Changes In Human Erythrocytes Research Articles

Cadmium chloride generates cytotoxic reactive species that cause oxidative damage and morphological changes in human erythrocytes.

Cadmium chloride (CdCl2) is a widely used industrial compound that exhibits multiple organ toxicity. Cadmium is transported through blood where erythrocytes are exposed to its action. Here the effect of CdCl2 on human erythrocytes was examined under in vitro conditions. Human erythrocytes were treated with 0.01-0.5mM CdCl2 for 24h at 37 °C. Lysates were made from CdCl2 treated and untreated (control) cells and used for further analysis. CdCl2 treatment resulted in marked hemolysis of erythrocytes and oxidation of hemoglobin to methemoglobin. This will result in anemia and also reduce the oxygen carrying ability of erythrocytes. Hemoglobin oxidation was accompanied by degradation of heme and release of free ferrous iron moiety. Further analysis showed elevated lipid hydroperoxides and formation of advanced oxidation protein products along with reduction in total sulfhydryl content, indicating the generation of oxidative stress condition in the cell. Incubation of erythrocytes with CdCl2 enhanced generation of reactive oxygen and nitrogen species, decreased the antioxidant power and inhibited pathways of glucose metabolism. Plasma membrane was damaged as indicated by enhanced osmotic fragility and inhibition of membrane bound enzymes. This was confirmed by electron microscopy which showed formation of echinocytes. These results show that CdCl2 generates reactive species which impair the antioxidant system resulting in oxidative damage to erythrocytes.

Olive Oil Phenols Prevent Mercury-Induced Phosphatidylserine Exposure and Morphological Changes in Human Erythrocytes Regardless of Their Different Scavenging Activity.

Phosphatidylserine (PS) translocation to the external membrane leaflet represents a key mechanism in the pathophysiology of human erythrocytes (RBC) acting as an “eat me” signal for the removal of aged/stressed cells. Loss of physiological membrane asymmetry, however, can lead to adverse effects on the cardiovascular system, activating a prothrombotic activity. The data presented indicate that structurally related olive oil phenols prevent cell alterations induced in intact human RBC exposed to HgCl2 (5–40 µM) or Ca2+ ionophore (5 µM), as measured by hallmarks including PS exposure, reactive oxygen species generation, glutathione depletion and microvesicles formation. The protective effect is observed in a concentration range of 1–30 µM, hydroxytyrosol being the most effective; its in vivo metabolite homovanillic alcohol still retains the biological activity of its dietary precursor. Significant protection is also exerted by tyrosol, in spite of its weak scavenging activity, indicating that additional mechanisms are involved in the protective effect. When RBC alterations are mediated by an increase in intracellular calcium, the protective effect is observed at higher concentrations, indicating that the selected phenols mainly act on Ca2+-independent mechanisms, identified as protection of glutathione depletion. Our findings strengthen the nutritional relevance of olive oil bioactive compounds in the claimed health-promoting effects of the Mediterranean Diet.

Laser Raman tweezer spectroscopy to explore the bisphenol A-induced changes in human erythrocytes.

The dermal penetration of bisphenol-A (BPA) from thermal papers into the human skin is a matter of major health concern due to its extensive use in developing countries like India, one of its largest users in the world. Bisphenol A is widely used in the manufacture of many consumer goods like polycarbonate water bottles, baby bottles, food containers, home appliances, thermal papers used in billing and tickets, the inner lining of food cans, etc. BPA can be easily adsorbed into the blood rapidly. The integration of optical tweezers with Raman spectroscopic techniques has realized avenues for interpreting single cell investigations. In the present work, the impact of BPA from thermal papers on individual human erythrocytes (red blood cells) has been investigated using micro-Raman spectroscopy. Significant intensity variations were noticed for hemoglobin oxygenation markers in the Raman spectra of red blood cells (RBCs). Raman spectral variations supporting RBC hemoglobin depletion were also found in the presence of BPA. Evident morphological changes are also observed in RBCs due to BPA in vitro exposures, which ultimately lead to cell bursting at higher concentrations.

