Antimicrobial Activity of Quercetin, Naringenin and Catechin: Flavonoids Inhibit Staphylococcus aureus-Induced Hemolysis and Modify Membranes of Bacteria and Erythrocytes.

Search for novel antimicrobial agents, including plant-derived flavonoids, and evaluation of the mechanisms of their antibacterial activities are pivotal objectives. The goal of this study was to compare the antihemolytic activity of flavonoids, quercetin, naringenin and catechin against sheep erythrocyte lysis induced by α-hemolysin (αHL) produced by the Staphylococcus aureus strain NCTC 5655. We also sought to investigate the membrane-modifying action of the flavonoids. Lipophilic quercetin, but not naringenin or catechin, effectively inhibited the hemolytic activity of αHL at concentrations (IC50 = 65 ± 5 µM) below minimal inhibitory concentration values for S. aureus growth. Quercetin increased the registered bacterial cell diameter, enhanced the fluidity of the inner and surface regions of bacterial cell membranes and raised the rigidity of the hydrophobic region and the fluidity of the surface region of erythrocyte membranes. Our findings provide evidence that the antibacterial activities of the flavonoids resulted from a disorder in the structural organization of bacterial cell membranes, and the antihemolytic effect of quercetin was related to the effect of the flavonoid on the organization of the erythrocyte membrane, which, in turn, increases the resistance of the target cells (erythrocytes) to αHL and inhibits αHL-induced osmotic hemolysis due to prevention of toxin incorporation into the target membrane. We confirmed that cell membrane disorder could be one of the direct modes of antibacterial action of the flavonoids.

Hydrolysable tannins change physicochemical parameters of lipid nano-vesicles and reduce DPPH radical - Experimental studies and quantum chemical analysis.

Tannins belong to plant secondary metabolites exhibiting a wide range of biological activity. One of the important aspects of the realization of the biological effects of tannins is the interaction with lipids of cell membranes. In this work we studied the interaction of two hydrolysable tannins: 1,2,3,4,6-penta-O-galloyl-ß-d-glucose (PGG) and 1,2-di-O-galloyl-4,6-valoneoyl-ß-d-glucose (T1) which had the same number of both aromatic rings (5) and hydroxyl groups (15) but differing in flexibility due to the presence of valoneoyl group in the T1 molecule with DMPC (dimyristoylphosphatidylcholine) lipid nano-vesicles (liposomes). Tannins-liposomes interactions were investigated using fluorescence spectroscopy, differential scanning calorimetry, laser Doppler velocimetry, dynamic light scattering and Fourier Transform Infra-Red spectroscopy. It was shown that more flexible PGG molecules stronger decreased the microviscosity of the liposomal membranes and increased the values of negative zeta potential in comparison with the more rigid T1. Both compounds diminished the phase transition temperature of DMPC membranes, interacted with liposomes via PO groups of head of phospholipids and their hydrophobic regions. These tannins neutralized DPPH free radicals with the stoichiometry of the reaction equal 1:1. The effects of the studied compounds on liposomes were discussed in relation to tannin quantum chemical parameters calculated by molecular modeling.

Comparative analysis of molecular properties and reactions with oxidants for quercetin, catechin, and naringenin.

Flavonoids, a large group of secondary plant phenolic metabolites, are important natural antioxidants and regulators of cellular redox balance. The present study addressed evaluation of the electronic properties of some flavonoids belonging to different classes such as quercetin (flavonols), catechin (flavanols), and naringenin (flavanones) and their interactions with oxidants in model systems of DPPH reduction, flavonoid autoxidation, and chlorination. According to our ab initio calculations, the high net negative excess charges of the C rings and the small positive excess charges of the B rings of quercetin, catechin, and naringenin make these parts of flavonoid molecules attractive for electrophilic attack. The 3'-OH group of the B ring of quercetin has the highest excess negative charge and the lowest energy of hydrogen atom abstraction for the flavonoids studied. The apparent reaction rate constants (s-1, 20 °C) and the activation energies (kJ/mol) of DPPH reduction were 0.34 ± 0.06 and 23.0 ± 2.5 in the case of quercetin, 0.09 ± 0.02 and 32.5 ± 2.5 in the case of catechin, respectively. The stoichiometry of the DPPH-flavonoid reaction was 1:1. The activation energies (kJ/mol) of quercetin and catechin autoxidations were 50.8 ± 6.1 and 58.1 ± 7.2, respectively. Naringenin was not oxidized by the DPPH radical and air oxygen (autoxidation) and the flavonoids studied effectively prevented HOCl-induced hemolysis due to direct scavenging of hypochlorous acid (flavonoid chlorination). The best antioxidant quercetin had the highest value of HOMO energy, a planar structure and optimal electron orbital delocalization on all the phenolic rings due to the C2=C3 double bond in the C ring (absent in catechin and naringenin).

