Incubation Of Red Blood Cells Research Articles

Neutrophil Diversity (Immature, Aged, and Low-Density Neutrophils) and Functional Plasticity: Possible Impacts of Iron Overload in β-Thalassemia.

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal), although data on this are limited. In this study, blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16- CD62L+) and aged (senescent) neutrophils (CD16+ CD62L-) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations, some of the indicators, including the flow cytometry markers (CD11b, CD62L, CD66b, CD63, apoptosis, and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence), were lower than the control. Additionally, low-density neutrophils (LDNs), which are found in the peripheral blood mononuclear cell (PBMC) fraction, were observed in Beta-thal patients but not in the control group. The expression of CD11b, CD66b, CD63, arginase I, and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion, iron overload induces neutrophil diversity along with some dysfunctions.

Isoscutellarein from Bixa orellana Leaves Uncompetitively Inhibits Recombinant Human Aldose Reductase and Prevents Sorbitol Accumulation and Lens Opacity

Introduction: Enhanced aldose reductase activity results in increased accumulation of sorbitol. Therefore, inhibition of aldose reductase is an effective strategy to prevent or delay certain diabetic complications. Method: Various extracts of the leaves of B. orellana were tested for their inhibitory activity on the aldose reductase. Ethyl acetate extract that showed maximum inhibition was further fractionated and the inhibitor was identified as isoscutellarein by spectroscopic methods. IC50 of recombinant human aldose reductase by isoscutellarein was found to be 14 μM and the mode of inhibition was uncompetitive with a decrease in both Km and Vmax. Isoscutellarein was bound to the active site of aldose reductase (3RX3), namely to Ala-299, Leu-300, Leu-301, His-110 and Tyr-48. Result: Docking results exhibited a binding energy of -9.15 kJ/mol. Conclusion: The incubation of red blood cells with high glucose concentrations mimicking hyperglycemic conditions promoted sorbitol accumulation, which was effectively inhibited by isoscutellarein. Further, xylose-induced opacity of the lens was effectively inhibited by isoscutellarein.

Integration of Trehalose Lipids with Dissociative Trehalose Enables Cryopreservation of Human RBCs.

Cryopreservation of red blood cells (RBCs) is imperative for transfusion therapy, while cryoprotectants are essential to protect RBCs from cryoinjury under freezing temperatures. Trehalose has been considered as a biocompatible cryoprotectant that naturally accumulates in organisms to tolerate anhydrobiosis and cryobiosis. Herein, we report a feasible protocol that enables glycerol-free cryopreservation of human RBCs by integration of the synthesized trehalose lipids and dissociative trehalose through ice tuning and membrane stabilization. Typically, in comparison with sucrose monolaurate or trehalose only, trehalose monolaurate was able to protect cell membranes against freeze stress, achieving 96.9 ± 2.0% cryosurvival after incubation and cryopreservation of human RBCs with 0.8 M trehalose. Moreover, there were slight changes in cell morphology and cell functions. It was further confirmed by isothermal titration calorimetry and osmotic fragility tests that the moderate membrane-binding activity of trehalose lipids exerted cell stabilization for high cryosurvival. The aforementioned study is likely to provide an alternative way for glycerol-free cryopreservation of human RBCs and other types of cells.

Red Blood Cell Membrane Cholesterol May Be a Key Regulator of Sickle Cell Disease Microvascular Complications.

