Substrate-Specific Reduction of Tetrazolium Salts by Isolated Mitochondria, Tissues, and Leukocytes.

Tetrazolium salts are commonly used in cytochemical and biochemical studies as indicators of metabolic activity of cells. Formazans, formed by reduction of tetrazolium salts, behave as pseudo-solutions during initial incubation, which allows monitoring their optical density throughout incubation. The criteria and conditions for measuring oxidative activity of mitochondria and dehydrogenase activity in reduction of nitroblue tetrazolium (NBT) and methyl thiazolyl tetrazolium (MTT) in suspensions of isolated mitochondria, tissue homogenates, and leukocytes were investigated in this work. We found that the reduction of these two acceptors depended on the oxidized substrate - NBT was reduced more readily during succinate oxidation, while MTT - during oxidation of NAD-dependent substrates. Reduction of both acceptors was more sensitive to dehydrogenase inhibitors that to respiratory chain inhibitors. The reduction of NBT in isolated mitochondria, in leukocytes in the presence of digitonin, and in liver and kidney homogenates was completely blocked by succinate dehydrogenase inhibitors - malonate and TTFA. Based on these criteria, activation of succinate oxidation was revealed from the increase in malonate-sensitive fraction of the reduced NBT under physiological stress. The effect of progesterone and its synthetic analogs on oxidation of NAD-dependent substrates by mitochondria was investigated using MTT. Both acceptors are also reduced by superoxide anion; the impact of this reaction is negligible or completely absent under physiological conditions, but can become detectable on generation of superoxide induced by inhibitors of individual enzyme complexes or in the case of mitochondrial dysfunction. The results indicate that the recording of optical density of reduced NBT and MTT is a highly sensitive method for evaluation of metabolic activity of mitochondria applicable for different incubation conditions, it offers certain advantages in comparison with other methods (simultaneous incubation of a large set of probes in spectral cuvettes or plates); moreover, it allows determination of activity of separate redox-dependent enzymes when selective inhibitors are available.

[Genetic system for maintaining the mitochondrial human genome in yeast Yarrowia lipolytica].

For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.

[Redox-dependent ferric oxide nanoparticles loaded with doxorubicin and their influence on the functions of mitochondria].

The spectral, fluorescent and functional properties of ferric oxide and ferric hydroxide nanoparticles loaded with doxorubicin and stabilized with citric acid or lysine were studied in comparison with free doxorubicin. Their effect on the opening of calcium-induced mitochondrial pore and the possibility of the controlled release of doxorubicin under the influence of redox stimuli were investigated. The data show that the effect of nanoparticles on mitochondria depends on the type of a stabilizer. The spectral and fluorescence methods used allow us to estimate the presence or absence of free doxorubicin in solution of nanoparticles and the:concentration of bound doxorubicin. It is shown that the dithiotreitol and glutathione increase the amplitude of absorption and fluorescence of doxorubicin during incubation with nanoparticles. It is assumed that this effect may be associated with the reduction of the oxidized iron by thiols with subsequent release of doxorubicin.

[Changes in gene expression and content of titin (connectin) in striated muscles of chronically etihanol-fed rats].

Changes in gene expression and isoform composition of giant sarcomeric protein titin (connectin) in cardiac muscle, as well as changes of its isoform composition in skeletal muscle (m. soleus) of chronically ethanol-fed rats have been studied using real-time RT-PCR and low percentage SDS-gel electrophoresis. The decrease of titin content in examined muscles and the decrease in titin gene expression in myocardium of chronically ethanol-fed rats have been shown. These changes indicate the development of pathologic process.

[The role of thiol antioxidants in restoring mitochondrial functions, modified by microbial metabolites].

The effects of phenolic acids of microbial origin on mitochondrial functions and the possibility of removing their effects by thiol antioxidants dithiotreitol and N-acethylcysteine were studied. The action of some phenolic acids on the redox state of NADH, the membrane potential and calcium capacity of mitochondria is due to their interaction with thiol groups. The partial restoration of mitochondrial functions occurred in the presence of dithiotreitol and N-acethylcysteine, the full recovery (short-term duration) was promoted by the combined action of dithiotreitol and menadione (vitamin K3). It was found that the protective effect of thiol antioxidants became prooxidant one, if the medium contained free iron and compounds with a quinone structure, capable of entering into a redox cycle with thiols. It is shown that the interaction of thiols with iron and menadione is accompanied by absorption of oxygen to form superoxide anion. Prooxidant effect of thiol antioxidants may explain the absence of the protective effect at the later stages of sepsis and systemic inflammatory syndrome.