A chip calorimetry-based method for the real-time investigation of metabolic activity changes in human erythrocytes caused by cell sickling

A new calorimetric technique is described which allows the measurement of metabolic heat rates in biological materials which are triggered by changes in the oxygen tension. It uses the high permeability for oxygen of a thin-walled Teflon flow channel of a chip calorimeter in order to adjust variable but defined oxygen concentrations in the samples. Using this technique, for the first time, the heat production rate of sickle cell erythrocytes (SS-RBCs) in sickled and non-sickled state could be compared. To measure heat rate changes in real time when sickled SS-RBCs return to the non-sickled state, the heat dissipation was measured during the re-oxygenation of the cell samples which had been treated before under anoxic conditions. We found higher heat production rate of the blood cells in non-sickled state, which is in agreement with the reported increased glucose uptake.

Sodium chlorite increases production of reactive oxygen species that impair the antioxidant system and cause morphological changes in human erythrocytes.

Sodium chlorite (NaClO2 ) is used in the production of chlorine dioxide for bleaching and stripping of textiles, pulp, and paper. It is also used as disinfectant in municipal water treatment and as a component in therapeutic rinses and gels. The effect of NaClO2 on human erythrocytes has been studied under in vitro conditions. Incubation of 5% suspension of erythrocytes with NaClO2 (0.1-2.0 mM) at 37°C for 30 min resulted in marked cell lysis (1.2-3.8 fold) and increased their osmotic fragility. Several parameters were assayed in cell lysates prepared from NaClO2 -treated and -untreated (control) erythrocytes. Compared to controls, exposure to NaClO2 caused significant increase in protein oxidation (1.1-8.07 fold), lipid peroxidation (1.08-4.95 fold) with decrease in total sulfhydryl (-5 to -61%), and glutathione levels (-7 to -86%). Methemoglobin content was tremendously increased, by 5-52 fold when compared to control, while methemoglobin reductase activity decreased (-17 to -93%) upon NaClO2 treatment. NaClO2 enhanced the generation of reactive oxygen species by 3-21 fold and lowered the metal reducing and free radical quenching ability of erythrocytes. It also caused an increase in nitric oxide levels (2.7-15.4 fold) showing generation of nitrosative stress too. The activities of major antioxidant and membrane bound enzymes were significantly altered. Gross morphological changes, from discocytes to echinocytes, were seen in NaClO2 -treated erythrocytes under electron microscope. These results show that NaClO2 induces oxidative stress in human erythrocytes, damages the membrane, and impairs the cellular antioxidant defence system. This oxidative damage can shorten the life span of erythrocytes in blood resulting in red cell senescence. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1343-1353, 2017.

Eryptosis-inducing activity of bisphenol A and its analogs in human red blood cells (in vitro study)

Bisphenols are important chemicals that are widely used in the manufacturing of polycarbonates, epoxy resin and thermal paper, and thus the exposure of humans to these substances has been noted. The purpose of this study was to assess eryptotic changes in human erythrocytes exposed (in vitro) to bisphenol A (BPA) and its selected analogs, i.e.,bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF). The erythrocytes were incubated with compounds studied at concentrations ranging from 1 to 250μg/mL for 4, 12 or 24h. The results showed that BPA and its analogs increased cytosolic calcium ions level with the strongest effect noted for BPAF. It has also been revealed that all bisphenols analyzed, and BPAF and BPF in particular increased phosphatidylserine translocation in red blood cells, which confirmed that they exhibited eryptotic potential in this cell type. Furthermore, it was shown that BPA and its analogs caused significant increase in calpain and caspase-3 activities, while the strongest effect was noted for BPAF. BPS, which is the main substituent of bisphenol A in polymers and thermal paper production exhibited similar eryptotic potential to BPA. Eryptotic changes in human erythrocytes were provoked by bisphenols at concentrations, which may influence the human body during occupational exposure or subacute poisoning with these compounds.