Cell membrane lipid composition, especially cholesterol, affects many functions of embedded enzymes, transporters and receptors in red blood cells (RBC). High membrane cholesterol content affects the RBCs' main vital function, O2 and CO2 transport and delivery, with consequences on peripheral tissue physiology and pathology. A high degree of deformability of RBCs is required to accommodate the size of micro-vessels with diameters significantly lower than RBCs. The potential therapeutic role of high-density lipoproteins (HDL) in the removal of cholesterol and its activity regarding maintenance of an optimal concentration of RBC membrane cholesterol have not been well investigated. On the contrary, the focus for HDL research has mainly been on the clearance of cholesterol accumulated in atherosclerotic macrophages and plaques. Since all interventions aiming at decreasing cardiovascular diseases by increasing the plasma level of HDL cholesterol have failed so far in large outcome studies, we reviewed the potential role of HDL to remove excess membrane cholesterol from RBC, especially in sickle cell disease (SCD). Indeed, abundant literature supports a consistent decrease in cholesterol transported by all plasma lipoproteins in SCD, in addition to HDL, low- (LDL) and very low-density lipoproteins (VLDL). Unexpectedly, these decreases in plasma were associated with an increase in RBC membrane cholesterol. The concentration and activity of the main enzyme involved in the removal of cholesterol and generation of large HDL particles-lecithin cholesterol ester transferase (LCAT)-are also significantly decreased in SCD. These observations might partially explain the decrease in RBC deformability, diminished gas exchange and tendency of RBCs to aggregate in SCD. We showed that incubation of RBC from SCD patients with human HDL or the HDL-mimetic peptide Fx5A improves the impaired RBC deformability and decreases intracellular reactive oxygen species levels. We propose that the main physiological role of HDL is to regulate the cholesterol/phospholipid ratio (C/PL), which is fundamental to the transport of oxygen and its delivery to peripheral tissues.

Comparative toxic effect of bulk zinc oxide (ZnO) and ZnO nanoparticles on human red blood cells

Abstract Metal oxide nanoparticles (NPs) are a member of nanomaterials family that have been produced on a large scale and their toxicity affected by their high surface-to-volume ratio. The hemolytic toxic effects of metal oxide NPs may be completely different from those observed in their bulk counterparts. So, the main purpose of this research was to measure the difference between the hemolytic effects of ZnO (NPs and bulk) on isolated human red blood cells (RBCs). Hemolysis was measured after incubation of human RBCs with 0.01–1 mM of ZnO (bulk and NPs) for 6 h. For measurement of other variables, human RBCs were treated with 0.1 mM of ZnO (NPs and bulk) for 1, 2, and 3 h. Our results demonstrate that bulk ZnO did not show any toxic effects in the concentrations tested, while ZnO NPs caused toxic hemolytic effects through formation of ROS, lipid peroxidation, and glutathione depletion.

Red blood cells induce endothelial dysfunction in patients with ST-elevation myocardial infarction and elevated C-reactive protein

Abstract Background The important role of inflammation in atherosclerotic plaque progression and instability leading to myocardial infarction has been widely demonstrated. C-reactive protein (CRP) has been shown to be of predictive value in atherosclerotic cardiovascular disease. The red blood cell (RBC) is an important regulator of cardiovascular function through nitric oxide bioactivity and oxidative stress in ischemic heart disease. Also, arginase-1 has been shown to greatly influence nitric oxide bioactivity in RBCs and to cause endothelial dysfunction. However, the mechanisms by which RBCs regulate vascular function in patients with myocardial infarction and its relation to inflammation and arginase-1 remain unknown. Objective The study aimed to investigate the effect of RBCs on endothelial function in patients with ST-elevation myocardial infarction (STEMI) and its possible association with systemic inflammation and arginase-1. Material and methods Blood samples were collected from patients with STEMI within 36 h after admission and from age-matched healthy controls. RBCs were incubated with isolated rat aortic segments for 18 h after which the aortas were assessed for endothelium-dependent and endothelium-independent relaxations using wire myographs and application of acetylcholine and nitroprusside, respectively. The vascular response was evaluated in relation to the level of inflammation defined as CRP <2 and ≥2 mg/L at admission. The levels of 4-hydroxynonenal (4-HNE; a marker of oxidative stress formed by lipid peroxidation) and the expression of arginase-1 were visualized in rat aortas following incubation with RBCs by immunohistochemistry. All handling and procedures regarding human subjects and sampling were performed according to the Declaration of Helsinki. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996). Results RBCs from patients with STEMI and elevated CRP (≥2 mg/L, mean of 9.6 mg/L) induced significant impairment of endothelium-dependent relaxation compared to RBCs from patients with STEMI and low CRP (<2 mg/L, mean of 1.0 mg/L) and to the healthy controls (Fig. 1). Endothelium-independent relaxations did not differ between the groups. Immunohistochemical staining of the aorta revealed that incubation with RBCs from patients with STEMI and high CRP increased the expression of 4-HNE and arginase-1 compared to incubation of RBCs from healthy controls and patients with STEMI and low CRP (Fig. 2). Conclusion RBCs from patients with STEMI and elevated CRP induce endothelial dysfunction and increase the expression of 4-HNE and arginase-1, indicating that inflammation is involved in the mechanism by which RBCs induce endothelial dysfunction in STEMI. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung foundation

