Active oxygen chemistry within the liposomal bilayer. Part IV: Locating 2',7'-dichlorofluorescein (DCF), 2',7'-dichlorodihydrofluorescein (DCFH) and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) in the lipid bilayer

2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) is commonly used to detect the generation of reactive oxygen intermediates and for assessing the overall oxidative stress in toxicological phenomenon. It has been suggested that DCFH-DA crosses the cell membrane, subsequently undergoing deacetylation by intracellular esterases. The resulting 2',7'-dichlorodihydrofluorescein (DCFH) is proposed to react with intracellular hydrogen peroxide or other oxidizing ROS to give the fluorescent 2',7'-dichlorofluorescein (DCF). Using an NMR chemical shift-polarity correlation, we have determined that DCFH-DA and DCFH are located well within the lipid bilayer and certainly not at the interface. These results, therefore, put into serious question the proposed ability of DCFH to come in contact with the aqueous phase and thereby interact with aqueous intracellular ROS and components. However, H2O2 and superoxide can cross or at least penetrate the lipid bilayer and react with certain lipophilic substrates. This may well describe the mode of reaction of these and other ROS with DCFH.     

Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions

We examined intra- and extracellular H(2)O(2) and NO formation during contractions in primary rat skeletal muscle cell culture. The fluorescent probes DCFH-DA/DCFH (2,7-dichlorofluorescein-diacetate/2,7-dichlorofluorescein) and DAF-2-DA/DAF-2 (4,5-diaminofluorescein-diacetate/4,5-diaminofluorescein) were used to detect H(2)O(2) and NO, respectively. Intense electrical stimulation of muscle cells increased the intra- and extracellular DCF fluorescence by 171% and 105%, respectively, compared with control nonstimulated cells (p <.05). The addition of glutathione (GSH) or Tiron prior to electrical stimulation inhibited the intracellular DCFH oxidation (p <.05), whereas the addition of GSH-PX + GSH inhibited the extracellular DCFH oxidation (p <.05). Intense electrical stimulation also increased (p <.05) the intra- and extracellular DAF-2 fluorescence signal by 56% and 20%, respectively. The addition of N(G)-nitro-L-arginine (L-NA) completely removed the intra- and extracellular DAF-2 fluorescent signal. Our results show that H(2)O(2) and NO are formed in skeletal muscle cells during contractions and suggest that a rapid release of H(2)O(2) and NO may constitute an important defense mechanism against the formation of intracellular (*)OH and (*)ONOO. Furthermore, our data show that DCFH and DAF-2 are suitable probes for the detection of ROS and NO both intra- and extracellularly in skeletal muscle cell cultures.     

Cell separation in the buffy coat

One of the most rapid methods to determine cell counts in whole blood is by way of layer thickness measurements of the buffy coat. The purpose of this study was to determine the separation and purity of blood cells in the different layers of the buffy coat. Blood samples were centrifuged at 10,000 g in microhematocrit tubes with an inserted float to expand the buffy coat region. Whole blood from normal laboratory individuals separates by density into four regions: platelets, a layer of lymphocyte and monocytes, granulocytes and erythrocytes. A thin band of highly swollen red cells was discovered between the buffy coat layers of many normal volunteers and patients. Stereological analysis of electron micrographs showed that mixing of formed elements within the layers is less than 2% with the exception of some erythrocytes, which can make up a higher volume fraction in the lymphocyte/monocyte and granulocyte layers. The red cell column contains about 95.7% erythrocytes and is depleted of platelets and leukocytes. In approximately 5% of hospital blood samples, the granulocyte-erythrocyte interface was feathered and undetectable, and a significantly higher volume fraction of red cells was found among the granulocytes. Cell mass density determinations indicate that the erythrocytes in these abnormal granulocyte layers have a lowered mass density, overlapping with that of the granulocytes.     

Evidence for free radical formation during the oxidation of 2'-7'-dichlorofluorescin to the fluorescent dye 2'-7'-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements.

