Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Background

DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.

Aim

To investigate the effect of red blood cells on H₂O₂-induced DNA damage and repair in human peripheral blood mononuclear cells.

Methods

DNA breaks were induced in peripheral blood mononuclear cells by H₂O₂ in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by ³H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.

Results

Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.

Conclusion

Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.     

Some aspects of zinc transport across eel (Anguilla anguilla) red blood cell membranes.

Zinc movement across eel and human red blood cell membranes was measured by atomic absorption spectrophotometry. It was observed that:

• 1) In human red blood cells zinc uptake is twice as rapid as in fish red blood cells over a temperature range of 10-40°C. The low rate of zinc uptake in eel red blood cell may be simply the side effect of different surface area to volume ratios for the differences in cell size or, it may be due to the low permeability of bicarbonate through the red blood cell membranes.
• 2) Zinc uptake measured in eel and human red blood cells treated and untreated with external trypsin shows different features. The zinc uptake was reduced by about 40% in treated eel red blood cells with respect to the total uptake of untreated red blood cells. Human red blood cells treated and untreated with trypsin do not show any differences in the amount of zinc transported.
• 3) In fish red blood cells, zinc uptake in NANO₃ medium is markedly reduced, compared with that measured in NaCl medium. The [Zn²⁺_i slightly increases in the presence of bicarbonate. In human red blood cells in NANO₃ medium the zinc uptake is strongly reduced and the presence of bicarbonate marginally increases the zinc influx.
• 4) In eel red blood cells there seem to be two independent pathways for zinc uptake: one DIDS-sensitive and the other DIDS-insensitive. DIDS 10 μM inhibits only 64% of the total zinc transported. Iincreasing the DIDS concentration did not give more inhibition. In human red blood cells only one DIDS-sensitive pathway for zinc transported seems to exist, because, 2,5 μM DIDS inhibits 97% of zinc uptake.

     

Human red blood cells with normal or improved oxygen transport function prepared and frozen in the primary polyvinyl chloride plastic blood collection container.

This is a report a a new system for freezing human red blood cells in the same polyvinyl chloride plastic container in which the blood is collected and separated into components. This polyvinyl chloride plastic collection bag with integrally attached transfer packs for blood collection, component separation, red blood cell biochemical modification, freezing, storage, and post-thaw dilution before washing, represents a major advancement in the freeze-preservation process. The label with the donor's blood type and identification number affixed to the bag at the time of collection remains in place throughout the freezing and thawing process. The transfused red blood cells are of superior quality, and the processing cost is less than with other methods of freeze-preservation. There is a lower risk of contamination with these red blood cells because manipulation of the product is kept at a minimum. "Rejuvenation", a bioengineering process by which outdated red blood cells can be salvaged, can be incorporated into the preservation process using one of the attached transfer packs of the primary collection bag. This process has been introduced as a possible means of alleviating the dramatic blood shortages which occur periodically. Red blood cells may also be "rejuvenated" after storage in the liquid state to increase their 2,3 DPG and ATP levels to 150 to 200% of normal, and these red blood cells with improved oxygen transport function have been administered to anemic patients with and without cardiopulmonary insufficiency, patients undergoing cardiopulmonary bypass and treatment with hypothermia during cardiac surgery, and in instances where nonhemolytic transfusion reactions might be expected.     

Proteolytic cleavage of band 3 protein in relation to anion transport in fish (Oncorhynchus mykiss) red blood cells

• 1.1. The effects of trypsin and chymotrypsin on HCO₃⁻/Cl⁻ exchange through red blood cell membranes of humans and trout were studied.
• 2.2. To measure the anion exchange we used a right-angle light-scattering technique by applying the Jacobs-Stewart cycle in ammonium solution and the osmotiration method at constant cell volume.
• 3.3. The Cl⁻ flux in human red blood cells remained unaltered after treatment with external trypsin and chymotrypsin while in trout red blood cells the flux decreased.
• 4.4. This partial inhibition of anion transport in fish, ranging from 30 to 40%,suggest that one or several of the cleavage sites in band 3 protein, essential for anion transport function, are exposed in fish red blood cells.
• 5.5. In human red blood cells the fragments of band 3 which are affected by proteolytic digestion, retain their tertiary structure because there is no influence on anion transport.