The inhibition of glucose uptake to erythrocytes: microwave dielectric response.

Dielectric spectroscopy has been used in the study and development of non-invasive glucose monitoring (NIGM) sensors, including the range of microwave frequencies. Dielectric relaxation of red blood cell (RBC) cytosolic water in the microwave frequency band has been shown to be sensitive to variations in the glucose concentration of RBC suspensions. It has been hypothesized that this sensitivity stems from the utilization of D-glucose by RBCs. To verify this proposition, RBCs were pretreated with inhibitors of D-glucose uptake (cytochalasin B and forskolin). Then their suspensions were exposed to different D-glucose concentrations as measured by microwave dielectric spectroscopy (MDS) in the 500MHz-40GHz frequency band. After incubation of RBCs with either inhibitor, the dielectric response of water in the cytoplasm, and specifically its relaxation time, demonstrated minimal sensitivity to the change of D-glucose concentration in the medium. This result allows us to conclude that the sensitivity of MDS to glucose uptake is associated with variations in the balance of bulk and bound RBC cytosolic water due to intracellular D-glucose metabolism, verifying the correctness of the initial hypothesis. These findings represent a further argument to establish the dielectric response of water as a marker of glucose variation in RBCs.

The Influence of Selected Antipsychotic Drugs on Biochemical Aspects of Alzheimer's Disease.

The aim of this study was to assess the potency of selected antipsychotic drugs (haloperidol (HAL), bromperidol (BRMP), benperidol (BNP), penfluridol (PNF), pimozide (PIM), quetiapine (QUET) and promazine (PROM)) on the main pathological hallmarks of Alzheimer’s disease (AD). Binary mixtures of donepezil and antipsychotics produce an anti-BuChE effect, which was greater than either compound alone. The combination of rivastigmine and antipsychotic drugs (apart from PNF) enhanced AChE inhibition. The tested antipsychotics (excluding HAL and PNF) significantly reduce the early stage of Aβ aggregation. BRMP, PIM, QUET and PROM were found to substantially inhibit Aβ aggregation after a longer incubation time. A test of human erythrocytes hemolysis showed that short-term incubation of red blood cells (RBCs) with QUET resulted in decreased hemolysis. The antioxidative properties of antipsychotics were also proved in human umbilical vein endothelial cells (HUVEC); all tested drugs were found to significantly increase cell viability. In the case of astrocytes, BNP, PNF, PIM and PROM showed antioxidant potential.

Cytoprotective effect of taurine against sodium chlorate-induced oxidative damage in human red blood cells: an ex vivo study.

Sodium chlorate (NaClO3) is a common non-selective herbicide that is also used in paper and pulp mills and is produced as a by-product during drinking water disinfection by chlorine dioxide. Here, we report the effect of dietary antioxidant taurine on NaClO3-induced cytotoxicity in human red blood cells (RBC). RBC were treated with 5mM NaClO3, either alone or in presence of 1, 2.5 and 5.0mM taurine. Incubation of RBC with NaClO3 alone caused hemolysis, increased oxidation of lipids and proteins, methemogobin level and decreased total sulfhydryl and glutathione content. It lowered the activities of antioxidant enzymes thioredoxin reductase, glutathione peroxidase, catalase and glutathione reductase, while Cu-Zn superoxide dismutase activity was increased. The antioxidant capacity of RBC was impaired. This strongly suggests that NaClO3 causes the induction of oxidative stress condition in RBC. The specific activities of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and plasma membrane bound enzymes, were also greatly altered. However, prior treatment of RBC with taurine conferred significant protection against NaClO3-induced oxidative damage and also improved the antioxidant defence system of cells. These results were supported by electron microscopy images of RBC. Treatment with NaClO3 alone converted the normal biconcave discoidal RBC to acanthocytes and echinocytes but this transformation was greatly prevented in the presence of taurine. Thus, taurine mitigates the cytotoxicity of NaClO3 in human RBC and can function as an effective chemoprotectant.

