Intracellular Hydrogen Peroxide Concentration Research Articles

Genetically Encoded Biosensor HyPer as a Tool for Quantification of Intracellular Hydrogen Peroxide Concentrations

This mini-reviewsystematizes information on methods for quantitative assessment of intracellular hydrogenperoxide concentration based on the use of a genetically encodedperoxide sensor HyPer. Two approaches are being considered: 1) calibrationof the biosensor using exogenous hydrogen peroxide, based on assessingthe rate of peroxide penetration into cells and intracellular peroxidaseactivity; 2) direct determination of the intracellular peroxide content, basedon measuring the level of oxidation of the biosensor, theoxidation reaction constant and the reduction reaction constant of HyPerin the cells. The use of these methods makes itpossible to solve a wide range of tasks in cellularredox biology—to determine the range of physiological anddamaging concentrations of hydrogen peroxide in cells, to evaluate theeffectiveness of the antioxidant defense system in various cellular compartmentsunder conditions of oxidative stress, to determine the contribution ofvarious enzymatic systems to the peroxidase activity of cells, andto characterize antioxidant defense systems in various biological contexts (inthe process of cellular senescence, differentiation, reprogramming, during the developmentof pathologies). The described methods can be adapted for othergenetically encoded hydrogen peroxide biosensors.

Ferritin-nanocaged copper arsenite minerals with oxidative stress-amplifying activity for targeted cancer therapy

We report copper(II) arsenite-encapsulated ferritin nanoparticles (CuAS-FNs) as oxidative stress-amplifying anticancer agents. The CuAS-FNs were fabricated through CuAS mineralization in the cavity of the FNs. The formation of crystalline CuAS complex minerals in the FNs was systematically identified using various analytical tools, including X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM)-associated energy-dispersive X-ray spectroscopy (TEM-EDS). The CuAS-FNs showed pH-dependent release behavior, in which the CuAS mineral was effectively retained at physiological pH, in contrast, at lysosomal pH, the CuAS complex was dissociated to release arsenite and Cu2+ ions. At lysosomal pH, the release rate of arsenite (HAsO32-) and Cu2+ ions from the CuAS-FNs more accelerated than at physiological pH. Upon transferrin receptor-1-mediated endocytosis, the CuAS-FNs simultaneously released arsenite and Cu2+ ions in cells. The released arsenite ions can increase the intracellular concentration of hydrogen peroxide (H2O2), with which the Cu2+ ions can elevate the level of hydroxyl radicals (·OH) via Fenton-like reaction. Thus, the CuAS-FNs could target cancer cell through the recognizing ability of FNs and kill cancer cells by amplifying the ·OH level through the synergistic activity of Cu2+ and arsenic ions. Importantly, MCF-7 tumors were effectively suppressed by CuAS-FNs without systemic in vivo toxicity. Therefore, the CuAS-FNs is a promising class of Fenton-like catalytic nanosystem for cancer treatment.

Improve the cytotoxic effects of megavoltage radiation treatment by Fe3O4@Cus–PEG nanoparticles as a novel radiosensitizer in colorectal cancer cells

BackgroundTo enhance the performance of radiotherapy, emerging nanoparticles that can professionally enhance X-ray irradiation to destruct cancer cells are extremely necessary. Here, we examined the potential of PEG-coated magnetite copper sulfide hetero-nanoparticles (Fe3O4@Cus–PEG) as a radiosensitizer agent.MethodsFe3O4@Cus–PEG nanoparticles were synthesized and characterized. The toxicity of nanoparticles on HT-29 colorectal cancer cells was assessed by the MTT assay. The radio-sensitizing effects of Fe3O4@Cus–PEG nanoparticles on HT-29 cancer cells were investigated by the MTT and colony formation assays. Moreover, the underlying mechanisms for Fe3O4@Cus–PEG nanoparticles to improve the radiation sensitivity of cells were evaluated.ResultsThe results demonstrated that nanoparticles enhanced the effects of X-ray irradiation in a dose-dependent manner. The effects of combined treatments (nanoparticles and X-ray radiation) were strongly synergistic. The sensitizing enhancement ratio (SER) of nanoparticles was 2.02. Our in vitro assays demonstrated that the nitric oxide production, the intracellular hydrogen peroxide concentration, and the expression level of Bax and Caspase-3 genes significantly increased in the cells treated with the combination of nanoparticles and radiation. Whereas, the Glutathione peroxidase enzyme activity and the expression level of the Bcl-2 gene in the combined treatment significantly decreased compared to the radiation alone.ConclusionsOur study suggests that Fe3O4@Cus–PEG nanoparticles are the promising nano radio-sensitizing agents for the treatment of cancer cells to enhance the efficacy of radiation therapy through increasing the reactive oxygen species generation, nitric oxide production, and inducing apoptosis.Graphical