     

Bicarbonate permeability and immunological evidence for an anion exchanger-like protein in the red blood cells of the sea lamprey,Petromyzon marinus

Physiological and immuno-blotting experiments were used to determine whether the red blood cell membrane of a primitive vertebrate, the sea lamprey Petromyzon marinus, contained a counterpart similar to the vertebrate anion exchange protein known as AE1 or band 3. Results of the physiological experiments which measured CO₂ production after adding H¹⁴CO ₃ ^- to the extracellular saline, indicated significant transmembrane bicarbonate movement in lamprey blood which unlike that in most vertebrates, was insensitive to inhibition by 4,4 diisothiocyanatostilbene-2,2 disulfonic acid. The present study also showed that lamprey red blood cells possess acetazolamide-sensitive carbonic anhydrase which is an important component of CO₂ production by vertebrate red blood cells. Polyclonal immunoglobulins against a 12 amino acid domain in the C-terminus of the mouse AE1 recognized a trout red blood cell membrane protein with a relative molecular mass of 97 kDa, but failed to immunoreact with any membrane proteins from the red blood cells of lamprey. Antibodies against trout AE1 immunoreacted with trout red blood cell membrane proteins of approximately 97 kDa, 200 kDa and >200 kDa. Interestingly, only a 200-kDa membrane protein from the red blood cells of the primitive lamprey immunoreacted with the trout anti-AE1 immunoglobulin proteins. Therefore, lamprey red blood cells appear to possess an AE1-like protein that may be physiologically different than that in most other vertebrates.     

Association between transfusion of whole blood and recurrence of cancer.

Transfusion affects the immune response to renal transplantation and may be associated with recurrence of various human neoplasms. Data from patients with colonic, rectal, cervical, and prostate tumours showed an association between transfusion of any amount of whole blood or larger amounts of red blood cells at the time of surgery and later recurrence of cancer. Recipients of one unit of whole blood had a significantly higher incidence of recurrence (45%) than recipients of a single unit of red cells (12%) (p = 0.03). Recipients of two units of whole blood also had a higher rate of recurrence (52%) than those receiving two units of red cells (23%) (p = 0.03). Recipients of any amount of whole blood had similar recurrence rates (38-52%). Recipients of four or more units of red blood cells had a higher rate of recurrence (55%) than those receiving three or fewer units of red blood cells (20%) (p = 0.005). Mortality due to cancer in patients receiving three or fewer units of red blood cells (2%) was similar to that in patients who did not have transfusions (7%) and significantly lower than that observed in patients receiving three or fewer units of whole blood (20%) (p = 0.003). A proportional hazards risk analysis showed that transfusion of any whole blood or more than three units of red blood cells was significantly associated with earlier recurrence and death due to cancer. These data support an association between transfusion and recurrence of cancer. They also suggest that some factor present in greater amounts in whole blood, such as plasma, may contribute to the increased risk of recurrence in patients who have undergone transfusion. Until the questions raised by retrospective studies of cancer recurrence and transfusion can be answered by prospective interventional trials with washed red blood cells, red blood cells should be transfused to patients with cancer in preference to whole blood when clinically feasible.     

A new method to study shape recovery of red blood cells using multiple optical trapping.

In this new method for studying the shape recovery of deformed red blood cells, three optical traps ("optical tweezers") induce a parachute-shaped red cell deformation, which is comparable to the deformation in small capillaries. The shape recovery is recorded, and a relaxation time is obtained for each individual red blood cell. The sensitivity of this technique for the detection of differences in relaxation times is demonstrated on subpopulations of density-separated red blood cells: "young" cells have shorter (162 ms) and "old" cells have longer (353 ms) relaxation times compared with the total population (271 ms). The relaxation time is remarkably shorter (114 ms) when the plasma surrounding the cells is replaced by a phosphate-buffered saline solution. The main advantages of this technique are the relatively short measuring and preparation time and the physiological type of deformation and shape recovery in which all relevant cell properties play a role. Therefore, especially when automated further, the technique may be a powerful tool for the study of (sub)populations of pathological red blood cells.     