Role of mitochondrial calcium in hypochlorite induced oxidative damage of cells.

Hypochlorite (HOCl) is one of the most important mediators of inflammatory processes. Recent evidence demonstrates that changes in intracellular calcium pool play a significant role in the damaging effects of hypochlorite and other oxidants. Mitochondria are shown to be one of the intracellular targets of hypochlorite. But little is known about the mitochondrial calcium pool changes in HOCl-induced mitochondrial dysfunction. Using isolated rat liver mitochondria, we showed the oxidative damage of mitochondria (GSH oxidation and mixed protein-glutathione formation without membrane lipid peroxidation) and alterations in the mitochondrial functional parameters (decrease of respiratory activity and efficiency of oxidative phosphorylation, NADH and FADH coenzyme levels, and membrane potential) under hypochlorite action (50-300 µM). Simultaneously, the mitochondrial calcium release and swelling were demonstrated. In the presence of EGTA, the damaging effects of HOCl were less pronounced, reflecting direct involvement of mitochondrial Ca2+ in mechanisms of oxidant-induced injury. Furthermore, exposure of HeLa cells to hypochlorite resulted in a considerable increase in cytoplasmic calcium concentrations and a decrease in mitochondrial ones. Applying specific inhibitors of calcium transfer systems, we demonstrated that mitochondria play a key role in the redistribution of cytoplasmic Ca2+ ions under hypochlorite action and act as mediators of calcium release from the endoplasmic reticulum into the cytoplasm.

Flavonoids modulate liposomal membrane structure, regulate mitochondrial membrane permeability and prevent erythrocyte oxidative damage.

In the present work, we investigated the interaction of flavonoids (quercetin, naringenin and catechin) with cellular and artificial membranes. The flavonoids considerably inhibited membrane lipid peroxidation in rat erythrocytes treated with tert-butyl hydroperoxide (700 µM), and the IC50 values for prevention of this process were equal to 9.7 ± 0.8 µM, 8.8 ± 0.7 µM, and 37.8 ± 4.4 µM in the case of quercetin, catechin and naringenin, respectively, and slightly decreased glutathione oxidation. In isolated rat liver mitochondria, quercetin, catechin and naringenin (10-50 µM) dose-dependently increased the sensitivity to Ca2+ ions - induced mitochondrial permeability transition. Using the probes TMA-DPH and DPH we showed that quercetin rather than catechin and naringenin strongly decreased the microfluidity of the 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomal membrane bilayer at different depths. On the contrary, using the probe Laurdan we observed that naringenin transfer the bilayer to a more ordered state, whereas quercetin dose-dependently decreased the order of lipid molecule packing and increased hydration in the region of polar head groups. The incorporation of the flavonoids, quercetin and naringenin and not catechin, into the liposomes induced an increase in the zeta potential of the membrane and enlarged the area of the bilayer as well as lowered the temperature and the enthalpy of the membrane phase transition. The effects of the flavonoids were connected with modification of membrane fluidity, packing, stability, electrokinetic properties, size and permeability, prevention of oxidative stress, which depended on the nature of the flavonoid molecule and the nature of the membrane.

Spectroscopic, Zeta-potential and Surface Plasmon Resonance analysis of interaction between potential anti-HIV tannins with different flexibility and human serum albumin.

