Experimental evaluation of 2-merkaptobenztyazol radioprotective capacities on cell cultures test system. / EKSPERYMENTAL'NA OTsINKA RADIOPROTEKTORNYKh VLASTYVOSTEi 2-MERKAPTOBENZTIAZOLU U TEST-SYSTEMI KUL'TURY KLITYN.

OBJECTIVE: To study the radioprotective properties of 2-merkaptobenztyazol on proliferating cell culture test system. MATERIALS AND METHODS: cytogenetic, cytological, statistics. RESULTS: Analysis of cytogenetic effects of 2-merkaptobenztiazol by frequency aberrations criteria and aberrant cells damage criteria showed that upon entire investigated range of reagent concentrations the frequency of aberrations was not significantly higher than the value of this indicator in control, but there was a tendency of reduction of mitotic activity of root meristem cells. Upon the incubation of inoculated cell line L929 with 2-merkaptobenztiazolom in the concentration range 0.03 - 3.00 mg/ml statistically significant change (r≤0,05) of cell population density in monolayer cultures was found. At the same time for all the applied reagent concentrations stimulation of mitotic activity in the terminal (5 days) stage of cultivation was observed. Exposure of cells by gamma quanta of 60Co in doses of 1, 5 and 10 Gy led to dose-dependant morphological changes in cell culture. Exposure of cells in the presence of 2-merkaptobenztiaz significantly reduced the negative impact of radiation on cell viability parameters in cultureConclusions. Quantifying radioprotective properties of 2-merkaptobenztyazol in the test system - cell cultureL929 - showed that the highest rates of protection factor (0, 31 - 0.36) reagent had at a concentration of 3 mg/ml under the irradiation at a 1Gy dose. However, dose reduction factor (DRF) calculated by LD50 for concentrations of 0.03 and 0.30 mg/ml had a value of 1.5 and 1.8 respectively, and the concentration of 3.00 mg/ml DRF was the maximum - 4.The literature study and the results of our own research showed that 2 merkaptobenztiazol can be considered to be reagent with radioprotective properties for cells in vitro.

Features of biological effects in cell culture in the combined exposure to ionizing radiation and copper ions. / Osobennosti biologicheskix e'ffektov v kul''ture kletok pri kombinirovannom dejstvii ioniziruyushhego izlucheniya i ionov medi.

The objective of the work was to research the features of monoand combined impact of ionizing radiation and copper salts on cell viability in vitro. Materials and methods. The studies were performed on a passaged line L929 cell culture. Copper ions of different concentrations were added to the culture medium 1 hour after exposure to irradiation at doses of 0.5; 5.0 and 10.0 Gy. There were evaluated the structure of the cell populations, the proliferative and mitotic activity and apoptosis. Results and conclusions. After incubation of the cells with copper ions of various concentrations there were seen both the activation and inhibition of proliferative and mitotic activity. Cell survival and mitotic activity were decreased in exposure to increasing doses of radiation but the polykaryocyte index was increased. After the combined in vitro exposure to ionizing radiation and copper ions there were seen morphofunctional changes of cell properties, which were not in direct proportional dependence on the dose or the copper ions concentration.

Impact of gadolinium-157 containing nanoscale magnetosensitive composites on morfofunctional properties of cells in vitro. / Vplyv nanorozmirnyh magnitochutlyvyh kompozytiv, shho mistjat'' gadolinij-157, na morfofunkcional''ni vlastyvosti klityn IN VITRO.

Objective - to investigate the morphofunctional cells properties under the action of magneticallybased nanocomposites containing gadolinium-157. Materials and methods. Experimental studies are performed in cell culture line L929 Nanocomposites based on magnetite modified by functional amino groups chemically fixed by diethylenetriaminepentaacetic acid (DTPA) and gadolinium - (Fe3O4/γ-APS/DTPA-Gd) were studied (1), also by meso-2,3-dimercaptosuccinic acid (DMSA) - (Fe3O4/DMSA-Gd), which binds to the hydroxyl group of magnetite surface (2); gadolinium was adsorbed from a solution of gadolinium sulfate. Reagent 3 - magnetic substance Fe3O4 with sodium oleate. Morphofunctional characteristics of cell culture were evaluated in various terms by standard indicators of sustainability: proliferative and mitotic activity and the number of giant multinuclear cells, apoptosis. Results and conclusions. We established that magnetdriven nanocomposites with gadolinium modified by DTPA and DMSA, were more biocompatibile to cells: incubation of cells with neutron capture agents (NCA) in the studied range of concentrations showed no toxicity, except maximum concentration, while decreasing cells adhesive properties. For all nanocomposites we observed decrease in mitotic activity in the background of the control cell population density, which may indicate synchronization of cell division. We found that the stabilized by sodium oleate ferrite caused destructive changes in cell culture only at concentrations of 500 µg / ml, but reduced mitotic activity in cell culture in 3-5 times in the whole range of concentrations. It is shown that magnetdriven nanocomposites induce different levels of apoptosis in cultured cells depending on the concentration of the reactants.