Inhibition of adrenergic proton extrusion in rainbow trout red cells by nitrite-induced methaemoglobinaemia

Summary The effects of nitrite-induced methaemoglobinaemia on adrenergic proton extrusion from rainbow trout red blood cells were studied using the pH-stat method. In control conditions adrenergic proton extrusion was completely inhibited by amiloride and was greater in deoxygenated than in oxygenated erythrocytes. Nitrite-induced methaemoglobinaemia was associated with a pronounced reduction in the catecholamine-stimulated proton efflux from both deoxygenated and oxygenated erythrocytes. In deoxygenated erythrocytes the initial proton efflux upon catecholamine stimulation decreased by 60–70%, while the percentage of methaemoglobin in the red cells increased from the control level of 1–3% to 20%. In oxygenated erythrocytes the decrease was 30% at the same methaemoglobin percentage range. It is suggested that the pronounced influence of nitriteinduced methaemoglobinaemia on adrenergic proton efflux results from an inhibition of the red cell sodium/proton exchanger by the R-like haemoglobin conformations.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - RBC red blood cell     

透析式洗涤冰冻红细胞去除白细胞和血小板的方法

目的：用低速离心的方法减少悬浮红细胞中自细胞和血小板的数量,使透析式洗涤机洗涤的冰冻红细胞中白细胞和血小板的残留量小于1％。方法：分别选用低速离心（700和500r／min）和常规离心（3800r／min）全血制备悬浮红细胞,计算白细胞和血小板的清除率;甘油低温冰冻保存,透析式冰冻红细胞洗涤机洗涤冰冻红细胞,对洗涤后的红细胞进行质量检测。结果：500r／min离心后白细胞和血小板的清除率（％）分别为53.82±6.83和85.23±4.21;洗涤后的冰冻红细胞中白细胞和血小板残留量（％）分别为0.843±0.058和0.903±0.035。700r／min离心后白细胞和血小板的清除率（％）分别为23.38±2.36和62.61±3.82;洗涤后的冰冻红细胞中白细胞和血小板残留量（％）分别为0.983±0.024和1.021±0.045。3800r／min离心后白细胞和血小板的清除率（％）分别为9.82±4.12和6.39±3.26;洗涤后的冰冻红细胞中白细胞和血小板残留量（％）分别为3.839±2.896和3.528±2.689。结论：用500r／min离心全血制备的悬浮红细胞,经甘油低温冻存,透析式洗涤机洗涤后,血小板和白细胞的残留量等各项指标均符合国家标准。     

Blood clam (Anadara inflata) red cells. partition in aqueoues two-polymer phase systems

Anadara inflata is a clam which has red blood cells in its hemolymph. Furthermore, the nucleated red blood cells contain two structurally distinct hemoglobins. Clam red cells were subjected to partition in aqueous dextran-polyethylene glycol two-phase systems with the following results:

1.

1. Clam red cells are the largest cells (about 20 μm in diameter) so far studied in two-polymer phases. It is shown that not only can such cells be partitioned in dextran-polyethylene glycol phase systems, but that countercurrent distribution resolves the clam red cell population into more and less metabolically active cells. The distribution of these cells in relation to the whole population is similar to that of young and old red cells from mammals.     

Total sialic acid content of glycophorins during senescence of human red blood cells.

Glycophorins extracted from membranes of young and old human red blood cells have within an error of +/- 1.5% the same sialic acid content when referred to a relative measure of the number of glycophorins. The degree of surface iodination in glycophorins, which was shown to be the same in young and old cells, served as this relative measure. This finding implies that senescent human red blood cells hardly reveal desialylated surface proteins (less than or equal to 3%). However, the sialic acid content per cell was repeatedly reported to be 10 to 15% lower in old than in young cells. Therefore, we conclude 1) that human red blood cells lose intact glycophorin together with membrane during red blood cell senescence, and 2) that removal of desialylated and senescent red blood cells from the circulation proceeds by different routes.     

