Gloria Celedón

Hypoxia-related lipid peroxidation: evidences, implications and approaches.

Hypoxia may be intensified by concurrent oxidative stress. Lack of oxygen in relation to aerobic ATP requirements, as hypoxia has been defined, goes along with an increased generation of reactive oxygen species (ROS). Polyunsaturated fatty acids (PUFAs) range among the molecules most susceptible to ROS. Oxidative breakdown of n-3 PUFAs may compromise not only membrane lipid matrix dynamics, and hence structure and function of membrane-associated proteins like enzymes, receptors, and transporters, but also gene expression. Eicosapentaenoic acid depletion, products of lipid peroxidation (LP), as well as, lack of oxygen may combine in exacerbating activity of nuclear factor kappa B (NFkappaB), an ubiquitous pro-inflammatory and anti-apoptotic transcription factor. Field studies at high altitude show malondialdehyde (MDA) content in exhaled breath condensate (EBC) of mountaineers to correlate with Lake Louis score of acute mountain sickness. A pathogenic role of LP in hypoxia can therefore be expected. By control of LP, some species seem to cope more efficiently than others with naturally occurring hypoxia. Limitation of potential pro-inflammatory effects of hypoxia-related LP by an adequate provision of n-3 PUFAs and antioxidants may contribute to increase survival under conditions where oxygen is lacking in relation to aerobic ATP requirements. A need for antioxidant intervention, however, should be weighed against the ROS requirement for triggering adaptive processes in response to an increased demand of oxygen.

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.

Contribution of haemoglobin and membrane constituents modification to human erythrocyte damage promoted by peroxyl radicals of different charge and hydrophobicity.

We have investigated the influence of the free radical initiator characteristics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2′-Azobis(2-amidinopropane) (AAPH) and 4,4′-azobis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilicity, as evaluated from octanol/water partition constants. At physiological pH, they are a di-cation and a di-anion, respectively. AAPH and ACV readily oxidise purified oxyhemoglobin in a very efficient free radical-mediated process, particularly for ACV-derived radicals, where nearly one heme moiety was modified per radical introduced into the system, suggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-compounds lead to different oxidation products. Methemoglobin, hemichromes and choleglobin were produced in AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominantly form hemichromes, with very low production of choleglobin. Red cell damage was evaluated at the level of hemoglobin and membrane constituents modification, and was expressed in terms of free radical doses. Before the onset of the lytic process, ACV leads to more lipid peroxidation than AAPH, and induces a moderate oxidation of intracellular Hb. This intracellular oxidation is markedly increased if ACV hydrophilicity is decreased by lowering the pH. On the other hand, AAPH-derived radicals are considerable more efficient in promoting protein band 3 modification and cell lysis, without significant intracellular hemoglobin oxidation. These results show that the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant factor leading to red blood cell lysis.

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( − )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.

Protein degradation in red cells exposed to 2,2'-azo-bis(2-amidinopropane) derived radicals.

Extensive proteolysis is observed when red blood cells are exposed to free radicals produced in the thermolysis of 2,2′‐azo‐bis(2‐amidinopropane). It is evaluated that nearly one amino terminal group is produced by each free radical introduced into the system. These groups are considered to arise mainly from band 3 fragmentation due to the action of red cell proteinases. Protein fragmentation takes place prior to significant hemolysis or lipid peroxidation, as evaluated by thiobarbituric acid‐reactive substances measurements.

Stycholysin II, a cytolysin from the sea anemone Stichodactyla helianthus promotes higher hemolysis in aged red blood cells

We have investigated the relationship between the status of red blood cells (RBCs) and their susceptibility to toxin sticholysin II (StII) hemolytic activity; we have evaluated this effect in different RBC ensembles, comprising young and old cells, and in cells partially damaged by their pre-exposition to a free radical source. Upon action of StII, young cell populations are less prone to hemolysis than the whole population, while old cell populations and peroxyl-oxidized red cells are lysed faster than the whole population. Cell K(+) content was higher in young cells and lower in both senescent cells and in peroxyl-damaged cells relative to whole cell population. The relevance of cell K(+) content in St II-induced lysis was shown when external Na(+) was partially replaced by K(+); under this condition, RBC lysed faster in the presence of St II but no difference was observed among young cells, whole cells population and peroxyl-damaged cells; only old cells lysed faster that the whole population, response that can be due to an enhanced St II-induced pore formation as supported by evaluation of St II irreversible binding to RBC. It is concluded that this factor and the amount of intracellular K(+) are the dominant parameters that modulate the resistance of RBC to St II-induced lysis.

Peroxyl-oxidized Erythrocyte Membrane Band 3 Protein with Anion Transport Capacity is Degraded by Membrane-bound Proteinase

Human red blood cells anion exchange protein (band 3) exposed to peroxyl radicals produced by thermolysis of 2,2′-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through band 3 was smaller indicating that removal achieved by proteinases includes oxidized band 3 which still retain transport ability. Proteinases recognize band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.

Thiol-induced hemoglobin oxidation.

Addition of cysteine in the mM range to purified oxyhemoglobin, red blood cell lysate or red blood cell suspensions leads to oxidation of the hemoprotein. The rate and extent of the process depend on the initial hemoglobin and cysteine concentrations, and the reaction is limited by the total destruction of the sulfhydryl groups. Similar results are obtained employing glutathione, but the rate of the process is considerably slower. Oxidation of the purified hemoprotein is faster than in the red blood cell lysate. This difference is mainly due to the inhibitory effect of catalase present in the lysate. Addition of sodium azide increases the rate of oxyhemoglobin oxidation in the lysate, while addition of catalase reduces the rate of oxidation of the purified hemoprotein. The results are interpreted in terms of a mechanism comprising the oxidation of the oxyhemoglobin by the -SH group, with concomitant formation of superoxide anion and hydrogen peroxide. These species further contribute to the oxyhemoglobin oxidation. A chain oxidation of the thiol, catalyzed by the hemoprotein, explains the extensive cysteine destruction.

Effect of pre-exposure of human erythrocytes to oxidants on the haemolytic activity of Sticholysin II. A comparison between peroxynitrite and hypochlorous acid

Abstract Stichodactyla heliantus II (St II) is a haemolytic toxin whose activity depends of the characteristics of red blood cells (RBC). Among the factors that may tune the response of the RBC to the toxin activity stand the oxidative status of the cell. This study investigates how pre-oxidation of RBC modifies St II activity employing two oxidants, peroxynitrite and hypochlorous acid. Results show that peroxynitrite-treated RBC are more resistant to St II activity. On the other hand, hypochlorous acid-treated RBC become more susceptible to St II. This contrasting behaviour of both oxidants is related to the modifications elicited in RBC by both oxidant agents. Peroxynitrite does not modify RBC osmotic fragility but reduces anion transport through band 3 protein. This effect, together with an increase in K+ efflux, can explain the increased resistance to the toxin activity. On the other hand, results obtained with hypochlorous acid can be explained in terms of a disruption of the membrane organization without the compensating effect of a reduction in band 3-mediated anion transport. The present results, obtained employing the effect of a model haemolytic toxin on RBC, emphasize the specificity of the RBC response to different endogenous oxidative agents.