zealand Black (NZB) mice release O2- and H2O2 after adherence to a plastic surface without any chemical or particulate stimulant. This phenomenon is age dependent and more pronounced in animals with sever autoimmune disease. Significant differences were observed between the high and low breakage NZB sublines (HB and LB), which were previously developed by selective matings on the basis of chromosome breakage rates. The LB subline differs significantly from the HB subline with respect to autoimmune hemolytic anemia and tumor incidence. When the macrophages were stimulated with the tumor promoter TPA, the number of "responders" was higher in the HB than in the LB subline and correlated with the degree of splenomegaly, that is, with the severity of the disease. A negative response to agonist stimulation and very low spontaneous production of active oxygen species was observed in NZW and Swiss mice, which is the normal finding for resident macrophages according to data from the literature. The increased superoxide and hydrogen peroxide production by macrophages of NZB mice is discussed with respect to autoimmune disease and cancer.     

The cellular production of hydrogen peroxide

1. The enzyme–substrate complex of yeast cytochrome c peroxidase is used as a sensitive, specific and accurate spectrophotometric H₂O₂ indicator. 2. The cytochrome c peroxidase assay is suitable for use with subcellular fractions from tissue homogenates as well as with pure enzyme systems to measure H₂O₂ generation. 3. Mitochondrial substrates entering the respiratory chain on the substrate side of the antimycin A-sensitive site support the mitochondrial generation of H₂O₂. Succinate, the most effective substrate, yields H₂O₂ at a rate of 0.5nmol/min per mg of protein in state 4. H₂O₂ generation is decreased in the state 4→state 3 transition. 4. In the combined mitochondrial–peroxisomal fraction of rat liver the changes in the mitochondrial generation of H₂O₂ modulated by substrate, ADP and antimycin A are followed by parallel changes in the saturation of the intraperoxisomal catalase intermediate. 5. Peroxisomes supplemented with uric acid generate extraperoxisomal H₂O₂ at a rate (8.6–16.4nmol/min per mg of protein) that corresponds to 42–61% of the rate of uric acid oxidation. Addition of azide increases these H₂O₂ rates by a factor of 1.4–1.7. 6. The concentration of cytosolic uric acid is shown to vary during the isolation of the cellular fractions. 7. Microsomal fractions produce H₂O₂ (up to 1.7nmol/min per mg of protein) at a ratio of 0.71–0.86mol of H₂O₂/mol of NADP⁺ during the oxidation of NADPH. H₂O₂ is also generated (6–25%) during the microsomal oxidation of NADH (0.06–0.025mol of H₂O₂/mol of NAD⁺). 8. Estimation of the rates of production of H₂O₂ under physiological conditions can be made on the basis of the rates with the isolated fractions. The tentative value of 90nmol of H₂O₂/min per g of liver at 22°C serves as a crude approximation to evaluate the biochemical impact of H₂O₂ on cellular metabolism.     

Detection of hydrogen peroxide production in the isolated rat lung using Amplex red

Abstract

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (H₂O₂) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured H₂O₂ using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. AR’s highly fluorescent oxidation product resorufin was measured in the perfusate. Experiments were carried out without and with rotenone (complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), antimycin A (complex III inhibitor), potassium cyanide (complex IV inhibitor), or diohenylene iodonium (inhibitor of flavin-containing enzymes, e.g. NAD(P)H oxidase or NOX) added to the perfusate. We also evaluated the effect of acute changes in oxygen (O₂) concentration of ventilation gas on lung rate of H₂O₂ release into the perfusate. Baseline lung rate of H₂O₂ release was 8.45?±?0.31 (SEM) nmol/min/g dry wt. Inhibiting mitochondrial complex II reduced this rate by 76%, and inhibiting flavin-containing enzymes reduced it by another 23%. Inhibiting complex I had a small (13%) effect on the rate, whereas inhibiting complex III had no effect. Inhibiting complex IV increased this rate by 310%. Increasing %O₂ in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O₂-dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial H₂O₂, and that most of acute hyperoxia-enhanced lung rate of H₂O₂ release is from nonmitochondrial rather than mitochondrial sources.     