Tannins belong to secondary metabolites of plants that exhibit a variety of biological activities, including antiviral one. In this research, we studied the interaction of human serum albumin (HSA) with two ellagitannins: 2,4-valoneoyl-3,6-hexahydroxydiphenoyl-ß-d-glucose (T1) and 1,2-di-O-galloyl-3,6-valoneoyl-ß-d-glucose (T2) from Euphorbia species having antiviral potential against HIV and differing in molecular flexibility due to the presence of valoneoyl- and hexahydroxydiphenoyl groups. A fluorescence analysis demonstrated that the tannins studied strongly interacted with HSA and quenched tryptophan (Trp) fluorescence in the range of 0.25-4 µM. The quenching occurred by a static mechanism. The logKb for more flexible T2 was generally higher in comparison with stiffer T1 (4.94 ±â€¯0.82 vs. 4.12 ±â€¯0.31 and 4.94 ±â€¯0.53 vs. 4.07 ±â€¯0.45 for 296 K and 303 K respectively). The difference was also in the nature of the forces participating in the interaction with HSA. The stiff T1 reacted with HSA via hydrophobic forces, whereas the flexible T2 interacted with the protein by van der Waals forces and hydrogen bonds. The nature of the bonds was also confirmed by a study of the hydrophobicity of the compounds. Zeta-potential measurements showed slightly modifications of albumin electric charge but without significant changes in the surface structure of protein. Surface Plasmon Resonance imaging (SPRi) revealed that the used tannins fully saturated a 3 ng/mL solution of albumin at the concentrations of above 15 ng/mL. Our experiments clearly showed that the tannins used formed complexes with HSA and that the flexibility of the tannins was an important factor determining their interaction with the protein.

Calcium-Induced Mitochondrial Permeability Transitions: Parameters of Ca2+ Ion Interactions with Mitochondria and Effects of Oxidative Agents.

We evaluated the parameters of Ca2+-induced mitochondrial permeability transition (MPT) pore formations, Ca2+ binding constants, stoichiometry, energy of activation, and the effect of oxidative agents, tert-butyl hydroperoxide (tBHP), and hypochlorous acid (HOCl), on Ca2+ -mediated process in rat liver mitochondria. From the Hill plot of the dependence of MPT rate on Ca2+ concentration, we determined the order of interaction of Ca2+ ions with the mitochondrial sites, n = 3, and the apparent Kd = 60 ± 12 µM. We also found the apparent Michaelis-Menten constant, Km, for Ca2+ interactions with mitochondria to be equal to 75 ± 20 µM, whereas that in the presence of 300 µM tBHP was 120 ± 20 µM. Using the Arrhenius plots of the temperature dependences of apparent mitochondrial swelling rate at various Ca2+ concentrations, we calculated the activation energy of the MPT process. ΔEa was 130 ± 20 kJ/mol at temperatures below the break point of the Arrhenius plot (30-34 °C) and 50 ± 9 kJ/mol at higher temperatures. Ca2+ ions induced rapid mitochondrial NADH depletion and membrane depolarization. Prevention of the pore formation by cyclosporin A inhibited Ca2+-dependent mitochondrial depolarization and Mg2+ ions attenuated the potential dissipation. tBHP (10-150 µM) dose-dependently enhanced the rate of MPT opening, whereas the effect of HOCl on MPT depended on the ratio of HOCl/Ca2+. The apparent Km of tBHP interaction with mitochondria in the swelling reaction was found to be Km = 11 ± 3 µM. The present study provides evidence that three calcium ions interact with mitochondrial site with high affinity during MPT. Ca2+-induced MPT pore formations due to mitochondrial membrane protein denaturation resulted in membrane potential dissipation. Oxidants with different mechanisms, tBHP and HOCl, reduced mitochondrial membrane potential and oxidized mitochondrial NADH in EDTA-free medium and had an effect on Ca2+-induced MPT onset.

Cranberry flavonoids prevent toxic rat liver mitochondrial damage in vivo and scavenge free radicals in vitro.

