Pro-oxidative, genotoxic and cytotoxic properties of uranyl ions.

It is demonstrated that hydroxyl radicals and hydrogen peroxide are formed under the action of uranyl ions in aqueous solutions containing no reducing agents. In the presence of uranyl ions, formation of 8-oxoguanine in DNA and long-lived protein radicals are observed in vitro. It is shown that the pro-oxidant properties of uranyl at micromolar concentrations mostly result from the physico-chemical nature of the compound rather than its radioactive decay. Uranyl ions lead to damage in DNA and proteins causing death of HEp-2 cells by necrotic pathway. It is revealed that the uranyl ions enhance radiation-induced oxidative stress and significantly increase a death rate of mice exposed to sublethal doses of X-rays.

Burst of succinate dehydrogenase and α-ketoglutarate dehydrogenase activity in concert with the expression of genes coding for respiratory chain proteins underlies short-term beneficial physiological stress in mitochondria.

Conditions for the realization in rats of moderate physiological stress (PHS) (30-120 min) were selected, which preferentially increase adaptive restorative processes without adverse responses typical of harmful stress (HST). The succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (KDH) activity and the formation of reactive oxygen species (ROS) in mitochondria were measured in lymphocytes by the cytobiochemical method, which detects the regulation of mitochondria in the organism with high sensitivity. These mitochondrial markers undergo an initial 10-20-fold burst of activity followed by a decrease to a level exceeding the quiescent state 2-3-fold by 120 min of PHS. By 30-60 min, the rise in SDH activity was greater than in KDH activity, while the activity of KDH prevailed over that of SDH by 120 min. The attenuation of SDH hyperactivity during PHS occurs by a mechanism other than oxaloacetate inhibition developed under HST. The dynamics of SDH and KDH activity corresponds to the known physiological replacement of adrenergic regulation by cholinergic during PHS, which is confirmed here by mitochondrial markers because their activity reflects these two types of nerve regulation, respectively. The domination of cholinergic regulation provides the overrestoration of expenditures for activity. In essence, this phenomenon corresponds to the training of the organism. It was first revealed in mitochondria after a single short-time stress episode. The burst of ROS formation was congruous with changes in SDH and KDH activity, as well as in ucp2 and cox3 expression, while the activity of SDH was inversely dependent on the expression of the gene of its catalytic subunit in the spleen. As the SDH activity enhanced, the expression of the succinate receptor decreased with subsequent dramatic rise when the activity was becoming lower. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaption and therapy.

[Study of the mechanisms of cytotoxic effect of uranyl nitrate].

The mechanisms of cytotoxic effect of uranyl nitrate were studied. It was shown that uranyl nitrate induced HEp-2 cell death, mainly by necrotic way. In the experiments in vitro, uranyl nitrate caused an appearance of 8-oxoguanine in DNA, indicating the induction of oxidative stress. The experiments with isolated rat liver mitochondria revealed that 1 mM uranyl nitrate decreased the respiration rates of mitochondria in state 3 and DNP-induced respiration. At the same time, uranyl nitrate had no influence on the opening of the mitochondrial permeability transition pore and decreased the rate of formation of H2O2 by mitochondria. Possible molecular mechanisms of uranyl-induced necrosis are discussed.

[A method for the detection and characterization of GABA(A) receptors by calcium-sensitive fluorescent probes].

A method for the detection and characterization of GABA(A) receptors of neurons has been developed, which is based on the measurement of the activity of potential-dependent calcium channels using the fluorescence of the two-wavelength calcium-sensitive probe Fura-2. The method makes it possible to detect the ligands of GABA(A) receptors and determine the constants of activation and inhibition as well as the type of inhibition. The object of investigation was a young (two- to four-day-old) rat hippocampal cell culture in which GABA induces the depolarization and a transient increase in Ca2+ concentration in the cytosol of neurons due to the activation of potential-dependent calcium channels. It was shown that a short-time application of GABA induces a decrease in the amplitude of calcium responses to subsequent addition of the depolarizing agents GABA or KCl. However, at low amplitudes of calcium responses to the addition of GABA, this reducing effect on the subsequent addition of KCl was insignificant. It was found that the amplitudes of calcium responses to KCl and GABA are linearly dependent on the angular coefficient b = 3.41. This enabled one to develop a method of normalizing calcium signals, which makes it possible to compare experiments performed on different days and different cultures. By using this normalization technique, the values of EC50 = 2.21 +/- 0.14 ?M and the Hill coefficient = 1.9 +/- 0.2 were estimated. The blocker of potential-dependent calcium channels nifedipine suppressed simultaneously the amplitudes of calcium responses to the addition of KCl and GABA. In this case, the linear relationship between the amplitudes of calcium responses to the addition of KCl and GABA was retained. To verify the validity of the method, the constant of inhibition of a calcium signal and the type of inhibition for known noncompetitive and competitive antagonists of GABA(A) receptors were determined.

