Effect of hypervitaminosis A on hemolysis and lipid peroxidation in the rat

Erythrocytes from rats fed large doses of Vitamin A alone, or large doses of vitamin A and vitamin E or diphenyl-p-phenylene diamine (DPPD) were studied for H2O2-induced hemolysis. The vitamin A-dosed rats were more susceptible than normal rats to H2O2-induced hemolysis. Hemolysis was not accompanied by lipid peroxidation. Nevertheless, the antioxidants vitamin E and DPPD inhibited hemolysis in erythrocytes from vitamin A-dosed rats. These antioxidants had the same inhibitory effect when they were included in the diet or added to erythrocyte suspensions in vitro. Erythrocytes from vitamin A-dosed rats with or without added vitamin E or DPPD were less susceptible than the erythrocytes from normal rats to osmotic challenge, showing that vitamin A was present in levels sufficient to alter the structure of the erythrocyte membrane. These studies show that oxidative hemolysis occurs when the erythrocyte membrane is modified. Furthermore, this oxidative hemolysis is unrelated to lipid peroxidation.     

Lipid peroxidation is not the cause of lysis of human erythrocytes exposed to inorganic or methylmercury

Exposure of human erythrocytes in a 50% hematocrit to 0.5-1 mM Hg2+ initiated immediate hemolysis which proceeded at a constant rate without any formation of lipid hydroperoxides. Treatment of 0.03% hematocrits with 0.4 ppm of Hg2+ or 40 ppm of methylmercury caused rapid hemolysis after a short lag period. The kinetics of the process were unaltered by saturation of the cell suspensions with oxygen, by its replacement with He or CO, or by variation in the level of vitamin E in the membranes. The results show that peroxidation of erythrocyte membrane lipids is not the cause of hemolysis induced by either Hg2+ or methylmercury.     

Studies on the role of goat heart galectin-1 as an erythrocyte membrane perturbing agent

Galectins are β-galactoside binding lectins with a potential hemolytic role on erythrocyte membrane integrity and permeability. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its hemolytic actions on erythrocyte membrane. When exposed to various saccharides, lactose and sucrose provided maximum protection against hemolysis, while glucose and galactose provided lesser protection against hemolysis. GHG-1 agglutinated erythrocytes were found to be significantly hemolyzed in comparison with unagglutinated erythrocytes. A concentration dependent rise in the hemolysis of trypsinized rabbit erythrocytes was observed in the presence of GHG-1. Similarly, a temperature dependent gradual increase in percent hemolysis was observed in GHG-1 agglutinated erythrocytes as compared to negligible hemolysis in unagglutinated cells. The hemolysis of GHG-1 treated erythrocytes showed a sharp rise with the increasing pH up to 7.5 which became constant till pH 9.5. The extent of erythrocyte hemolysis increased with the increase in the incubation period, with maximum hemolysis after 5 h of incubation. The results of this study establish the ability of galectins as a potential hemolytic agent of erythrocyte membrane, which in turn opens an interesting avenue in the field of proteomics and glycobiology.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Vitamin E: inhibition of retinol-induced hemolysis and membrane-stabilizing behavior.

