Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate

The role of trace metals in the generation of free radical mediated oxidative stress in normal human red cells was studied. Ascorbate and either soluble complexes of Cu(II) or Fe(III) provoked changes in red cell morphology, alteration in the polypeptide pattern of membrane proteins, and significant increases in methemoglobin. Neither ascorbate nor the metal complexes alone caused significant changes to the cells. The rate of methemoglobin formation was a function of ascorbate and metal concentrations, and the chemical nature of the chelate. Cu(II) was about 10-times more effective than Fe(III) in the formation of methemoglobin. Several metals were tested for their ability to compete with Cu(II) and Fe(III). Only zinc caused a significant inhibition of methemoglobin formation by Fe(III)-fructose. These observations suggest that site-specific as well as general free radical damage is induced by redox metals when the metals are either bound to membrane proteins or to macromolecules in the cytoplasm. The Cu(II) and Fe(III) function in two catalytic capacities: (1) oxidation of ascorbate by O2 to yield H2O2, and (2) generation of hydroxyl radicals from H2O2 in a Fenton reaction. These mechanisms are different from the known damage to red cells caused by the binding of Fe(III) or Cu(II) to the thiol groups of glucose-6-phosphate dehydrogenase. Our system may be a useful model for understanding the mechanisms for oxidative damage associated with thalassemia and other congenital hemolytic anemias.     

Structural modification of erythrocyte membranes during oxidative stress and activity of membrane bound NADH-methemoglobin reductase

The activity of NADH-methemoglobin reductase (metHb-reductase) in membranes isolated from human erythrocytes treated with phenylhydrazine at its sublytic concentration was studied. A decrease in the activity of membrane-bound metHb-reductase was shown to depend on the concentration of phenylhydrazine. Simultaneously, an increase in the level of membrane-bound methemoglobin and a change in the fluorescence parameters of membrane-bound 4,4'-diisothiocy-anatostilbene-2,2'-disulfonic acid were registered. In the case when Hb-free erythrocyte ghosts were treated with 0.2-2.0 mM phenylhydrazine, the activity of metHb-reductase did not change. The obtained results indicate that the inhibition of the activity of membrane-bound metHb-reductase by phenylhydrazine-induced oxidative stress in human erythrocytes is not caused by the direct action of the oxidant on the enzyme. The reason for this is the interaction of the products of hemoglobin oxidation with erythrocyte membrane (protein band 3) and structural changes in membrane proteins.     

In vitro effects of 50 Hz magnetic fields on oxidatively damaged rabbit red blood cells

The aim of this study was to investigate the effects of 50 Hz magnetic fields (0.2–0.5 mT) on rabbit red blood cells (RBCs) that were exposed simultaneously to the action of an oxygen radical-generating system, Fe(II)/ascorbate. Previous data obtained in our laboratory showed that the exposure of rabbit erythrocytes or reticulocytes to Fe(II)/ascorbate induces hexokinase inactivation, whereas the other glycolytic enzymes do not show any decay. We also observed depletion of reduced glutathione (GSH) content with a concomitant intracellular and extracellular increase in oxidized glutathione (GSSG) and a decrease in energy charge. In this work we investigated whether 50 Hz magnetic fields could influence the intracellular impairments that occur when erythrocytes or reticulocytes are exposed to this oxidant system, namely, inactivation of hexokinase activity, GSH depletion, a change in energy charge, and hemoglobin oxidation. The results obtained indicate that a 0.5 mT magnetic field had no effect on intact RBCs, whereas it increased the damage in an oxidatively stressed erythrocyte system. In fact, exposure of intact erythrocytes incubated with Fe(II)/ascorbate to a 0.5 mT magnetic field induced a significant further decay in hexokinase activity (about 20%) as well as a twofold increase in methemoglobin production compared with RBCs that were exposed to the oxidant system alone. Although further studies will be needed to determine the physiological implications of these data, the results reported in this study demonstrate that the effects of the magnetic fields investigated are able to potentiate the cellular damage induced in vitro by oxidizing agents. Bioelectromagnetics 18:125–131, 1997. © 1997 Wiley-Liss, Inc.     

