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.

Chlorogenic Acid Protects against Advanced Alcoholic Steatohepatitis in Rats via Modulation of Redox Homeostasis, Inflammation, and Lipogenesis.

The aim of this study was to evaluate the therapeutic effects of chlorogenic acid (CGA) in rats with advanced alcoholic steatohepatitis. The rats were fed on a high-fat diet and gavaged with ethanol (4 g/kg) for 8 weeks. The livers of ethanol-treated rats showed steatosis; necrosis and mononuclear infiltration; and significant upregulation of the mRNA expression of the prooxidant (Cyp2e1, iNos), lipogenic (Srebp1, Acc), proinflammatory (Tlr4, Nf-κb, TnfA, Il-1B, and Il-6), and profibrogenic (TgfB, Col1, VegfA) genes. Simultaneously, a downregulation of level of Sod and Nrf2 was observed, which was accompanied by increased serum transaminase, TnfA, and serum and liver triglycerides levels. CGA administration (40 and 80 mg/kg, 8 weeks) to ethanol-fed group reduced the liver expression levels of Cyp2e1 and iNos, whereas it markedly enhanced the expression of Sod, Nrf2, and Ho-1. CGA at both doses downregulated the expressions of lipogenic, proinflammatory, and profibrogenic genes, while the expression of Tlr4 was lowered only after the higher dose of CGA. The higher dose of CGA efficiently prevented the progression of alcohol-induced steatosis and reduced inflammation through regulation of the expression of genes encoding the proteins involved in the Tlr4/Nf-κB signaling pathway and fibrosis. The study revealed hepatoprotective and anti-inflammatory effects of CGA through the regulation of expression of genes encoding Cyp2e1/Nrf2 involved in oxidative stress modulation. These results demonstrate CGA as a therapeutic candidate for the prevention and treatment of alcoholic steatohepatitis.

Thermodynamic Studies of Interactions between Sertraline Hydrochloride and Randomly Methylated ß-Cyclodextrin Molecules Supported by Circular Dichroism Spectroscopy and Molecular Docking Results.

The interaction between sertraline hydrochloride (SRT) and randomly methylated ß-cyclodextrin (RMßCD) molecules have been investigated at 298.15 K under atmospheric pressure. The method used-Isothermal Titration Calorimetry (ITC) enabled to determine values of the thermodynamic functions like the enthalpy (ΔH), the entropy (ΔS) and the Gibbs free energy (ΔG) of binding for the examined system. Moreover, the stoichiometry coefficient of binding (n) and binding/association constant (K) value have been calculated from the experimental results. The obtained outcome was compared with the data from the literature for other non-ionic ßCD derivatives interacting with SRT and the enthalpy-entropy compensation were observed and interpreted. Furthermore, the connection of RMßCD with SRT was characterized by circular dichroism spectroscopy (CD) and complexes of ßCD derivatives with SRT were characterized through the computational studies with the use of molecular docking (MD).

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.

Studies of the Formation and Stability of Ezetimibe-Cyclodextrin Inclusion Complexes.

In the presented studies, the interactions between ezetimibe (EZE) and selected cyclodextrins were investigated. α-Cyclodextrin (αCD), ß-cyclodextrin (ßCD) and its modified derivatives, hydroxypropyl-ß-cyclodextrin (HPßCD) and sulfobutylether-ß-cyclodextrin (SBEßCD), were selected for the research. Measurements were carried out using calorimetric and spectroscopic methods. Additionally, the Hirshfeld surface and biochemical analysis were achieved. As a result of the study, the inclusion complexes with 1:1 stoichiometry were obtained. The most stable are the complexes of ß-cyclodextrin and its derivatives. The comparison of ßCD with its derivatives shows that the modifications have an affect on the formation of more durable and stable complexes.

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.

Mechanism of Stiff Substrates up-Regulate Cultured Neuronal Network Activity.