For the elucidation of the mechanism of membrane stabilization by vitamin E, the effects of alpha-tocopherol and its model compounds on either retinol-induced hemolysis of rabbit erythrocytes or the permeability and fluidity of liposomal membranes have been studied. Retinol-induced rabbit erythrocyte hemolysis has been found not to be caused by the oxidative disruption of erythrocyte membrane lipids initiated by retinol oxidation, but rather to arise from physical damage of the membrane micelle induced by penetration of retinol molecules. In suppressing hemolysis, alpha-tocopherol was more effective than other naturally occurring tocopherols. alpha-Tocopheryl acetate, nicotinate, and 6-deoxy-alpha-tocopherol were more effective than alpha-tocopherol itself. The inhibitory effects of alpha-tocopherol model compounds having side chains with at least two isoprene units or a long straight chain instead of the isoprenoid side chain were similar to those of alpha-tocopherol. These data suggest that for protection of membranes against retinol-induced damage, the hydroxyl group of alpha-tocopherol is not critical, but rather the chroman ring, three methyl groups on the aromatic ring, and the long side chain are necessary. To verify the mechanism of the inhibitory effect on hemolysis, not only the effect of vitamin E and its model compounds on the membrane permeability and fluidity, but also the mobility of alpha-tocopherol molecule in membranes has been investigated using bilayer liposomes as the model membranes. Addition of alpha-tocopherol to membranes produced a greater decrease in the permeability and fluidity of rat liver phosphatidylcholine liposomes compared with egg yolk phosphatidylcholine liposomes. In dipalmitoylphosphatidylcholine liposomes, however, alpha-tocopherol was less effective, that is, the more unsaturated the lipids, the more they interact with alpha-tocopherol. 2,2,5,7,8-Pentamethyl-6-chromanol with no isoprenoid side chain and phytol without the chromanol moiety had no effect. The measurement of 13C NMR relaxation times revealed that the mobility of methyl groups on the aromatic ring of alpha-tocopherol in membranes is significantly restricted. In contrast, the methyl groups at positions 4'a and 8'a on the isoprenoid side chain have high degrees of motional freedom in the lipid core of membranes. Furthermore, it was found that alpha-tocopherol in membranes interacts with chromate ions added as potassium chromate outside the membranes, resulting in an increase in membrane fluidity. These results are compatible with those of the inhibitory effect on retinol-induced erythrocyte hemolysis. On the basis of the results obtained here, a possible mechanism for membrane stabilization by vitamin E is proposed.     

Lidocaine: An inhibitor in the free‐radical‐induced hemolysis of erythrocytes

Lidocaine was reported to protect erythrocytes from hemolysis induced by 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH). Since AAPH‐induced hemolysis was a convenient in vitro experimental system to mimic erythrocytes undergoing peroxyl radicals attack, the aim of this work was to investigate the antioxidant effect of lidocaine on AAPH‐induced hemolysis by chemical kinetics. As a result, one molecule of lidocaine can only trap 0.37 radical, much lower than melatonin. Meanwhile, lidocaine cannot protect erythrocytes from hemolysis induced by hemin, which the mechanism of hemolysis was due to the erythrocyte membrane destroyed by hemin. Accordingly, lidocaine protected erythrocytes by scavenging radicals preferentially rather than by stabilizing membrane. Moreover, the interactions of lidocaine with two radical species, including 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonate) radical cation (ABTS^+?) and 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH), indicated that lidocaine can reduce ABTS^+? with 260 µM as the 50% inhibition concentration (IC₅₀) and cannot react with DPPH. Thus, lidocaine served as a reductant rather than a hydrogen donor to interact with radicals. Finally, the quantum calculation proved that, compared with the melatonin radical, the stabilization of N‐centered radical of lidocaine was higher than the amide‐type N‐centered radical but lower than the indole‐type N‐centered radical in melatonin. These results provided basic information for lidocaine to be an antiradical drug. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:81–86, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20267     

羟自由基对人红细胞磷脂酰乙醇胺脂质体相变性质的影响

研究了过氧化氢与亚铁离子体系产生的羟自由基对人红细胞膜磷脂酸乙醇胺(PE)脂质体相变性质的影响.结果表明,羟自由基导致脂质体不饱和脂肪酸链的含量明显降低和丙二醛(MDA)含量显著升高,同时其膜流动性随之下降.在室温下,羟自由基诱使PF脂质体冰冻断裂面出现脂质颗粒,说明羟自由基通过脂质过氧化作用可促进PE脂质体从脂双层转变为非双层结构.     

羟自由基对人红细胞磷脂酰乙醇胺脂质体相变性质的影响

研究了过氧化氢与亚铁离子体系产生的羟自由基对人红细胞膜磷脂酸乙醇胺(PE)脂质体相变性质的影响.结果表明,羟自由基导致脂质体不饱和脂肪酸链的含量明显降低和丙二醛(MDA)含量显著升高,同时其膜流动性随之下降.在室温下,羟自由基诱使PF脂质体冰冻断裂面出现脂质颗粒,说明羟自由基通过脂质过氧化作用可促进PE脂质体从脂双层转变为非双层结构.     

Influence of superoxide generating system, vitamin E, and superoxide dismutase on radiation consequences.

