2-Hexadecenal inhibits growth of C6 glioma cells.

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.

2-Hexadecenal Regulates ROS Production and Induces Apoptosis in Polymorphonuclear Leucocytes.

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 Ca2+ in the cytoplasm, a decrease in ROS production, and a decline in mitochondrial potential. Both of these processes are accompanied by cytoskeleton reorganization.

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.

Modification of redox processes in astroglial cells induced by microbial and viral infections.

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.

Effects of peroxynitrite and lipopolysaccharide on mitotic activity of C6 glioma cells.

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.

Activation of redox-systems of monocytes by hydrogen peroxide.

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.

Influence of neopterin on generation of reactive species by myeloperoxidase in human neutrophils.

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.

Influence of neopterin on the generation of reactive oxygen species in human neutrophils.

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.