Trypanosoma evansi: Effect of experimental infection on the osmotic fragility, lipid peroxidation and calcium-ATPase activity of rat red blood cells

Trypanosoma evansi is the causative agent of equine trypanosomoses. The disease is characterized by fever, anemia, and cachexia. Peroxidative damage of the red blood cells caused by the parasite, may contribute to the pathogenesis of the anemia seen in trypanosomoses. Consequently, we evaluated the hematocrit, the osmotic fragility of the red blood cells, the level of lipid peroxidation and the activity of the Ca-ATPase of red blood cell ghosts from rats experimentally infected with T. evansi. After 72 h inoculation, the hematocrit decreased from 49.5% to 33%; the osmotic fragility of the red blood cells was approximately 40% higher as compared to the healthy animals; and the red blood cell ghosts showed a higher level of lipid peroxidation and a lower Ca-ATPase activity than the red cell ghosts from the healthy animals. In vitro incubations of red blood cells from healthy animals with T. evansi, produced also a significant increase of the osmotic fragility of the red blood cells.     

A comparison of transketolase assay and transketolase and lactate dehydrogenase activity levels in whole blood and red cell hemolysates and in leukocytes

1. A study was made of transketolase activity in red and white blood cells and of conditions for assay for transketolase activity and for assessment of the "TPP effect" in human and rat blood. 2. The ratio of the transketolase activity in white cells to that in red cells varied between 23 and 93. 3. Red cells or white cells can both be used for assessment of transketolase activity and the "TPP effect", but the best source for evaluation of transketolase activity and the percent change on addition of thiamin diphosphate appears to be whole blood.     

Margination of red blood cells infected by Plasmodium falciparum in a microvessel

We investigated numerically the mechanism of margination of Plasmodium falciparum malaria-infected red blood cells (Pf-IRBCs) in micro-scale blood flow. Our model illustrates that continuous hydrodynamic interaction between a Pf-IRBC in the trophozoite stage (Pf-T-IRBC) and healthy red blood cells (HRBCs) results in the margination of the Pf-T-IRBC and, thus, a longer duration of contact with endothelial cells. The Pf-T-IRBC and HRBCs first form a "train". The volume fraction of RBCs is then locally increased, to approximately 40%, and this value is maintained for a long period of time due to the formation of a long train in high-hematocrit conditions. Even in low-hematocrit conditions, the local volume fraction is instantaneously elevated to 40% and the Pf-T-IRBC can migrate to the wall. However, the short train formed in low-hematocrit conditions does not provide continuous interaction, and the Pf-T-IRBC moves back to the center of the channel.     

Method for removing leukocytes and thrombocytes from preserved blood and packed red cells

A method has been described for removing leukocytes and thrombocytes from preserved ACD blood and from packed red cells of the same type of blood. A system has been used comprising two connected plastic bags model "Fenval". The leukocyte and thrombocyte removal is performed by two consecutive procedures, each comprising centrifugation with subsequent fast sedimentation of erythrocytes. No filters are needed. 97.3 +/- 3.04% leukocyte removal and 96.90 +/- 5.96% thrombocyte removal as regards the preserved blood and 95.65 +/- 8.70% leukocyte removal and 98.44 +/- 0.70% thrombocyte removal as regards the packed red cells have been achieved.     

The temperature dependence of erythrocyte water diffusion permeability

1. The activation energy of the diffusion water exchange in red blood cells increases with temperature. 2. Fetal blood has a higher activation energy for diffusion water exchange than adult blood. 3. Treatment of red cells with p-chloromercuribenzoate alters the activation energy and apparently allows a lipid and a protein pore pathway to be resolved. The permeability and activation energy of the treated cells is in the range found for lipid membranes; and the difference between treated and untreated cells, the "protein" pathway, has a diffusion activation energy comparable with that of free water. 4. A resolution of the discrepancies between the NMR methods of measuring diffusion water exchange is suggested.     

What befalls the proteins and water in a living cell when the cell dies?