Changes in rodent-erythrocyte methemoglobin reductase system produced by two malaria parasites, viz. Plasmodium yoelii nigeriensis and Plasmodium berghei

The methemoglobin reductase system plays a vital role in maintaining the equilibrium between hemoglobin and methemoglobin in blood. Exposure of red blood cells to oxidative stress (pathological/physiological) may cause impairment to this equilibrium. We studied the status of erythrocytic methemoglobin and the related reductase system during Plasmodium yoelii nigeriensis infection in mice and P. berghei infection in mastomys. Malaria infection was induced by intraperitoneal inoculation with 10⁶ infected erythrocytes. The present investigation revealed a significant decrease in the activity of methemoglobin reductase, with a concomitant rise in methemoglobin content during P. yoelii nigeriensis infection in mice erythrocytes. This was accompanied with a significant increase in reduced glutathione and ascorbate levels. The activity of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and glutathione reductase increased with a progressive rise in parasitemia. However, no methemoglobin or associated reductase activity was detected in normal and P. berghei-infected mastomys. P. berghei infection in mastomys resulted in an increase in the level of reduced glutathione and ascorbate in erythrocytes, and also in the activity of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and glutathione reductase. These results suggest that antioxidants/antioxidant enzymes may prevent or reduce the formation of methemoglobin in the host and thereby protect the host from methemoglobinemia.     

Erythrocyte alterations in hemoglobin H disease

This study on erythrocytes in hemoglobin H (Hb H) disease reveals that unstable Hb H is bound to membranes to a greater extent, especially when it forms methemoglobin or is precipitated as inclusion body. The methemoglobin content of these erythrocytes is elevated in spite of a higher activity of NADH-methemoglobin reductase. The ATPase activity is doubled, and the ATP is presumably used for phosphorylation of membrane proteins, which leads to cross-linking of membrane proteins. This assumption could be supported by the observed decrease in non-electrolyte permeability, by increased binding of hemoglobin to the membrane and by polymerisation of membrane proteins detected by SDS-polyacrylamide gel electrophoresis. By means of electron microscopy, it could also be shown that the inclusion bodies are bound to the inner surface of membrane and cause its protrusion. This linkage might be responsible for the observed inhibition of the lateral movement of intramembrane particles.     

Oxidation of iron-nitrosyl-hemoglobin by dehydroascorbic acid releases nitric oxide to form nitrite in human erythrocytes

The reaction of deoxyhemoglobin with nitric oxide (NO) or nitrite ions (NO 2 (-)) produces iron-nitrosyl-hemoglobin (HbNO) in contrast to the reaction with oxyhemoglobin, which produces methemoglobin and nitrate (NO 3 (-)). HbNO has not been associated with the known bioactivities of NO. We hypothesized that HbNO in erythrocytes could be an important source of bioactive NO/nitrite if its oxidation was coupled to the ascorbic acid (ASC) cycle. Studied by absorption and electron paramagnetic resonance (EPR) spectroscopy, DHA oxidized HbNO to methemoglobin and liberated NO from HbNO as determined by chemiluminescence. Both DHA and ascorbate free radical (AFR), the intermediate between ASC and DHA, enhanced NO oxidation to nitrite, but not nitrate; nor did either oxidize nitrite to nitrate. DHA increased the basal levels of nitrite in erythrocytes, while the reactions of nitrite with hemoglobin are slow. In erythrocytes loaded with HbNO, HbNO disappeared after DHA addition, and the AFR signal was detected by EPR. We suggest that the ASC-AFR-DHA cycle may be coupled to that of HbNO-nitrite and provide a mechanism for the endocrine transport of NO via hemoglobin within erythrocytes, resulting in the production of intracellular nitrite. Additionally, intracellular nitrite and nitrate seem to be largely generated by independent pathways within the erythrocyte. These data provide a physiologically robust mechanism for erythrocytic transport of NO bioactivity allowing for hormone-like properties.     

Inhibition by thiols of copper(II)-induced oxidation of oxyhemoglobin.