During studies of the mechanism by which hemolysis is induced in irradiated human erythrocytes in vitro, several inducements of membrane lipid peroxidation and protective effects of vitamin E (V.E) and superoxide dismutase (SOD) were investigated. Findings were: (1) Before hemolysis, K+ release from erythrocytes induced by radiation stimulated hemolysis but was inhibited by V.E or SOD. (2) Lipid peroxidation of mitochondria induced by Fe3+, ADP, and superoxide (O2-) generating system, and lipid peroxidation of microsome induced by O2- generating system, were also inhibited by V.E or SOD. (3) X-ray or 60Co gamma-ray radiation stimulated lipid peroxidation of liver homogenate, microsome, and liposome. Some of this peroxidation was inhibited by V.E. or SOD. These results suggest that O2- and/or OH formation by radiation induces membrane lipid peroxidation, which causes deterioration of membrane resulting in change of ion permeability and consequent hemolysis.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

Abstract

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (^*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H₂O₂; ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Molecular mechanism of action of chlorogenic acid on erythrocyte and lipid membranes

Abstract

The high antioxidant capacity of chlorogenic acid (CGA) in respect to biological systems is commonly known, though the molecular mechanism underlying that activity is not known. The aim of the study was to determine that mechanism at the molecular and cell level, in particular with regard to the erythrocyte and the lipid phase of its membrane. The effect of CGA on erythrocytes and lipid membranes was studied using microscopic, spectrophotometric and electric methods. The biological activity of the acid was determined on the basis of changes in the physical parameters of the membrane, in particular its osmotic resistance and shapes of erythrocytes, polar head packing order and fluidity of erythrocyte membrane as well as capacity and resistivity of black lipid membrane (BLM). The study showed that CGA becomes localized mainly in the outer part of membrane, does not induce hemolysis or change the osmotic resistance of erythrocytes, and induces formation of echinocytes. The values of generalized polarization and fluorescence anisotropy indicate that CGA alters the hydrophilic region of the membrane, practically without changing the fluidity in the hydrophobic region. The assay of electric parameters showed that CGA causes decreased capacity and resistivity of black lipid membranes. The overall result is that CGA takes position mainly in the hydrophilic region of the membrane, modifying its properties. Such localization allows the acid to reduce free radicals in the immediate vicinity of the cell and hinders their diffusion into the membrane interior.     

Effects of Proteus mirabilis lipopolysaccharides with different O-polysaccharide structures on the plasma membrane of human erythrocytes

The effects of O33 and O49 P. mirabilis lipopolysaccharides (LPSs) on human erythrocyte membrane properties were examined. Physical parameters of the plasma membrane, such as membrane lipid fluidity, physical state of membrane proteins, and osmotic fragility, were determined. The fluidity of the lipids was estimated using three spin-labeled stearic acids of doxyl derivatives: 5-doxylstearic acid, 12-doxylstearic acid, and 16-doxylstearic acid. All the applied labels locate to different depths of the lipid layer and provide information on the ordering of phospholipid fatty acyl chain mobility. LPSs O49 increased the membrane lipid fluidity in the polar region of the lipid bilayer as indicated by spin-labeled 5-doxylstearic acid. An increase in fluidity was also observed in the deeper region using 12-doxylstearic acid only for O33 LPSs. The highest concentration of O33 LPSs (1 mg/ml) increased the motion of membrane proteins detected by the spin-label residue of iodoacetamide. These results showed different actions of O33 and O49 LPSs on the plasma membrane due to the different chemical structures of O-polysaccharides. P. mirabilis O33 and O49 LPSs did not induce changes in the membrane cytoskeleton, osmotic fragility and lipid peroxidation of erythrocytes. On the other hand a rise in the content of carbonyl compounds was observed for the highest concentrations of O33 LPS. This result indicated protein oxidation in the erythrocyte membrane. Lipid A, the hydrophobic part of LPS, did not change the membrane lipid fluidity and osmotic fragility of erythrocytes. Smooth and rough forms of P. mirabilis LPSs were tested for their abilities for complement-mediated immunohemolysis of erythrocytes. Only one out of seven LPSs used was a potent agent of complement-mediated hemolysis. It was rough, Ra-type of P. mirabilis R110 LPS. The O-polysaccharide-dependent scheme of reaction is presented.     

