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.     

Comparison of membrane structure,osmotic fragility,and morphology of multiple sclerosis and normal erythrocytes

Erythrocyte membranes from multiple sclerosis (MS) patients and normal individuals were studied by electron spin resonance spectroscopy, osmotic fragility tests, scanning electron microscopy (SEM) and fatty acid analysis of membrane lipids. There was no significant difference in the membrane fluidity between MS and normal erythrocytes using fatty acid spin labels with the nitroxide moiety on carbons 5, 12, or 16 from the carboxyl group. Linoleic acid, which has been reported to decrease the absolute electrophoretic mobility of only MS erythrocytes, increased the fluidity of MS and normal erythrocyte membranes to a similar extent. The osmotic fragility of MS erythrocytes obtained from outpatients was similar to normal control cells but the osmotic fragility of erythrocytes obtained from hospitalized MS patients was greater than normal. Scanning electron microscopy of MS erythrocytes revealed no gross abnormalities. Cells incubated with linoleic acid had transformed from discocytes into sphero-echinocytes with prominent membrane surface indentations but MS and normal erythrocytes appeared identical. Of the fatty acid content of the total lipid extract, erythrocytes from most, but not all, MS hospitalized patients and some patients with other demyelinating diseases had relatively less (P<.001) 18:2 than the normal cells. These results indicate that at least some of the abnormalities reported in MS erythrocytes may only be found in hospitalized patients and may be due to other complications of the disease. They also indicate that the reported abnormal effects of linoleic acid on the electrophoretic mobility of MS erythrocytes may be caused by some other mechanism than an effect on the fluidity of the bilayer.     

The role of oxidative stress in alterations of hematological parameters and inflammatory markers induced by early hypercholesterolemia

Aims

The investigation of the effects of a high cholesterol diet (HD) for a short-time period on hematological parameters and the potential role of oxidative stress and inflammation markers.

Main methods

Rabbits were fed either a control diet or a diet containing 1% cholesterol (HD) for 5–6 weeks. The plasma lipid levels, C reactive protein (CRP), total red blood cells (RBC), total white blood cells (WBC), platelet count, packed cell volume (PCV) and leukocyte formula were determined. Oxidative stress was evaluated by the thiobarbituric acid reactive substances (TBARS), total glutathione and GSH serum level measurements. The osmotic fragility and the membrane fluidity of erythrocytes were determined. The levels of total cholesterol and TBARS were also measured in the erythrocyte membrane suspension.

Key findings

A decrease in the RBC and PCV was observed in rabbits fed on HD. The membrane rigidity and osmotic fragility were increased, and the morphological changes caused by the HD and TBARS levels in the erythrocyte membrane may account for this phenomenon. The inflammatory markers as the CRP levels, the platelet count, the WBC and the neutrophils were increased. The TBARS and GSH levels in the serum were increased and decreased, respectively.

Significance

This study shows that feeding rabbits an HD for a short time induces hematological alterations, disturbances in the oxidant–antioxidant balance and an increase of inflammatory markers. These findings support the importance of the early correction or prevention of high cholesterol levels to disrupt the process leading to the development of cardiovascular diseases.     

Spin label studies of erythrocytes with abnormal lipid composition: comparison of red cells in a hereditary hemolytic syndrome and lecithin: Cholesterol acyltransferase deficiency

Erythrocytes from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency have been shown to exhibit an increase in membrane fluidity which is surprisingly small in view of the extensive alterations both in membrane lipicl composition (namely, an elevation in cholesterol and phosphatidylcholine contents as well as a decrease in phosphatidylethanolamine) and in the functional properties of these cells. In the hope of deriving some information concerning the interrelationship between the structural and functional abnormalities, we have used the spin probe 5-doxyl stearic acid to investigate the temperature-dependent fluidity properties of red cells from two patients with a hereditary hemolytic syndrome (HHS) whose red cells are also characterized by qualitatively similar alterations in phosphatidylcholine and phosphatidylethanolamine but, unlike those in LCAT deficiency, have relatively normal levels of membrane cholesterol. A small increase in membrane fluidity of HHS erythrocytes equivalent to that previously observed in LCAT deficiency was found, indicating that membrane cholesterol level does not exert an important modulatory influence on membrane fluidity in these cells. It is concluded that while the distinct patterns of structural and functional erythrocyte alterations in these two disorders cannot be explained on the basis of differences in bulk membrane fluidity, the marginally increased fluidity may underlie the abnormalities in osmotic fragility and membrane p-nitrophenylphosphatase activity which are shared in common by both types of modified red cells.     