The oxidation of 2'-7'-dichlorofluorescin (DCFH) to the fluorescent 2'-7'-dichlorofluorescein (DCF) by horseradish peroxidase (HRP) was investigated by fluorescence, absorption, and electron spin resonance spectroscopy (ESR). As has been previously reported, HRP/H2O2 oxidized DCFH to the highly fluorescent DCF. However, DCF fluorescence was still observed when H2O2 was omitted, although its intensity was reduced by 50%. Surprisingly, the fluorescence increase, in the absence of exogenous H2O2, was still strongly inhibited by catalase, demonstrating that H2O2 was present and necessary for DCF formation. H2O2 was apparently formed during either chemical or enzymatic deacetylation of 2'-7'-dichlorofluorescin diacetate (DCFH-DA), probably by auto-oxidation. Spectrophotometric measurements clearly showed that DCFH could be oxidized either by HRP-compound I or HRP-compound II with the obligate generation of the DCF semiquinone free radical (DCF*-). Oxidation of DCF*- to DCF by oxygen would yield superoxide (O2*-). ESR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of both superoxide and hydroxyl radicals in the DCFH/H2O2/HRP system. Both radicals were also detected in the absence of added H2O2, although the intensities of the resultant adducts were decreased. This work demonstrates that DCF fluorescence cannot be used reliably to measure O2*- in cells because O2*- itself is formed during the conversion of DCFH to DCF by peroxidases. The disproportionation of superoxide forms H2O2 which, in the presence of peroxidase activity, will oxidize more DCFH to DCF with self-amplification of the fluorescence. Because the deacetylation of DCFH-DA, even by esterases, can produce H2O2, the use of this probe to measure H2O2 production in cells is problematic.     

Altered oxidative metabolism in selenium-deficient rat granulocytes

Rats fed a selenium-deficient diet for 12 to 15 wk became selenium-depleted, measured by the selenium content of liver and granulocytes. The activity of granulocyte glutathione peroxidase, a selenoenzyme, in deficient rats was 11% of the activity in replete rat granulocytes. When stimulated with an H2O2 generating system, the HMPS activity of the deficient granulocytes was 50% of replete; however, when stimulated with methylene blue, the HMPS activity in deficient and replete granulocytes was the same. When granulocytes were incubated with PMA or OPZ, deficient granulocytes initially had the same O-2-generating activity as replete granulocytes; however, with increasing duration of stimulation, granulocytes from deficient rats generated less O-2 than replete rats. After 20 min in an H2O2-generating system, deficient granulocytes stimulated with PMA or OPZ generated less O-2 than replete granulocytes. These results indicate that deficient granulocytes did not metabolize H2O2 as well as replete granulocytes and that H2O2 caused damage to the O2-generating system. Measurement of O-2 generation in membrane-enriched particles showed the above effects were due to inactivation of the NADPH-dependent O2-generating system. Deficient granulocytes stimulated with OPZ for 20 min had 70% less membrane O-2-generating activity than controls. In addition, when membrane-enriched particles were made from cells that had been stressed with an H2O2-generating system, NADPH-dependent O-2-generating activity in deficient granulocytes was 50% of replete. In selenium-deficient granulocytes with low GSH-Px activity, prolonged incubation with stimulants and prior incubations with an H2O2-generating system caused loss of activity of the membrane-bound, NADPH-dependent, O-2-generating system.     

Measurement of Crassostrea gigas hemocyte oxidative metabolism by flow cytometry and the inhibiting capacity of pathogenic vibrios

A flow cytometric method to measure the production of oxidative metabolism products was adapted for use with Crassostrea gigas hemocytes. The method is based upon the oxidation, by hydrogen peroxide (H2O2), of intracellular 2',7'-dichlorofluorescin (DCFH) to green-fluorescent dichlorofluorescein. Activation of the respiratory burst (RB) was tested using phorbol myristate acetate with no success. By contrast, activation by zymosan particles increased oxidation of DCFH in C. gigas hemocytes, mainly granulocytes, and optimization tests showed a good response with 20 zymosan particles per hemocyte. Anti-aggregant solution, used to prevent hemocytes from clumping during bleeding, inhibited the RB activity measured by DCFH oxidation. The flow cytometric method developed during this work was used to evaluate the DCFH oxidation-inhibiting capacity of four strains of vibrio bacteria, known or suspected to be pathogenic for bivalves.     

