Study of the effect of dose-rate on radiation-induced damage to human erythrocytes

Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit of 2%) were irradiated with γ-rays at three dose-rates of 66.7, 36.7, 25 Gy min⁻¹ in order to investigate the influence of the dose-rate on radiation-induced membrane damage, hemoglobin oxidation and loss of reduced glutathione.The obtained results showed that such processes as erythrocyte hemolysis, lipid and protein destruction depend on the radiation dose-rate. The parameter values describing these processes showed an inverse dose-rate effect.     

Erythrocyte response to near-infrared radiation

The effects of NIR (near-infrared radiation 700-2,000 nm) on bovine erythrocytes in plasma was studied as a continuation of earlier studies. Cell shape was observed and the changes of ratio of hemolysis and electrokinetic potential measured as a function of irradiation time. After 10 min of irradiation, the shape of erythrocyte cells was mainly echinocytic. When these cells were incubated at 311 K for 24 h they regained their initial shape, but fresh erythrocytes that were irradiated for 30 min and aged in vitro did not. These phenomena are due to: (1) the absorption of NIR excitation by hemoglobin; the primary photochemical process being the photo-dissociation of oxyhemoglobin to deoxyhemoglobin. Resulting shape and ratio of hemolysis, structural changes and oxidative stress follow higher deoxyhemoglobin concentration. (2) The absorption of the NIR excitation by proteins, water and lipids. After NIR absorption the membrane surface dehydrates, leading to enhanced protonation and dissociation of hydrogen-bonded complexes. This in turn leads to a change in electrokinetic potential.     

PHOTOSENSITIZATION BY 2-CHLORO-3, 11-TRIDECADIENE-5, 7, 9-TRIYN-1-OL: DAMAGE TO ERYTHROCYTE MEMBRANES, Escherichia coli, AND DNA

The natural product 2-chloro-3,11-tridecadiene-5,7,9-triyn-1-ol (1) photosensitized the inactivation of Escherichia coli in the presence of near-ultraviolet light (320-400 nm; NUV) under both aerobic and anaerobic conditions. A series of E. coli strains differing in DNA repair capabilities and catalase proficiency exhibited indistinguishable inactivation kinetics following treatment with the chemical plus NUV. The presence of carotenoids did afford some protection to E. coli against inactivation under aerobic conditions, consistent with the involvement of singlet oxygen. The photosensitized hemolysis of human erythrocytes occurred more rapidly in the absence than in the presence of oxygen. Aerobically, the onset of hemolysis was partially inhibited by NaN3 and by 2,6-di-t-butyl-4-methylphenol (BHT) but not by superoxide dismutase (SOD). The aerobic lipid peroxidation observed in the membranes of erythrocyte ghosts was completely inhibited by BHT, and partially by NaN3, but not by SOD. These results suggest that either lipid peroxidation of the membrane is not the main cause of photohemolysis or that BHT has insufficient access to intact erythrocyte lipids to protect them. Aerobically, crosslinking of membrane proteins was also observed; it was not affected by SOD, but was partially inhibited by BHT and NaN3. The anaerobic photosensitized hemolysis of erythrocytes was more rapid; a radical mechanism was suggested since BHT inhibited the hemolysis to a greater extent than under aerobic conditions. Neither lipid peroxidation nor protein crosslinking was observed under conditions believed to be anaerobic. A light-dependent electron transfer to cytochrome c was obtained under argon but not under oxygen. Although induced mutations were not observed in the experiments with E. coli, 1 was capable of damaging both supercoiled pBR322 and Haemophilus influenzae transforming DNA in a manner that seemed to be equivalent under aerobic and anaerobic conditions. In conclusion, 1 can behave as typical photodynamic molecule under aerobic conditions but, in contrast to most photodynamic molecules, it is also phototoxic under anaerobic conditions. The extent to which the radical reactions detected under anaerobic reactions compete with the photodynamic processes when oxygen is present is not known.     