The influence of metmyoglobin and ferrylmyoglobin on the human erythrocyte membrane

Preliminary experiments revealed that ferrylmyoglobin decayed more slowly in the absence than in the presence of intact erythrocytes and erythrocyte membranes. This suggested the existence of interactions between FerrylMb and the erythrocyte membrane. Subsequent studies examined the influence of FerrylMb on the membrane of intact erythrocytes and on isolated erythrocyte membranes. The incubation of intact erythrocytes with FerrylMb did not influence their osmotic fragility or the fluidity of their membranes; the level of peroxidation of the membrane lipids increased only slightly (there was only a slight increase in the level of membrane lipid peroxidation). The activity of acetylcholinesterase significantly increased after 15 minutes of incubation, whereas longer incubation did not lead to any changes in the activity of this enzyme. The incubation of isolated erythrocyte membranes with FerrylMb resulted in an increase in their fluidity and a significant rise in the level of lipid peroxidation.     

Damage to the erythrocyte membrane caused by chlorophenoxyacetic herbicides

We studied the damage caused to erythrocyte membranes by chlorophenoxyacetic herbicides. An increase in haemolysis was observed. The compounds investigated caused lipid bilayer damage by lipid peroxidation, as well as an increase in membrane fluidity at the 16th carbon atom of fatty acids was observed. Metabolites caused damage to membrane proteins - the free SH group content was increased. Higher toxicity of metabolites compared to basic compounds was observed.     

Alterations in human red blood cell membrane properties induced by the lipopolysaccharide from Proteus mirabilis S1959

The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant (P<0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly (P<0.05) decreased the relative rotational correlation time of ISL and significantly (P<0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h(w)/h(s) ratio. These results indicated increased membrane protein mobility, mainly in the spectrin-actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.     

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.     

Carbamylation of proteins leads to alterations in the membrane structure of erythrocytes

The effect of the sodium cyanate-induced carbamylation (carbamoylation) of proteins in erythrocytes was studied using spin labelling and spectrophotometric methods. The experiments were conducted in whole blood and in erythrocytes in phosphate buffer using 25 mmol/L of sodium cyanate. Lipid membrane fluidity was determined using three spin-labelled fatty acids: 5-, 12- and 16-doxylstearic acids (5-DS, 12-DS, 16-DS). Internal viscosity was measured with Tempamine, using also EPR spectroscopy. Osmotic fragility was determined spectrophotometrically. Incubation of whole blood with sodium cyanate led to an increase in lipid membrane fluidity in the deeper region of the lipid layer, indicated by 12- and 16-doxylstearic acid, and a decrease near the surface (5-DS). Statistically significant results were obtained for the internal viscosity and osmotic fragility of erythrocytes. An increase in internal viscosity and increase in osmotic fragility were found in erythrocytes after incubation of whole blood, as well as in erythrocytes incubated with sodium cyanate in buffer. Alterations in internal viscosity were stronger in erythrocytes incubated with sodium cyanate in blood than in erythrocytes in the buffer. On the other hand, higher osmotic fragility was observed for erythrocytes in the buffer.     

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.     

Electron spin resonance, hematological, and deformability studies of erythrocytes from patients with Huntington's disease.

Electron spin resonance, hematologic, and deformability studies of erythrocytes from patients with Huntington's disease have been performed A decreased deformability of Huntington's disease erythrocytes compared to normal controls was demonstrated. No difference in erythrocyte hematologic indices, osmotic fragility, reticulocyte counts, or intracellular Na+ concentration was found. Huntington's disease serum had no demonstrable effect on electron spin resonance parameters of a protein-specific spin label attached to membrane proteins in control erythrocytes compared to the effect of control serum. This finding suggests that under the conditions employed no serum component or circulating factor is responsible for the changes in the physical state of membrane proteins in Huntington's disease erythrocytes (Butterfield, D.A., Oeswein, J.Q. and Markesbery, W.R. (1977) Nature 267, 453--455). No alteration in lipid fluidity of Huntington's disease erythrocyte membranes could be discerned suggesting that the underlying molecular defect in Huntington's disease involves a membrane protein. The results of the present studies on erythrocytes strongly support the concept that Huntington's disease is associated with a generalized membrane abnormality.     

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.     

