The quantitative measurement of H2O2 generation in isolated mitochondria

Adequate methods to measure the rate of mitochondrial oxygen radical generation are needed since oxygen radicals are involved in many pathologies. A fluorometric method appropriate to measure the rate of generation of H₂O₂ in intact mitochondria is described. Just after isolation of functional mitochondria from fresh tissues, rates of generation of H₂O₂ are kinetically measured by fluorometry in the presence of homovanillic acid and horseradish peroxidase. The method is specific for H₂O₂ and is sensitive enough to assay mitochondrial H₂O₂ generation in the presence of respiratory substrate without inhibitors of the respiratory chain. Simultaneous measurement of mitochondrial oxygen consumption allows calculation of the free radical leak: the percentage of electrons out of sequence which reduce oxygen to oxygen radicals along the mitochondrial respiratory chain instead of reducing oxygen to water at the terminal cytochrome oxidase. The method shows instantaneous response to H₂O₂. This makes it appropriate to study the quick effects of different inhibitors and modulators on the rate of mitochondrial oxygen radical production. Its application to the localization of the sites where caloric restriction decreases mitochondrial oxygen radical generation in heart mitochondria is described.     

Oxidation products of acylated anthocyanins under acidic and neutral conditions

Acylated anthocyanidin-3,5-diglucosides are oxidized with H₂O₂ under acidic conditions to acylated ortho-benzoyloxyphenylacetic acid esters. When the same reaction is carried out under neutral conditions, the reaction product is the 3-O-acyl-glucosyl-5-O-glucosyl-7-hydroxy coumarin.     

Atherogenic levels of low-density lipoprotein increase hydrogen peroxide generation in cultured human endothelial cells: Possible mechanism of heightened endocytosis

Cultured human umbilical vein endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels have augmented reactive oxygen species generation. Confluent EC were incubated with 30–330 mg/dl LDL cholesterol and cellular hydrogen peroxide (H₂O₂) generation measured. EC incubated with 30 and 90 mg/dl LDL cholesterol showed similar low level H₂O₂ production. In contrast, EC exposed to 180 and 330 mg/dl LDL cholesterol have a marked, dose-related elevation in H₂O₂ generation. Subsequent studies have explored if direct EC exposure to H₂O₂ promotes cellular functional changes similar to those induced by high LDL levels (> 160 mg/dl cholesterol). Confluent EC were incubated with 0.1–10 mM H₂O₂ for 30 minutes and endocytosis measured and cytoskeletal structure examined. H₂O₂ exposure (0.5 and 1 mM) promoted heightened EC endocytosis, which similarly occurs with high LDL exposure. Likewise, cytoskeletal examination of EC perturbed with 1 mM H₂O₂ reveals structural remodeling with a marked increase in stress fibers, which similarly happens with high LDL levels. The above observations that high LDL levels cause increased EC H₂O₂ production, and direct H₂O₂ exposure promotes cellular functional changes similar to those induced by high LDL concentrations, suggest a modulatory role for reactive oxygen species. Thus LDL-induced reactive oxygen species generation may contribute mechanistically to endothelial perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis. © 1996 Wiley-Liss, Inc.     

Hydrogen peroxide production by roots and its stimulation by exogenous NADH

H₂O₂ production by roots of young seedlings was monitored using a non-destructive in vivo assay at pH 5.0. A particularly high rate of H₂O₂ production was measured in the roots of soybean (Glycine max L. cv. Labrador) seedlings which were used for further investigation of the physiological and enzymological properties of apoplastic H₂O₂ production. In the soybean root H₂O₂ production can be stimulated 10-fold by exogenous NADH or NADPH. This response displays typical features of a peroxidase-catalyzed oxidase reaction using NAD(P)H as electron donor for the reduction of O₂ to H₂O₂. Comparative measurements showed that the NADH-induced H₂O₂ production of the roots resembles the H₂O₂-forming activity of horseradish peroxidase with respect to NADH and O₂ concentration requirements and sensitivity to inhibition by KCN, NaN₃, superoxide dismutase and catalase. NADH-induced H₂O₂ production can be observed with similar intensity in all regions of the root, in agreement with the distribution of apoplastic peroxidase activity. In contrast, the activity responsible for the basal H₂O₂ production in the absence of exogenous NADH was mainly confined to a short subapical zone of the root and differs from the NADH-induced reaction by insensitivity to inhibition by superoxide dismutase and a strikingly lower requirement for O₂. It is concluded that the basal H₂O₂ production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O₂^?-producing oxidase.     