The present study was undertaken for further elucidation of the mechanisms of flavonoid biological activity, focusing on the antioxidative and protective effects of cranberry flavonoids in free radical-generating systems and those on mitochondrial ultrastructure during carbon tetrachloride-induced rat intoxication. Treatment of rats with cranberry flavonoids (7 mg/kg) during chronic carbon tetrachloride-induced intoxication led to prevention of mitochondrial damage, including fragmentation, rupture and local loss of the outer mitochondrial membrane. In radical-generating systems, cranberry flavonoids effectively scavenged nitric oxide (IC50 = 4.4 ± 0.4 µg/ml), superoxide anion radicals (IC50 = 2.8 ± 0.3 µg/ml) and hydroxyl radicals (IC50 = 53 ± 4 µg/ml). The IC50 for reduction of 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) was 2.2 ± 0.3 µg/ml. Flavonoids prevented to some extent lipid peroxidation in liposomal membranes and glutathione oxidation in erythrocytes treated with UV irradiation or organic hydroperoxides as well as decreased the rigidity of the outer leaflet of the liposomal membranes. The hepatoprotective potential of cranberry flavonoids could be due to specific prevention of rat liver mitochondrial damage. The mitochondria-addressed effects of flavonoids might be related both to radical-scavenging properties and modulation of various mitochondrial events.

Oxidative damage of rat liver mitochondria during exposure to t-butyl hydroperoxide. Role of Ca²âº ions in oxidative processes.

AIMS: The present study was designed for further evaluation of the biochemical mechanism of hepatic mitochondrial dysfunction under oxidative damages induced by organic hydroperoxide, tert-butyl hydroperoxide (tBHP), for estimation of the molecular targets impaired during oxidative stress, and for investigation of the role of Ca(2+) ions in mitochondrial oxidative reactions and of the protective effect of melatonin during mitochondrial peroxidative damage. MAIN METHODS: Mitochondria were isolated by differential centrifugation from the rat liver. The effects of tBHP exposure, EDTA, Ca(2+) ions and melatonin on mitochondrial respiratory activity, mitochondrial enzyme activities and redox status were measured. KEY FINDINGS: The present study provides evidence that tBHP (at low concentrations of 0.02-0.065mM, in EDTA-free medium) induced uncoupling of the oxidation and phosphorylation processes and decreased the efficiency of the phosphorylation reaction. This effect depended on the respiratory substrate used. The presence of EDTA prevented oxidative impairment of mitochondrial respiration, but Ca(2+) ions in the medium enhanced oxidant-induced mitochondrial damage considerably. In the presence of 0.5mM EDTA, tBHP (at high concentrations, 0.5-2mM) considerably oxidized mitochondrial reduced glutathione, enhanced accumulation of membrane lipid peroxidation products and mixed protein-glutathione disulfides and led to an inhibition of oxoglutarate dehydrogenase and succinate dehydrogenase. SIGNIFICANCE: Direct oxidative modification of enzymatic complexes of the respiratory chain and mitochondrial matrix, mitochondrial reduced glutathione depletion, protein glutathionylation, membrane lipid peroxidation and Ca(2+) overload are the main events of mitochondrial peroxidative damages. Experiments in vitro demonstrated that melatonin inhibited the mitochondrial peroxidative damage, preventing redox-balance changes and succinate dehydrogenase inactivation.

Corrections by melatonin of liver mitochondrial disorders under diabetes and acute intoxication in rats.

The aim of the present work was to investigate the mechanisms of oxidative damage of the liver mitochondria under diabetes and intoxication in rats as well as to evaluate the possibility of corrections of mitochondrial disorders by pharmacological doses of melatonin. The experimental (30 days) streptozotocin-induced diabetes mellitus caused a significant damage of the respiratory activity in rat liver mitochondria. In the case of succinate as a respiratory substrate, the ADP-stimulated respiration rate V3 considerably decreased (by 25%, p < 0·05) as well as the acceptor control ratio (ACR) V3/V2 markedly diminished (by 25%, p < 0·01). We observed a decrease of the ADP-stimulated respiration rate V3 by 35% (p < 0·05), with glutamate as substrate. In this case, ACR also decreased (by 20%, p < 0·05). Surprisingly, the phosphorylation coefficient ADP/O did not change under diabetic liver damage. Acute rat carbon tetrachloride-induced intoxication resulted in considerable decrease of the phosphorylation coefficient because of uncoupling of the oxidation and phosphorylation processes in the liver mitochondria. The melatonin administration during diabetes (10 mg·kg⁻¹ body weight, 30 days, daily) showed a considerable protective effect on the liver mitochondrial function, reversing the decreased respiration rate V3 and the diminished ACR to the control values both for succinate-dependent respiration and for glutamate-dependent respiration. The melatonin administration to intoxicated animals (10 mg·kg⁻¹ body weight, three times) partially increased the rate of succinate-dependent respiration coupled with phosphorylation. The impairment of mitochondrial respiratory plays a key role in the development of liver injury under diabetes and intoxication. Melatonin might be considered as an effector that regulates the mitochondrial function under diabetes.