Phosphorylation of Histone H2AX on Ser 139 and Activation of ATM During Oxidative Burst in Phorbol Ester-Treated Human Leukocytes

Oxidative burst is a defense mechanism used by specialized phagocytes such as granulocytes or monocytes to kill the invading microorganisms through generation of superoxide anions. Oxidative burst also results in DNA damage of the phagocytes. Phagocytes are terminally differentiated and some of very short life-span cells. We could find no reports whether oxidative burst-mediated DNA damage triggers in such cells histone H2AX-Ser139 phosphorylation and activation of Ataxia Telangiectasia Mutated (ATM), the signals otherwise used to activate DNA repair and checkpoint pathways in proliferating cells. We now present the evidence that induction of oxidative stress in human peripheral blood leukocytes by phorbol myristate acetate (PMA) was associated with intense phosphorylation of histone H2AX and with ATM activation, seen already 60 min after exposure to PMA. The modifications of H2AX and ATM in individual granulocytes, monocytes and lymphocytes were detected prior to caspases activation and thus were unrelated to induction of apoptosis. A large intercellular variation in response was observed, and only a fraction of cells in these subpopulations showed H2AX and ATM modifications. The data are compatible with the earlier observations of DNA damage during oxidative burst and suggest that even in terminally differentiated cells that have a short life-span, DNA damage triggers recruitment of the DNA repair machinery. The observed H2AX phosphorylation in lymphocytes may reflect their DNA damage by the superoxide ions propagating from the neighboring granulocytes and/or monocytes.     

Effect of catalase on the proliferation of human lymphocytes to phorbol myristate acetate

Phorbol esters have been documented to stimulate the proliferation of human blood mononuclear cell cultures. In addition, these agents are also known to stimulate the production and release of reactive oxygen species by monocytes. We demonstrated previously that H2O2, one of these oxygen metabolites, impairs the proliferative capacity of human blood lymphocytes. Therefore, in these experiments, we determined whether or not the H2O2 released by monocytes after activation by PMA modifies the proliferation of lymphocytes to this agent. Human blood mononuclear cells (80% lymphocytes and 20% monocytes) were incubated with PMA, and lymphoblastic transformation (LBT) was quantitated at 3 and 5 days by pulsing the cultures with thymidine. Initial experiments established that the concentration of PMA required for optimal LBT was 50 ng/ml. We then demonstrated that this concentration of PMA also induces a burst in hexose monophosphate shunt activity and H2O2 production of mononuclear cells as indicated by the enhanced oxidation of 14C-glucose and 14C-formate, respectively. The amount of H2O2 released into the medium was substantial. Our measurements indicate that the concentration of H2O2 could reach values as high as 0.008 mM during the first 2 hr of the cultures. The addition of catalase to PMA-treated cultures in concentrations sufficient to scavenge the H2O2 released by the monocytes was associated with an enhanced thymidine uptake (mean 79%). These results indicate that the hydrogen peroxide released by the monocytes modifies the response of lymphocytes to the PMA. Paradoxically, mononuclear cell cultures depleted of monocytes also had a lower proliferation to PMA than mononuclear cell cultures. This observation indicates that monocytes also produce factors required for lymphocyte proliferation to PMA such as an interleukin. In contrast, to PMA cultures, catalase did not alter the proliferation of mononuclear cell cultures stimulated by PHA. We previously documented that PHA does not stimulate an immediate burst in the oxidative metabolism of mononuclear cultures. Therefore, the effect of catalase in these two culture systems appears to correlate with the capacity of the mitogen to stimulate the oxidative metabolism of mononuclear cells. These observations suggest that the release of reactive oxygen species by monocytes may modify the response of lymphocytes to antigens both in vitro and in vivo.     