The solvency of solutes of varying molecular size in the intracellular water of freshly-killed Ehrlich carcinoma cells fits the same theoretical curve that describes the solvency of similar solutes in a 36% solution of native bovine hemoglobin--a protein found only in red blood cells and making up 97.3% of the red cell's total intracellular proteins. The merging of the two sets of data confirms the prediction of the AI Hypothesis that key intracellular protein(s) in dying cells undergo(es) a transition from: (1) one in which the polypeptide NHCO groups assume a fully-extended conformation with relatively strong power of polarizing and orienting the bulk-phase water in multilayers; to (2) one in which most of the polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformations (see below for definition) with much weaker power in polarizing-orienting multilayers of bulk-phase water. This concordance of the two sets of data also shows that what we now call native hemoglobin--supposedly denoting hemoglobin found in its natural state in living red blood cells--, in fact, more closely resembles the water-polarizing, and -orienting intracellular proteins in dead cells. Although in the dead Ehrlich carcinoma cells as well as in the 36% solution of native hemoglobin, much of the protein's polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformation (Perutz 1969; Weissbluth 1974), both systems produce a weak but nonetheless pervasive and "long-range" water polarization and orientation. It is suggested that in both the dead Ehrlich carcinoma ascites cells and in the 36% native bovine hemoglobin solution, enough polypeptide NHCO groups assume the fully-extended conformation to produce the weak but far-reaching multilayer water polarization and orientation observed.     

Inhibition of the immune response by membrane-bound antibody.

Cells from the spleens of "normal" swine, which were pretreated with pronase to remove surface membrane-bound immunoglobulin, gave an enhanced hemolytic plaque-forming cell response to sheep red blood cells in vitro in comparison with untreated controls. The enhancement could be abrogated by preincubating pronase-treated spleeen cells in preparations containing antibody to sheep red blood cells. This effect was demonstrated by autologous sera, immune sera, and all three known classes of porcine serum immunoglobulins, including IgM, IgA, and IgG and could be removed by absorption with sheep red blood cells. Surface membrane-bound antibody exerted its effect by binding to the nonadherent cell population. The response of normal spleen cells was unaffected by antibody treatment. Pronase-treatment was not mitogenic, did not function as a polyclonal B cell activator, and did not selectively eliminate T or B cells. The results indicate that removal of antibody from the surface of lymphoid cells enhanced the humoral immune response invitro and confirm that membrane-bound antibody can inhibit response to antigen.     

Red cell distribution and the recruitment of pulmonary diffusing capacity.

The distribution of red blood cells in alveolar capillaries is typically nonuniform, as shown by intravital microscopy and in alveolar tissue fixed in situ. To determine the effects of red cell distribution on pulmonary diffusive gas transport, we computed the uptake of CO across a two-dimensional geometric capillary model containing a variable number of red blood cells. Red blood cells are spaced uniformly, randomly, or clustered without overlap within the capillary. Total CO diffusing capacity (DLCO) and membrane diffusing capacity (DmCO) are calculated by a finite-element method. Results show that distribution of red blood cells at a fixed hematocrit greatly affects capillary CO uptake. At any given average capillary red cell density, the uniform distribution of red blood cells yields the highest DmCO and DLCO, whereas the clustered distribution yields the lowest values. Random nonuniform distribution of red blood cells within a single capillary segment reduces diffusive CO uptake by up to 30%. Nonuniform distribution of red blood cells among separate capillary segments can reduce diffusive CO uptake by >50%. This analysis demonstrates that pulmonary microvascular recruitment for gas exchange does not depend solely on the number of patent capillaries or the hematocrit; simple redistribution of red blood cells within capillaries can potentially account for 50% of the observed physiological recruitment of DLCO from rest to exercise.     

Red blood cell mechanics and capillary blood rheology.

Blood contains a high vol fraction of erythrocytes (red blood cells), which strongly influence its flow properties. Much is known about the mechanical properties of red cells, providing a basis for understanding and predicting the rheological behavior of blood in terms of the behavior of individual red cells. This review describes quantitative theoretical models that relate red cell mechanics to flow properties of blood in capillaries. Red cells often flow in single file in capillaries, and rheological parameters can then be estimated by analyzing the motion and deformation of an individual red cell and the surrounding plasma in a capillary. The analysis may be simplified by using lubrication theory to approximate the plasma flow in the narrow gaps between the cells and the vessels walls. If red cell shapes are assumed to be axisymmetric, apparent viscosities are predicted that agree with determinations in glass capillaries. Red cells flowing in microvessels typically assume nonaxisymmetric shapes, with cyclic "tank-treading" motion of the membrane around the interior. Several analyses have been carried out that take these effects into account. These analyses indicate that nonaxisymmetry and tank-treading do not significantly influence the flow resistance in single-file or two-file flow.