Resistance to H2O2-induced oxidative stress in human cells of different phenotypes

Application of genetically encoded biosensors of redox-active compounds promotes the elaboration of new methods for investigation of intracellular redox activities. Previously, we have developed a method to measure quantitatively the intracellular concentration of hydrogen peroxide (H2O2) in living cells using genetically encoded biosensor HyPer. In the present study, we refined the method and applied it for comparing the antioxidant system potency in human cells of different phenotypes by measuring the gradient between the extracellular and cytoplasmic H2O2 concentrations under conditions of H2O2-induced external oxidative stress. The measurements were performed using cancer cell lines (K-562 and HeLa), as well as normal human cells – all expressing HyPer in the cell cytoplasm. As normal cells, we used three isogenic lines of different phenotypes – mesenchymal stem/stromal cells (MSCs), induced pluripotent stem cells (iPSCs) derived from MSCs by reprogramming, and differentiated iPSC progenies with the phenotype resembling precursory MSCs. When exposing cells to exogenous H2O2, we showed that at low oxidative loads (<50 μM of H2O2) the gradient depended on extracellular H2O2 concentration. At high loads (>50 μM of H2O2), which caused the exhaustion of thioredoxin activity in the cell cytoplasm, the gradient stabilized, pointing out that it is the functional status of the thioredoxin-depended enzymatic system that drives the dependence of the H2O2 gradient on the oxidative load in human cells. At high H2O2 concentrations, the cytoplasmic H2O2 level in cancer cells was found to be several hundred times lower than the extracellular one. At the same time, in normal cells, extracellular-to-intracellular gradient amounted to thousands of times. Upon reprogramming, the potency of cellular antioxidant defense increased. In contrast, differentiation of iPSCs did not result in the changes in antioxidant system activity in the cell cytoplasm, assuming that intensification of the H2O2-detoxification processes is inherent to a period of early human development.

Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H2O2 biosensor HyPer

In this study, we propose a method for quantification of average hydrogen peroxide concentration within a living cell that is based on the use of genetically encoded H2O2 biosensor HyPer. The method utilizes flow cytometric measurements of HyPer fluorescence in H2O2-exposed cells to analyze the biosensor oxidation kinetics. Fitting the experimental curves with kinetic equations allows determining the rate constants of HyPer oxidation/reduction which are used further for the calculation of peroxide concentrations in the cells of interest both in the presence and absence of external H2O2. Applying this method to K562 cells, we have estimated the gradient as about 390-fold between the extracellular and intracellular level of exogenous H2O2 in cells exposed to the micromole doses of peroxide, as well as the average basal level of H2O2 in the cytosol of undisturbed cells (). The method can be extended to other H2O2-sensitive redox probes or to procedures in which, rather than adding external peroxide, intracellular production of peroxide is triggered, providing a tool to quantitate not only basal average H2O2 concentrations but also the concentration of peroxide build up in the vicinity of redox probes.

Calculated cell-specific intracellular hydrogen peroxide concentration: Relevance in cancer cell susceptibility during ascorbate therapy.