Features of cellular effects under combined effects of nickel ions and ionizing radiation.

UNLABELLED: Under current conditions the probability of simultaneous effects of radiation and chemical factors on biological objects increases, and that's why the issue of combined influence of factore different by their nature becomes progressively important. The objective was to study the cytotoxicity of nickel ions in a test-system of cell culture and to detect the cellular reactions under a combined impact of nickel ions and ionizing radiation. MATERIALS AND METHODS: Studies were performed on inoculated cell culture line L929. Cells were cultivated according to standard methods of treating the cell strains. After 24 h cultivation the cells were γ-irradiated at the unit "Teratron" (Canada) at doses of 0.5, 5.0 and 10.0 Gy. The water-soluble salt of nickel acetate was added to the culture medium 1 h prior to irradiation in the final concentration of 10, 1, 0.1, or 0.01 µmol/L. Morfological and functional characteristics of cells in a culture were determined at different periods of cultivation. Proliferative cell activity was assessed by growth kinetics: the total number of cells, the number of mitoses and giant multi (2 or more nuclei) cells were counted within grid area of 0.05 mm2. Simultaneously the number of apoptotic cells was determined. STUDY RESULTS: As a result , it was found that nickel ions exhibit the cytotoxic effects on cells in vitro: an increase in concentration of the element caused the cell death intencification with proliferative and mitotic activity decrease. Moreover, at low concentrations (0.01-0.1 µmol/L) the number of atypical polykaryocytes increased as well as the level of apoptosis. When nickel ions concentration increased the cell death took place through apoptosis and necrosis. Under the combined influence of radiation and nickel ions the nonlinear cellular responses were observed: under the low-dose radiation (0.5 Gy) a sensitization of cells to radiation was noted, and the sublethal doses (10 Gy) caused a particular radioprotection. Number of apoptotic cells under these conditions was significantly higher vs. control. CONCLUSIONS: Studies have shown that nickel ions have significant toxic effects on proliferation and mitotic activity of cells in vitro. Under the combined effects of ionizing radiation the cell sensitization to nickel ion was detected till following exposure to low dose (0.5 Gy) and reduction of cells tolerance to sublethal doses of irradiation (10 Gy).

[Phospholipid composition of normal and transformed cells cultivated with N-acylethanolamines].

Effects of N-stearoylethanolamine (NSE), N-oleoilethanolamine (OEA) and mixture of more than 20 saturated and unsaturated N-acylethanolamines on phospholipids composition of normal and transformed fibroblasts in the culture were compared in the work. It was shown that cultivation of cells NSE decreases percent content of phosphatidylinositol (PI), phosphatidylserine (PS) and sphingomyeline (SM) - important mediators of cell signaling. Under the influence of OEA the level of SM decreases as well, but at the same time PI and PS levels increase. NAEs mixture decreases PS and PI levels in cells and causes phosphatidylcholine (PC) amount increase. Moreover NSE and NAEs mixture decrease the content of main mitochondria membrane lipid - diphosphatidylglicerole (DPG). OEA increases DPG amount. In transformed fibroblasts (line L929) NAE modulate lipid composition as well: NSE decreases the level of phosphatidylethanolamine (PE), OEA and NAEs mixture increase sphingomyeline levels. It is shown that response of normal and transformed fibroblasts on NAE application is different, depending on substance structure and cell-target type.

[Effects of N-stearoyl- and N-oleoylethanolamine on cardiac voltage-dependent sodium channels].

The group of N-acylethanolamines (NAE) includes lipids that are capable of modulating plasma membrane ion channels without involvement of cannabinoid receptors. However, the action of various members of NAE on voltage-gated Na+ channels (VGSC) in cardiac tissue is still not fully elucidated. Here using patch-clamp technique we have studied the modulation of biophysical properties of VGSC of neonatal cardiomyocytes by saturated N-stearoylethanolamine (NSE) and monounsaturated N-oleoylethanolamine (OEA). NSE in 1-200 tM concentration range did not significantly alter the amplitude of inward Na+ current (I(Na)), but 100 microM NSE shifted its steady-state activation and inactivation curves in hyperpolarization direction by 2.4 mV and 10.6 mV, respectively. Activation kinetics of the current was not changed by NSE, but its inactivation was accelerated by about 1.2-fold in the -60 - -30 mV range of membrane potentials. Unlike NSE, OEA dose-dependently inhibited I(Na) with kappa(D) = 11.4 +/- 1.6 microM and maximal block at saturating concentration of 30 +/- 3%. It also stronger than NSE shifted current's steady-state activation and inactivation curves (-6.4 mV and -14.0 mV, respectively, at 100 microM) in hyperpolarization direction. The effect of OEA on I(Na) activation kinetics was negligible, but it more pronouncedly than NSE accelerated inactivation of the current. Thus, both members of NAE influence the voltage-dependence of activation, inactivation and kinetics of I(Na). These effects were more prominent for monounsaturated OEA, which also partially blocked I(Na). The discovered effects of NSE and OEA on VGSCs may in part be responsible for the decrease of cardiomycytes' excitability by these lipids under normal as well as pathologic conditions.