Interaction of pentoxifylline with human erythrocytes. I. Interaction of xanthine derivatives with human erythrocyte ghosts

The interaction of pentoxifylline and other xanthine derivatives with human erythrocyte ghosts was studied. By fluorescence spectroscopy it was found that xanthine derivatives have two modes of binding to erythrocyte ghosts. One is a high-capacity binding to erythrocyte membranes. It seems that the 5-oxohexyl side chain of pentoxifylline is important for this. The second type may be a binding to proteins on the membranes and is specific for pentoxifylline and caffeine. From the circular dichroism spectra, it was presumed that the second binding mode of pentoxifylline occurs at hydrophobic regions of beta-structure of the membrane proteins. The relative high specificity in the interaction of pentoxifylline with erythrocytes should be related to its unique physiological activity on erythrocytes.     

Red blood cell ghosts and intact red blood cells as complementary models in photodynamic cell research

Recent research on erythrocytes as model cells for photodynamic therapy showed differing behaviour of certain photosensitisers in erythrocytes compared to other cells. Differences of dye accumulation in the cell membrane were proposed to be the reason for the distinct photodynamic effects. Using pheophorbide a as an example, the combination of erythrocyte ghosts as models to follow the dye accumulation in the cell membrane and intact erythrocytes as model cells to show the photodynamic damage is provided. Evidence for the correctness of the combination of erythrocyte ghosts and intact erythrocytes as a functioning model system in photodynamic cell research is provided using the confocal laser scanning microscopy on intact, pheophorbide a loaded erythrocytes.     

Kinetic regularities of erythrocyte hemolysis and hemoglobin oxidation under the action of sulfur-nitrosyl iron complexes as nitric oxide donors

The kinetics of erythrocyte hemolysis and intra-erythrocyte hemoglobin oxidation under the action of synthetic sulfur-nitrosyl iron complexes was studied. The complexes capable of releasing nitric oxide due to spontaneous hydrolytic decomposition was studied. The addition of these complexes to a 0.2% suspension of mouse erythrocytes results in hemolysis. The kinetic curves of hemolysis exhibit an induction period, whose duration is different for each complex. The hemolysis is preceded by hemoglobin oxidation with nitric oxide penetrating into the cell. The oxidation of hemoglobin follows the first-order rate equation. The apparent first-order rate constants characterizing the NO-donating ability of each complex were determined. The hemolytic effect of the studied complexes is suggested to be related to the formation of peroxynitrite inside erythrocytes. Peroxynitrite is the cytotoxic product of interaction of nitric oxide and the superoxide radical anion.     

Insulin binding and degradation by human erythrocytes and erythrocyte membranes

Conclusions By the current results there is evidence that the insulin degrading enzyme activity of the erythrocytes is not located on the inner or outer surface of the plasma membrane but is exclusively cytosolic. On the other hand, specific insulin binding to erythrocytes and erythrocyte membranes has been demonstrated and it could be supposed that the insulin binding region of the plasma membrane is not associated with an insulin degrading activity.In comparison to erythrocytes unsealed and sealed ghosts bound an equal amount of ¹²⁵I-insulin. Since there is binding but not degradation of insulin the membranes of erythrocytes might be a useful tool for the investigation of insulin internalization.

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Insulin-Bindung und -Abbau durch menschliche Erythrocyten und Erythrocyten-Membranen

     

Rb+跨人红细胞膜的研究

The erythrocyte hemolysis was examined to assure Rb^＋ to have a weak toxicity toward human body. The way of Rb^＋ transporting into human erythrocytes was determined, and the factors to affect this transport process were evaluated. The effects of extracellular concentration of Rb^＋, incubation temperature, incubation time, medium pH, and specific inhibitors were investigated via flame atomic absorption spectrometry. The results indicated that the membrane transport of Rb^＋ through human erythrocytes was controlled mainly by both active transport and simple diffusion. Every mentioned factor took a positive effect on the Rb^＋ uptake by human erythrocytes, however neither DIDS nor nefidpine could inhibit the uptake of Rb^＋.     

The exchange of erythrocyte membrane phospholipids with rat liver extracts in vitro.