Free fatty acid determination by peroxidase catalysed luminol chemiluminescence

A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl-CoA oxidase. The H₂O₂ formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C₁₀-C₁₈) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H₂O₂ standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase-luminol reaction.     

Determination of free cholesterol based on a novel flow‐injection chemiluminescence method by immobilizing enzyme

A novel flow injection chemiluminescence method is proposed for determination of cholesterol in this paper. The cholesterol oxidase was immobilized onto sol–gel and prepared as an enzymatic reaction column. The determination of cholesterol was performed by quantitative determination of hydrogen peroxide produced from an enzymatic reaction. The luminol–H₂O₂–metal chelate diperiodatocuprate(III) system ensured that the method was highly sensitive and selective. Free cholesterol was determined over the range 5.0 × 10^–8 mol/L–5.0 × 10^–7 mol/L, with a limit of detection (3σ) of 1.9 × 10^–8 mol/L. The relative standard deviation (RSD) for 2.5 × 10^–7 mol/L was 2.7% (n = 7). The proposed method offered the advantages of sensitivity, selectivity, simplicity and rapidity for free cholesterol determination, and was successfully applied to the direct determination of free cholesterol in serum. Copyright © 2008 John Wiley & Sons, Ltd.     

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.     

Aquopentacyanoferrate (II): an effective probing electron donor in the conversion of oxyhemoglobin to methemoglobin and peroxide.

HbA O₂ reacts readily with Fe^II(CN)₅H₂O^3? to form aquometHb and peroxide via a second order process: rate=k[HbO₂][Fe^II(CN)₅H₂O^3?]. A slight enchancement in the rate of metHb formation due to the H₂O₂ produced can be prevented by addition of catalase. The reaction is free from complications exhibited by other reductants. The hexacyanide, ferrocyanide, reacts with HbA O₂ but at only ca. 0.02% the rate and with formation of cyanometHb. Reductants such as phenols and sulfa drugs may produce radicals that can enter into side reactions. Fe^II(CN)₅H₂O^3? shows promise as an effective probing reagent for the characterization of H₂O₂ production from oxygenated heme and other proteins.     

An EPR Investigation of Human Methaemoglobin Oxidation by Hydrogen Peroxide: Methods to Quantify all Paramagnetic Species Observed in the Reaction

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglabin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H₂O₂ was added at 3- and 10-fold molar excess to 100 μM metHb in 50 mM phosphate buffer, pH 7.4, 37°C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H₂O₂ the EPR signals (- 196°C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (- 196°C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g° = 2.035 and g° = 2.0053), and the other an isotropic singlet (g = 2.0042, AH = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic Pehaviour of both peroxyl (ROO^*) and nonperoxyl (P^*) free radicals were similar, we concluded that ROO^* radicals are not formed from P^* radicals by addition of O₂. The time courses for both radicals showed a steady state during the time required for H₂O₂ to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 ± 10^-3 s^-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H₂O₂. The formation of the rhombic Fe(III) non-haemcentres with g = 4.35 was found. Their yield was proportional to the H₂O₂ concentration used and the centers were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at —196°C were used.     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

