Cytotoxic and genotoxic effects of tert-butyl hydroperoxide on Chinese hamster B14 cells

The organic hydroperoxide, tert-butyl hydroperoxide (t-BHP), is a useful model compound to study mechanisms of oxidative cell injury. In the present work, we examined the features of the interactions of this oxidant with Chinese hamster B14 cells. The aim of our study was to reveal a possible role of structural modifications in membranes and loss of DNA integrity in t-BHP-induced cell injury and death. The tert-butyl hydroperoxide treatment (100-1000 microM, 37 degrees C for 1h) did not decrease cell viability (as measured by cell-specific functional activity with an MTT test), but completely prevented cell growth. We observed intracellular reduced glutathione (GSH) oxidation and total glutathione (GSH+GSSG) depletion, a slight increase in the level of lipid-peroxidation products, an enhancement of membrane fluidity, intracellular potassium leakage and a significant decrease of membrane potential. At oxidant concentrations of 100-1500 microM, a significant damage to DNA integrity was observed as revealed by the Comet assay. The inhibition of cell proliferation (cell-growth arrest) may be explained by genotoxicity of t-BHP, by disturbance of the cellular redox-equilibrium (GSH oxidation) and by structural membrane modifications, which result in ion-non-selective pore formation. The disturbance in passive membrane permeability and the DNA damage may be the most dramatic cell impairments induced by t-BHP treatment. The presence of another oxidant, hypochlorous acid (HOCl), completely prevented t-BHP-induced DNA strand breaks, perhaps due to extracellular oxidation of t-BHP by HOCl.     

Oxidative Processes Induced by <Emphasis Type="Italic">tert</Emphasis>-Butyl Hydroperoxide in Human Red Blood Cells: Chemiluminescence Studies

The erythrocyte is a good model for investigation of the mechanisms of cell damage induced by oxidizing agents. Oxidative damage to cell components and cellular metabolism results in impaired rheological properties of circulating red blood cells and is involved in the development of some pathologies. The aim of the present study was to elucidate further the oxidative processes induced by tert-butyl hydroperoxide (tBOOH) in erythrocytes, identify cellular targets damaged by the oxidant, as well as estimate the energy and stoichiometry of the reactions that occur. The generation of free radicals in the cell was registered using the chemiluminescence technique. The products of oxyhemoglobin (oxyHb) oxidation, changes in intracellular glutathione (GSH) pool, and accumulation of the stable products of membrane lipid peroxidation were concurrently measured. The oxidative processes induced by tBOOH in red blood cells can be described as follows: 1) rapid GSH oxidation (30-60 sec) by glutathione peroxidase; 2) formation of radicals in the reaction between tBOOH and cellular Hb, which are then immediately consumed in lipid peroxidation reactions; 3) generation of chemiluminescence by the radicals formed. Several stages of the oxidative processes can be revealed. The order of the chemiluminescence reaction (n) with respect to oxidant was estimated to be equal to 2.5 at oxidant concentrations less than 0.5 mM and equal to 1.0 at higher oxidant concentrations. The order of the reaction of membrane lipid peroxidation was found to be n = 2.2 at 0.25-0.6 mM tBOOH and n = 0.5 at higher oxidant concentrations. The apparent activation energy of membrane lipid peroxidation was 55.8 +/- 6.4 kJ/mol, and that of oxyHb oxidation was 108 +/- 16 kJ/mol. It is shown that the interaction of tBOOH and HOCl in erythrocytes is accompanied by changes in both the total number of radicals generated in the cell and the time corresponding to the maximal rate of radical generation.     

Study of heat denaturation of human serum albumin in water-alcohol and water-salt solutions in the presence of organic ligands

The method of differential scanning microcalorimetry was used to show a decrease in heat stability of serum albumin in the presence of aliphatic alcohols. In aqueous-alcohol media, the melting temperature, denaturation transition enthalpy were decreased, and the protein intermolecular aggregation enhanced. When the alcohol concentration in aqueous solution was elevated, the number of epsilon-amino groups of lysine residues in human serum albumin exposed to the solvent rose from 6-7 in aqueous solution to maximum 20 groups in the aqueous-alcohol solution, respectively. The elevation of ionic strength also induced an increase in the number of exposed lysine residues and was accompanied by an enhancement of protein aggregation. The modification of six amino groups by pyridoxal phosphate or three by glucose in the initial albumin stabilized the protein incubated at 65 degrees-70 degrees C both in the aqueous-alcohol media. At the given concentration and temperature the native protein was denatured and fully aggregated. Aliphatic alcohols displaced fatty acids from the binding sites on the molecule of serum albumin, which resulted in a change in the number of peaks of the melting curve.     