Intact rat or human erythrocytes and their isolated (ghost) membranes were incubated with the high speed supernatant fraction of homogenates derived from 32P-labeled rat livers. Phospholipid molecules were transferred between the red cell membranes and the liver extracts, as reflected by the convergence of their specific radioactivities with time. Whereas ghosts usually approached isotopic equilibrium with the liver supernatant fraction during a few hours of incubation at 37 degrees C, the exchange of phospholipids by intact cells was no more than one-half, even after 18 hr. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin were all exchanged in both intact cells and ghosts, albeit to different extents. (A control experiment, incubating 32P-labeled rat erythrocytes or ghosts with unlabeled rat liver extracts, also demonstrated the exchange of all four major phospholipids.) These data may signify that the phospholipids on the cytoplasmic side of the membrane of intact erythrocytes do not exchange with the phospholipids in exogenous liver extracts. If so, all four major phospholipid classes would appear to be present to some extent at both membrane surfaces. The first inference is in agreement with several other studies on this membrane, while the second inference is not.     

LONG-WAVELENGTH UVA RADIATION INDUCES OXIDATIVE STRESS,CYTOSKELETAL DAMAGE and HEMOLYSIS*

Abstract— We investigated the ability of the different wavelength regions of UV radiation, UVA(320–400 nm), UVB(290–320 nm) and UVC(200–290 nm), to induce hemolysis. Sheep erythrocytes were exposed to radiation from either a UVA1 (>340 nm) sunlamp, a UVB sunlamp, or a UVC germicidal lamp. The doses used for the three wavelength regions were approximately equilethal to the survival of L5178Y murine lymphoma cells. Following exposure, negligible hemolysis was observed in the UVB- and UVC-irradiated erythrocytes, whereas a decrease in the relative cell number (RCN), indicative of hemolysis, was observed in the UVA 1-exposed samples. The decrease in RCN was dependent on dose(0–1625 kj/m²), time(0–78 h postirradiation) and cell density (10⁶-10⁷ cells/mL). Hemolysis decreased with increasing concentration of glutathione, hemoglobin or cell number, while the presence of pyruvate drastically enhanced it. Because scanning spectroscopy(200–700 nm) showed that hemoproteins and nicotinamide adenine dinucleotides were oxidized, cytoplasmic oxidative stress was implicated in the lytic mechanism. Further evidence of oxidation was obtained from electron micrographs, which revealed the formation of Heinz bodies near the plasma membrane. The data demonstrate that exposure of erythrocytes to UVA1, but not UVB or UVC, radiation causes oxidation of cytoplasmic components, which results in cytoskeletal damage and hemolysis.     

Interaction of liposomes with human erythrocytes

Interaction of liposomes (phospholipid vesicles) with human erythrocytes was studied by means of a spectroscopic method. Transfer of hemoglobin between liposomes and erythrocytes was observed. This transfer was mediated by a migration of band 3 proteins. In this case, a transfer of band 4.5 also was observed by means of electrophoresis. An interaction of lipid monomers from the liposomes with the erythrocyte membranes seemed to be closely correlated to the transfer of these proteins. It was presumed that this interaction induced some changes in the molecular organization of the cell membranes around band 3, resulting in release of the proteins from the erythrocyte membranes.     

Temperature-dependent changes in erythrocytes' cytosol state during natural and artificial hypobiosis

At present, the question of how the structural state of the erythrocyte cytosol is arranged to maintain essential permeabilities successfully both at normal temperature and during periods with a significant body temperature reduction during hypobiosis remains unanswered. In the present work, we performed comparative investigations of temperature-dependent changes in the cytosol state of erythrocytes from animals subjected to natural (winter hibernating ground squirrels) or artificial hypobiosis. The cytosol state was evaluated by the ESR method of spin probes (TEMPON) within the temperature range of 0-50 degrees C. Erythrocyte resistance to acid hemolysis, which is limited by the permeability of membranes for protons and the state of the anion channel, were determined using the method described by Terskov and Getelson [Biofizika 2 (1957) 259]. A change in cytosol microviscosity of erythrocytes was found as well as a temperature-dependent increase in acid resistance of erythrocytes. Our investigations allow us to conclude that physiological changes occurring in a mammalian organism during natural and artificial hypobiosis are accompanied by structural modifications of the erythrocyte cytosol. The temperature range where these modifications are observed (8, 15, 40 degrees C) suggests that the most probable modifying link is spectrin and/or the sites of its interaction with membrane. The interaction of cytoskeletal components with the cell membrane plays a key role in regulation of membrane permeability, suggesting an important role of this interaction in the adaptive reactions of erythrocytes.     