[Alteration of brown adipocyte Ca2+ responses in culture by adrenergic activation].

Thermogenic capability of brown adipose tissue is controlled by norepinephrine. Interaction of norepinephrine with adipocyte at- and P3-adrenergic receptors results in the increase of Ca2+ and cAMP concentrations. The [Ca2+]i changes initiated by norepinephrine and selective agonists of alpha1- and beta-adrenergic receptors, cirazolin and isoproterenol, were recorded in single cells of primary culture on the 1st, 3rd and 6th days in vitro. On the first day, isoproterenol-induced [Ca2+]i changes as compared to cirazolin-induced ones were characterized by greater amplitude and lesser impulse duration over the entire range of physiological concentrations used. These differences were negligible after 3 days and kinetic differences were practically absent after 6 days of cultivation. The agonist-induced [Ca2+]i changes in proliferating and differentiated cells differed significantly: in the process of cell growth in culture, the amplitude of calcium response increased, the duration of impulse signal decreased and the sensitivity to adrenergic agonists increased. The Ca2+ store in endoplasmic reticulum increased during the cell growth and development in culture, according to thapsigargin-induced Ca2+ response amplitude increase in Ca2+ free medium. The rate of Ca2+ pumping out of cell characterizing PMCA-activity also increased.

[Destabilization of the cytosolic calcium level and cardiomyocyte death in the presence of long-chain fatty acid derivatives].

It has been shown using the fluorescent microscopy technique that long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, exert the most toxic effect on rat ventricular cardiomyoctes. The addition of 20-50 microM acylcarnitines increases calcium concentration in cytoplasm ([Ca2+]i) and causes cell death after the 4-8 min lag-period. This effect is independent on extracellular calcium and L-type calcium channel inhibitors. Free acids (myristic and palmitic acids) at a concentration of 300-500 microM have a little effect on [Ca2+]i within 30 min. We suggest that the toxic effect is due to the activation of sarcoplasmic reticulum calcium channels by acylcarnitines and resulting acyl-CoA. Mitochondria play a role of calcium-buffer system in these conditions. The calcium capacity of this buffer determines the lag-period. Phosphate increases the calcium capacity of mitochondrial and the lag-period. In the presence of rotenone and oligomycin the elevation of [Ca2+]i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondrial leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.

[Studies of interaction of intracellular signalling and metabolic pathways under inhibition of mitochondrial aconitase with fluoroacetate].

Mitochondrial aconitase has been shown to be inactivated by a spectrum of substances or critical states. Fluoroacetate (FA) is the most known toxic agent inhibiting aconitase. The biochemistry of toxic action of FA is rather well understood, though no effective therapy has been proposed for the past six decades. In order to reveal novel approaches for possible antidotes to be developed, experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells and cardiomyocytes, exposed to FA or fluorocitrate in vitro. The effect of FA developed at much higher concentrations in comparison with fluorocitrate and was dependent upon respiratory substrates in experiments with mitochondria: with pyruvate, FA induced a slow oxidation and/or leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides was prevented by incubation of mitochondria with cyclosporin A. Studies of the pyridine nucleotides level and calcium response generated in Ehrlich ascite tumor cells under activation with ATP also revealed a loss of pyridine nucleotides from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H under exposure to FA. An increase of cytosolic [Ca2+] was observed in the cell lines exposed to FA and is explained by activation of plasma membrane calcium channels; this mechanism, could have an impact on amplitude and rate of Ca2+ waves in cardiomyocytes. Highlighting the reciprocal relationship between intracellular pyridine nucleotides and calcium balance, we discuss metabolic pathway modulation in the context of probable development of an effective therapy for FA poisoning and other inhibitors of aconitase.