Structure, stability, and antiplatelet activity of O-acyl derivatives of salicylic acid and lipophilic esters of acetylsalicylate.

The anti-thrombotic activity of acetylsalicylic acid (ASA) has been shown to be due to specific irreversible acetylation of blood platelet cyclooxygenase. The aim of our study was to investigate the associations between the antiplatelet activities of derivatives of both ASA and salicylic acid (SA), as well as the structure, stability, and molecular properties of these compounds. Homologous series of O-acyl derivatives of salicylic acid (propionyl-, butyrylsalicylic acids, PSA, BSA) and lipophilic dodecyl (C12)-, hexadecyl (C16)-, and cholesteryl acetylsalicylates were synthesized and tested for structure-activity relationships. The molecular properties (heat of formation, molecular surface area, dipole moment) of ASA and SA derivatives obtained by theoretical calculations changed with the increasing length of the acyl or alkyl residue. The inhibition of whole blood platelet aggregation and the reduction in thromboxane (TX) generation by O-acyl derivatives were concentration-dependent and decreased along with increasing the length of acyl hain. These effects correlated with the extent of platelet reactivity and P-selectin expression inhibition in collagen-activated platelets. In contrast to ASA and O-acyl derivatives of SA, none of the lipophilic ASA derivatives had a significant inhibitory effect on platelet aggregation. In conclusion, all SA and ASA derivatives studied under in vitro conditions showed much lower antiplatelet activities than ASA itself, despite their higher affinity to plasma proteins or membrane components and their equivalent ability to acetylate protein free amino groups.We suggest the significance of the carboxylic group, dipole moment, geometry, and size of these pharmaceuticals in their ability to bind to the active site of cyclooxygenase and their antiplatelet efficacy.

Melatonin attenuates metabolic disorders due to streptozotocin-induced diabetes in rats.

Enhanced oxidative stress and impairments in nitric oxide synthesis and bioavailability are of considerable importance in the pathogenesis of diabetic vascular diseases. The aim of the present work was to evaluate the metabolic effects of pharmacological doses of the melatonin, a known antioxidant, on streptozotocin-induced diabetic damage in rats. We investigated the indolamine's influence on the cellular redox-balance, nitric oxide (NO) level, and the activities of antioxidative defence enzymes, as well as the activities of enzymes involved in phase II detoxication and NADPH-generating pentose phosphate pathway. Blood glucose, glycated hemoglobin, bilirubin, as well as plasma alanine aminotransferase activities increased and body weight was reduced in rats with streptozotocin-induced (60 mg/kg, i.p.) diabetes (25 days). The NO level was markedly increased in diabetic plasma (by 50%) and aortic tissue (by 30%). The hyperglycemia resulted in reduced activities of glutathione peroxidase (by 25%), catalase (by 20%), glucose-6-phosphate dehydrogenase (by 55%) and transketolase (by 40%) in liver tissue of diabetic animals. Melatonin treatment (10 mg/kg, 18 days) did not influence the level of hyperglycemia or glycated hemoglobin and it had little effect on the activities of antioxidative enzymes. However, melatonin markedly reversed the activities of glucose-6-phosphate dehydrogenase and transketolase in liver tissue of diabetic rats. The most pronounced effect of the melatonin administration was the prevention of an increase in nitric oxide levels in blood plasma and aortic tissue during diabetes. In in vitro experiments, nitrosomelatonin formation in the presence of nitrosodonors was observed. This implies that melatonin might operate as an NO scavenger and carrier. Thus, melatonin treatment may have some beneficial effects in controlling diabetic vascular complications.

Cytotoxic and genotoxic effects of tert-butyl hydroperoxide on Chinese hamster B14 cells.