Adaptation of the dichlorofluorescein assay for detection of radiation-induced oxidative stress in cultured cells

The oxidation of 2'7'-dichlorofluorescin (DCFH) to 2'7'-dichlorofluorescein (DCF), a fluorescent DCFH oxidation product, is a highly sensitive indicator that is used to measure oxidative stress in cells. In the present study, a DCF assay has been adapted to quantify oxidative stress in human breast epithelial cell cultures after exposure to gamma rays. The results demonstrate that the sensitivity and specificity of the DCF assay is strongly influenced by the timing of DCFH diacetate (DCFH-DA) substrate loading in relation to radiation exposure and by the matrix in which the cells were loaded with DCFH-DA substrate. Under the conditions optimized in this study, the DCF assay is capable of detecting increased DCFH oxidation in cell cultures irradiated with gamma rays at a dose as low as 1.5 cGy. The increase in fluorescence was directly proportional to the radiation dose, which ranged from 0 to 2 Gy, and a minimal level of fluorescence was observed in sham-irradiated cells. These results indicate that the DCF assay optimized in this study is highly sensitive, linear and specific for measuring oxidative stress in irradiated cells.     

Augmentation of activities of peripheral granulocytes and of resistance against bacterial infection after administration of G-CSF into mice]

We evaluated the metabolic capability of murine peripheral granulocytes after administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) by quantitative flow cytometric assay for H2O2-dependent oxidative product formation. Intraperitoneal administration of a daily dose of 10 micrograms of rhG-CSF for 5 days induced doubling of the leukocyte population. Differential counting of peripheral leukocytes and scattergram by flow cytometry showed an increased mature granulocyte population. After stimulation with phorbol myristate acetate, the granulocytes of the rhG-CSF-administered mice demonstrated some hyperresponsive population and an increased H2O2 production. The hyperresponsive population showed H2O2 production 4-6 times higher than did normal cells. Granulocytes from the G-CSF-treated mice revealed an augmented phagocytic activity and an increased expression of Mac-1 molecules. Moreover, mice treated with G-CSF showed an enhanced resistance against intravenous infection with a lethal dose of E. coli. Granulocytes showing such markedly increased oxidative metabolism may be a significant component of the host defence to various infective organisms.     

Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry

Phagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations. In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis. In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4-diacetoxy-2,3-dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria. The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays. The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement. The calculated mean values were transferred into the self-learning database of the DIAGNOS1-program system. The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states. The assays require 1 ml of heparinized whole blood and the results are available within 1 hour.     

Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry.

The novel calcium indicator fura red and the oxidative burst indicator dihydrorhodamine (both excited at 488 nm) were used in combination with multiparameter flow cytometry to allow simultaneous kinetic measurements of calcium fluxes and oxidative bursts in monocytes and granulocytes. Using this method it was possible to obtain direct evidence for the following cell type- and stimulus-specific differences in signal transduction pathways: 1) n-formyl-methionyl-leucyl-phenylalanine (FMLP)/cytochalasin B-induced oxidative burst is several-fold higher in granulocytes than in monocytes although the calcium fluxes have similar amplitudes in the two cell types; 2) stimulus-induced calcium fluxes in granulocytes are mainly due to release from intracellular stores, whereas monocytes mobilize calcium mainly by influx from the medium; 3) the FMLP/cytochalasin B-induced calcium flux in monocytes is less sensitive to the G-protein inhibitor pertussis toxin than the flux in granulocytes; 4) in contrast to FMLP/cytochalasin B, the protein kinase C activator phorbol myristate acetate (PMA) induces an oxidative burst that is not preceded by a cytoplasmic calcium flux; 5) the PMA-induced oxidative burst can be triggered in monocytes and granulocytes that are depleted of intracellular calcium ions, whereas that induced by FMLP/cytochalasin B can not; 6) the G-protein inhibitor pertussis toxin blocks an early event in the signal transduction pathway of FMLP/cytochalasin B, as shown by inhibition of both calcium fluxes and oxidative burst; and 7) 100 nM of the protein kinase inhibitor staurosporine blocks the FMLP/cytochalasin B-induced respiratory burst by interfering with a step downstream to cytoplasmic calcium fluxes, whereas only 10-20 nM is necessary to block PMA-induced oxidative burst.     