The Effect of Glucose and Poloxamer 188 on Red-Blood-Cell Aggregation.

Glucose metabolism disorders contribute to the development of various diseases. Numerous studies show that these disorders not only change the normal values of biochemical parameters but also affect the mechanical properties of blood. To show the influence of glucose and poloxamer 188 (P188) on the mechanical properties of a red-blood-cell (RBC) suspension, we studied the aggregation of the cells. To show the mechanisms of the mechanical properties of blood, we studied the effects of glucose and poloxamer 188 (P188) on red-blood-cell aggregation. We used a model in which cells were suspended in a dextran 70 solution at a concentration of 2 g/dL with glucose and P188 at concentrations of 0–3 g/dL and 0–3 mg/mL, respectively. RBC aggregation was determined using an aggregometer, and measurements were performed every 4 min for 1 h. Such a procedure enabled the incubation of RBCs in solution. The aggregation index determined from the obtained syllectograms was used as a measure of aggregation. Both the presence of glucose and that of P188 increased the aggregation index with the incubation time until saturation was reached. The time needed for the saturation of the aggregation index increased with increasing glucose and P188 concentrations. As the concentrations of these components increased, the joint effect of glucose and P188 increased the weakening of RBC aggregation. The mechanisms of the observed changes in RBC aggregation in glucose and P188 solutions are discussed.

Comparative evaluation of cadmium-induced oxidative stress in camel and bovine erythrocytes

ObjectivesErythrocytes or red blood cells (RBCs) are the most abundant cells in blood. They transport oxygen from lungs to tissues and bring back carbon dioxide from tissues to lungs. Being non-nucleated and lacking the cellular organelles, they have been widely used for in vitro toxicity studies. The present study was carried out to study the comparative in vitro toxicity of cadmium (Cd) on RBCs of camel and cow. MethodsBoth cow and camel whole blood samples were exposed to 0, 10, 50 and 100 μM of cadmium chloride (CdCl2) and the tubes were incubated at 37 °C for 24 h. The erythrocytes from both the species were separated and used for biochemical determinations for markers of oxidative stress. ResultsCd caused a concentration dependent decrease in bovine glutathione (GSH) content and superoxide dismutase (SOD) activity. However, in the case of camel RBCs, the decrease in GSH content and SOD activity were observed at medium and high concentrations only. Cd exposure also resulted in significant inhibition of glutathione peroxidase (GPx) and glutathione S-transferase in both bovine and camel RBCs. The GPx in camel RBCs was inhibited only at a high concentration of Cd. Various concentrations of Cd inhibited the catalase in bovine RBCs only, without any significant inhibition of catalase in camel RBCs. Medium and high concentrations of Cd caused an increase in lipid peroxidation in bovine RBCs. In the case of camel RBCs, none of the used concentrations of Cd caused lipid peroxidation. ConclusionsThe present study concludes that in vitro incubation of camel and bovine RBCs with Cd causes an impairment of their antioxidant system and therefore increases their susceptibility to hemolysis. However, the camel RBCs appear to have an increased resistance to oxidative stress generated by cadmium exposure.