Comparative study of the effect of BPA and its selected analogues on hemoglobin oxidation, morphological alterations and hemolytic changes in human erythrocytes

Bisphenol A (BPA) has been shown to provoke many deleterious impacts on human health, and thus it is now successively substituted by BPA analogues, whose effects have been poorly investigated. Up to now, only one study has been realized to assess the effect of BPA on human erythrocytes, which showed its significant hemolytic and oxidative potential. Moreover, no study has been conducted to evaluate the effect of BPA analogues on red blood cells. The purpose of the present study was to compare the impact of BPA and its selected analogues such as bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF) on hemolytic and morphological changes and hemoglobin oxidation (methemoglobin formation) of human erythrocytes. The erythrocytes were incubated with different bisphenols concentrations ranging from 0.5 to 500μg/ml for 1, 4 and 24h. The compounds examined caused hemolysis in human erythrocytes with BPAF exhibiting the strongest effect. All bisphenols examined caused methemoglobin formation with BPA inducing the strongest oxidative potential. Flow cytometry analysis showed that all bisphenols (excluding BPS) induced significant changes in erythrocytes size. Changes in red blood cells shape were conducted using phase contrast microscopy. It was noticed that BPA and BPAF induced echinocytosis, BPF caused stomatocytosis, while BPS did not provoke significant changes in shape of red blood cells. Generally, the results showed that BPS, which is the main substituent of bisphenol A in polymers and thermal paper production, exhibited significantly lower disturbance of erythrocyte functions than BPA.

Studies of the structure and functions of blood cells in senile patients with pneumonia on the biological model of hypoxia by scanning probe microscopy.

Biological model of hypoxia can be used for the diagnosis of functional changes in human erythrocytes under the effect of the hypoxic factor. The use of this model together with modern methods of scanning probe microscopy for evaluation of the severity of pulmonological disease in senile patients will help to predict treatment efficiency and outcome.

Oxidative damage to human red blood cells treated with chlorfenvinphos, an organophosphate insecticide (in vitro)

Chlorfenvinphos (CFVF) is an organophosphorus insecticide, which was used to control insect pest on livestock and household pests such as flies, fleas, and mites. The molecular basis of toxic properties of CFVF in animals has been insufficiently studied. Blood can transport oxygen and nutrients as well as toxic compounds. Xenobiotics can enter to red blood cells and cause damage. Therefore, investigation of the toxicity of different compounds to erythrocytes is very important. The purpose of the present experiment was to evaluate the effect of this compound on human erythrocytes. We have evaluated the hemolysis, hemoglobin oxidation (met-Hb formation) and lipid peroxidation in human erythrocytes. Moreover, the changes in the level of reactive oxygen species (ROS) were assessed using flow cytometry as well as those in morphological changes of erythrocytes using phase contrast microscopy. This study describes the interaction of low concentrations of CFVF with human erythrocytes as well as the concentrations, which may enter human organism as a result of acute poisoning (0.5–250 μM). It was shown that CFVF only at high concentration induced changes in human erythrocytes. We have observed hemolysis (at 250 μM), changes in morphological parameters including echinocytes formation (at 250 μM), as well as increase in lipid peroxidation in erythrocytes (at 250 μM), ROS formation (at 100 μM) in red blood cells treated 1 hour with CFVF. Additionally, CFVF after 4 h of incubation oxidized hemoglobin, however, to a lower degree.

Influence of iodine-containing radiographic contrast media on the phenotype of erythrocytes from different laboratory animal species