The high extracellular hydrogen peroxide (H2O2) concentrations generated during pharmacological ascorbate (P-AscH-) therapy has been shown to exhibit a high flux into susceptible cancer cells leading to a decrease in clonogenic survival. It is hypothesized that the intracellular H2O2 concentration for susceptibility is independent of cell type and that the variation observed in dosing is associated with differences in the cell-specific overall steady-state intracellular H2O2 concentration values. The steady-state variation in intracellular H2O2 concentration is coupled to a number of cellular specific transport and reaction factors including catalase activity and membrane permeability. Here a lumped-parameter mathematical modeling approach, assuming a catalase-dominant peroxide removal mechanism, is used to calculate intracellular H2O2 concentration for several cell lines. Experimental measurements of critical parameters pertaining to the model are obtained. The cell lines investigated are normal pancreatic cells, H6c7, the pancreatic cancer cell line, MIA PaCa-2 and the glioblastoma cell lines, LN-229, T98G, and U-87; all which vary in susceptibility. The intracellular H2O2 concentration estimates are correlated with the clonogenic surviving fraction for each cell line, in-vitro. The results showed that, despite the fact that the experimental parameters including catalase concentration and plasma membrane permeability demonstrated significant variability across cell lines, the calculated steady-state intracellular to extracellular H2O2 concentration ratio did not vary significantly across cell lines. Thus, the calculated intracellular H2O2 concentration is not unique in characterizing susceptibility. These results imply that, although intracellular H2O2 concentration plays a key role in cellular susceptibility to P-AscH- adjuvant therapy, its overall contribution in a unifying mechanism across cell types is complex.

P 117 - HyPer biosensor to monitor intracellular hydrogen peroxide in skeletal muscle cells

Hydrogen peroxide (H2O2) is one of the reactive oxygen species (ROS) that seems to play an essential role in cellular signalling pathways coupled to frequent pathophysiological processes. However, using traditional methodology it is virtually impossible to identify and quantify H2O2 flux in cells. We have developed methodological approaches based on the use of a hydrogen peroxide biosensor, HyPer, in skeletal muscle cells: myoblasts and myotubes C2C12, and individual matured muscle fibres isolated from the mouse muscle. Using transfection techniques with chemical agents and microinjection/electroporation techniques, we have achieved the expression of HyPer biosensor in those cells. In combination with live cell fluorescence microscopy image analysis we have monitored the intracellular flow of H2O2 in situ and in real time in those skeletal muscle cells that expressed HyPer. The HyPer fluorescence emitted by cells was registered during a period in which cells were exposed either to extracellular hydrogen peroxide or to a reducing agent, dithiothreitol, in the medium. Conclusion: i) it is possible the expression of hydrogen peroxide biosensor HyPer in myoblasts and myotubes C2C12 and in single isolated skeletal muscle fibres, and ii) HyPer biosensor is functional and detects changes in the intracellular concentration of hydrogen peroxide in situ and in real time in skeletal muscle cells.

Is the microtubule disruption-induced alteration of peroxide concentration a factor inhibiting the assembly of ribonucleoprotein stress granules?

It has been examined whether the destruction of cell microtubules affects the increase in the intracellular hydrogen peroxide concentration caused by sodium arsenite, which induces the formation of stress ribonucleoprotein granules. As expected, sodium arsenite caused a 50% increase in hydrogen peroxide concentration in HeLa cells; on the other hand, another stress granule inducer tert-buthylhydroquinone did not affect the peroxide concentration. The disruption of microtubules by nocodazole or vinblastine also resulted in some increase in the intracellular peroxide concentration,y taxol did not affect it. The combined treatment of cells with and the microtubule stabilization by taxol did not affect it. The combined treatment of cells with arsenite and antimicrotubule drugs caused an additive effect, and the peroxide concentration increased twice or more. Thus, the inhibition of stress granule formation after microtubule disruption cannot be explained by a decrease in peroxide concentration as compared with the affect of arsenite.

Mammalian Numb-interacting Protein 1/Dual Oxidase Maturation Factor 1 Directs Neuronal Fate in Stem Cells

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production. The neuronal differentiation in nip1-overexpressing P19 cells was achieved in a retinoic acid-independent manner and was corroborated by an increase in the expression of the neuronal basic helix-loop-helix transcription factors and neural-lineage cell markers. Furthermore, depletion of nip1 by short hairpin RNA led to a decrease in the expression of neuronal basic helix-loop-helix transcription factors and ROS. However, inhibition of ROS production in nip1-overexpressing P19 cells restricted but did not extinguish neuronal differentiation. Microarray and mass spectrometry analysis identified intermediate filaments as the principal cytoskeletal elements affected by up-regulation of nip1. We show here the first evidence for a functional interaction between Nip1 and a component of the nuclear lamina, lamin A/C. associated with a neuronal-specific phenotype. Taken together, our data reveal an important role for Nip1 in the guidance of neuronal differentiation through ROS generation and modulation of intermediate filaments and implicate Nip1 as a novel intrinsic regulator of neuronal cell fate.