3,4-Dihydroxybenzaldehyde mitigates fluoride-induced cytotoxicity and oxidative damage in human RBC

BackgroundFluoride is an essential micronutrient that is needed for mineralization of bones and formation of dental enamel. It is a widely dispersed environmental pollutant and chronic exposure to it is toxic, resulting in malignancies and hematological damage in humans. Blood is a major and early target of environmental pollutants and toxicants like fluoride. Fluoride generates reactive oxygen species and free radicals which induce oxidative stress in target cells and mediate its toxic effects. The aim of this study was to determine the mitigating effect of plant antioxidant 3,4-dihydroxybenzaldehyde (DHB) on sodium fluoride (NaF) induced oxidative damage and cytotoxicity in isolated human red blood cells (RBC) MethodIsolated human RBC were treated with 0.5 mM NaF, in absence or presence of different concentrations of DHB (0.1–2.5 mM). Several biochemical parameters were analyzed in cell lysates and whole cells. ResultsTreatment of RBC with NaF increased the formation of reactive oxygen and nitrogen species. It oxidized thiols, proteins and lipids and generated their peroxidative products. Methemoglobin level, heme degradation and lipid peroxidation were increased but cellular antioxidant status declined significantly in NaF alone treated RBC, compared to the control. NaF inhibited antioxidant, membrane bound and glycolytic enzymes in RBC. However, prior incubation of RBC with DHB significantly attenuated the NaF-induced alterations in all these parameters in a DHB concentration-dependent manner. ConclusionThese results show that DHB mitigates NaF-induced oxidative damage in human RBC, probably because of its antioxidant character.

Attenuation of Hg(II)-induced cellular and DNA damage in human blood cells by uric acid.

Mercury (Hg) is a widespread environmental pollutant and toxicant that induces multiple organ damage in humans and animals. Hg toxicity is mediated by the induction of oxidative stress in the target cells. We used uric acid (UA), a potent antioxidant found in biological fluids, to protect human red blood cells (RBC) and lymphocytes against Hg-mediated cell, organelle, and genotoxicity. RBCs were incubated with mercuric chloride (HgCl2), an Hg(II) compound, either alone or in the presence of UA. Incubation of RBCs with only HgCl2 increased the production of nitrogen and oxygen radical species, enhanced methemoglobin levels, heme degradation, free ferrous iron, oxidation of proteins and membrane lipids, and reduced the antioxidant capacity of cells. UA enhanced the antioxidant capacity of RBCs and restored metabolic, plasma membrane-bound, and antioxidant enzyme activities. Scanning electron microscopy showed that UA prevented HgCl2-mediated morphological changes in RBCs. HgCl2 dissipated the mitochondrial membrane potential and increased lysosomal membrane damage in lymphocytes, but UA pre-treatment attenuated these effects. Genotoxicity analysis by comet assay showed that UA protected lymphocyte DNA from HgCl2-induced damage. Importantly, UA itself did not exhibit any deleterious effects on RBCs or lymphocytes. Thus, UA protects human blood cells from Hg(II)-mediated oxidative damage, reducing the harmful effects of this extremely toxic metal. We suggest that UA has a similar protective role in plasma against heavy metal toxicity.

Abstract 1888: Anti-CD38 interference with blood compatibility testing: Differentiating isatuximab and daratumumab via functional epitope mapping