Permeabilization of biological and artificial membranes by a bacterial dirhamnolipid produced by Pseudomonas aeruginosa

Pseudomonas aeruginosa, when cultured under the appropriate conditions, secretes rhamnolipids to the external medium. These glycolipids constitute one of the most interesting classes of biosurfactants so far. A dirhamnolipid fraction was isolated and purified from the crude biosurfactant, and its action on model and biological membranes was studied. Dirhamnolipid induced leakage of internal contents, as measured by the release of carboxyfluorescein, in phosphatidylcholine unilamellar vesicles, at concentrations below its CMC. Membrane solubilization was not observed within this concentration range. The presence of inverted cone-shaped lipids in the membrane, namely lysophosphatidylcholine, accelerated leakage, whereas cone-shaped lipids, like phosphatidylethanolamine, decreased leakage rate. Increasing concentrations of cholesterol protected the membrane against dirhamnolipid-induced leakage, which was totally abolished by the presence of 50 mol% of the sterol. Dirhamnolipid caused hemolysis of human erythrocytes through a lytic mechanism, as shown by the similar rates of K⁺ and hemoglobin leakage, and by the absence of effect of osmotic protectants. Scanning electron microscopy showed that the addition of the biosurfactant changed the usual disc shape of erythrocytes into that of spheroechinocytes. The results are discussed within the frame of the biological actions of dirhamnolipid, and the possible future applications of this biosurfactant.     

Radiation damage to human erythrocytes. Relative contribution of hydroxyl and chloride radicals in N2O-saturated buffers

The erythrocyte suspensions in Na-phosphate buffered isotonic NaCl solution (PBS) or Na-phosphate isotonic buffer (PB) (hematocrit 1%) were irradiated with the dose of 400 Gy under N₂O. Erythrocytes were incubated in the medium in which the cells were irradiated or in fresh PBS. The level of damage to cells was estimated on the basis of the course of post-radiation hemolysis and hemoglobin (Hb) oxidation. The medium in which the cells were irradiated and incubated influenced the course of the post-radiation hemolysis and Hb oxidation as well as some other parameters. We discussed the contribution of hydroxyl and chloride radicals in the initiation of erythrocyte damage and oxygen modification of these processes.     

STUDY OF THE EFFECTS OF ULTRAVIOLET LIGHT ON BIOMEMBRANES—IV. THE EFFECT OF OXYGEN ON UV-INDUCED HEMOLYSIS AND LIPID PHOTOPEROXIDATION IN RAT ERYTHROCYTES AND LIPOSOMES

Abstract— Hemolysis induced by irradiation with ultraviolet (UV) light at 254 nm showed a pronounced oxygen effect: under irradiation in vacuum, the rate of hemolysis was decreased by an order of magnitude. Irradiation at 254 nm in air but not under vacuum caused the peroxidation of erythrocyte membrane lipids. These results suggest that membrane lipid photoperoxidation is one of the causative factors of UV hemolysis. Irradiation at different wavelengths showed that UV-induced lipid photoperoxidation in erythrocyte membranes developed while the antioxidant α-tocopherol was directly photooxidized. It is shown that the process of lipid photolysis in erythrocyte membranes involves sensitization, possibly by protoporphyrin, whose presence in liposomes accelerates the photoperoxidation at 254 and 365 nm of unsaturated fatty acid residues in lecithin. Possible mechanisms of photochemical damage to erythrocyte membranes are discussed.     

Proton and methanol transport behavior of SPEEK/TPA/MCM-41 composite membranes for fuel cell application

This paper reports proton and methanol transport behavior of composite membranes prepared for use in the direct methanol fuel cell (DMFC). The composite membranes were prepared by embedding various proportions (10–30 wt.%) of inorganic proton conducting material (tungstophosphoric acid (TPA)/MCM-41) into sulfonated poly(ether ether ketone) (SPEEK) polymer matrix. The results indicate that the proton conductivity of the membranes increases with increasing loading of solid proton conducting material. The highest conductivity value of 2.75 mS/cm was obtained for the SPEEK composite membrane containing 30 wt.% solid proton conducting material (50 wt.% TPA in MCM-41). The methanol permeability and crossover flux were also found to increase with increasing loading of the solid proton conducting material. Lowest permeability value of 5.7 × 10⁻⁹ cm² s⁻¹ was obtained for composite membrane with 10 wt.% of the solid proton conducting material (40 wt.% TPA in MCM-41). However, all the composite membranes showed higher selectivity (ratio between the proton conductivity and the methanol permeability) compared to the pure SPEEK membrane. In addition, the membranes are thermally stable up to 160 °C. Thus, these membranes have potential to be considered for use in direct methanol fuel cell.     