[Effect of steroid hormones on production of reactive oxygen species in mitochondria].

Among the targets of the steroid hormones are mitochondria, which as the main source of reactive oxygen species (ROS) in the cell play a central role in the development of various pathologies. We studied the effect of progesterone and its synthetic analogues on mitochondrial ROS production. It was found that progesterone activates the formation of superoxide anion and hydrogen peroxide in mitochondria during oxidation of complex I substrates of the respiratory chain and exerts no influence on production of ROS during oxidation of succinate, complex II substrate of the respiratory chain. Synthetic analogues of progesterone - medroxyprogesterone acetate, buterol, acetomepregenol, megestrol acetate, have different effects on ROS production, depending on their chemical structure. By the effectiveness of impact on ROS production in mitochondria all the steroids tested can be classified in the descending order as follows: progesterone > buterol > or = atsetomepregenol > medroxyprogesterone acetate > megestrol acetate. Activation of ROS production by progesterone and buterol has different mechanisms: progesterone acts as an inhibitor of NAD-dependent respiration, while buterol and acetomepregenol form noncovalently associated complexes by hydrogen bonds between the ester carbonyl at C3 and SH-groups of the respective targets.

[Effect of ethanol on synthesis of serine and exchange of methyl groups in hepatocytes by NMR spectroscopy].

The method of NMR spectroscopy was used to investigate the role of voltage-dependent anion channels in the outer mitochondrial membrane in the mechanism of ethanol hepatotoxicity using the synthesis of serine and exchange of methyl groups in hepatocytes metabolizing 13C-labeled glycine. Here we present and describe a methodological approach developed for the independent monitoring of the synthesis of serine in two intracellular compartments: the cytoplasm and mitochondria of intact hepatocytes, and quantification of different serine isotopomers synthesized in hepatocytes from 13C-labeled glycine. The data obtained indicate that the treatment of cells with ethanol as well as cysteamine (specific inhibitor of mitochondrial synthesis of serine) suppressed the level of mitochondria but not cytoplasmic serine isotopomers. It is concluded that the decrease in the production of mitochondrial serine isotopomers in hepatocytes exposed to ethanol can be caused not only by decreased permeability of the outer mitochondrial membrane due to the closure of voltage-dependent anion channels and suppression of the exchange of substrates of serine synthesis in mitochondria but also by the restoration of the cytoplasmic and/or mitochondrial pool of pyridine nucleotides (NADH) during the oxidation of ethanol. Our work reveals a new mechanism of action of ethanol (alcohol intoxication) in hepatocytes through the regulation of glycine metabolism and opens new possibilities in the treatment of alcohol poisoning.

[Mitochondria and hepatotoxicity of ethanol].

The current understanding of the effects of alcohol intoxication on the basic mitochondrial functions has been presented. Both, the direct toxic effect of ethanol on biological membranes and various cellular systems and the toxicity of acetaldehyde and reactive oxygen species (the products of ethanol oxidation) are discussed, with emphasis on the effect of ethanol on the basic functions of mitochondria and Ca(2+)-dependent mitochondrial permeability transition. Based on the available experimental data, it is demonstrated that acute alcohol intoxication causes a global mitochondrial dysfunction in the liver, resulting in considerable disturbance of the whole cellular metabolism. Alcohol poisoning of the liver leads to a decreased ability of cells to withstand oxidative stress, to support the synthesis of vital metabolic intermediates (e.g., methyl groups), as well as to produce urea from ammonia, due to a decreased permeability of the outer membrane and impaired exchange of substrates between the cytoplasm and the mitochondrial matrix. This review emphasizes the role of the voltage-dependent anion channels of the outer mitochondrial membrane in ethanol-mediated disturbances of basic mitochondrial functions and its consequences for the entire cell metabolism in the liver.

[The role of the voltage-dependent anion channels in the outer membrane of mitochondria in the regulation of cellular metabolism].