The ability of thiols, 2-imidazolethiones and uric acid to protect bovine oxyhemoglobin from copper(II)-induced oxidation to methemoglobin was investigated. The oxidation of oxyhemoglobin by Cu(II) proceeded in two phases: (1) an initial rapid reaction (less than 30 s) followed by (2) a slower reaction that carried it to completion. Thiols, including N-acetyl-L-cysteine, DL-dithiothreitol, reduced glutathione, DL-homocysteine, 2-mercaptoethanol and 2- and 3-mercaptopropionic acid, whose sulfhydryl groups were slowly oxidized by Cu(II) (with the exception of 2-mercaptopropionic acid), protected oxyhemoglobin in both phases of the reaction. Other thiols, including L-cysteine, cysteamine, and D-penicillamine, whose sulfhydryl groups were readily oxidized by Cu(II), protected hemoglobin initially, but within 2-4 min, the rate of methemoglobin formation was the same as Cu(II)-treated oxyhemoglobin. 2-Mercaptoimidazole and 1-methyl-2-mercaptoimidazole, which complex Cu(II) and inhibit Cu(II)-catalyzed oxidation of ascorbic acid, also protected hemoglobin in the initial phase, but not in the second phase. Uric acid, L-ergothioneine, and thiourea did not protect oxyhemoglobin in either the fast or slow phase. Cu(II) may have a coordination site involved in the oxidation of hemoglobin that is not blocked by the 2-imidazolethiones, uric acid, or the oxidized thiols. It is concluded that certain thiols that complex Cu(II) and are not rapidly oxidized will protect oxyhemoglobin from Cu(II)-induced oxidation, but the thiols are no longer effective once they are oxidized.     

Reactivity studies of the Fe(III) and Fe(II)NO forms of human neuroglobin reveal a potential role against oxidative stress

Neuroglobin, recently discovered in the brain and in the retina of vertebrates, belongs to the class of hexacoordinate globins, in which the distal histidine coordinates the iron center in both the Fe(II) and Fe(III) forms. As for most other hexacoordinate globins, the physiological function of neuroglobin is still unclear, but seems to be related to neuronal survival following acute hypoxia. In this study, we have addressed the question whether human neuroglobin could act as a scavenger of toxic species, such as nitrogen monoxide, peroxynitrite, and hydrogen peroxide, which are generated at high levels in the brain during hypoxia; we have also investigated the kinetics of the reactions of its Fe(III) (metNGB) and Fe(II)NO forms with several reagents. Binding of cyanide or NO* to metNGB follows bi-exponential kinetics, showing the existence of two different protein conformations. In the presence of excess NO*, metNGB is converted into NGBFe(II)NO by reductive nitrosylation, in analogy to the reactions of NO* with metmyoglobin and methemoglobin. The Fe(II)NO form of neuroglobin is oxidized to metNGB by peroxynitrite and dioxygen, two reactions that also take place in hemoglobin, albeit at lower rates. In contrast to myoglobin and hemoglobin, metNGB unexpectedly does not generate the cytotoxic ferryl form of the protein upon addition of either peroxynitrite or hydrogen peroxide. Taken together, our data indicate that human neuroglobin may be an efficient scavenger of reactive oxidizing species and thus may play a role in the cellular defense against oxidative stress.     

The basis for EDTA-stimulation of methemoglobin reduction in hemolysates of human erythrocytes.

We have studied the stimulation by EDTA of methemoglobin reduction in hemolysates of human erythrocytes. The EDTA effect has been shown not to be the result of an allosteric interaction of EDTA with hemoglobin or the result of a photochemical reduction. The effect does not appear to be due to a direct interaction of free EDTA with either of the catalytic components of the erythrocyte methemoglobin reduction system. The EDTA stimulation seen in hemolysates is due to the formation of an iron-EDTA complex, which transfers electrons from the reductase to methemoglobin.     