Relationship of hydrogen peroxide production by Mycoplasma pulmonis to virulence for catalase-deficient mice

Mycoplasma pulmonis, an etiological agent of murine pneumonia, produced about 0.065 mumoles of hydrogen peroxide (H(2)O(2)) per hr per 10(10) colony-forming units. When glucose was present at a concentration of 0.01 m, H(2)O(2) production was increased by 50%. To determine if H(2)O(2) production by M. pulmonis could be correlated with virulence, normal, acatalasemic, and acatalatic mice were infected with the organism. Three days after infection with M. pulmonis significantly more acatalatic mice had pneumonia than did normal or acatalasemic mice. The pneumonia in acatalatic mice was also more severe than in the other two groups. Five days after infection, pneumonia in the acatalatic mice was resolved, whereas normal mice were severely affected. The presence of pneumonia and the severity were correlated with the recovery of M. pulmonis from the lesions. In vitro studies of the effect of catalase on M. pulmonis showed that exogenously supplied catalase stimulated the growth of M. pulmonis at 37 C and prolonged its survival at 25 C. Hemolysis of sheep blood, guinea pig blood, rabbit blood, and normal and acatalasemic mouse blood by M. pulmonis was inversely related to the catalase activity of the erythrocytes. These findings suggest that H(2)O(2) secretion contributes to the virulence of M. pulmonis and to the death of the microorganism in the absence of host catalase.     

Ammonium-dependent hydrogen peroxide production by mitochondria

NADH-supported generation of H2O2 by permeabilized rat heart mitochondria was partially prevented by the specific complex I-directed inhibitor, NADH-OH, and was significantly stimulated by ammonium. Ammonium did not affect H2O2 production by complex I in coupled submitochondrial particles. The soluble mitochondrial matrix protein fraction catalyzed NADH-dependent H2O2 production, which was greatly (approximately 10-fold) stimulated by ammonium. We conclude that complex I is not the major contributor to mitochondrial superoxide (hydrogen peroxide) generation and that there are specific ammonium-sensitive NADH:oxygen oxidoreductase(s) in the mitochondrial matrix which are responsible for mitochondrial H2O2 production.     

Spectrofluorometric analysis of hydrogen peroxide

The pH of maximum fluorescence (above pH 7) and the optimal excitation and emission wavelengths (468 nm and 519 nm, respectively) were determined for 2′,7′-dichlorofluorescein (DCF). The stoichiometry after hydrolysis of the oxidation of the stable nonfluorescent compound 2′,7′-dichlorofluorescin diacetate (LDADCF) was determined and found to be 2 moles of DCF produced per mole of hydrogen peroxide used.     

Enzymatic production of hydrogen peroxide and acetaldehyde in a pressure reactor

Alcohol oxidase, an enzyme which exhibits relatively weak substrate specificity among short chain alcohols, forms the corresponding aldehyde and hydrogen peroxide as coproduct. The ability of alcohol oxidase from Pichia pastoris yeast to convert ethanol to acetaldehyde and hydrogen peroxide was examined in an oxygen pressure reactor under conditions, such that oxygen availability was sufficient to permit rapid catalysis. Hydrogen peroxide levels of approximately 1.8/M (6% w/w) were attained in 2-3 h with 2.8 muM enzyme, corresponding to a productivity of approximately 30 g peroxide/g enzyme. Optimal conditions (within equipment limitations) were 900 psi oxygen, 2.6M ethanol, at 4 degrees C. Similar levels of products were reached in the reactor using enzyme immobilized covalently on controlled pore glass and noncovalently on an anion exchange support. Recycle of covalently immobilized enzyme was not possible as a result of enzyme inactivation after a single run. Limited recycle of noncovalently immobilized enzyme was accomplished with substantial decreases in levels of product attainable on each cycle.     