The organic hydroperoxide, tert-butyl hydroperoxide (t-BHP), is a useful model compound to study mechanisms of oxidative cell injury. In the present work, we examined the features of the interactions of this oxidant with Chinese hamster B14 cells. The aim of our study was to reveal a possible role of structural modifications in membranes and loss of DNA integrity in t-BHP-induced cell injury and death. The tert-butyl hydroperoxide treatment (100-1000 microM, 37 degrees C for 1h) did not decrease cell viability (as measured by cell-specific functional activity with an MTT test), but completely prevented cell growth. We observed intracellular reduced glutathione (GSH) oxidation and total glutathione (GSH+GSSG) depletion, a slight increase in the level of lipid-peroxidation products, an enhancement of membrane fluidity, intracellular potassium leakage and a significant decrease of membrane potential. At oxidant concentrations of 100-1500 microM, a significant damage to DNA integrity was observed as revealed by the Comet assay. The inhibition of cell proliferation (cell-growth arrest) may be explained by genotoxicity of t-BHP, by disturbance of the cellular redox-equilibrium (GSH oxidation) and by structural membrane modifications, which result in ion-non-selective pore formation. The disturbance in passive membrane permeability and the DNA damage may be the most dramatic cell impairments induced by t-BHP treatment. The presence of another oxidant, hypochlorous acid (HOCl), completely prevented t-BHP-induced DNA strand breaks, perhaps due to extracellular oxidation of t-BHP by HOCl.

Hypochlorous acid-induced oxidative stress in Chinese hamster B14 cells: viability, DNA and protein damage and the protective action of melatonin.

This study provides further evidence for the toxicity of hypochlorous acid (HOCl) in mammalian cells. Using the Chinese hamster B14 cell line, a significant decrease in cell viability was demonstrated after exposure to 100-200 microM HOCl for 1 h. Loss of viability was accompanied by a slight increase in DNA damage as shown by the Comet assay and by oxidation of cellular thiols. Exposure of B14 cells, erythrocyte membranes and human serum albumin to HOCl resulted in an extensive protein carbonyl accumulation. Thus, the cytotoxicity of HOCl may be due to both protein damage (carbonyl formation and oxidation of protein thiol groups) and DNA damage. The well-known antioxidant melatonin interacted with the oxidant and significantly protected cells during HOCl exposure, diminishing its cytotoxic effects and reducing protein carbonyl generation.

Cell survival, DNA, and protein damage in B14 cells under low-intensity near-infrared (810 nm) laser irradiation.

OBJECTIVE: The aim of this study was to reveal the possible cytotoxic and genotoxic effects of low-intensity (200 mW) near-infrared (810 nm) laser irradiation, using B14 cell line. BACKGROUND DATA: Laser therapy is widely used in biomedical treatment of many diseases, but the possible molecular mechanisms of laser actions remain unclear and the damaging effects of laser irradiation are still controversial. The side effects of laser therapy involve the generation of reactive oxygen and nitrogen species which in turn initiate lipid peroxidation, protein damage or DNA modification. METHODS: B14 cells and suspension of human erythrocyte membranes were irradiated with near-infrared (810 nm) therapy laser at different radiant exposures (3.75-15.0 J/cm(2)) and light power (fluency rate) 200 mW at 22 degrees C. Laser induced cellular oxidative damage was measured in terms of cell survival, DNA damage, measured using the method of single cell gel electrophoresis (Comet assay), protein damage measured as protein carbonyls formation. RESULTS: No substantial changes of cell survival under B14 cells irradiation at radiant exposures 3.75-11.25 J/cm(2) were observed. Similarly, neither considerable light-induced DNA damage or protein carbonyls accumulation was revealed. On the contrary, laser irradiation has led to decrease of cell protein carbonyl groups level in a dose-dependent manner. Additionally, using human red blood cell membranes as model membranes and biological oxidant HOCl we observed that laser irradiation resulted in a decrease of the level of membrane protein carbonyl groups accumulated under oxidative HOCl treatment. CONCLUSIONS: We can conclude that laser irradiation used (810 nm, 200 mW, 3.75-11.25 J/cm(2)) did not produce any considerable cytotoxic or genotoxic effects in B14 cells. Moreover, laser irradiation reduced cellular protein damage (protein carbonyl groups) produced by biological oxidant HOCl.

Hypochlorous acid-induced oxidative damage of human red blood cells: effects of tert-butyl hydroperoxide and nitrite on the HOCl reaction with erythrocytes.