Cu,Zn-superoxide dismutase is an intracellular catalyst for the H(2)O(2)-dependent oxidation of dichlorodihydrofluorescein

Dichlorodihydrofluorescein (DCFH(2)) is a widely used probe for intracellular H(2)O(2). However, H(2)O(2) can oxidize DCFH(2) only in the presence of a catalyst, whose identity in cells has not been clearly defined. We compared the peroxidase activity of Cu,Zn-superoxide dismutase (CuZnSOD), cytochrome c, horseradish peroxidase (HRP), Cu(2+), and Fe(3+) under various condi-tions to identify an intracellular catalyst. Enormous increase by bicarbonate in the rate of DCFH(2) oxidation distinguished CuZnSOD from cytochrome c and HRP. Cyanide inhibited the reaction catalyzed by CuZnSOD but accelerated that by Cu(2+) and Fe(3+). Oxidation of DCFH(2) by H(2)O(2) in the presence of a cell lys-ate was also enhanced by bicarbonate and inhibited by cyanide. Confocal microscopy of H(2)O(2)-treated cells showed enhanced DCF fluorescence in the presence of bicarbonate and attenuated fluorescence for the cells pre-incubated with KCN. Moreover, DCF fluorescence was intensified in CuZnSOD-transfected HaCaT and RAW 264.7 cells. We propose that CuZnSOD is a potential intracellular catalyst for the H(2)O(2)-dependent oxidation of DCFH(2).     

Resistin inhibits essential functions of polymorphonuclear leukocytes

The serum levels of resistin, a 12-kDa protein primarily expressed in inflammatory cells in humans, are increased in patients with chronic kidney disease and in those with diabetes mellitus. Both groups of patients have an increased risk of infections mainly as a result of disturbed polymorphonuclear leukocyte (PMNL) functions. Therefore, we investigated the influence of resistin on human PMNLs. Serum resistin concentrations were determined with a sandwich enzyme immunoassay. Using PMNLs from healthy subjects, chemotaxis was tested by the under-agarose method. Flow cytometric assays to measure oxidative burst and phagocytosis were conducted in whole blood. The uptake of deoxyglucose was determined as measure of the PMNL activation state. The activity of intracellular kinases was assessed by Western blotting and by in vitro kinase assays. Resistin inhibited PMNL chemotaxis and decreased the oxidative burst stimulated by Escherichia coli and by PMA, but did not influence PMNL phagocytosis of opsonized E. coli and PMNL glucose uptake. The inhibition of PMNLs by resistin was observed at concentrations found in serum samples of uremic patients, but not in concentrations measured in healthy subjects. Experiments with specific signal transduction inhibitors and measurements of intracellular kinases suggest that PI3K is a major target of resistin. In conclusion, resistin interferes with the chemotactic movement and the stimulation of the oxidative burst of PMNL, and therefore may contribute to the disturbed immune response in patients with increased resistin serum levels such as uremic and diabetic subjects.     

F1651 fimbriae of the P fimbrial family inhibit the oxidative response of porcine neutrophils

The F165(1) fimbrial system has been associated with the resistance of Escherichia coli O115:K"V165" to phagocytic killing by porcine polymorphonuclear leukocytes (PMNLs). One mechanism of this resistance seemed to be inhibition of the oxidative response as observed following induction of PMNLs by phorbol myristate acetate (PMA) and treatment with bacteria possessing the F165(1) fimbriae. In order to confirm whether or not the F165(1) fimbriae are involved in this inhibition, we evaluated the effect of F165(1)-positive strains (a pathogenic wild-type strain 5131, and a recombinant strain HB101(pCJ7)) or an F165(1)-negative strain HB101 (used as negative control) on the oxidative response of porcine neutrophils (pNs) stimulated with PMA. Incubation of pNs with pathogenic E. coli strain 5131 resulted in significant inhibition of the oxidative response as compared to that observed for pNs incubated without bacteria, as assessed by hydrogen peroxide (H2O2) and superoxide anion (O2-) release from the phagocytes, and by the chemiluminescence assay. Similarly, incubation of pNs with the F165(1)-producing cloned strain HB101(pCJ7) resulted in significant inhibition of the pN oxidative response as compared to that observed for pNs incubated without bacteria or with strain HB101. In contrast, addition of purified F165(1) fimbriae to the pNs had no effect on the oxidative response.     

Cyclosporin A inhibits phorbol ester-induced activation of superoxide production in resident mouse peritoneal macrophages.