Different cellular targets for Cu- and Fe-catalyzed oxidation observed using a Cu-compatible thiobarbituric acid assay

The widely used thiobarbituric acid (TBA) assay for oxidative damage to biomolecules fails in Cu2(+)-containing solutions due to the formation of a cloudy precipitate. The chelation of Cu2+ ions with EDTA or Chelex was investigated. Both prevented precipitate formation, but only Chelex allowed proper color development in the TBA assay. The Chelex modified assay could be adapted to a variety of systems, and was applied to the detection of Cu2+/ascorbate dependent deoxyribose breakdown and oxidative damage in erythrocyte ghosts, lysates and whole cells. Using this method, it was shown that Cu2+/ascorbate caused membrane damage in ghosts but not in whole red blood cells (RBC). Fe3+/ascorbate, on the other hand, caused formation of TBA-reactive products even in whole RBC. When Cu2+ and Fe3+ were presented to isolated hemoglobin as their 1:1 nitrilotriacetate complexes, the protein bound 10-12 cupric ions per molecule, but no ferric ions. It is suggested that oxidative damage catalyzed by copper or iron ions has different cellular targets, determined by the different binding properties of the two metals to various cellular components.     

Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats.

Oxidant stress is one of the factors proposed to be responsible for damaged erythrocytes observed during and after exercise. The impact of exertional oxidant stress after acute exhaustive treadmill running on erythrocyte damage was investigated in sedentary (Sed) and exercise-trained (ET) rats treated with or without antioxidant vitamins C and E. Exhaustive exercise led to statistically significant increments in the levels of thiobarbituric acid-reactive substance (TBARS) and H2O2-induced TBARS in Sed rats and resulted in functional and structural alterations in erythrocytes (plasma hemoglobin concentrations, methemoglobin levels, and rise in osmotic fragility of erythrocytes with decrease in erythrocyte deformability). Administration of antioxidant vitamin for 1 mo before exhaustive exercises prevented lipid peroxidation (TBARS, H2O2-induced TBARS) in Sed rats without any functional or structural alterations in erythrocytes. Parameters indicating erythrocyte lipid peroxidation and deterioration after exhaustive exercise in rats trained regularly with treadmill running for 1 mo were not different from those in Sed controls. Erythrocyte lipid peroxidation (TBARS) increased in exhausted-ET rats compared with ET controls; however, the plasma hemoglobin, methemoglobin levels, and erythrocyte osmotic fragility and deformability did not differ. Exhaustive exercise-induced lipid peroxidation in ET rats on antioxidant vitamin treatment was prevented, whereas functional and structural parameters of erythrocytes were not different from those of the ET controls. We conclude that exertional oxidant stress contributed to erythrocyte deterioration due to exercise in Sed but not in ET rats.     

Spectral analysis of Fe(III)-complex reduction by hemoglobin: Possible mechanisms of interaction

• 1.1. Hemoglobin is capable of electron transfer to Fe(III)-complexes of ATP, EDTA, NTA, and citrate leading to formation of reduced Fe(II) and its concurrent release from these chelating compounds as evident in the formation of a Fe(II)-Tris 2,2' bipyridine complex.
• 2.2. Multi-component analysis of kinetic spectra in the visible region (700–500 nm) has permitted a determination of the effect of various chelating molecules bound to Fe(III), pH, the effects of ionic strength, temperature, and the molecular nature of the Fe(III)-complex on reaction rates.
• 3.3. We have examined and compared the reactivities of normal adult hemoglobin A (α₂ β₂) to reduce these Fe(III)-complexes and suggest possible mechanism(s) for the electron transfer process.

     

Damage to erythrocytes caused by the interaction of nitrite-ions with hemoglobin

The formation of two hemoglobin forms (methemoglobin and nitrite methemoglobin) in native human erythrocytes in the presence of sodium nitrite in suspension was shown. In normal erythrocytes, the interaction of intracellular oxyhemoglobin with nitrite ions results in the formation of methemoglobin, whereas in metabolically exhausted erythrocytes, this leads predominantly to the formation of nitrite methemoglobin. The nitrite methemoglobin reacts with hydrogen peroxide to form reactive intermediates (e.g. peroxynitrous acid) and the products of hemoglobin destruction. During the storage of erythrocyte suspensions containing methemoglobin and modified nitrite methemoglobin, differences in the forms of erythrocytes and the degree of their hemolysis were revealed. It is assumed that the formation of methemoglobin leads to the destruction of erythrocytes.     