Hypochlorous acid, one of the most powerful biological oxidants, is believed to be important in the pathogenesis of some diseases. The purpose of this study was to further characterise the membrane and intracellular events which resulted in HOCl-induced oxidative impairments and haemolysis of human erythrocytes and interaction of different oxidative agents, which accumulated during respiratory burst, in the process of RBS oxidation. The sequence of cellular events after red blood cell exposure to HOCl: cell morphological transformations, oxidation of cellular constituents, enzyme modifications, and haemolysis have been evaluated. It was shown that HOCl-treated cells underwent colloid-osmotic haemolysis, preceded by rapid morphological transformations and membrane structural transitions. The activation energy of the process of haemolysis (after removal of the excess of oxidative agent) was estimated to be 146+/-22 kJ/mol at temperatures above the break point of Arrhenius plot (31-32 degrees C). This value corresponds to the activation energy of the process of protein denaturation. Modification of erythrocytes by HOCl inhibited membrane acetylcholinesterase (uncompetitive type of inhibition), depleted intracellular glutathione, activated intracellular glutathione peroxidase, but did not induce membrane lipid peroxidation. The presence of other oxidants, nitrite or tert-butyl hydroperoxide (t-BHP), promoted the oxidative damage induced by HOCl and led to new oxidative reactions.

Hypochlorous acid-induced membrane pore formation in red blood cells.

The hyperproduction of hypochlorous acid (HOCl), an extremely toxic biological oxidant generated by neutrophils and monocytes, is involved in the pathogenesis of many diseases. In these studies, we attempted to determine the membrane and cellular events associated with HOCl-induced erythrocyte impairment and haemolysis. In vitro human erythrocyte exposure to HOCl (0.1-1.0 mM) resulted in rapid oxidation of reduced glutathione, an increase in cell osmotic fragility and the formation of transient membrane pores. The process of glutathione oxidation depended on the [oxidant]/[cell number] ratio. The HOCl-induced haemolysis observed was apparently mediated by pore formation and altered membrane electrolyte permeability. The estimated pore radius was approximately 0.7 nm and the average number per cell was 0.01. The rate constant of HOCl-produced haemolysis depended on pH. There were significant differences in haemolysis of HOCl-treated erythrocytes which had maximal stability at pH 7.2-7.3.

The mechanism of Zn-phthalocyanine photosensitized lysis of human erythrocytes.

The phthalocyanines have recently been suggested as one of most effective possible sensitizers for photodynamic therapy and the blood viral inactivation. The further characterisation of the mechanism of human red blood cell lysis and membrane alterations upon photodynamic treatment in the presence of Zn-phthalocyanine was the aim of this study. It was found that there were (2.7+/-0.4).10(7) dye binding sites per red blood cell with the association constant equal to (1.4+/-0.3).10(4) M(-1). Two types of the photosensitized haemolysis: haemolysis during irradiation ("light" haemolysis) and post-irradiation haemolysis ("dark" haemolysis) were studied. The erythrocyte membrane hyperpolarisation, membrane fluidisation and cell swelling preceded the "light" haemolysis. The modification of the erythrocyte membrane band 3 protein by DIDS (an inhibitor of anion exchange) increased the rate of the "light" haemolysis. The rate of "dark" haemolysis was higher and that of "light" haemolysis was lower in potassium media in comparison to sodium ones. The rates of photohaemolysis depended on the erythrocyte membrane potential: a decrease of membrane potential inhibited both types of haemolysis. The cell shrinkage in the presence of sucrose (up to 15 mM) inhibited the "dark" haemolysis but significantly increased the "light" haemolysis. Oxidation of intracellular oxyHb to metHb by nitrite, which drastically decreases intracellular oxygen concentration, as well as GSH concentration, inhibited the rate of the "light" haemolysis. The results allow for the conclusion that the mechanism of photochemical ("light") haemolysis is not of a colloid-osmotical type, in contrast to the post-irradiation ("dark") haemolysis. The photochemical oxidation or denaturation of band 3 protein plays a significant role in the formation of haemolytic holes. The membrane lipid peroxidation, as well as glutathione oxidation, does not participate in the process of photosensitized haemolysis. From the inhibition of "dark" haemolysis by sucrose the apparent pore radius was estimated to be about 1.1 nm. The pores appear to be transient short-lived ones, the average pore number per cell was 0.02.