Fatty acid dependent hydrogen peroxide production in Lactobacillus

Lactobacillus leichmanii growing in complex medium supplemented with decanoic acid accumulated high concentrations of hydrogen peroxide in the culture. The H2O2-generating system was specifically induced by one of the saturated fatty acids from 4:0 to 16:0 or oleic acid. The induction of this system was associated with the presence of a fatty acyl-CoA-dependent H2O2-generating activity in the cell-free extracts. This activity is shown for the first time in a procaryote organism.     

Detecting hydrogen peroxide in leaves in vivo – a comparison of methods

Four hydrogen peroxide detecting probes, 3,3′‐diaminobenzidine (DAB), Amplex Red (AR), Amplex Ultra Red (AUR) and a europium–tetracycline complex (Eu₃Tc) were infiltrated into tobacco leaves and tested for sensitivity to light, toxicity, subcellular localization and capacity to detect H₂O₂ in vivo. In the absence of leaves, in water solutions, AUR was very much sensitive to strong light, AR showed slight light sensitivity, while DAB and Eu₃Tc were insensitive to irradiation. When infiltrated into the leaves, the probes decreased the photochemical yield (Φ_PSII) in the following order of effect AR > DAB > AUR > Eu₃Tc. With the exception of Eu₃Tc, all probes stimulated the build‐up of non‐photochemical quenching either temporally (DAB, AUR) or permanently (AR), showing that their presence may already limit the photosynthetic capacity of leaves, even in the absence of additional stress. This should be taken into account when using these probes in plant stress experiments. Confocal laser scanning microscopy studies with the three fluorescent H₂O₂ probes showed that the localizations of Eu₃Tc and AUR were mainly intercellular. AR partly penetrated into leaf chloroplasts but probably not into the thylakoid membranes. Photosynthesis‐related stress applications of AR seem to be limited by the low availability of internal leaf peroxidases. Applications of AR for kinetic H₂O₂ measurements would require a co‐infiltration of external peroxidase, imposing another artificial modifying factor and thus taking experiments further from ideal, in vivo conditions. Our results suggest that the studied H₂O₂ probes should be used in leaf studies with caution, carefully balancing benefits and artifacts.     

The production of hydroxyl radical from hydrogen peroxide

The formation of hydroxyl radical (OH·) from the oxidation of glutathione, ascorbic acid, NADPH, hydroquinone, catechol, and riboflavin by hydrogen peroxide was studied using a range of enzymes and copper and iron complexes as possible catalysts. Copper-1,10-phenanthroline appears to catalyze the production of OH· from hydrogen peroxide without superoxide radical being formed as an intermediate, and without the involvement of a catalyzed Haber-Weiss (Fenton) reaction. Superoxide radical is involved, however, in the Cu²⁺ -catalyzed decomposition of hydrogen peroxide, and in the oxidation of glutathione by atmospheric oxygen. For this latter oxidation, copper-4,7-dimethyl-1,10-phenanthroline was found to be a much more effective catalyst than the copper complex of 1,10-phenanthroline, which is normally used. Mechanisms for these reactions are proposed, and the toxicological significance of the ability of a variety of biological reductants to provide a prolific source of OH· when oxidized by hydrogen peroxide is discussed.     

Sodium acetate enhances hydrogen peroxide production in Weissella cibaria

Aims:  To investigate hydrogen peroxide production by lactic acid bacteria (LAB) and to determine the key factors involved.
Methods and Results:  Six strains of Weissella cibaria produced large amounts (2·2–3·2 mmol l⁻¹) of hydrogen peroxide in GYP broth supplemented with sodium acetate, but very low accumulations in glucose yeast peptone broth without sodium acetate. Increased production of hydrogen peroxide was also recorded when strains of W. cibaria were cultured in the presence of potassium acetate, sodium isocitrate and sodium citrate. Oxidases and peroxidases were not detected, or were present at low levels in W. cibaria . However, strong nicotinamide adenine dinucleotide (NADH) oxidase activity was recorded, suggesting that the enzyme plays a key role in production of hydrogen peroxide by W. cibaria .
Conclusions:  Weissella cibaria produces large quantities of hydrogen peroxide in aerated cultures, in a process that is dependent on the presence of acetate in the culture medium. NADH oxidase is likely the key enzyme in this process.
Significance and Impact of the Study:  This is the first study showing that sodium acetate, normally present in culture media of LAB, is a key factor for hydrogen peroxide production by W. cibaria . The exact mechanisms involved are not known.     