Abstract Introduction: Two anti-CD38 monoclonal antibodies (mAb) are approved for the treatment of multiple myeloma: isatuximab (Isa) and daratumumab (Dara); binding to CD38 on red blood cells (RBCs) results in false positive agglutination on indirect antiglobulin tests (IATs). Compared to Dara, which interferes with IATs in nearly 100% of patients, Isa interfered in 67/99 (67%) patients in the Phase 3 ICARIA-MM (NCT02990338) study and 95/150 (63%) patients in the Phase 3 IKEMA (NCT0327585) study. We sought to understand differences between the binding abilities of Isa and Dara to CD38 on RBCs. Experimental Procedures: In vitro binding experiments in RBCs were performed following pretreatment of RBCs isolated from healthy donors +/- a mouse anti-human CD38 mAb (clone HB-7 or AT13/5) or a nicotinamide adenine dinucleotide (NAD) analog (irreversible CD38 inhibitor) before incubation with Isa, Dara, or control human IgG (hIgG). RBC surface bound CD38 antibodies were detected with fluorophore conjugated secondary antibodies and quantified by flow cytometry, imaging or mass spectrometry. Binding of Isa to recombinant human CD38 (rhCD38) pretreated with HB-7 or an NAD analog was quantified by Surface Plasmon Resonance (SPR). Ex vivo IAT was performed on plasma samples spiked with Isa or Dara. Results: In vitro testing in the absence of human plasma showed that incubation of RBCs with Dara but not Isa resulted in a dose-dependent increase in RBC binding. The binding specificity of Dara was demonstrated by treatment with HB-7, an anti-CD38 mAb that recognizes epitopes that overlap with Dara but not Isa binding. Flow cytometry, confocal microscopy, SPR and mass spectrometry showed HB-7 had a competitive effect on Dara binding and induced Isa to bind to CD38 without competition. Pretreatment of RBCs with another anti-CD38 mAb AT13/5 or an NAD analog also induced Isa binding to RBCs. The NAD analog is an irreversible CD38 inhibitor that stabilizes CD38 dimer conformation. SPR demonstrated that pretreatment with HB-7 or the NAD analog may enhance dimer formation increasing Isa binding affinity to rhCD38. These results indicate that CD38 conformational changes caused by anti-CD38 antibodies or the NAD analog may facilitate Isa-binding to RBC-expressing CD38. In the ex vivo IAT, except for gel testing in which both Isa and Dara were panreactive, including all other IAT methods Dara reacted with many more cells, and reactions were stronger than Isa at all concentrations tested. Conclusions: In this study, we confirmed that both Isa and Dara interfere with IATs but at different magnitudes. This reflects distinct mechanisms of CD38 binding. In the absence of human plasma, CD38 on RBCs can be directly recognized by Dara while Isa requires a cofactor, such as an anti-CD38 antibody or an NAD analog. Future studies are essential to characterize the impact of human plasma on Isa binding to RBCs-expressing CD38. Citation Format: Zhili Song, Olivier Bedel, Bailin Zhang, Joern Hopke, Gejing Deng, Sandrine Macé, Marielle Chiron, Francisco Adrian, Taro Fukao, Frank Basile, France Pirenne, Makoto Okuda, Morvarid Moayeri, Btissam Chami, Yoko Hidaka, Ashok Nambiar, Thomas Martin, Chen Zhu. Anti-CD38 interference with blood compatibility testing: Differentiating isatuximab and daratumumab via functional epitope mapping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1888.

β-Carotene-Induced Alterations in Haemoglobin Affinity to O2.

β-Carotene (β-Crt) can be dispersed in hydrophobic regions of the membrane of red blood cells (RBC). Its location, orientation and distribution strongly depend on carotenoid concentration. In the present pilot trial (six human subjects involved), it is demonstrated that incubation of RBCs with β-Crt (1.8 × 107 β-Crt molecules per RBC, 50 μmol/L) results in expansion of the membrane of RBCs and slight elongation of the cell. The changes are of statistical significance, as verified by the Wilcoxon test at p < 0.05. They indicate (i) a highly random orientation and location of β-Crt inside the membrane and (ii) a tendency for its interaction with membrane skeleton proteins. The accompanying effect of decreased RBC resistance to lysis is possibly a result of the incorrect functioning of ion channels due to their modification/disruption. At higher β-Crt concentrations, its clustering inside membranes may occur, leading to further alterations in the shape and size of RBCs, with the most pronounced changes observed at 1.8 × 108 β-Crt molecules per RBC (500 μmol/L). Due to the reduced permeability of ions, such membranes exhibit increased resistance to haemolysis. Finally, we show that interactions of β-Crt with the membrane of RBCs lead to an alteration in haemoglobin-oxygen affinity, shifting the oxyhaemoglobin dissociation curve toward higher oxygen partial pressures. If the impact of β-Crt on a curve course is confirmed in vivo, one may consider its role in the fine tuning of O2 transportation to tissues. Hence, at low concentrations, providing unchanged elastic and functional properties of RBCs, it could serve as a beneficial agent in optimising heart performance and cardiovascular load.

