The aim of the present investigation was to verify the effect of H2O2-induced oxidative stress on SO4= uptake through Band 3 protein, responsible for Cl-/HCO3- as well as for cell membrane deformability, due to its cross link with cytoskeletal proteins. The role of cytoplasmic proteins binding to Band 3 protein has been also considered by assaying H2O2 effects on hemoglobin-free resealed ghosts of erythrocytes. Oxidative conditions were induced by 30 min exposure of human erythrocytes to different H2O2 concentrations (10 to 300 μM), with or without GSH (glutathione, 2 mM) or curcumin (10 μM), compounds with proved antioxidant properties. Since SO4= influx through Band 3 protein is slower and better controllable than Cl- or HCO3- exchange, the rate constant for SO4= uptake was measured to prove anion transport efficiency, while MDA (malondialdehyde) levels and –SH groups were estimated to quantify the effect of oxidative stress. H2O2 induced a significant decrease in rate constant for SO4= uptake at both 100 and 300 μM H2O2. This reduction, observed in erythrocytes but not in resealed ghosts and associated to increase in neither MDA levels nor in –SH groups, was impaired by both curcumin and GSH, whereas only curcumin effectively restored H2O2-induced changes in erythrocytes shape. Our results show that: i) 30 min exposure to 300 μM H2O2 reduced SO4= uptake in human erythrocytes; ii) oxidative damage was revealed by the reduction in rate constant for SO4= uptake, but not by MDA or –SH groups levels; iii) the damage was produced via cytoplasmic components which cross link with Band 3 protein; iv) the natural antioxidant curcumin may be useful in protecting erythrocytes from oxidative injury; v) SO4= uptake through Band 3 protein may be reasonably suggested as a tool to monitor erythrocytes function under oxidative conditions possibly deriving from alcohol consumption, use of drugs, radiographic contrast media administration, hyperglicemia or neurodegenerative diseases.
The objective of this study was to assess the effects of nickel chloride on human and rainbow trout erythrocytes in vitro. The cells were incubated with 0, 0.5 and 1 mM nickel chloride for 1 h at pH 7.40 and 25 degrees C, then K(+) efflux, SO (4) (2-) uptake and GSH and GSSG concentrations were measured. In both kind of cells, "high concentration" nickel treatment increased KCl efflux with respect to the control. The SO (4) (2-) uptake was not significantly different at "low nickel concentration" but was lower in erythrocytes treated with 1 mM nickel chloride; the rate constant of SO (4) (2-) uptake decreased by 35% in human erythrocytes and by 44% in fish erythrocytes. Nickel chloride also acts on cellular metabolism and in particular on erythrocyte glutathione peroxidase with consequent increase in oxidative stress; the data show a significant decrease in intracellular GSH in both human (25%) and fish erythrocytes (18%) after treatment with nickel chloride, with concomitantly high GSSG concentrations and lower GSH/GSSG ratios.
The morphological and functional effects of titanium (Ti) citrate on human erythrocytes were studied by scanning electron microscope (SEM), sulphate uptake via band 3 protein and by determining the reduced and oxidised glutathione (GSH and GSSG, respectively) concentrations. The rate constant for sulphate uptake was significantly lower after Ti citrate treatment. Ti citrate (0.001 and 0.0025 mM) significantly decreased erythrocyte GSH and increased GSSG concentrations. At 0.005 mM Ti citrate, the intracellular GSH could not be tested due to significant cellular damage. SEM of erythrocytes treated with 0.001 mM and 0.0025 mM Ti citrate showed structural membrane defects but almost normal cellular diameters. At even higher Ti citrate concentrations (0.005 mM), erythrocytes showed obvious morphological alteration and shape changes compromising the cells physiological functions. In conclusion, although the Ti concentrations used in our experiments are physiologically high, the cumulative effect of prolonged exposure to much lower doses of Ti, as might occur during total hip replacement, should be considered for further experimental testing
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