The effect of Pycnogenol on the erythrocyte membrane fluidity

In the present study, the in vitro effect of polyphenol rich plant extract, flavonoid--Pycnogenol (Pyc), on erythrocyte membrane fluidity was studied. Membrane fluidity was determined using 1-[4-trimethyl-aminophenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), 1,6-diphenyl-1,3,5-hexatriene (DPH) and 12-(9-anthroyloxy) stearic acid (12-AS) fluorescence anisotropy. After Pyc action (50 microg/ml to 300 microg/ml), we observed decreases in the anisotropy values of TMA-DPH and DPH in a dose-dependent manner compared with the untreated erythrocyte membranes. Pyc significantly increased the membrane fluidity predominantly at the membrane surface. Further, we observed the protective effect of Pyc against lipid peroxidation, TBARP generation and oxidative hemolysis induced by H2O2. Pyc can reduce the lipid peroxidation and oxidative hemolysis either by quenching free radicals or by chelating metal ions, or by both. The exact mechanism(s) of the positive effect of Pyc is not known. We assume that Pyc efficacy to modify effectively some membrane dependent processes is related not only to the chemical action of Pyc but also to its ability to interact directly with cell membranes and/or penetrate the membrane thus inducing modification of the lipid bilayer and lipid-protein interactions.     

Inhibition of oxidative hemolysis in erythrocytes by mitochondria-targeted antioxidants of SkQ series

In the present work we studied the effect of antioxidants of the SkQ1 family (10-(6′-plastoquinonyl)decyltriphenylphosphonium) on the oxidative hemolysis of erythrocytes induced by a lipophilic free radical initiator 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) and a water-soluble free radical initiator 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH). SkQ1 was found to protect erythrocytes from hemolysis, 2 μM being the optimal concentration. Both the oxidized and reduced SkQ1 forms exhibited protective properties. Both forms of SkQ1 also inhibited lipid peroxidation in erythrocytes induced by the lipophilic free radical initiator AMVN as detected by accumulation of malondialdehyde. However, in the case of induction of erythrocyte oxidation by AAPH, the accumulation of malondialdehyde was not inhibited by SkQ1. In the case of AAPH-induced hemolysis, the rhodamine-containing analog SkQR1 exerted a comparable protective effect at the concentration of 0.2 μM. At higher SkQ1 and SkQR1 concentrations, the protective effect was smaller, which was attributed to the ability of these compounds to facilitate hemolysis in the absence of oxidative stress. We found that plastoquinone in the oxidized form of SkQ1 could be reduced by erythrocytes, which apparently accounted for its protective action. Thus, the protective effect of SkQ in erythrocytes, which lack mitochondria, proceeded at concentrations that are two to three orders of magnitude higher than those that were active in isolated mitochondria.     

Role of membrane-bound calcium in changes in the ATPase activity, permeability and structural state of human erythrocyte membranes]

A study was conducted on the reconstituted erythrocytes obtained by the method of fast reversible hemolysis. The concentration of free Ca2+ ions in the reconstituted erythrocytes was supported by Ca-EGTA and Ca-nitrate buffers. Oubain-uninhibited ATPase component with a high affinity for Ca2+ (K0.5=4 micron) and alteration of passive and active K+-permeability in the region of free Ca2+ concentration up to 10 micron could be determined only when the content of membrane-bound Ca+ varied. Depletion of the inner side of the membrane of reconstituted erythrocyte is accompanied by alteration of hydrophobic character of the hydrocarbon region of the membrane. It is suggested that Ca+-induced alterations in the structure of the erythrocyte membrane may be a direct cause of the alterations in ATPase activity with a high Ca2+ affinity and permeability for univalent cations.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Genistein abrogates pre-hemolytic and oxidative stress damage induced by 2,2'-Azobis (Amidinopropane)

The pre-hemolytic mechanism induced by free radicals initiated from water-soluble 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) and its reversal by genistein was investigated in human erythrocytes. The time course of K+ efflux compared to the occurrence of hemolysis suggests that AAPH-induced hemolysis occurs indirectly via pore formation and band 3 oxidation as expected. However, genistein inhibited hemolysis, LDH release and membrane protein oxidation but not K+ efflux. This indicated that erythrocyte protein oxidation possibly in the hydrophobic core plays a significant role in the membrane pre-hemolytic damage. Chemiluminescence (CL) analysis carried out in non-lysed erythrocytes treated with AAPH showed a dramatic increase in CL indicating both reduced levels of antioxidants and increased membrane lipid peroxide. The V0 value was also increased up to 6 times, denoting a high degree of membrane peroxidation very early in erythrocyte membrane damage. The whole process was inhibited by genistein in a dose-dependent manner. These results indicate that the genistein inhibited both hemolysis and pre-hemolytic damage and also hindered membrane lipid peroxide formation and protein oxidation. In addition, it is suggested that pre-hemolytic damage is mediated mainly by the oxidation of both phospholipid and protein located in the deeper hydrophobic region of the membrane.     