THE OXIDATION OF SODIUM LACTATE BY HYDROGEN PEROXIDE

By means of a modification of the technique of the Osterhout apparatus it is possible to follow the production of CO₂ from sodium lactate when acted upon by H₂O₂. The results of this process indicate that the reaction is not a simple one but is of an autocatalytic type. This conclusion is borne out by the fact that the determinations of H₂O₂ during the reaction show an increased amount of peroxide during the earlier stages of the reaction. This is considered to be due to the formation of a peroxide by the oxidation of the acetaldehyde (formed by the interaction of H₂O₂ and sodium lactate) with the oxygen of the air. When the reaction is carried out in an atmosphere of nitrogen no increase is observed. Further experiments in nitrogen tend to show that acetaldehyde is the end-product of the action of H₂O₂ alone. The effect of FeCl₃ upon the reaction depends upon the previous treatment of the iron salt. If the iron solution is added to the H₂O₂ before mixing with the lactate there is an increased amount of CO₂. If, however, the iron is added to the lactate before the addition of the peroxide, the action tends to inhibit the production of CO₂. The reaction of H₂O₂ with sodium lactate is comparable to the action of killed yeast and methylene blue as determined by Palladin and his coworkers.     

Is functional manganese involved in hydrogen-peroxide-stimulated anomalous oxygen evolution in CACl2-washed photosystem II membranes?

When detergent-derived photosystem II (PSII) membranes are treated with CaCl₂ to remove the three extrinsic proteins associated with the O₂-evolving complex, the resulting membranes (CaPSII) can still catalyze water oxidation if sufficient Ca²⁺ and Cl^- are present. When CaPSII membranes are exposed to single turnover flashes on an O₂ rate electrode, anomalous O₂ is produced by the first two flashes. The addition of catalase to the membrane suspension completely inhibits O₂ produced by the first two flashes, but not by subsequent flashes. Exogenous H₂O₂ stimulates anomalous O₂ production by the first few flashes in CaPSII membranes, but not in control PSII membranes. Diuron (DCMU) does not inhibit H₂O₂-stimulated O₂ production by the first flash. However, it does inhibit the O₂ yield of all subsequent flashes, indicating that all flash-induced O₂ signals in CaPSII membranes are dependent on photosystem II electron transport. H₂O₂ stimulation of O₂ yields is inhibited in Tris-, heat-, and EDTA-(ethylenediaminetetraacetic acid)-treated CaPSII. In the presence of high salt, H₂O₂ (but not EDTA) treatment of CaPSII, extracts Mn functional in normal photosynthetic O₂ evolution. The addition of exogenous Mn²⁺ reconstitutes anomalous O₂ production in Tris-and H₂O₂/EDTA-treated CaPSII preparations but only in the presence of H₂O₂. Anomalous H₂O₂-stimulated O₂ production can be observed both with a Clark electrode (steady state) and an O₂ rate electrode (flash sequence). The mechanism involves electron donation from H₂O₂, mediated by free Mn²⁺, to PSII, and the 33-kDa extrinsic protein under some conditions can block this process. Since H₂O₂ can remove functional Mn from CaPSII membranes, its presence can convert functional Mn to the Mn²⁺ mediator state required for anomalous O₂ production. EDTA binds Mn in CaPSII disrupted by H₂O₂ and prevents anomalous O₂ evolution.Abbreviations CaPSII a PSII preparation washed with approximately 1M CaCl₂ - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EDTA ethylenediaminetetraacetic acid - MES 2-[N-morpholino]-ethanesulfonic acid - PSII a detergent-derived photosystem II membrane preparation - RC reaction center - Tris tris(hydroxymethyl)-aminomethane - Y_n oxygen rate electrode flash yield resulting from the nth flash of a sequence of single turnover flashes of light Operated by the Midwest Research Institute for the U.S. Department of Energy under contract DE-AC02-83CH10093.     

Salinity-induced subcellular accumulation of H2O2 in leaves of rice

The localization of salt-induced H₂O₂ accumulation in the leaves of rice was examined using 3,3-diaminobenzidine and CeCl₃ staining at ultrastructure level. When the 3-week-old rice plants were affected by 100 mM NaCl for 14 days, the swelling of thylakoids and the destruction of thylakoid membranes were observed. H₂O₂ accumulation was also observed in the chloroplast of the leaf treated with NaCl. The electron dense products of 3,3-diaminobenzidine and CeCl₃ were mainly observed especially around the swelling of thylakoids. H₂O₂ accumulation and any ultrastructural changes were not observed in the chloroplasts under dark condition. Furthermore, treatment with ascorbic acid suppressed both H₂O₂ accumulation and the changes in chloroplast ultrastructure. These results suggest that light-induced production of excess H₂O₂ under salinity is responsible for the changes in chloroplast ultrastructure. H₂O₂ accumulation was also observed in the mitochondria, peroxisomes, plasma membrane, and cell walls under light but not dark, suggesting that these organelles are also the source of H₂O₂ and the production is light dependent under salinity.     