[N-stearoylethanolamine inhibits the proliferation of transformed cells and modulates mitochondrial enzyme activity in normal and transformed cells].

The N-stearoylethanolamine (NSE) influence on the proliferation of different cell types and the activity of mitochondrial electron transport enzymes, succinatedehydrogenase (SDG) and glycerophosphate-dehydrogenase (GFDG), in transformed cells under the action of NSE was studied. The incubation of the cells of mouse leukemic lymphocyte cell line L1210 and transformed mouse fibroblasts L929 with NSE caused the inhibition of cell growth and decreased the survival level of cells, but this effect was not associated with apoptotic cell death. It was shown for the first time that NSE addition to the cultural medium decreased the SDG activity and increased the GFDG activity in L929 cells. That leads to the SDG/GFDG imbalance in transformed fibroblasts and affects the cell energy metabolism. The results of the work suggest that N-stearoylethanolamine inhibited the transformed cell proliferation due to modulation of the activity of electron transport enzymes.

[Regulation of the excitability of neonatal cardiomyocytes by N-stearoyl- and N-oleoyl-ethanolamines].

N-acylethanolamines (NAE) are biologically active lipids able of modulating ion transport through the cellular plasma membrane, however specific targets of their action and signalling mechanisms involved in cardiac tissue are still poorly understood. Physiological activity of NAEs is known to depend on the level of unsaturation. Therefore, here we investigated the effects of saturated N-stearylethanolamine (NSE) and monounsaturated N-oleylethanolamine on electric excitability of neonatal rat cardiomyocytes. 1 microM of either NSE or OEA decreased the duration of cardiac action potential (AP) from all parts of heart muscle. Shortening of AP was partially reversible, though the reversibility of AP duration upon washout of substances was more complete for endocardial ventricular compared to epicardial and atrial cardiomyocytes. I microM NSE depolarized resting membrane potential (RMP) of epicardial and of 65% of endocardial cells, whilst other cells types showed weakly reversible hyperpolarization. 1 microM OEA caused reversible RMP hyperpolarization of all studied cell types. NSE and OEA decreased the amplitude and upstroke velocity of AP that suggests their effect on sodium channels. NSE and to a lesser extent OEA inhibited the amplitude of AP phase 2 (plateau) which may indicate an inhibition of high-voltage-activated calcium channels. Effects of NSE and OEA on RMP and repolarization phase of AP (phase 3) depended on cardiac cell type suggesting differential regulation of inward rectifier Kir and voltage-gated delayed rectifier potassium channels by these lipids. We cannot also exclude interaction of NSE and OEA with anion channels, backgound K+ channels and ion transporters of the cardiomyocytes' plasma membrane. Overall, NSE-induced changes of AP parameters were less reversible than those induced by OEA, suggesting a slower degradation/ convertion of NSE in plasma membrane compared to OEA.

[Influence of N-acylethanolamines on embryonic cell culture survival under the effect of irradiation].

The effects of the N-stearoylethanolamine (NSE), N-oleoylethanolamine (OEA) and N-acylethanolamine (NAE) mixture on the cell survival, apoptosis and activity of mitochondrial succinate dehydrogenase (SDG) and glycerophosphate dehydrogenase (GFDG) in embryonic cell culture under normal conditions and irradiation were compared in the work. It was shown, that all investigated NAE were able to modulate the proliferative activity of intact cells as well as irradiation-exposed cells in concentration-depended manner. The most pronounced effect was observed under the NAE mixture action. It was established, that NAE prevented the damage effects of the irradiation and diminished the activation of apoptotic cell death. It was found that NSE and OEA decreased the activity of the SDG (42.3 and 44.14%, accordingly) and the GFDG activity (14.67 and 17.33%, accordingly) in embryonic cell culture, while addition of the NAE mixture decreased SDG activity by 20%, at the same time GFDG activity increased by 20%. Our findings suggested that antiproliferative effects of NAE depended on their influence on mitochondrial functioning.