Increase in vulnerability to oxidative damage in cholesterol-modified erythrocytes exposed to t-BuOOH

During the course of radical oxidation, cholesterol may exert seemingly contradictory effects. In order to gain a better understanding of the relationship between cholesterol levels and membrane susceptibility to oxidative damage induced by reactive oxygen species (ROS), here we analyze the integrity and structural stability of cholesterol-modified (enriched or depleted) and unmodified (control) erythrocytes exposed to tert-butyl hydroperoxide. The oxidant significantly increased ROS production, with almost complete oxidation of hemoglobin and a reduction in GSH content in the different erythrocyte groups at 2 mM concentration. These changes were accompanied by losses of cholesterol and total phospholipids, the main decreases being in phosphatidylethanolamine and phosphatidylcholine. The highest lipid loss was found in the cholesterol-depleted group. Fatty acid analyses revealed changes only in peroxidized cholesterol-modified erythrocytes, with decreases in linoleic and arachidonic acids. Fluorescence anisotropy studies showed an increase in the fluidity of the negatively charged surface of peroxidized control erythrocytes. Increased hemolysis and a positive correlation between cellular osmotic fragility and malondialdehyde contents were found in all peroxidized groups. These findings provide evidence that the modification of cholesterol levels in the erythrocyte membrane has provoking effects on peroxidation, with corresponding increases in oxidative damage in the treated cell, possibly as a consequence of lipid bilayer destabilization.     

The mechanism of the hemolytic activity of polyene antibiotics

The kinetics of the filipin-, amphotericin B- and nystatin-induced hemolysis of human erythrocytes were investigated. Filipin-induced hemolysis is of the damage type. It is an all-or-none process, partly inhibited by Ca2+ or Ba2+ but not by Mg2+, Na+ or SO42-. The hemolytic activity of filipin is explained by the formation of large aggregates within the erythrocyte membrane in the form of large perforations, permeable to substances of low molecular weight as well as to macromolecules, including hemoglobin. In isotonic KCl solution, both amphotericin B and nystatin, at low concentrations, form smaller aggregates within the membranes. As a result, the permeability of the membranes to KCl increases and hemolysis occurs. However, the kinetics of the hemolysis induced by the two polyenes is complex. The process shows some features of the permeability type and some of the damage type. It is suggested that amphotericin B and nystatin may simultaneously form a number of transport systems, differing in their molecular organisation and hemolytic activity. Their participation in erythrocyte membrane permeability can be modified by small changes in membrane organisation and the chemical composition of the incubation medium. In isotonic solutions of divalent cation chlorides, and at higher antibiotic concentration, additional aggregates, allowing divalent cations to permeate, appear. These structures do not permit SO4(2-) to permeate.     

Comparison of the effect of phenoxyherbicides on human erythrocyte membrane (in vitro)

The molecular mechanisms of phenoxyherbicides action in animals have been insufficiently studied. Now, we have investigated the interaction of sodium salts of phenoxyherbicides, e.g., 2,4-dichlorophenoxyacetic acid (2,4-D-Na), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T-Na) and 4-chloro-2-methylphenoxyacetic acid (MCPA-Na) with human erythrocytes. In this study, we evaluated the effect of these compounds on erythrocyte membrane fluidity as well as changes in membrane proteins content. It was observed that all of the compounds studied altered membrane fluidity, changed the size and shape of the erythrocytes and provoked echinocytes formation. It was also revealed that 2,4-D-Na and 2,4,5-T-Na changed the content of erythrocyte membrane proteins mainly by a decrease in the level of spectrin and low molecular weight proteins. The comparison of the action of phenoxyherbicides examined showed that 2,4,5-T-Na caused the greatest changes in the erythrocytes membrane, whereas MCPA-Na induced the lowest alterations in the incubated cells. It must be noted that changes of the investigated parameters were observed only at presence of significant concentrations of these compounds that may penetrate human organism only as a result of acute poisoning.