Galina Semenkova

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‐γ. 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 Earls solution (pH=7.4) at 37°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.

Free-radical Destruction of Sphingolipids Resulting in 2-hexadecenal Formation

The action of hypochlorous acid (HOCl) and γ-radiation on aqueous lysosphingolipid dispersions was found to produce 2-hexadecenal (Hex). This process includes the stages of formation of nitrogen-centered radicals from the starting molecules and the subsequent fragmentation of these radicals via the rupture of C–C and O–H bonds. These findings prove the existence of a nonenzymatic pathway of sphingolipid destruction leading to the formation of Hex, which possesses a wide spectrum of biological activity. Analysis of the effect of HOCl on transplantable rat glioma C6 cells and human embryonic kidney 293 cells points to the formation of Hex. This suggests that the described mechanism of free-radical destruction of sphingolipids may be replicated on cell culture under the stress of active chlorine forms.

Effects of hydrogen peroxide on neutrophil ability to generate reactive oxygen and chlorine species and to secrete myeloperoxidase in vitro

In this work, the effects of H2O2 at concentrations of 10−8–10−2 mol/l on the neutrophil ability to generate reactive oxygen and chlorine species (ROCS) and to secrete myeloperoxidase (MPO) were studied, as well as the H2O2 damaging action on neutrophils. It was found that H2O2 at concentrations of 2 × 10−3–10−2 mol/l led to disturbances of neutrophil membrane barrier properties and to a lactate dehydrogenase release. Incubation of neutrophils with an addition of 10−4–10−7 mol/l H2O2 was accompanied by an increase of the cell ability to generate ROCS during phagocytosis and a decrease of neutrophil ability to secrete MPO and ROCS into the extracellular medium during adhesion. Mechanisms of the H2O2 action are coupled with arachidonic acid metabolism. Inhibition of the 5-lipoxygenase or cyclooxygenase metabolism pathways produced an enhancement of the H2O2 destructive effect. Block of 5-lipoxygenase pathway led to elimination of the H2O2 action on MPO and ROCS secretion and to an enhancement of the H2O2 effect on the neutrophil ability to generate ROCS during phagocytosis. The obtained data indicate a high blood neutrophil resistance to the H2O2 destructive action and confirm the H2O2 regulatory role with respect to the neutrophil functions.

Morphometric characteristics of neutrophils stimulated by adhesion and hypochlorite

HighlightsFlattening and degranulation ensure neutrophils morphology changes at adhesion.Neutrophils exposure to sodium hypochlorite results in changes of morphology.Sodium hypochlorite induces the neutrophils actin cytoskeleton reorganization.Sodium hypochlorite leads to intensified degranulation of neutrophils. Abstract The aim of this work was to compare cell form, size and volume as well as the locomotor activity of polymorphonuclear leukocytes (PMNLs) stimulated by adhesion to glass and exposed to hypochlorous acid at non‐toxic dose. After 20 min of adhesion to a glass surface, volume, cell surface area and projection area of PMNLs were equaled to 143.1 ± 21.4 &mgr;m3, 288.8 ± 28.8 &mgr;m2 and 248.3 ± 32.3 &mgr;m2, respectively. Projection area of PMNLs exposed to NaOCl was noticeably enlarged as compared with control samples. The cell volume of 20 min adherent cells exposed to NaOCl was enlarged in comparison with both control cells and 5 min adhered exposed to NaOCl cells. NaOCl exposure induced a degranulation of PMNLs as measured by lysozyme release. Granules could be found both above the cell surface and on the substratum near the cell. The S/V ratio for PMNLs increased (from 1.52 to 2.02 &mgr;m−1) with the increasing of cell activation time. But at NaOCl addition the reverse tendency was observed (from 2.10 to 1.87 &mgr;m−1). In cells exposed to NaOCl the redistribution and decrease of concentration of F‐actin took place. This observation supports the hypothesis that the priming of PMNLs with hypochlorous acid modifies cell motility and morphology and reflects also on other functions.

Proliferation of cultured glioma cells mediated by coenzyme Q 10 under conditions of serum deprivation

The effect of coenzyme Q10 on glioma-cell proliferation under serum-deprived conditions has been studied. Our results have shown that the addition of coenzyme Q10 into a serum-free culture medium enhances cell viability, stimulates cell growth, restores mitochondrial potential, and increases the quantity of energized mitochondria. It is found that coenzyme Q10-induced glioma-cell proliferation in conditions of serum deficiency is a result of an intracellular reduced glutathione concentration with subsequent activation of protein kinase C, ERK1/2, and phosphoinositol-3-kinase.

Pteridine-dependent oxygen activation in neutrophils

We investigated the influence of neopterin and 7,8-dihydroneopterin on the myeloperoxidase activity and secretory degranulation in neutrophils and interaction of pteridines with its major substrate (hydrogen peroxide) and intermediate product of halogenation cycle (hypochlorous acid). It was shown that, in neutrophils, the redox-pair, neopterin and 7,8-dihydroneopterin, control oxygen activation, which is regulated by myeloperoxidase. Pteridines influence the secretion of myeloperoxidase depending on concentration and decrease the level of hydrogen peroxide and hypochlorous acid, which are the substrate and intermediate product of the enzyme, respectively. It was found that, in micromolar concentrations, 7,8-dihydroneopterin is a noncompetitive inhibitor of myeloperoxidase. We suppose that myeloperoxidase facilitates 7,8-dihydroneopterin oxidation by hypochlorous acid and results in an increase in neopterin concentration. These changes modify the concentration of intracellular and extracellular reactive oxygen species.

Regulation of Morphological and Functional Properties of Astrocytes by Hydrogen Peroxide

Effects of hydrogen peroxide on morphological characteristics, proliferation index, and menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H2O2 at 5 × 10−7−1 × 10−8 M concentrations acted as a regulator of morphological and functional properties of astrocytes, inducing their reactivation, which is manifested as cell body hypertrophy and an increase of proliferative activity and menadione-induced production of superoxide anion radicals (O2•−). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 × 10−6 M on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O2•− under the effect of menadione. Use of lipopolysaccharide B as a functional stimulator has shown that H2O2 modifies signaling pathways leading to an increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-dependent chemiluminescence of astrocytes under the action of menadione to the level of control values.

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.