Oxidative damages in erythrocytes of patients with metabolic syndrome

The aim of the study was to estimate the changes caused by oxidative stress in structure and function of membrane of erythrocytes from patients with metabolic syndrome (MS). The study involved 85 patients with MS before pharmacological treatment and 75 healthy volunteers as a control group. Cholesterol level, lipid peroxidation, glutathione level (GSH), and antioxidant enzyme activities in erythrocytes were investigated. The damage to erythrocyte proteins was also indicated by means of activity of ATPase (total and Na⁺,K⁺ ATPase) and thiol group level. The membrane fluidity of erythrocytes was estimated by the fluorescent method. The cholesterol concentration and the level of lipid peroxidation were significantly higher, whereas the concentration of proteins thiol groups decreased in the patient group. ATPase and GSH peroxidase activities diminished compared to those in the control group. There were no differences in either catalase or superoxide dismutase activities. The membrane fluidity was lower in erythrocytes from patients with MS than in the ones from control group. These results show changes in red blood cells of patients with MS as a consequence of a higher concentration of cholesterol in the membrane and an increased oxidative stress.     

Erythrocyte membrane glycerophospholipid organization is normal in multiple sclerosis

The phospholipid composition of erythrocyte membranes from patients with multiple sclerosis (MS) was found to be normal, in agreement with previous reports. The transbilayer asymmetry of the glycerophospholipids in MS red cells was probed using bee venom phospholipase A₂ and was also found not to be significantly different from normal. Abnormal membrane glycerophospholipid organisation is therefore not involved in the increased red cell size, osmotic fragility, and electrophoretic mobility associated with MS.     

Effect of 8-alkylberberine homologues on erythrocyte membrane

8-alkylberberine homologues (Ber-C8-n, where n indicates carbon atom number of gaseous normal alkyl at 8 position, n = 0, 2, 4, 6, 8, 10, 12, or 16) were synthesized and their effects on the hemolysis of rabbit erythrocyte, the fluidity of membrane and the fluorescence of membrane protein were investigated by fluorescence analysis technique. Ber-C8-n with mediate length alkyl (4 < n < 10) exhibited obvious hemolysis effect on rabbit erythrocyte when their concentration exceed 1.25 x10(-4) mol/L, and Ber-C8-8 displayed the highest hemolysis effect among all tested homologues. All of Ber-C8-n influenced the fluidity of erythrocyte membrane to different extents, which exhibited an obvious dose-effect relationship. The effect of Ber-C8-n on fluidity increased as the length of alkyl chain was elongated and decreased gradually when the alkyl carbon atoms exceeded 8. The fluorescence of erythrocyte membrane protein was quenched by Ber-C8-n, which showed a similar changing tendency on membrane fluidity. Experiments in vitro suggested that disturbing effects of Ber-C8-n on the conformation and function of membrane protein leaded to the changes of membrane fluidity and stability, and then the membrane was broken down.     

Lipopolysaccharide from Proteus mirabilis O29 induces changes in red blood cell membrane lipids and proteins

Alterations in red blood cell (RBC) plasma membranes, i.e. in lipids and proteins, and osmotic fragility of these cells after treatment with Proteus mirabilis O29 endotoxin (lipolysaccharide (LPS)) were examined using a spin labelling method. At the highest concentration of LPS, insignificantly decreased fluidity of membrane lipids was observed. Changes in conformation of membrane proteins were determined by two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (MSL) and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The analysis of spectra of MSL and ISL showed modifications in membrane proteins in red blood cells treated with the highest concentration of lipopolysaccharide. On the other hand, in the case of isolated membranes, disturbances in membrane were observed for all concentrations of LPS. The alterations in membrane lipids and proteins are paralleled in a significant rise in osmotic fragility of RBCs upon endotoxin treatment. These results provide experimental evidence that P. mirabilis O29 LPS causes deleterious changes in membranes of human red blood cells. They show that action of lipopolysaccharide mainly concerns the membrane cytoskeleton.     

Effects of temperature, oxygen and carbon dioxide on osmotic fragility of carp, Cyprinus carpio L., erythrocytes

The in vitro effects of gases and temperature on the osmotic fragility of carp erythrocytes were studied. At the three different temperatures analyzed (5, 11 and 20°C) there was no noticeable modification in erythrocyte membrane osmotic resistance. Osmotic fragility of red blood cells was altered by CO₂ and air treatment, as compared to the standard procedure. This suggests the need to take into account a possible moderate hypoxia that develops in the routine procedure of nucleated erythrocyte osmotic fragility tests.     

小鼠网织红细胞微观流变学特性的研究

按Bishop方法,在小鼠血液里诱导生成大量网织红细胞,然后提取网织红细胞,对其电泳率、渗透脆性、膜的流动性、细胞的变形能力和取向性进行了系统研究。研究结果表明网织红细胞在转变为成熟红细胞的短短时间内,其微观流变学特性发生了明显的变化：电泳率变小、渗透脆性变好、膜的流动性变大、细胞的变形能力变强、取向性变好,最终发育成具有全面功能的成熟红细胞。     

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.     

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.