Neutrophil oxidative metabolism in Down syndrome patients with congenital heart defects

Down syndrome (DS) occurs when an individual has three, rather than two, copies of the 21st chromosome. Cytosolic superoxide dismutase (SOD-1) is encoded by a gene on chromosome 21 and thus, SOD-1 activity is elevated in patients with DS. Forty percent of all cases with DS are associated with congenital heart defects (CHD). Although the contribution of SOD1 to disease phenotype is unknown, it is considered to be a "molecular marker" of the disease. It was hypothesized herein that the presence of CHD may alter the expression of SOD1 and oxidative metabolism in patients with DS. This hypothesis was tested via four experimental groups as follows: patients with DS without CHD, DS patients with CHD, CHD patients without DS and controls. Expression and activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), myeloperoxidase (MPO), and catalase (CAT) were determined in neutrophils from all experimental groups. Intracellular hydrogen peroxide concentration and superoxide release were also evaluated in neutrophils. A significant increase was observed in SOD and GPx amount and activity in patients with DS with and without CHD. No significant difference was found in the amount and activity of MPO and CAT among the different experimental groups. Intracellular hydrogen peroxide concentration was similar in all groups, whereas a prominent decrease was seen in superoxide release in cases with DS. Patients with DS with and without CHD showed no significant differences in any of the measured parameters. The data suggest that CHD observed in patients with DS does not result from altered redox metabolism associated with the disease.

Manganese superoxide dismutase overexpression changes plating efficiency bidirectionally according to change in redox for SaOS2 human osteosarcoma cell line

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that converts cytotoxic superoxide radicals into hydrogen peroxide. MnSOD activity is lower in tumor cells, and MnSOD overexpression reportedly ameliorates malignant phenotypes. We established stable MnSOD overexpressing cell lines from a human osteosarcoma cell line, SaOS2, and then investigated the effects of MnSOD overexpression on plating efficiency (PE) and the involvement of reactive oxygen species, including nitric oxide (NO) in those effects. The PE of SaOS2FM(L), a moderate MnSOD overexpression cell line, increased, while that of SaOS2FM(H), a high MnSOD overexpression cell line, decreased. Although we assessed PE using a colony-formation assay, time-lapse microscopic observation revealed that cells attached to the flasks had undergone neither apoptosis nor necrosis. Moreover, MnSOD overexpression did not affect cell doubling time. Therefore, MnSOD overexpression might correlate directly with cellular adhesion's effect on PE changes. When L-buthionine-[S,R]-sulfoximine (BSO) was administered to increase the intracellular concentration of hydrogen peroxide, the PEs of both cell lines decreased, and when hydrogen peroxide was eliminated by the administration of sodium pyruvate, only the PE of SaOS2FM(H) increased. The combination of BSO and NO (NOR4 or isosorbide 5-mononitrate) administration synergistically decreased PE in both cell lines. These findings suggest that changes in cellular adhesion properties correlate with the balance between increased hydrogen peroxide levels and decreased superoxide radical levels. This is the first report to indicate that PE and cellular adhesion properties change bidirectionally according to the levels of MnSOD overexpression: first increasing then decreasing as MnSOD activity increases. Our results indicate that PE changes might be decided by the balance between two cytotoxic compounds (decreased superoxide radical levels and increased hydrogen peroxide levels), and that NO loading and increased hydrogen peroxide synergistically reduce PE and cellular adhesion.

Increase in the Intracellular Hydrogen Peroxide Concentration in Escherichia coli Cells Treated with Ethanol and Its Relation to Cell Death.

When Escherichia coil cells were treated with 20-40% (v/v) ethanol solution, there was an increase in intracellular concentration of hydrogen peroxide accompanied by a decrease in the survival rate of the cells. Several mutants with higher catalase activity were more resistant and some other mutants with lower catalase activity were more sensitive to ethanol treatment than the parent strain K12. The concentration of hydrogen peroxide in the ethanol-treated E. coil K12 cells was 20 to160 times higher than that in the untreated cells. The catalase-gene kat E', kat G, and kat E cloned strains, having higher catalase activity, were more ethanol-resistant than E. coil K12, recipient strain UM1 and UM1 charomid 9-28. For the bactericidal mechanism of ethanol, it is suggested that ethanol treatment might cause some damage to the cellular aerobic respiratory system and as a result, hydrogen peroxide may be generated to result in cell death.