Effects of hydrogen peroxide in a keratinocyte-fibroblast co-culture model of wound healing

Recently, there has been renewed interest in the role of reactive oxygen species (ROS), especially H(2)O(2), in wound healing. We previously showed that H(2)O(2) stimulates healing in a keratinocyte scratch wound model. In this paper, we used a more complex and physiologically relevant model that involves co-culturing primary keratinocytes and fibroblasts. We found that the two main cell types within the skin have different sensitivities to H(2)O(2) and to the widely used "antioxidant"N-acetyl-l-cysteine (NAC). Keratinocytes were very resistant to the toxicity of H(2)O(2) (250 and 500 μM) or NAC (5 mM). However, the viability of fibroblasts was decreased by both compounds. Using the co-culture model, we also found that H(2)O(2) increases re-epithelialization while NAC retards it. Our data further illustrate the possible role of ROS in wound healing and the co-culture model should be useful for screening agents that may influence the wound healing process.     

Genes associated with leukocyte production in mice.

We have investigated the genes influencing leukocyte production in mice by selective breeding for differences in leukocyte count and developed two major lines differing about 8-fold. In these lines we have made counter selective breeding, crossbreeding between them, and developed methods of using marker genes to search for individual genes distributed on different chromosomes. We have revealed an overall dominance of genes for low leukocyte count over those for high counts, and effects of genes at the agouti locus, certain mutant genes, and a gene or genes in the XVI linkage group. We have commented on the application and approach of quantitative genetic experimentation from both a theoretical and practical point of view and, on the basis of present results, discussed the nature of polygenes in relation to genes causing polymorphisms.     

Cytochemical localization of hydrogen peroxide production in the rat uterus

A reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent H2O2-generating activity of the rat uterus was investigated both electron cytochemically and biochemically. We tried to cytochemically demonstrate H2O2 generation from the oxidation of reduced NADH or NADPH using the cerium method. NADPH oxidation resulted in electron-dense deposits on the apical plasma membrane covering the microvilli of the surface epithelium of the lightly fixed endometrium. In control specimens incubated in a medium from which substrate was omitted, no such deposits were observed. The reduction of ferricytochrome c due to NADH oxidation was spectrophotometrically detected in the lightly fixed uterus. Absorption at 550 nm increased with the addition of NADH, but not with that of NAD. The reaction was weakened by preheating and adversely affected by the addition of superoxide dismutase, but it was not inhibited by adding 50 mM sodium azide. These results suggest that a kind of NAD(P)H oxidase, generating H2O2 via superoxide formation, may possibly be present on the apical plasma membrane of the rat endometrial epithelium.     

Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.     

Reversibility of the ionomycin promoted synaptosomal hydrogen peroxide production

We previously reported on the release of hydrogen peroxide from guinea pig cerebral cortex synaptosomes (13). An important finding was that in glutathione depleted synaptosomes a linear release of hydrogen peroxide is rapidly induced on addition of the Ca++ -ionophore ionomycin (in the presence of Ca++) or upon depolarization of the plasma membrane. We report here that the ionomycin induced hydrogen peroxide is reversed following the addition of bovine serum albumin which strongly binds the ionophore, to be reactivated by further addition of excess ionomycin, or of the depolarizing agent KC1. Similarly, the effect of ionomycin is removed on decreasing the concentration of free Ca++. Bovine serum albumin, which counteracts the effect of ionomycin on the release of H2O2, also counteracts the effect of the ionophore on the movements of Ca++ and the release of gamma-aminobutyrate. These findings support the idea that the synaptosomal production of H2O2 is a carefully controlled important physiological event.