Effect of iron and phagocytosis on murine macrophage activation in vitro

Iron-exposed murine macrophages have a modified bactericidal activity as shown by previous observations. In order to assess the role of iron in macrophage activation, as measured by free radical production and by intracellular bacterial killing, murine peritoneal macrophages were cultivated in the presence of various sources of iron, human iron-saturated transferrin and ammonium ferric citrate, or iron chelators, Desferal, and human Apo-transferrin, and were infected with an enteropathogenic strain ofE. coli. The release of nitrite (NO₂ ^?), and the production of superoxide anion (O₂ ^?) and hydrogen peroxide (H₂O₂) by the phagocytes were measured and compared to the production by uninfected macrophages. The synergistic action with murine r.IFN-γ was also studied in the radical production reaction and for the bactericidal activity of macrophages. Our results show that in vitro phagocytosis ofE. coli induced elevated production of NO₂ ^? and H₂O₂ by macrophages, and that oxygen derivatives were released independently of the presence of added iron or chelator. Despite a phagocytosis-related enhancement of NO₂ ^? release, reactive nitrogen intermediates (RNI) are not directly involved in the bactericidal mechanism, as revealed by increased intracellular killing owing to RNI inhibitors. Moreover, bacterial killing may depend on oxygen derivatives, as suggested by the effect of the antioxidant sodium ascorbate leading to both a diminished H₂O₂ production and a decreased bactericidal activity of macrophages.     

Analysis of the lifetime and spatial localization of hydrogen peroxide generated in the cytosol using a reduced kinetic model

Hydrogen peroxide (H₂O₂) acts as a signaling molecule via its reactions with particular cysteine residues of certain proteins. Determining the roles of direct oxidation by H₂O₂ versus disulfide exchange reactions (i.e. relay reactions) between oxidized and reduced proteins of different identities is a current focus. Here, we use kinetic modeling to estimate the spatial and temporal localization of H₂O₂ and its most likely oxidation targets during a sudden increase in H₂O₂ above the basal level in the cytosol. We updated a previous redox kinetic model with recently measured parameters for HeLa cells and used the model to estimate the length and time scales of H₂O₂ diffusion through the cytosol before it is consumed by reaction. These estimates were on the order of one micron and one millisecond, respectively. We found oxidation of peroxiredoxin by H₂O₂ to be the dominant reaction in the network and that the overall concentration of reduced peroxiredoxin is not significantly affected by physiological increases in intracellular H₂O₂ concentration. We used this information to reduce the model from 22 parameters and reactions and 21 species to a single analytical equation with only one dependent variable, i.e. the concentration of H₂O₂, and reproduced results from the complete model. The reduced kinetic model will facilitate future efforts to progress beyond estimates and precisely quantify how reactions and diffusion jointly influence the distribution of H₂O₂ within cells.     

Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: Catalytic requirements and oxygen dependence

