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2-Hexadecenal (2-HD)-a biologically active long-chain fatty aldehyde formed in organism enzymatically or nonenzymatically in the reaction of free-radical destruction of sphingolipids under the action of hypochlorous acid, producing by myeloperoxidase. This research aimed to study 2-HD effects on polymorphonuclear leukocytes' (PMNLs) functions. It has been shown that at submicromolar concentrations, 2-HD causes an elevation in ROS production by PMNLs. It has been found that such effect is associated with signal transduction pathways modification and expressed in elevation of NADPH oxidase, MPO, and JNK-MAPK contributions to this process. At higher concentrations, 2-HD induces apoptosis, which correlates with a significant increase in free Ca 2+ in the cytoplasm, a decrease in ROS production, and a decline in mitochondrial potential. Both of these processes are accompanied by cytoskeleton reorganization., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 Ca 2+ 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 Ca 2+ ions under hypochlorite action and act as mediators of calcium release from the endoplasmic reticulum into the cytoplasm., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Thymoquinone (TQ) is a highly perspective chemotherapeutic agent against gliomas and glioblastomas because of its ability to cross the blood-brain barrier and its selective cytotoxicity for glioblastoma cells compared to primary astrocytes. Here, we tested the hypothesis that TQ-induced mild oxidative stress provokes C6 glioma cell apoptosis through redox-dependent alteration of MAPK proteins. We showed that low concentrations of TQ (20-50 μM) promoted cell-cycle arrest and induced hydrogen peroxide generation as a result of NADH-quinone oxidoreductase 1-catalyzed two-electron reduction of this quinone. Similarly, low concentrations of TQ efficiently conjugated intracellular GSH disturbing redox state of glioma cells and provoking mitochondrial dysfunction. We demonstrated that high concentrations of TQ (70-100 μM) induced reactive oxygen species generation due to its one-electron reduction. TQ provoked apoptosis in C6 glioma cells through mitochondrial potential dissipation and permeability transition pore opening. The identified TQ modes of action on C6 glioma cells open up the possibility of considering it as a promising agent to enhance the sensitivity of cancer cells to standard chemotherapeutic drugs.

2-Hexadecenal (2HD) formation in the organism occurs via irreversible enzymatic degradation of sphingosine-1-phosphate or nonenzymatic γ-, UV-, or HOCl-induced destruction of a number of sphingolipids including S1P. The current research focuses on the study of 2HD effects on C6 glioma cells growth. The results obtained show that 2HD causes a dose-dependent decrease in proliferative and mitotic indices. The change in the mitotic index is due to the redistribution of cells in the different phases of mitosis. These processes are accompanied by cytoskeleton rearrangement and changes in cell morphology, which are expressed in F-actin redistribution, change in the number and type of filopodia and fibrils, leading to cell shape changes, decrease in intercellular contacts and monolayer rarefaction. Cells treatment with 2HD leads to apoptosis induction and signalling pathways modification, including activation of JNK, p38, and ERK1/2 MAPK but not PI3K. The effects observed are not related to the cytotoxicity of 2HD. Significance of the study: 2HD-an unsaturated aldehyde, which level can rise under conditions of oxidative stress as a result of nonenzymatic sphingolipids' destruction. The mechanisms of 2HD action on various cell types have not been sufficiently studied. Therefore, the study on functional role of this aldehyde in different cell types that may be its target is relevant. This study demonstrated that 2HD inhibits growth of C6 glioma cells due to modification of intracellular processes of signal transduction, cytoskeleton rearrangement, change in the mitotic regimen and apoptosis induction., (© 2019 John Wiley & Sons, Ltd.)

Mechanisms of coenzyme Q10 effect on serum-deprived glioma cell proliferation have been studied. Our results have shown that the addition of coenzyme Q10 into serum-free culture medium leads to increase in cell viability, stimulation of cell growth, as well as restoration of mitochondrial potential and increase of quantity of energized mitochondria. It has been found out that coenzyme Q10-induced glioma cell proliferation under serum deficiency is a result of intracellular reduced glutathione concentration decrease with subsequent activation of proteinkinase C, ERK1/2 and phosphoinositol-3-kinase.

Background: The authors hypothesized that the cell redox state might be modified during microbial and viral infections. To detect and evaluate changes in astroglial cell redox state, rat C6 glioma cells after exposure to lipopolysaccharide (LPS) or after herpes simplex virus type 1 (HSV-1) inoculation were used. Redox state modification of glioma cells was determined by the change in menadione-induced superoxide yield., Material/methods: Menadione-induced superoxide formation was registered by the lucigenin-enhanced chemiluminescence (CL) method., Results: The results demonstrate that exposure of C6 glioma cells to LPS for 24 hours resulted in a dose-dependent increase in the mitotic index and integral intensity of menadione-induced lucigenin-enhanced CL. Menadione-induced ROS generation in C6 cells during HSV-1 infection changed depending on the time after HSV-1 inoculation., Conclusions: The redox state of astroglial cells is modified during microbial and viral infections. The use of redox-active quinones is an informative model for determining cell redox state change and analyzing cells' functional state.