3,4-Dihydroxybenzaldehyde attenuates pentachlorophenol-induced cytotoxicity, DNA damage and collapse of mitochondrial membrane potential in isolated human blood cells

Pentachlorophenol (PCP) is a chlorophenolic compound that is widely used as pesticide, biocide and as a wood preservative to treat utility poles and wharf pilings. PCP is rapidly absorbed through the gastrointestinal tract and enters the blood where it generates active oxygen species in target cells. We have, therefore, examined the protective effect of plant antioxidant 3,4-dihydroxybenzaldehyde (DHB) against PCP-induced cyto-and geno-toxicity in human red blood cells (RBC) and lymphocytes, respectively. Human RBC were incubated at 37°C with 0.75 mM PCP, either alone or in presence of different concentrations of DHB (0.05–2.0 mM). Several biochemical parameters were determined in whole cells and hemolysates. Incubation of RBC with PCP alone increased the formation of reactive oxygen and nitrogen species (ROS and RNS) that resulted in oxidation of proteins, lipids, cellular thiols and plasma membrane damage. The antioxidant defense system was impaired and glucose metabolism was inhibited. However, prior treatment of RBC with DHB lowered ROS and RNS generation and attenuated PCP-induced oxidative damage of cell components. DHB alone enhanced electron transport by the plasma membrane redox system and also prevented its inhibition by PCP. DHB significantly prevented PCP-induced transformation of RBC morphology from normal biconcave shape to spherocytes, spiculated acanthocytes and echinocytes. DHB protected human lymphocytes from PCP-induced DNA damage and strand breaks, lysosomal membrane damage and collapse of the mitochondrial membrane potential. These results show that DHB mitigates PCP-induced cytotoxicity and can potentially function as a chemoprotective agent against the harmful effects of PCP and possibly other chlorophenols.

Mitigation of Cu(II)-induced damage in human blood cells by carnosine: An in vitro study

Copper (Cu) is an essential micronutrient but human exposure to high level of this metal results in adverse health effects. Oxidative stress is assumed to play a major role in the mechanism of Cu-induced toxicity. The protective role of carnosine, an antioxidant and antiglycating agent, was examined against Cu-induced toxicity in isolated human blood cells. Red blood cells (RBC) were treated with 0.5 mM copper chloride (CuCl2), a Cu(II) compound, either alone or after treatment with carnosine. Incubation of RBC with CuCl2 increased protein oxidation, lipid peroxidation, methemoglobin formation and lowered glutathione content. The antioxidant defense system was impaired and production of reactive oxygen (ROS) and reactive nitrogen species (RNS) was enhanced. Pre-incubation of RBC with carnosine protected the cells against CuCl2-induced oxidative damage. It restored the activities of several antioxidant, membrane-bound and metabolic enzymes, decreased the generation of ROS and RNS, enhanced the antioxidant power of cells and prevented inactivation of plasma membrane redox system. Carnosine also protected human lymphocytes from CuCl2-induced DNA damage. The protective effects of carnosine were concentration-dependent while carnosine itself did not exhibit any adverse effect. Carnosine can, therefore, be used as a possible chemoprotectant against the harmful effects of this extremely redox active metal.

Reactive species generated by heme impair alveolar epithelial sodium channel function in acute respiratory distress syndrome

We previously reported that the highly reactive cell-free heme (CFH) is increased in the plasma of patients with chronic lung injury and causes pulmonary edema in animal model of acute respiratory distress syndrome (ARDS) post inhalation of halogen gas. However, the mechanisms by which CFH causes pulmonary edema are unclear. Herein we report for the first time that CFH and chlorinated lipids (formed by the interaction of halogen gas, Cl2, with plasmalogens) are increased in the plasma of patients exposed to Cl2 gas. Ex vivo incubation of red blood cells (RBC) with halogenated lipids caused oxidative damage to RBC cytoskeletal protein spectrin, resulting in hemolysis and release of CFH. Patch clamp and short circuit current measurements revealed that CFH inhibited the activity of amiloride-sensitive epithelial Na+ channel (ENaC) and cation sodium (Na+) channels in mouse alveolar cells and trans-epithelial Na+ transport across human airway cells with EC50 of 125 nM and 500 nM, respectively. Molecular modeling identified 22 putative heme-docking sites on ENaC (energy of binding range: 86–1563 kJ/mol) with at least 2 sites within its narrow transmembrane pore, potentially capable of blocking Na+ transport across the channel. A single intramuscular injection of the heme-scavenging protein, hemopexin (4 μg/kg body weight), one hour post halogen gas exposure, decreased plasma CFH and improved lung ENaC activity in mice. In conclusion, results suggested that CFH mediated inhibition of ENaC activity may be responsible for pulmonary edema post inhalation injury.