Our previous work provided compelling evidence showing that substrate stiffness is crucial for regulating synaptic connectivity and excitatory synaptic transmission among neurons in the neuronal network. However, the underlying mechanisms remain elusive. In our study, polydimethylsiloxane (PDMS) substrates with different stiffness have been fabricated to investigate the mechanisms by which the substrate stiffness upregulates the formation and activity of the cultured neuronal network. Here we report that stiff substrate increased both the number of synapses and the efficacy of excitatory synaptic transmission. More colocalization of synaptotagmin and PSD-95 was observed in the neuronal network on stiff substrate, which indicated the synapse number has increased. We also found that the increased synapse number was mediated by Hevin and SPARC that are secreted from astrocyte. The increased efficacy of excitatory synaptic transmission induced by stiff substrate was explored in three aspects. First, stiff substrate enhanced the presynaptic activity through increasing the vesicular release probability (Pr) of neurotransmitters as well as the calcium influx. Second, stiff substrate reduced voltage-dependent Mg2+ blockade to N-methyl-d-aspartate receptor (NMDAR) channels, which led to higher postsynaptic activity. Third, our work suggested that the increased excitatory synaptic transmission in the neural network on stiff substrate involved the upregulated synaptic glutamate concentration. Taken together, these findings may provide a molecular mechanism underlying substrate stiffness regulation of excitatory synaptic transmission in the cultured neural network.

Antidiabetic effects and erythrocyte stabilization by red cabbage extract in streptozotocin-treated rats.

The protective effect of red cabbage extract (RCE) was evaluated in rats with streptozotocin-induced diabetes, assessing a probable role of this extract in the prevention of erythrocyte impairments associated with a high risk of vascular complications in diabetes. RCE was analyzed by ultrahigh performance liquid chromatography and mass spectrometry, and 11 anthocyanins, 3 hydroxybenzoic acids and 9 hydroxycinnamic acids were identified. Type 1 diabetes was induced by streptozotocin (60 mg kg-1) in Wistar male rats (n = 8 per group). After 7 days of acclimatization, streptozotocin-treated rats were given RCE (800 mg kg-1) or vehicle by intragastric administration for 4 weeks. The RCE treatment lowered blood glucose, and glycated and fetal hemoglobin concentrations and improved glucose tolerance as well as considerably raised serum insulin, proinsulin and C-peptide levels in streptozotocin-treated rats. Simultaneously, RCE improved pancreatic islet morphology, increasing the amount of pancreatic ß-cells in diabetic animals. The RCE administration prevented anemia in rats with streptozotocin-induced diabetes, enhanced erythrocyte resistance to acid hemolysis, and normalized reticulocyte production as well as sialic acid content in erythrocyte membranes. The enhanced lectin-induced erythrocyte aggregation in diabetic rats was significantly lowered after the RCE treatment. RCE demonstrated a significant antioxidant effect, decreasing MDA and protein carbonyl contents and increasing catalase and glutathione peroxidase activities in erythrocytes. These results indicate that RCE can be considered as a promising candidate for use as a drug or a food supplement to alleviate diabetes and its vascular complications.

Ferutinin Induces Membrane Depolarization, Permeability Transition Pore Formation, and Respiration Uncoupling in Isolated Rat Liver Mitochondria by Stimulation of Ca2+-Permeability.

It is well known that the terpenoid ferutinin (4-oxy-6-(4-oxybenzoyloxy) dauc-8,9-en), isolated from the plant Ferula tenuisecta, considerably increases the permeability of artificial and cellular membranes to Ca2+-ions and produces apoptotic cell death in different cell lines in a mitochondria-dependent manner. The present study was designed for further evaluation of the mechanism(s) of mitochondrial effects of ferutinin using isolated rat liver mitochondria. Our findings provide evidence for ferutinin at concentrations of 5-27 µM to decrease state 3 respiration and the acceptor control ratio in the case of glutamate/malate as substrates. Ferutinin alone (10-60 µM) also dose-dependently dissipated membrane potential. In the presence of Ca2+-ions, ferutinin (10-60 µM) induced considerable depolarization of the inner mitochondrial membrane, which was partially inhibited by EGTA, and permeability transition pore formation, which was diminished partly by cyclosporin A, and did not influence markedly the effect of Ca2+ on mitochondrial respiration. Ruthenium Red, a specific inhibitor of mitochondrial calcium uniporter, completely inhibited Ca2+-induced mitochondria swelling and membrane depolarization, but did not affect markedly the stimulation of these Ca2+-dependent processes by ferutinin. We concluded that the mitochondrial effects of ferutinin might be primarily induced by stimulation of mitochondrial membrane Ca2+-permeability, but other mechanisms, such as driving of univalent cations, might be involved.

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.

Cytoprotection of pancreatic ß-cells and hypoglycemic effect of 2-hydroxypropyl-ß-cyclodextrin: sertraline complex in alloxan-induced diabetic rats.