Peritoneal resident macrophages from mice are sensitive to inhibition by cyclosporin A (CsA) of phorbol 12-myristate 13-acetate (PMA)-stimulated oxidative burst. Inhibition was assessed in terms of superoxide anion (O2.-) and H2O2 production. Key findings were as follows. (a) CsA inhibited in a dose-dependent manner the production of O2.- when cells were stimulated with PMA. CsA did not alter the respiratory burst induced by other stimuli (zymosan, concanavalin A and fMet-Leu-Phe). It was verified that CsA itself had no scavenger effect. (b) A concomitant decrease in H2O2 liberation following CsA exposure was found. This inhibition was observed both in the initial rate of synthesis and in the accumulation after 15 min of incubation. (c) NADPH oxidase activity in the crude supernatant was unaffected by the previous incubation of macrophages with CsA. CsA does not inhibit glucose transport measured as 14CO2 production. (d) The production of O2.- was strongly dependent on the glucose concentration. Sodium oleate also stimulated O2.- production in resident macrophages. These data might be correlated with the inhibitory effect of CsA upon other functions of macrophages.     

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body’s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).     

Bicarbonate enhances peroxidase activity of Cu,Zn-superoxide dismutase. Role of carbonate anion radical and scavenging of carbonate anion radical by metalloporphyrin antioxidant enzyme mimetics.

Much evidence exists for the increased peroxidase activity of copper, zinc superoxide dismutase (SOD1) in oxidant-induced diseases. In this study, we measured the peroxidase activity of SOD1 by monitoring the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF). Bicarbonate dramatically enhanced DCFH oxidation to DCF in a SOD1/H(2)O(2)/DCFH system. Peroxidase activity could be measured at a lower H(2)O(2) concentration ( approximately 1 microm). We propose that DCFH oxidation to DCF is a sensitive index for measuring the peroxidase activity of SOD1 and familial amyotrophic lateral sclerosis SOD1 mutants and that the carbonate radical anion (CO(3)) is responsible for oxidation of DCFH to DCF in the SOD1/H(2)O(2)/bicarbonate system. Bicarbonate enhanced H(2)O(2)-dependent oxidation of DCFH to DCF by spinal cord extracts of transgenic mice expressing SOD1(G93A). The SOD1/H(2)O(2)/HCO(3)(-)-dependent oxidation was mimicked by photolysis of an inorganic cobalt carbonato complex that generates CO(3). Metalloporphyrin antioxidants that are usually considered as SOD1 mimetic or peroxynitrite dismutase effectively scavenged the CO(3) radical. Implications of this reaction as a plausible protective mechanism in inflammatory cellular damage induced by peroxynitrite are discussed.     

Functions and oxidative stress status of leukocytes in patients with nephrotic syndrome

This study was conducted to establish the functions and oxidative stress status in leukocytes of adult patients with nephrotic syndrome. Thirty adult patients with nephrotic syndrome and 32 controls were included. Phagocytosis ability, the killing ability of the micro-organism phagosited of polymorphonuclear leukocytes (PMNL) and monocytes, along with oxidative stress parameters of PMNLs were assessed. There was no statistically significant difference in phagocytosis function of PMNLs and monocytes of patients when compared to those of controls. PMNL burst activities of the patient and control groups also showed no difference; however, the monocyte burst activities of patients were significant (p = 0.012). The glutathione peroxidase (GSH-Px) activities in PMNLs of the patients with nephrotic syndrome were significantly higher (p = 0.026) when compared to those of controls. In comparison with those of the control subjects, the patients had also higher selenium levels in their PMNLs (p < 0.001). Although PMNL malonyldialdehyde (MDA) levels of the patients seem to be higher than those of controls, the difference had no statistical significance (p = 0.071). Conclusively, in the patients with nephrotic syndrome, PMNLs appear to be exposed to an oxidative stress as indicated by their increased GSH-Px activities and selenium content. However, PMNLs in nephrotic syndrome patients seem to be coping with the insulting oxidative stress, as suggested by their near-normal MDA productions. Furthermore, these data suggest that nephrotic syndrome appears not to have an influence on phagocytosis and killing abilities of granulocytes and monocytes as long as these cells can overcome the oxidative stress to which they are exposed in this disease.