Effect of Antioxidant Preparation «Mexidol» on the Protein Components of the Erythrocyte Cytoplasmic Membrane in Patients with Ulcerative Colitis

Background . Ulcerative colitis is a colon disease accompanied by a systemic inflammatory response with the development of oxidative stress, which leads to structural and functional changes in cell membranes. There are no data on the qualitative and quantitative changes in the protein components of the cytoplasmic membrane in ulcerative colitis under the influence of the antioxidant, membrane-stabilizing drug Mexidol. The article presents the data of our own research, the purpose of which is to study the nature of changes in protein components in the erythrocyte membrane in vitro when exposed to Mexidol (the active substance is oxymethylethyl peridine succinate) in patients with ulcerative colitis during an acute attack. Materials and methods . The study involved 51 patients with ulcerative colitis during the exacerbation period and 30 clinically healthy individuals comparable by sex and age. Ten major protein components of the erythrocyte membrane were evaluated. Results . As a result of the regression analysis, it was shown that in the group of clinically healthy individuals, almost all structural proteins are to varying degrees linked to proteins by glucose transporters and the enzyme glutathione-S-transferase and in patients with ulcerative colitis, the connection of protein components of membrane channels with spectrines is lost. Incubation of red blood cells of patients with ulcerative colitis in a solution of Mexidol in vitro did not affect the protein components of the erythrocyte cytoplasmic membrane. At the same time, in ulcerative colitis patients there was a restoration of spectrin connections with anion transport proteins and a glucose transporter, which were not detected in the initial state. Conclusion . The effect of Mexidol in vitro on the erythrocyte membranes of patients with ulcerative colitis was accompanied by the restoration of structural and functional protein bonds, which can be considered as a beneficial effect of this drug on the erythrocyte membrane under the conditions of the studied pathology.

Complement Deposition on the Surface of RBC After Trauma Serves a Biomarker of Moderate Trauma Severity: A Prospective Study.

Activation of the complement system and complement deposition on red blood cells (RBCs) contribute to organ damage in trauma. We conducted a prospective study in subjects with traumatic injuries to determine the pattern of complement deposition on RBC and whether they are associated with clinical outcomes. A total of 124 trauma patients and 42 healthy controls were enrolled in this prospective study. RBC and sera were collected at 0, 6, 24, and 72 h from trauma patients and healthy controls during a single draw. Presence of C4d, C3d, C5b-9, phosphorylation of band 3 and production of nitric oxide were analyzed by flow cytometry. RBC from trauma patients at all time points up to 24 h displayed significantly higher deposition of C4d on their RBC membrane as compared with healthy donors. Incubation of normal RBC with sera from trauma patients resulted in significant increase of C4d deposition (at 0, 6, 24, and 72 h), C5b-9 deposition (at 0 and 6 h), phosphorylation of band 3 (at 0 and 24 h), and nitric oxide production up to 24 h compared with sera from healthy subjects. Deposition of C4d and C5b-9 in patients with an Injury Severity Score (ISS) of 9 and above remained elevated up to 72 h. Our study demonstrates that the presence of C4d, C3d, and C5b-9 on the surface of RBC is linked to increased phosphorylation of band 3 and increased production of nitric oxide. Deposition of C4d and C5b-9 decreased faster over course of 3-day study in subjects with ISS less than 9.