Sertraline, a selective inhibitor of serotonin reuptake, is widely used as antidepressant in diabetic patients for improvement of depression and glycemic control. Sertraline is poorly soluble in water, which limits its oral applicability. In this work we tried to improve the pharmaceutical properties of sertraline by complexation with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) and evaluated the efficacy of the HPßCD:sertraline complex in prevention of alloxan-induced lesions in rats. The solubility of sertraline increased in the presence of HPßCD and the association constant for sertraline and HPßCD was equal to 4000 ± 1000 M(-1). Two-week treatment of diabetic animals with the HPßCD:sertraline complex improved pancreatic islet morphology and ß-cell survival, which, in turn, reduced the severity of diabetes, as evidenced by lowering of blood glucose and glycated hemoglobin contents as well as normalization of serum insulin level and insulin sensitivity (HOMA-IR). The effect of the HPßCD:sertraline complex was strongly expressed in comparison with the antidiabetic effect of both the monopreparations, HPßCD and sertraline. It is suggested that the cyclodextrin derivative increased the pharmacological effect of sertraline, probably due to enhanced drug bioavailability.

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.

The effect of near-infrared MLS laser radiation on cell membrane structure and radical generation.

The therapeutic effects of low-power laser radiation of different wavelengths and light doses are well known, but the biochemical mechanism of the interaction of laser light with living cells is not fully understood. We have investigated the effect of MLS (Multiwave Locked System) laser near-infrared irradiation on cell membrane structure, functional properties, and free radical generation using human red blood cells and breast cancer MCF-4 cells. The cells were irradiated with low-intensity MLS near-infrared (simultaneously 808 nm, continuous emission and 905 nm, pulse emission, pulse-wave frequency, 1,000 or 2,000 Hz) laser light at light doses from 0 to 15 J (average power density 212.5 mW/cm(2), spot size was 3.18 cm(2)) at 22 °C, the activity membrane bound acetylcholinesterase, cell stability, anti-oxidative activity, and free radical generation were the parameters used in characterizing the structural and functional changes of the cell. Near-infrared low-intensity laser radiation changed the acetylcholinesterase activity of the red blood cell membrane in a dose-dependent manner: There was a considerable increase of maximal enzymatic rate and Michaelis constant due to changes in the membrane structure. Integral parameters such as erythrocyte stability, membrane lipid peroxidation, or methemoglobin levels remained unchanged. Anti-oxidative capacity of the red blood cells increased after MLS laser irradiation. This irradiation induced a time-dependent increase in free radical generation in MCF-4 cells. Low-intensity near-infrared MLS laser radiation induces free radical generation and changes enzymatic and anti-oxidative activities of cellular components. Free radical generation may be the mechanism of the biomodulative effect of laser radiation.

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.

Targeting the urine and plasma determinants of thromboxane A2 metabolism in detection of aspirin effectiveness.

Detection of reduced aspirin effectiveness has gained significant importance since clinical consequences of aspirin resistance were reported. Nevertheless, due to differentiated molecular basis of aspirin resistance, the conflicting choice of referential method for detection of acetylsalicylic acid ineffectiveness has become troublesome. This study, using a rat model of antiplatelet therapy, examines the aptitude of selected TXB2 metabolism-based methods in the detection of acetylsalicylic acid effectiveness. We hypothesized that ex-vivo whole blood spontaneous TXB2 generation assay could be, contrary to basal TXB2 and urine 11-dTXB2, a novel surrogate measure for impaired acetylsalicylic acid-dependent inhibition of thromboxane synthesis. To address this hypothesis, we evaluated the sensitivity of TXB2 generation assay in hirudinized whole blood to detect acetylsalicylic acid-mediated inhibition of cyclooxygenase activity in healthy rats and diabetic rats treated with acetylsalicylic acid. In diabetic and control animals, both acetylsalicylic acid drenches in the dose-independent manner contributed to significant attenuation of basal plasma TXB2 and urinary 11-dTXB2 formation. Urinary concentrations of 11-dTXB2 were, contrary to basal TXB2, significantly higher, regardless of acetylsalicylic acid dose, among all diabetic groups, compared with corresponding control groups. Determination of TXB2 generation in whole blood enabled sensitive detection of dose-related acetylsalicylic acid effect in both groups, as well as increased TXB2 formation in diabetes. We showed for the first time that evaluation of spontaneous generation of TXB2 in hirudinized whole blood enables, contrary to basal plasma TXB2 and urine 11-dTXB2 concentrations, to sensitively determine the acetylsalicylic acid effect in healthy and diabetic subjects.