CROSS-LINKING OF MEMBRANE PROTEINS AND PROTOPORPHYRIN-SENSITIZED PHOTOHEMOLYSIS*

Abstract— Irradiation of protoporphyrin-sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis. Extensive cross-linking of membrane proteins can be observed before hemolysis occurs (Girotti, 1976).
Facile oxidative hemolysis can be achieved without observable cross-linking of membrane proteins upon incubation (37°C) of red cells containing membrane-bound 3ß-hydroxy-5α-hydroperoxy-△⁶-cholcstene. Thus, protein cross-linking is not obligatory for oxidative lysis. Deoxygenation by Ar bubbling strongly retards the light-induced increase in osmotic fragility and strongly inhibits eventual hemolysis of protoporphyrin-sensitized erythrocytes. However, similar reduction in oxygen concentration only partially inhibits cross-linking of membrane proteins. These results suggest that membrane protein cross-linking and photohemolysis are not coupled processes.     

Phospholipids Bilayers as Molecular Models for Drug- Membrane Interactions. The Case of the Antiepileptics Phenytoin and Carbamazepine

With the aim to better understand the molecular mechanisms of the interaction of phenytoin and carbamazepine with cell membranes we utilized a well-established model consisting in intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of its membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidyl-ethanolamine (DMPE), representative of phospholipid classes respectively located in the outer and inner monolayers of erythrocytes and other cell membranes. This report presents the following evidence that phenytoin and carbamazepine interact with membrane phospholipids: a) X-ray diffraction and fluorescence spectroscopy showed that both drugs preferentially interacted with DMPC; b) in IUM, the drugs induced a disordering effect on the polar head groups and acyl chains of the eryhrocyte membrane lipid bilayers; c) electron microscopy observations of human erythrocytes showed the echinocyte formation, an effect due to phenytoin and carbamazepine insertion in the outer monolayer of the red cell membrane.     

Investigation of surface and shape changes accompanying the membrane alteration responsible for the heat-induced lysis of human erythrocytes

Heat induces several successive events in erythrocyte membrane; the denaturation of spectrin at about 50°C, thermoporation at 62°C and denaturation of the anion channel at 67°C. The heat denaturation of major membrane proteins, spectrin and the anion channel, is not needed for the thermoporation which is involved in thermohemolysis. This study reports about the surface and shape changes which are specific for thermoporated membranes with spectrin and anion channel preserved intact. Thermoporation was produced exposing human erythrocytes to 39.5°C for 3 min in isotonic medium containing 18% (v/v) ethanol as membrane fluidizer and sucrose as osmotic protectant which prevents hemolysis (Ivanov, J. Therm. Biol. 1996). The control cells were processed similarly except that they were incubated at 23°C, thus avoiding thermoporation. In control and porated membranes the overall structure of spectrin and the anion channel was retained inasmuch as their enthalpies and denaturation temperatures were microcalorimetrically found preserved. Nevertheless, irregular shape, grainy surface and asymmetric spicules were apparent in porated cells through scanning electron microscopy. A decrease in the number of binding sites for Alcian blue and an increased binding of eosine was established in the membranes of porated cells. After poration the hexane/aqueous partition coefficient K_d of cells increased from 5 to about 220 and the electrophoretic mobility of cells decreased by about 25% indicating marked increase in cell surface hydrophobicity and a decrease in surface charge, respectively. In addition, adhesivity to hydrophobic interfaces and aggregability in low ionic media strongly increased after poration. In contrast to intact and control cells, the porated ones (all prefixed with 0.2% glutaraldehyde) made molecular contacts with inclined hydrophobic interfaces at low (5 mM NaCl) but not at high (150 mM NaCl) ionic media. Thus, the microtopological shape changes and exposure of new hydrophobic and charge groups over the outer cell surface, without major thermal unfolding, possibly indicates an irreversible redistribution of membrane material and disturbed lipid–protein complementation during thermoporation.