Intracellular Acidification Triggered by Mitochondrial-derived Hydrogen Peroxide Is an Effector Mechanism for Drug-induced Apoptosis in Tumor Cells

We recently showed that two photoproducts of merocyanine 540, C2 and C5, triggered cytochrome C release; however, C5 was inefficient in inducing caspase activity and apoptosis in leukemia cells, unlike C2. Here we show that HL60 cells acidified upon exposure to C2 but not C5. The intracellular drop in pH and caspase activation were dependent upon hydrogen peroxide production, and were inhibited by scavengers of hydrogen peroxide. On the contrary, caspase inhibitors did not block hydrogen peroxide production. In turn, increased intracellular hydrogen peroxide concentration was downstream of superoxide anion produced within 2 h of exposure to C2. Inhibitor of NADPH oxidase diphenyleneiodonium neither inhibited superoxide production nor caspase activation triggered by C2. However, exposure of purified mitochondria to C2 resulted in significantly increased superoxide production. Furthermore, cytochrome C release from isolated mitochondria induced by C2 was completely inhibited in the presence of scavengers of hydrogen peroxide. Contrarily, scavenging hydrogen peroxide had no effect on the cyclosporin A-sensitive mitochondrial permeability transition induced by C5. Our data suggest a scenario where drug-induced hydrogen peroxide production induces intracellular acidification and release of cytochrome C, independent of the inner membrane pore, thereby creating an intracellular environment permissive for caspase activation.

Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli.

The exponential phase of aerobic growth is associated with risk of endogenous oxidative stress in which cells need to cope with an approximately 10-fold increase in the rate of H2O2 generation. We addressed this issue by studying the regulation of the intracellular concentration of H2O2 in aerobically growing Escherichia coli. Intracellular H2O2 was kept at an almost constant steady-state value of approximately 0.2 microM (variation, less than twofold) over a broad range of cell densities in rich medium. This regulation was achieved in part by a transient increase in the OxyR-dependent transcription of the catalase gene katG (monitored by using a katG::lacZ operon fusion) during exponential growth, directly correlated with the increased rate of H2O2 generation. The OxyR-regulated alkyl hydroperoxide reductase encoded by ahpFC did not detectably affect H2O2 or catalase activity levels. Induction of katG, ahpFC, and perhaps other genes prevented the accumulation of oxidatively modified lipids but may not have protected DNA: the spontaneous mutation rate was significantly increased in both wild-type and delta(oxy)R strains during exponential growth compared to that in these strains during lag or stationary phases. Strains lacking oxyR showed throughout growth an 8- to 10-fold-higher frequency of spontaneous mutation than was seen for wild-type bacteria. The ahpdelta5 allele also had a mutator effect half of that of delta(oxy)R in exponential and stationary phases and equal to that of deltaoxyR in lag phase, perhaps by affecting organic peroxide levels. These results show that oxyR-regulated catalase expression is not solely an emergency response of E. coli to environmental oxidative stress, but also that it mediates a homeostatic regulation of the H2O2 produced by normal aerobic metabolism. The activation of the oxyR regulon in this process occurs at much lower levels of H2O2 (approximately 10(-7)M) than those reported for oxyR activation by exogenous H2O2 (approximately 10(-5) M).

Requirement for Generation of H 2 O 2 for Platelet-Derived Growth Factor Signal Transduction

Stimulation of rat vascular smooth muscle cells (VSMCs) by platelet-derived growth factor (PDGF) transiently increased the intracellular concentration of hydrogen peroxide (H2O2). This increase could be blunted by increasing the intracellular concentration of the scavenging enzyme catalase or by the chemical antioxidant N-acetylcysteine. The response of VSMCs to PDGF, which includes tyrosine phosphorylation, mitogen-activated protein kinase stimulation, DNA synthesis, and chemotaxis, was inhibited when the growth factor-stimulated rise in H2O2 concentration was blocked. These results suggest that H2O2 may act as a signal-transducing molecule, and they suggest a potential mechanism for the cardioprotective effects of antioxidants.

Mechanism of chromium(VI) toxicity in Escherichia coli: is hydrogen peroxide essential in Cr(VI) toxicity?