It is well known that clinically relevant concentrations of iodine-containing radiographic contrast media (CM) induce morphological changes in human erythrocytes. However, there are only few reports about CM effects on erythrocytes of animals (e.g. mice, rats, rabbits, and pigs). Thus, two conventional iodine-containing CM (iodixanol, Visipaque™ 320; iomeprol, Iomeprol™ 350) were tested for their effects on the morphology of erythrocytes from these. After venous blood sampling and blood centrifugation, the autologous plasma was supplemented with 40 vol% CM. Then, a defined number of erythrocytes was incubated in this CM-supplemented plasma for 5 min at body temperature (37°C). Subsequently, 10 μL of the cell suspension were transferred to a purified glass slide and the number of discocytes, echinocytes, and acanthocytes was counted within a total number of 100 erythrocytes (40 fold primary magnification, transmitted light mode). Shape changes of the erythrocytes from all animal species strongly depended on the type of CM and compared to the effects which have already been described for human erythrocytes. Incubation in both CM resulted in morphological changes of the erythrocytes. Incubation in a iodixanol/plasma mixture induced the lowest echinocyte or acanthocyte formation. Porcine erythrocytes showed a much more distinct shape change than those of other animal species and humans. These results suggest erythrocytes from mice, rats, and rabbits are a suitable model system for a model system for human erythrocytes when CM effects on the cellular shape of erythrocytes have to be tested. The distinct deformation of the pig erythrocytes could be due to differences in the pig erythrocyte membrane or the physical and chemical constitution of pig erythrocytes.

Cesium's Affects on Morphological Changes of Human Erythrocytes

AbstractCesium could play a toxic role in several pathological processes. Atomic force microscopy (AFM) was used to study morphological changes of human erythrocytes after incubating with different concentrations of CsCl, and the Raman spectra were used to study the effects of CsCl on the chemistry components of erythrocyte membrane. The AFM images showed that the "domain structures" that appeared after incubation with higher concentration of CsCl (150 mmol·L−1), are featured by the particles aggregated to form ranges and the separations among them enlarged to gorges, and this change may increase the permeability of cell membranes. The Raman results showed that the polar part of membrane phospholipid become more order and with the increasing of the concentration of CsCl, the longitudinal order of nonpolar parts first decreased and then increased. It is concluded that the aggregation of membrane proteins and the order changes of the phospholipid cause a change in the distribution and conformation of the phospholipid membrane. And the effects of CsCl on the erythrocyte membrane are mainly dependent on its concentration.

Lead‐induced changes in human erythrocytes and lymphocytes

In the present work we studied, by chemiluminescence measurements, the influence of lead on the production of reactive oxygen species (ROS) in haemolysates obtained from human erythrocytes incubated in the presence of different concentrations of lead acetate. Moreover, we evaluated the modification of proteins and lipids in human erythrocyte and lymphocyte membranes by using the fluorescence probes N-(1-pyrene)maleimide (PM), laurdan and pyrene. No significant changes in chemiluminescence were detected for erythrocytes incubated with 1-10 microM lead acetate for 3 h at 37 degrees C. By increasing the lead acetate concentration in cell suspensions up to 50 microM for the same incubation time, the percentage of chemiluminescence inhibition was ca. 20%. It was shown that, after incorporating fluorescence probes in the membrane lipid bilayer of erythrocytes and lymphocytes treated with 10 and/or 50 microM lead acetate, the total fluorescence intensity and the excimer to monomer intensity ratio of PM decreased and the generalized fluorescence polarization of laurdan decreased by 10-15%. The pyrene excimerization coefficient (kappa(ex)) increased by 20% (in comparison with a magnitude of kappa(ex) for white membranes isolated from intact erythrocytes) with 6-10 microM lead acetate for 3 h at 37 degrees C. The data obtained suggest that the effect of low concentrations of lead acetate does not cause production of ROS in erythrocytes in vitro, but can change the physicochemical state of proteins and lipids in erythrocyte and lymphocyte membranes. This effect is important because it influences the enzymatic activity and the functionality of receptors and channels present at the plasma membrane level, thus modulating the molecular composition of the intracellular space and cell functions.

In vitro effects of high glucose concentrations on membrane protein oxidation, G-actin and deformability of human erythrocytes.