The ability of paraquat radicals (PQ^+.) generated by xanthine oxidase and glutathione reductase to give H₂O₂-dependent hydroxyl radical production was investigated. Under anaerobic conditions, paraquat radicals from each source caused chain oxidation of formate to CO₂, and oxidation of deoxyribose to thiobarbituric acid-reactive products that was inhibited by hydroxyl radical scavengers. This is in accordance with the following mechanism derived for radicals generated by γ-irradiation [H. C. Sutton and C. C. Winterbourn (1984) Arch. Biochem. Biophys.235, 106–115] PQ^+. + Fe³⁺ (chelate) → Fe²⁺ (chelate) + PQ⁺⁺ H₂O₂ + Fe²⁺ (chelate) → Fe³⁺ (chelate) + OH^? + OH.. Iron-(EDTA) and iron-(diethylenetriaminepentaacetic acid) (DTPA) were good catalysts of the reaction; iron complexed with desferrioxamine or transferrin was not. Extremely low concentrations of iron (0.03 μm) gave near-maximum yields of hydroxyl radicals. In the absence of added chelator, no formate oxidation occurred. Paraquat radicals generated from xanthine oxidase (but not by the other methods) caused H₂O₂-dependent deoxyribose oxidation. However, inhibition by scavengers was much less than expected for a reaction of hydroxyl radicals, and this deoxyribose oxidation with xanthine oxidase does not appear to be mediated by free hydroxyl radicals. With O₂ present, no hydroxyl radical production from H₂O₂ and paraquat radicals generated by radiation was detected. However, with paraquat radicals continuously generated by either enzyme, oxidation of both formate and deoxyribose was measured. Product yields decreased with increasing O₂ concentration and increased with increasing iron(DTPA). These results imply a major difference in reactivity between free and enzymatically generated paraquat radicals, and suggest that the latter could react as an enzyme-paraquat radical complex, for which the relative rate of reaction with Fe³⁺ (chelate) compared with O₂ is greater than is the case with free paraquat radicals.     

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.     

Detection of hydrogen peroxide by lactoperoxidase-mediated dityrosine formation

The aim of this work was to study the dityrosine-forming activity of lactoperoxidase (LPO) and its potential application for measuring hydrogen peroxide (H₂O₂). It was observed that LPO was able to form dityrosine at low H₂O₂ concentrations. Since dityrosine concentration could be measured in a simple fluorimetric reaction, this activity of the enzyme was utilized for the measurement of H₂O₂ production in different systems. These experiments successfully measured the activity of NADPH oxidase 4 (Nox4) by this method. It was concluded that LPO-mediated dityrosine formation offers a simple way for H₂O₂ measurement.     

Intracellular catalase function: analysis of the catalatic activity by product formation in isolated liver cells

Intracellular catalase activity was measured in isolated rat hepatocytes by adding H₂O₂ under anaerobic conditions and measuring O₂ evolution. Hydrogen peroxide was introduced either by continuous infusion or by pulse injection. Continuous infusion at a rate similar to the endogenous H₂O₂ production rate provided results that 60–70% of the H₂O₂ was metabolized by the catalatic reaction. Comparison of rates of O₂ evolution to estimated rates of H₂O₂ metabolism obtained by the methanol-titration method (H. Sies and B. Chance, 1970, FEBS Lett.11, 172–176) indicated that the contribution of the peroxidatic reaction of catalase was small. The intracellular activity of glutathione peroxidase was estimated as the catalase-independent metabolism and used to determine the rate of intracellular H₂O₂ metabolism by the peroxidase. The results provide a quantitative basis for analysis of the physiological and toxicological aspects of H₂O₂ metabolism by liver.     

Localization changes of endogenous hydrogen peroxide during cell division cycle of Xanthomonas

Production and localization of endogenous hydrogen peroxide (H₂O₂) were investigated in strains of Xanthomonas by histochemical analysis under electron microscopy. Even though the levels of endogenous H₂O₂ production were different among various strains, the produced H₂O₂ was localized in the cell wall of all Xanthomonas strains tested. The impairment of the level of endogenous H₂O₂ accumulation resulted in a significantly decreased growth rate of bacteria, regardless if the difference of the H₂O₂ level is originally present between wild type strains or caused by mutation of the ahpC gene of Xanthomonas. The endogenous accumulation of H₂O₂ positively correlates with the cell division. Interestingly, the accumulated H₂O₂ was also localized in the mesosome-like structure and nucleoids during the cell division cycle. Furthermore, results revealed quantitative and dimensional changes of H₂O₂ accumulation in the two additional locations. These findings indicated that the additional locations of the accumulated H₂O₂ were closely associated with the process of cell division. Together, these results suggest that the endogenous H₂O₂ production plays an important role in cell proliferation of Xanthomonas.