It has been shown by the method of fluorescent analysis that the rate of hydrogen peroxide generation in human U251 glioma cells under the effect of lipophilic (menadione) or hydrophilic (vikasol) analogues of vitamin K3 was different. Analyzing experimental data we can conclude that menadione underwent one- and two-electron reduction by intracellular reductases in glioma cells. Reduced forms of menadione interact with molecular oxygen leading to reactive oxygen species (ROS) generation. The theoretical model of ROS generation including two competitive processes of one- and two-electron reduction of menadione has been proposed. Rate constants of ROS generation mediated by one-electron reduction process have been estimated.

We investigated the influence ofneopterin and 7, 8-dihydroneopterin on the activity and secretory degranulation of myeloperoxidase in neutrophils and the ability of pteridines to interact with the main substrate of this enzyme, hydrogen peroxide, and with the intermediate product of halogenation cycle--hypochlorous acid. It was shown that neopterin and 7, 8-dihydroneopterin while being a redox-pair regulated the process of oxygen activation in neutrophils by functioning of myeloperoxidase. Depending on concentration, pteridines can influence the secretion of myeloperoxidase into intracellular medium and decrease the level of hydrogen peroxide and hypochlorous acid that are a substrate and an intermediate product of the enzyme respectively. It was shown that 7, 8-dihydroneopterin in micromolar concentration appeared to be noncompetitive inhibitor of myeloperoxidase. We suppose that myeloperoxidase assists 7, 8-dihydroneopterin oxidation by hypochlorous acid that leads to neopterin concentration increase. These changes modify the concentration of reactive oxygen species in intracellular and extracellular media.

The influence of H2O2 at concentrations of 10(-8)--10(-2) mol/l on neutrophil ability to generate the reactive oxygen and chlorine species (ROCS) and secrete myeloperoxidase (MPO) was studied, and H202 injurious effect on neutrophils was also investigated in this work. It was revealed that H2O2 at concentrations of 2 x 10(-3)--2 x 10(-2) mol/l induced disturbance of the neutrophil membrane barrier properties and lactate dehydrogenase release. The incubation of the neutrophils with the addition of 10(-4)--10(-7) mol/l H2O2 led to an increase in the cell ability to generate ROCS during phagocytosis and decreased neutrophil ability to secrete MPO and ROCS in extracellular medium during adhesion. The mechanisms of H2O2 effect are coupled with arachidonic acid metabolism. Inhibition of metabolic pathways of 5-lipoxygenase or cyclooxygenase increased the destructive effect of H2O2 on the cells. Five-lipoxygenase way prohibition led to cancellation of H2O2 influence on MPO and ROCS secretion and to enhancement of H2O2 effect on neutrophil ability to generate ROCS during phagocytosis. The data obtained testify to the high neutrophil resistance to destructive effect of H2O2 and confirm the regulatory role of H2O2 with respect to the neutrophil functions.

Peroxynitrite is one of the most potent neurotoxic agents with multiple targets in neurons and glial cells. This study addressed a question of whether peroxynitrite-mediated cytotoxicity can be prevented by Escherichia coli lypopolisaccharide (LPS) due to its mitogenic activity towards C6 glioma cells. A number of characteristic morphological changes (processes impairments, nuclei modifications, cytoplasm vacuolization) and apoptotic cells were observed in the cell culture after 24-h treatment with 3-morpholinosyndnonimine (SIN-1), a well-known donor of peroxynitrite. These morphological changes were clearly associated with a SIN-1 dose-dependent increase in the number of pathological mitoses as well as with SIN-1 inhibition of the menadione-induced, lucigenin-enhanced chemiluminescence of C6 glioma cells, an independent indicator of mitotic activity of these cells. The mitotic index of C6 glioma cells increased in response to LPS and underwent non-uniform changes depending on SIN-1 concentrations. At a mitogenic concentration of 100 ng/ml, LPS reduced significantly the toxicity of SIN-1 determined as the accumulation of pathological mitoses, thus acting as a protective agent. Taken together, our findings indicate that SIN-1 specifically impairs the mitotic process in C6 glioma cells, and provide the first evidence that antimitotic effects of peroxynitrite can be restored by LPS.