The object of this study was to examine the effect of elevated in vitro glucose concentrations on protein modification and functional changes in human erythrocytes. Groups were exposed to 5-45 mM glucose concentrations. The time effect of any changes was also evaluated. In erythrocyte ghosts, protein glycation and oxidation were evaluated using spectrophotometric methods. G-actin was measured by a DNase I inhibition assay in cell lysates. Erythrocyte deformability was assessed using a cell transit analyser. At 24 h, a significant protein oxidation (at 25 and 45 mM glucose; p < 0.05), and G-actin increase was observed for all concentrations (p < 0.05). At 48 h, a significant increase in glycation (25 and 45 mM glucose; p < 0.05), protein oxidation (p < 0.05), and G-actin (p < 0.05) was observed in all groups. A significant positive correlation was observed between glucose /protein oxidation, glucose/G-actin and protein oxidation/G-actin at 24 and 48 h. Our findings show that the oxidative effect of glucose on erythrocytes depends on concentration and incubation time. We also present the first evidence of increased G-actin in human erythrocytes exposed to high glucose concentrations as a diabetes model.

Phenol and catechol induce prehemolytic and hemolytic changes in human erythrocytes

The toxic potency of two industrially used compounds (phenol and catechol) was studied in human blood cells in vitro. Catechol was found to be a more harmful toxin than phenol, since it provokes statistically significant changes in the function of erythrocytes even at low doses. Most of the changes was statistically significant for the doses of 50 ppm of catechol and 250 ppm of phenol. Both compounds induced methaemoglobin formation, glutathione depletion and conversion of oxyhaemoglobin to methaemoglobin, which is associated with superoxide anion production and lead to formation of ferryl hemoglobin, hydrogen peroxide or hydroxyl radicals. It is known that oxidation of catechol leads to formation of semiquinone radicals. Semiquinones are able to bind to nucleophilic residues like SH or NH 2 of proteins and these macromolecules may undergo inactivation. We observed among especially susceptible to action of catechol are catalase (CAT) (100 ppm) and superoxide dismutase (SOD) (250 ppm). Decrease of the activity of catalase and SOD by catechol induced radical species formation. This lead to inhibition of another protective enzymes such as glutathione- S-transferase (500 ppm), glutathione reductase (1000 ppm), glucose-6-phosphate dehydrogenase activity (1000 ppm). Cytotoxicity of phenol or catechol was noted as hemolysis. Haemoglobin liberated from erythrocytes in this process may further generate oxygen free radicals and subsequently initiate enzymes damage. It seems to be essential that in phenol and catechol toxicity special role play damages of heme proteins and other proteins molecule, and damages of lipids are not so important.

Glycophorin A-mediated haemolysis of normal human erythrocytes: evidence for antigen aggregation in the pathogenesis of immune haemolysis.

The inexplicable severity of anti-Pr autoimmune haemolytic anaemia led us to test the hypothesis that the haemolysis was primarily due to a change in the function of glycophorin A, on which the Pr antigen is located. The lectins Maclura pomifera and wheat germ agglutinin that bind to glycophorin A induced the haemolysis of normal erythrocytes in vitro. Lectin binding led to an increase in erythrocyte membrane permeability to sodium and potassium, the former resulting in an influx of water and subsequent haemolysis. The response was glycophorin A specific as Concanavalin A, which binds to band 3, did not cause haemolysis and peanut agglutinin only did so after removal of erythrocyte sialic acid. The lectin-induced cation leak was not mediated by activation of cation channels as the inhibitors, tetrodotoxin, amiloride and 4,4' disothiocyanate stilbene 2,2'disulphonate, had no effect, suggesting that the haemolysis was due to exacerbation of the inherent cation permeability of the erythrocyte membrane. A human IgAK anti-Pr autoantibody and a mouse anti-human glycophorin A antibody increased erythrocyte permeability to sodium. The role of glycophorin A in stabilizing and, upon aggregation, destabilizing the phospholipid bilayer is discussed. Our findings may help explain the severity of anti-Pr autoimmune haemolytic anaemia and other pathophysiological changes in human erythrocytes.

Regulation of erythrocyte ghost membrane mechanical stability by chlorpromazine

Chlorpromazine (CPZ), a widely used tranquilizer, is known to induce stomatocytic shape changes in human erythrocytes. However, the effect of CPZ on membrane mechanical properties of erythrocyte membranes has not been documented. In the present study we show that CPZ induces a dose-dependent increase in mechanical stability of erythrocyte ghost membrane. Furthermore, we document that spectrin specifically binds to CPZ intercalated into inside-out vesicles depleted of all peripheral proteins. These findings imply that CPZ-induced mechanical stabilization of the erythrocyte ghost membranes may be mediated by direct binding of spectrin to the bilayer. Membrane active drugs that partition into lipid bilayer can thus induce cytoskeletal protein interactions with the membrane and modulate membrane material properties.