To investigate the role of hydrogen peroxide in Cr(VI) toxicity in vivo toward bacterial cells, we examined the effect of Cr(VI), hydrogen peroxide, sodium azide, and mannitol on the viability of Escherichia coli. Bacterial cells were incubated for 1 h with shaking in the presence of Cr(VI), hydrogen peroxide, sodium azide as catalase inhibitor, and/or mannitol as radical scavenger. The colony-forming ability and double-strand DNA degradation were examined. The viability assays revealed that Cr(VI) toxicity depended on hydroxyl radicals generated in the reaction involving hydrogen peroxide and chromium. Moreover, incubation of E. coli cells with 10 mM Cr(VI) and 3 mM hydrogen peroxide caused the degradation of double-strand DNA in vivo, which was suppressed by the addition of mannitol. These results indicated that hydroxyl radicals generated in the incubation degraded DNA of E. coli cells, resulting in cell death. In the absence of added hydrogen peroxide, the intracellular concentration of hydrogen peroxide in E. coli was low (below 1 microM). A catalase-defective strain incubated in the absence of added hydrogen peroxide remained fully viable after 1 h but showed decreased viability after prolonged incubation (4-8 h). The addition of mannitol suppressed this decrease, suggesting that hydroxyl radicals may be involved in the expression of Cr(VI) toxicity even without added hydrogen peroxide.

Exogenous protoporphyrin inhibits Hep G2 cell proliferation, increases the intracellular hydrogen peroxide concentration and causes ultrastructural alterations

Ultrastructural hepatocellular abnormalities in early stages of erythropoietic protoporphyria lead to hepatobiliary changes that cause overt liver disease in 5-10% of patients, not infrequently progressing to fulminant hepatic failure. This cannot be attributed solely to protoporphyrin crystal deposition in the liver. Hepatic redox systems have therefore been postulated as an equivalent for the photoreaction of protoporphyrin. We studied the dark effects of protoporphyrin and hematoporphyrin on HL60 and Hep G2 cells. Cell proliferation and intracellular H2O2 concentrations were assessed and related to the ultrastructural morphology. The incubation with protoporphyrin and hematoporphyrin resulted in a dose- and time-dependent inhibition of proliferation indices of Hep G2 cells. Flow cytometric analyses of intracellular H2O2 concentrations demonstrated a dose-dependent increase in both cell lines upon incubation with protoporphyrin. Hep G2 cells displayed ultrastructural alteration of the endoplasmatic reticulum and plasma membranes. Also 'cell blebbing' occurred. We conclude that exogenous protoporphyrin increases the intracellular H2O2 concentration and exerts a cytotoxic dark effect. This may contribute to the liver injury observed in erythropoietic protoporphyria.

Increased hydrogen peroxide concentration in human tumor cells due to a nitroxide free radical

Evidence is presented that the nitroxide free radical, TEMPO, at concentrations commonly used to prevent oxidative damage, increases the intracellular hydrogen peroxide concentration. To investigate the origin of this increased hydrogen peroxide concentration, we have incubated various human tumor cell lines with compounds interfering with the generation of active oxygen metabolites. Sodium azide, inhibitor of the respiratory chain, the iron-chelating agent desferrioxamine, superoxide dismutase and catalase had no effect on the hydrogen peroxide concentration. Metyrapone, inhibitor of the cytochrome P450 system, was demonstrated to decrease, but not completely prevent, the hydrogen peroxide production. N-ethylmaleimide, a sulphydryl-bond alkylating agent, was able to completely prevent the increased hydrogen peroxide production. We conclude that, by increasing the cellular hydrogen peroxide concentration, TEMPO exerts a pro-oxidant effect. This increase in hydrogen peroxide production seems to be mediated by the induction of oxidase activity in the cytochrome P450 system, but other cellular systems involved in electron transport may also play a role.

ROLE OF CATALASE AND PEROXIDASE IN THE METABOLISM OF LEUCOCYTES.

LEUCOCYTES are known to be rich in catalase1 and Myeloperoxidase1–3, yet very little is known concerning the role of these enzymes in leucocyte metabolism. In a. recent investigation by Iyer et al.4 evidence was presented for the formation of hydrogen peroxide by guinea pig exudate leucocytes. An obvious physiological function of catalase or peroxidase, therefore, might be to regulate the intracellular concentration of hydrogen peroxide which, if allowed to accumulate, could become toxic to the cells.