Neutrophil-mediated methemoglobin formation in the erythrocyte. The role of superoxide and hydrogen peroxide

Human neutrophils incubated with phorbol myristate acetate oxidized hemoglobin within the intact erythrocyte by a mechanism dependent on cell-cell contact but independent of phagocytosis. Spectrophotometric examination of the erythrocyte lysates revealed that the major component formed was methemoglobin along with small amounts of a species with spectral characteristics similar to choleglobin. Methemoglobin formation was directly related to the neutrophil concentration and the time of incubation. The addition of superoxide dismutase or catalase modestly inhibited the formation of methemoglobin, while a combination of the enzymes provided the most dramatic protection. Methemoglobin of hydroxyl radical or hypochlorous acid scavengers. Apparently, either O2.- or H2O2 alone was capable of mediating methemoglobin formation in the intact erythrocyte. Maintenance of the intraerythrocytic hemoglobin in its oxygenated state or its derivatization to carbon monoxyhemoglobin markedly inhibited methemoglobin formation. Blockade of the anion channels in the intact erythrocyte with sulfonated stilbenes inhibited O2.- but not H2O2 from oxidizing intracellular hemoglobin. It appears that neutrophil-derived O2.- and H2O2 can cross the erythrocyte membrane through the anion channel or diffuse directly into the intracellular space and react with oxyhemoglobin or deoxyhemoglobin to form a mixture of hemoglobin oxidation products within the intact cell.     

The special behavior of equine erythrocytes connected with the methemoglobin regulation

Erythrocytes from thoroughbred horses were submitted to total (80-90%) and partial (25-40%) oxidation of hemoglobin by sodium nitrite. The ability of these cells to reduce methemoglobin to hemoglobin in the presence of either glucose, glucose plus methylene blue or lactate was investigated. The results were compared with those ones obtained for human erythrocytes. Under total oxidation: the horse erythrocytes need longer incubation time with glucose or glucose plus methylene blue than human erythrocytes for reducing the methemoglobin; methylene blue did not enhance methemoglobin reduction in the equine erythrocytes, as occurred in human erythrocytes; for horses, lactate was a more efficient substrate in promoting methemoglobin reduction. The reduction of methemoglobin by equine erythrocytes under partial oxidation was very quick in any of the incubation media. The results can explain the incongruity between the previously reported inability of equine erythrocytes to reduce methemoglobin and the lack of methemoglobinemias in equine veterinary practice.     

Hemoglobin redox reactions and oxidative stress

The role of hemoglobin in transporting oxygen is dependent on the reversible binding of oxygen to Fe(II) hemoglobin with molecular oxygen released at reduced oxygen pressures. The partially oxygenated hemoglobin formed with the release of oxygen from hemoglobin is susceptible to redox reactions where the functional Fe(II) heme is oxidized to Fe(III) and the substrate is reduced. In this article, we review two important redox reactions of hemoglobin and discuss the ramifications of these reactions. The reduction of oxygen to superoxide starts a cascade of oxidative reactions, which are a source for red cell-induced oxidative stress. The reduction of nitrite to nitric oxide produces a labile form of nitric oxide that can be a source for oxidative stress, but can also have important physiological functions.     

Nitrite uptake and metabolism and oxidant stress in human erythrocytes

Nitric oxide, when released into the bloodstream, is quicklyscavenged by Hb in erythrocytes or oxidized to nitrite. Nitrite canalso enter erythrocytes and oxidize Hb. The goals of this work were todetermine the mechanism of erythrocyte nitrite uptake and whether thisuptake causes oxidant stress in these cells. Erythrocytes took up 0.8 mM nitrite with a half-time of 11 min. Nitrite uptake was sensitive totemperature and to the pH and ionic composition of the medium but wasnot inhibited by the specific anion-exchange inhibitor DIDS. About 25%of nitrite uptake occurred on the sodium-dependent phosphatetransporter and the rest as diffusion of nitrous acid or other speciesacross the plasma membrane. Methemoglobin formation increased inproportion to the intracellular nitrite concentration. Nitritereacted with erythrocyte ascorbate, but ascorbate loading of cellsdecreased nitrite-induced methemoglobin formation only at high nitriteconcentrations. In conclusion, nitrite rapidly enters erythrocytes andreacts with oxyhemoglobin but does not exert a strong oxidant stress onthese cells.