Electrical and morphological changes of human erythrocytes under high hydrostatic pressure followed by dielectric spectroscopy.

The electrical and morphological properties of human erythrocytes under high hydrostatic pressure up to 500 MPa have been studied by dielectric spectroscopy. The pressure-induced changes in the dielectric behavior of erythrocyte suspensions indicate that hydrostatic pressure causes the change in cell shape from discoidal to spherical and hemolysis at 200-300 MPa, the formation of buds and spicular processes followed by vesiculation at 300-400 MPa, and the increase in the membrane capacitance at 400-500 MPa.

Membrane lipid diffusion and band 3 protein changes in human erythrocytes due to acute hypobaric hypoxia.

Because it has been reported that hypoxia in rats may promote lipid peroxidation and other free radical reactions that could modify membrane lipids and proteins, the effect of acute hypobaric hypoxia on human erythrocyte membranes was investigated. 12-(1-Pyrene)dodecanoic acid fluorescent probe was used to assess short-range lateral diffusion status in the membrane bilayer. Membrane protein modification was detected by SDS-PAGE. Healthy young men were exposed for 20 min to the hypobaric hypoxia, simulating an altitude of 4,500 m. Under this condition, erythrocyte membrane lipids reached a state of higher lateral diffusivity with respect to normobaric conditions and membrane band 3 protein was modified, becoming more susceptible to membrane-bound proteinases. These observations suggest that acute hypobaric hypoxia may promote an oxidative stress condition in the erythrocyte membrane.

Morphological Changes of Human Erythrocytes Induced by Cholesterol Sulphate

Morphological alterations of human erythrocytes induced by cholesterol sulphate (5-cholesten-3 beta-ol sulphate, CS) were studied. Influence of CS on red blood cell stability (in isotonic conditions) by simultaneous application of flow cytometry and scanning electron microscopy was studied. In isotonic medium CS induces erythrocyte size and shape changes in dose-and time-dependent manner. Incubation (in vitro) of erythrocytes with CS concentrations from 4 x 10(-5) mol/dm3 to 8 x 10(-5) mol/dm3 led to a progressive sphero-echinocitic shape transformation accompanied by a cell size decrease. In contrast to this, for CS content equal to 1 x 10(-5) mol/dm3 the maintenance of the normal biconcave shape of red blood cells was observed. The results suggest that CS, similarly to numerous evaginating amphiphilic agents, induces a transformation of the erythrocyte normal discoid shape to echinocytic form. This effect may be caused, at least partly, by an asymmetric expansion of the membrane lipid bilayer due to asymmetric distribution of CS incorporated into the membrane. The echinocytic shape transformation of erythrocytes indicated that CS intercalates in the outer hemileaflet of the lipid bilayer leading to membrane externalization.

Scanning electron microscopic study of erythrocyte shapes artificially jetted through tubes at different pressures by 'in vitro cryotechnique for erythrocytes'.

A new cryotechnique for examining morphological changes of human erythrocytes at different jet pressures was developed in the present study. Human fresh or stored erythrocytes passing through a tube were jetted into precooled isopentane-propane mixture (-193 degrees C), which was named as 'in vitro cryotechnique for erythrocytes'. After the cryotechnique procedure, the routine freeze-substitution method and subsequent t-butyl alcohol freeze-drying method were used for preparing the scanning electron microscopic specimens. At 100 mmHg or higher pressures of jetting, lamellar-arranged erythrocytes were observed to have elongated shapes. The more the jetting pressure increased, the fewer discoid shapes of erythrocytes were observed. This cryofixation technique could preserve the morphology of erythrocytes jetting from tubes, and provide the three-dimensional image of erythrocyte surfaces, as followed by scanning electron microscopy.