Effects of hydrogen peroxide on morphological characteristics, proliferation index, menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H2O2 at 1 x 10(-8) - 5 x 10(-7) M concentrations acts as a regulator of morphological and functional properties of astrocytes by inducing their reactivation that is manifested as a cell body hypertrophy and an increase of proliferative activity and of menadione-dependent production of superoxide (O2- ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 microM on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O2- under menadione action. Using lipopolysaccharide B as a functional stimulator it has been shown that H2O2 modifies signaling pathways leading to the increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-enhanced chemiluminescence of astrocytes under menadione action to the level of control values.

In this work the influence of H2O2 on the ability of human blood monocytes to generate ROS upon stimulation of cells by adhesion to glass surface and fMLP was studied using the luminol-dependent chemiluminescence (LDCL) method. Pretreatment of cells with H2O2 increased the adhesiveness of monocytes and ROS generation. Superoxide generation by cells in response to fMLP depended on the duration of pretreatment and the concentration of H2O2. The stimulatory effect on fMLP-induced LDCL of cells further depended on the Ca2+ concentration in the medium and on the activities of phospholipase A2, cyclooxygenases, and Mek1/2.

Processes of oxygen activation in monocytes stimulated with adhesion to glass were studied by methods of luminol-dependent and lucigen-independent chemiluminescence. It was shown that monocyte chemiluminescence was caused by cell adhesion to glass surface. Generation of reactive oxygen species at monocyte adhesion to glass was dependent on calcium ion concentration in the medium. The increase in the level of cytosolic calcium, as the extracellular calcium concentration elevated, was accompanied by the activation of phospholipase A2, 5-lypoxygenase and cycloxygenases. Magnesium ions exerted no influence on oxygen activation by cells. Incubation of cells in glucose-free medium, or the addition of glycolysis blocker (2-deoxy-D-glucose) to cell suspension led to a decrease in chemiluminescence intensity. By means of inhibitory analysis, it has been established that processes of oxygen activation are related to arachidonic acid metabolism, and depend on the activity of phospholipase A2.

Increased neopterin concentrations in human serum indicate activation of cell-mediated immune response. Earlier we have shown that neopterin enhanced generation of singlet oxygen, hydroxyl radical and nitric oxide in human peripheral blood neutrophils by NADPH-independent pathways. To further investigate a participation of neopterin in reactive species production by neutrophils, we studied its influence on myeloperoxidase (MPO) activity. MPO was isolated from human peripheral blood neutrophils from healthy donors. Generation of reactive species by MPO/H(2)O(2) in Earl's solution (pH=7.2) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol as light emitter. In the MPO/H(2)O(2) system, neopterin increased singlet oxygen in a concentration-dependent manner, but it decreased formation of other oxidizing species. Comparing several oxygen scavengers, formation of reactive species was totally blocked by sodium azide (NaN(3)), both in the presence and in the absence of neopterin. Superoxide dismutase (SOD) and d-mannitol insignificantly decreased chemiluminescence of this reaction, but diazabicyclo[2.2.2]octane (DABCO) strongly inhibited it. We conclude that the effects of neopterin on neutrophils' MPO are directed to increase singlet oxygen and to decrease other reactive species via inhibition of MPO and/or scavenging of reactive species.

Neopterin is synthesized by human monocyte-derived macrophages primarily upon stimulation with the cytokine interferon-gamma. We studied the influence of neopterin on the generation of reactive oxygen species (ROS) in human peripheral blood neutrophils. Radical formation was measured using a biochemiluminometer. Neutrophils were isolated from peripheral blood of healthy donors. The generation of ROS by neutrophils suspended in Earl's solution (pH=7.4) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol and lucigenin as light emitters. Neopterin induced chemiluminescence in suspensions of neutrophils in the presence of luminol, but not of lucigenin. Neopterin affected only adhesive cells. Addition of neopterin into the suspension of the cells involving D-mannitol, L-histidine and diazabicyclo[2.2.2]octane (DABCO) decreased luminol-dependent chemiluminescence (LDCL) of the neutrophils. The action of superoxide dismutase (SOD) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reduced neopterin-induced LDCL of neutrophils. Data suggest that neutrophils respond on exposure to neopterin with additional generation of singlet oxygen, hydroxyl radical and nitric oxide by nicotinamide adenine dinucleotide phosphate (NADPH)-independent pathways.

Cells of neuroglia--the astrocytes are of interest from the point of view of their participation in phagocytosis. Phagocyte ability to generate active oxygen forms (AOF) as used as the basic criterion of the estimation of their functional activity. For the purpose to clear up molecular and cellular mechanisms of phagocytosis a study of astrocyte redox-systems, participating in production of AOF, was undertaken. Registration of AOF in astrocytes was carried out using a method of luminol-dependent chemiluminescence. Primary culture of guinea pig astrocytes was used. Spontaneous chemiluminescence of low intensity was found for the astrocytes at the presence of luminol. The destruction of the cells was accompanied by a significant growth of the intensity of spontaneous chemiluminescence. Suspension of endocutosis inductors, particle of latex and phytohemagglutinin, added to astrocytes did not result in formation of AOF, characteristic for other cells, possessing phagocytosis. It was established, that addition of hydrogen peroxide destroys astrocytes at the presence of luminol and gives rise to the emission. Chemiluminescence was not observed in similar experiments with intact cells. A conclusion was made that inside astrocytes there are structures, which show peroxidase-like activity.