     

Hemoglobin redox reactions and oxidative stress

Abstract

The role of hemoglobin in transporting oxygen is dependent on the reversible binding of oxygen to Fe(II) hemoglobin with molecular oxygen released at reduced oxygen pressures. The partially oxygenated hemoglobin formed with the release of oxygen from hemoglobin is susceptible to redox reactions where the functional Fe(II) heme is oxidized to Fe(III) and the substrate is reduced. In this article, we review two important redox reactions of hemoglobin and discuss the ramifications of these reactions. The reduction of oxygen to superoxide starts a cascade of oxidative reactions, which are a source for red cell-induced oxidative stress. The reduction of nitrite to nitric oxide produces a labile form of nitric oxide that can be a source for oxidative stress, but can also have important physiological functions.     

Inactivation of enzymes and an enzyme inhibitor by oxidative modification with chlorinated amines and metal-catalyzed oxidation systems.

Oxidative inactivation of various key enzymes and alpha-1-proteinase inhibitor (alpha-1-PI) was studied by treatment with N-chloramines and the metal-catalyzed oxidation (MCO)-systems ascorbate/Fe(III) and ascorbate/Cu(II). Chlorinated amines completely inhibited alpha-1-PI, fructose-1,6-bis phosphatase (Fru-P2ase) and glyceraldehyde phosphate dehydrogenase (GAPD) at a low molar excess, and glucose-6-phosphate dehydrogenase (G6PD) at a high molar excess, but did not impair beta-N-acetylglucosaminidase (beta-NAG), alkaline phosphatase (AP) or lactate dehydrogenase (LDH). MCO-systems affected the activities of Fru-P2ase, GAPD, AP, LDH and G6PD, but not those of beta-NAG or alpha-1-PI. EDTA prevented inactivation of Fru-P2ase, G6PD and LDH by ascorbate/Cu(II) and of Fru-P2ase by ascorbate/Fe(III) suggesting a site-specific oxidation catalyzed by a protein-bound metal ion. In conclusion, N-chloramines and MCO-systems exhibited different properties with regard to oxidative inactivation, sulfhydryl-enzymes were susceptible to both systems, but other enzymes were only susceptible to one or neither system.     

Hematological responses of the Neotropical teleost matrinxã (Brycon cephalus) to environmental nitrite

Environmental increase in nitrite impairs the function of several aquatic species, including fishes. Nitrite reacts with hemoglobin yielding the non-functional methemoglobin (metHb), and many physiological disturbances can arise. The physiological mechanisms to cope with nitrite are still unclear in fish. Hematological parameters, the role of NADH-methemoglobin reductase system and the electrolytic balance were studied in the freshwater teleost Brycon cephalus (matrinx?) exposed to 0.2, 0.4 and 0.6 mg/L of nitrite N-NO(2) for 24 and 96 h. Hematocrit, total hemoglobin and the red blood cell (RBC) number decreased. Methemoglobin content increased from 1% to 69% for 24 h of exposure and drastically from 5-6% to 90% for 96 h. The activity of NADH-methemoglobin reductase system displayed a tendency of increase in response to nitrite concentration or time of exposure. In the plasma, nitrite was accumulated to values 30-fold higher than the environmental concentration. The plasma K(+) concentration increased only in fish exposed to NO(2) for 24 h. No changes in plasma protein and Na(+) were observed during nitrite exposure but Cl-presented a punctual increase at 0.2 mg/L N-NO(2)-96 h. The hematological data suggest that nitrite caused functional and hemolytic anemia. Furthermore, the electrolytic balance was relatively undisturbed, and the nitrite clearance in matrinx? is likely depending on other factors than NADH-methemoglobin reductase system.