Changes in sleep characteristics of rat preclinical model of Parkinson’s disease based on attenuation of the ubiquitin—proteasome system activity in the brain

Numerous experimental and epidemiological data indicate a high significance of environmental neurotoxins, specifically, inhibitors of the ubiquitin–proteasome system, in pathogenesis of Parkinson’s disease (PD). To develop a preclinical model of PD in rats we used a technique of intranasal administration of lactacystin, a natural proteasome inhibitor, into the brain. It was found that three weeks after the first lactacystin administration it induced a little degeneration of dopaminergic neurons in the olfactory bulb and substantia nigra pars compacta without any olfactory dysfunction and motor behavior disorders. Besides, its effect led to the appearance of some signs of sleep disorders: increased somnolence (especially in the dark, active daily phase), fragmentation of slow-wave sleep, decreased EEG delta rhythm during slow-wave sleep. These signs share some similarity with PD and could be useful in clinical studies for the quick search for polysomnographic markers of the early PD stage.     

Effects of exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> on the content of endogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript>, activities of catalase and hydrolases,and cell ultrastructure in tobacco leaves

It was shown that tobacco leaf treatment with 100 mM H₂O₂ increased their content of endogenous H₂O₂ and activities of catalase and hydrolases (acid phosphatase, proteases, and RNase) and also caused various changes in the cell structure. In this case, programmed cell death (PCD) occurred in some cells, which was observed as chromatin condensation, cytoplasm collapse, etc. In the meantime, many cells displayed organelle activation rather than PCD. It is suggested that cells that undergo H₂O₂-dependent PCD release signaling molecules inducing protective mechanisms against oxidative stress in neighboring cells not exhibiting PCD.     

Medium-scale mapping of vegetation in mountains of southern Siberia

An ecophytocenotic map has been compiled for southern Siberia at a scale of 1 : 1 000 000. The legend for mountainous areas includes information on the vegetation of the mapped units and environmental conditions (Σ _t > 10°, annual precipitation, altitudes above sea, and topographic features).     

Vegetation characteristics and eco-hydrological processes in a pristine mire in the Ob River valley (Western Siberia)

Relations between vegetation characteristics and eco-hydrological processes were assessed in a pristine mire in the valley of the Ob River (Western Siberia). Along a transect from the terrace scarp to the river, field data were collected on vegetation composition, peat stratigraphy, peat chemistry, hydrology and hydrochemistry. Based on floristic composition, eight vegetation communities were distinguished. Hydraulic head measurements were used to obtain an indication of groundwater flow directions. The water balance of the mire was calculated with a two-dimensional steady-state numerical groundwater model. Water types were defined based on cluster analysis of hydrochemical data. The results revealed that the dominant hydrological factor in the Ob mire is the discharge of groundwater, which supplies about threefold more water than net precipitation. Although the discharge flux decreases with increasing distance from the terrace scarp, high water levels and a “groundwater-like” mire water composition were observed in the major part of the study site. Precipitation and river water play only a minor role. Despite dilution of discharging groundwater with rainwater, spatial differences in pH and solute concentrations of the surficial mire water are small and not reflected in the vegetation composition. Although small amounts of silt and clay were found in the peat in the proximity of the river, indicating the occurrence of river floods in former times, no river-flood zone could be recognized based on hydrochemical characteristics or vegetation composition. A comparison of the Ob mire with well-studied and near-natural mires in the Biebrza River valley (Poland) revealed substantial differences in both vegetation characteristics and the intensity and spatial pattern of eco-hydrological processes. Differences in the origin and ratios of water fluxes as well as a dissimilar land use history would seem to be key factors explaining the differences observed.     

Effect of tobacco mosaic virus strains on the ultrastructure of tobacco leaf parenchymal cells

Accumulation of considerable amounts of viral particles has been demonstrated in parenchymal cells of young leaves in tobacco cultivar Samsun systemically infected with any of studied tobacco mosaic virus (TMV) strains isolated from pepper (TMV-p), tomato (TMV-t), and eggplant (TMV-e). Abnormal (swollen and thin) virions were found, which points to their destruction. Cell infection with all studied strains was accompanied by the activation of the lysosomal compartment manifested as formation of nascent dictyosomes, elements of smooth endoplasmic reticulum, cytoplasmic vacuoles, various vesicles, invaginated mitochondria, and multivesicular bodies. The studied viral strains could be arranged in the following sequence according to the degree of lysosomal compartment stimulation and induction of intracellular lytic processes mediating the destruction of viral particles and cell structures: TMV-p > TMV-e > TMV-t.