It was shown that neutrophil aggregation is caused by all lectins with different specificity for carbohydrates but generation of active oxygen forms is induced only by some lectins. Polyspecific lectins-erythroagglutinin and phytohemagglutinins have the greatest activity in relation to both processes.

As revealed by investigations made by the use of the method of luminol-dependent chemiluminescence and light dispersion, the addition of concanavalin A (ConA) to the suspension of human peripheral blood lymphocytes and subsequent incubation with recombinant interleukin-1 beta leads to a sharp increase in the yield of chemiluminescence and the rate of cell aggregation in comparison with similar parameters obtained for cells, not treated with interleukin-1 beta. The study revealed the potentiating action of recombinant interleukin-1 beta on ConA-induced proliferative response of thymocytes in the culture. The mechanisms of the priming action of recombinant interleukin-1 beta on ConA-induced reaction of lymphocytes are discussed.

Arachidonate-induced aggregation and generalization of active oxygen forms (OAF) by peripheral blood neutrophils in donors were studied in donors and Hodgkin's disease patients. Leukocytes of the latter had incomplete ability to produce AOF in response to cell stimulation with arachidonic acid. The study of arachidonate-induced aggregation of neutrophils indicated no differences in the speed of the process in the patients and donors. AOF catchers did not act on the rate of leukocyte aggregation in the patients though accelerated the process in the donors. It is inferred that Hodgkin's disease is associated with dysfunction of oxygen activation by neutrophils. The findings suggest that defects in leukocytes ability to activate oxygen in Hodgkin's disease may entail deranged regulation of other processes essential for functional activity of polymorphonuclear leukocytes.

The effects of culture media of various compositions on chemiluminescence developing in peroxidase oxidation of luminol with hydrogen peroxide were under study. The findings evidence that the presence of carbonate and bicarbonate ions in the medium results in a two-staged chemiluminescence kinetics and in more intensive chemiluminescence in the peroxidase-luminol-hydrogen peroxide system. This fact has brought the authors to a conclusion that carbonate and bicarbonate-containing media are more effective for the detection of low peroxidase concentrations by the chemiluminescence technique.

The method of luminol-dependent chemiluminescence was used to detect oxygen activation during interaction of HLA antibodies with HLA antigens expressed on lymphocyte surface. Introduction of specific anti-HLA serum into the lymphocyte suspension leads to a rapid decrease of intensity in chemiluminescence intensity follows. The maximum yield of induced chemiluminescence in case of using a specific antiserum is significantly lower than in the control. The method could be used for detection of HLA antigens on lymphocyte surface.

It has been established that oxygen activation by neutrophils and human blood lymphocytes at adhesion to glass and under the action of concanavalin A differently depends on pH. It has been suggested that oxygen activation by neutrophils and lymphocytes occurs through different mechanisms.

The influence of the liposome ozonolysis products on ionic permeability and stability of the planar bilayer lipid membrane ( PBLM ) was studied by electroconductivity . Addition of ozonized liposomes and PBLM preparation from the products of the ozonized lipids results in a decrease of the PBLM resistance. It is suggested that the enhancement of PBLM stability from ozonized lipids may be caused by production during lipid peroxidation of the intermolecular bonds between lipid molecules and by an increase of the lipid bilayer rigidity.

A comparative study of the interaction of cumene hydroperoxide with cytochrome P-450LM2 and with cytochrome C has been undertaken using the chemiluminescence method in the presence of luminol. Considerable differences in the mechanisms of action of both hemoproteins have been revealed with various inhibitors of radical stages, i.e. superoxide dismutase, mannitol, sodium azide, and alpha-tocopherol. It is shown that molecular oxygen participates in the process of hemoprotein-catalyzed hydroperoxide oxidation of luminol.

Oxygen activation by neutrophils and human blood lymphocytes at adhesion to glass have been studied by luminol--dependent chemiluminescence. It has been established that the cell interaction with glass leads to the formation of O2-., O2', .OH and H2O2. Chemiluminescence kinetics and the excited--oxygen forms ratio at adhesion were different for neutrophils and lymphocytes. The desorption of cells resulted in a decrease of the chemiluminescent response to neutrophils and lymphocytes when they again adhered to glass and in practically complete inhibition of chemiluminescence induced by adding concanavalin A. It has been determined that at adhesion of neutrophils and lymphocytes to glass different mechanisms of oxygen activation take place.