The role of the voltage-dependent anion channels (VDAC) harbored in the outer membrane of mitochondria in the regulation of cellular metabolism was investigated using an experimental model of ethanol toxicity in cultured hepatocytes. It was demonstrated that ethanol inhibits State 3 and uncoupled mitochondrial respirations, decreases the accessibility of mitochondrial adenylate kinase localized in the intermembrane space of mitochondria, and suppresses ureagenic respiration and synthesis of urea in cultured hepatocytes. Increasing the permeability of the outer mitochondrial membrane with closed VDAC with high concentrations of digitonin (> 80 microM), which creates pores in the membrane, allowing the alternative bypass of closed VDAC, and restores all reactions suppressed with ethanol. It is concluded that the effect of ethanol in hepatocytes leads to global loss of mitochondrial functions due to the closure of VDAC, which limits the free diffusion of metabolites into the intermembrane space of mitochondria. Our studies demonstrated that ethanol affects the main mitochondrial functions and revealed the role of VDAC channels in the outer mitochondrial membrane in the regulation of liver specific intracellular processes such as ureagenesis. The data obtained can be used for the development of pharmaceutical drugs that prevent the closure of VDAC in mitochondria of ethanol oxidizing liver, thus protecting liver tissue from the hepatotoxic action of alcohol.

[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.

Inhibition by Ca2+ of the hydrolysis and the synthesis of ATP in Ehrlich ascites tumour mitochondria: relation to the Crabtree effect.

Phosphorylation of ADP and hydrolysis of ATP by isolated mitochondria from Ehrlich ascites tumour cells is greatly reduced when the mitochondria have been preloaded with Ca2+ (50 nmol/mg protein or more). Translocation of ADP is diminished in Ca(2+)-loaded mitochondria. However, ATPase in toluene-permeabilized mitochondria and in inside-out submitochondrial particles is also strongly inhibited by micromolar concentrations of Ca2+, indicating that, independently of adenine nucleotide transport, F1Fo-ATPase is also affected. ATP hydrolysis by submitochondrial particles depleted of the inhibitory subunit of F1Fo-ATPase (the Pullman-Monroy protein inhibitor) is insensitive to Ca2+; however, this sensitivity is restored when the particles are supplemented with the inhibitory subunit isolated from beef heart mitochondria. In view of the previous observations that glucose elicits in Ehrlich ascites tumour cells an increase of cytoplasmic free Ca2+ (Teplova, V.V., Bogucka, K., Czyz, A., Evtodienko, Yu.V., Duszynski, J. and Wojtczak, L. (1993) Biochem. Biophys. Res. Commun. 196, 1148-1154) and that this calcium is then taken up by mitochondria, resulting in a strong inhibition of coupled respiration (Evtodienko, Yu.V., Teplova, V.V., Duszynski, J., Bogucka, K. and Wojtczak, L. (1994) Cell Calcium 15, 439-446), the present results are discussed in terms of the mechanism of the Crabtree effect in tumour cells.

The role of cytoplasmic [Ca2+] in glucose-induced inhibition of respiration and oxidative phosphorylation in Ehrlich ascites tumour cells: a novel mechanism of the Crabtree effect.

The effect of Ca2+ on energy-coupling parameters of Ehrlich ascites carcinoma was studied in digitonin-permeabilized cells. In nominally Ca-free medium the permeabilized cells respond to the addition of ADP by increased oxygen uptake with externally added respiratory substrates (succinate or pyruvate), decrease of the mitochondrial membrane potential (delta psi) and alkalinization of the medium. This typical behaviour is drastically changed if Ca2+ is added. The subsequent addition of ADP induces neither State 3 respiration, nor decrease of delta psi, nor alkalinization of the medium, indicating a complete block of ATP synthesis. These effects are produced by both a single pulse of 100 microM Ca2+ and a preincubation for 2 min with 0.4-1.0 microM Ca2+. Preincubation of the cells with glucose or deoxyglucose prior to permeabilization makes them sensitive to Ca2+ concentrations as low as 0.3 microM. In view of the previous finding that glucose and deoxyglucose produce an increase of cytoplasmic [Ca2+] in Ehrlich ascites cells [Teplova VV. Bogucka K. Czyz A. Evtodienko YuV. Duszynski J. Wojtczak L. (1993) Biochem. Biophys. Res. Commun., 196, 1148-1154; Czyz A. Teplova VV. Sabala P. Czarny M. Evtodienko YuV. Wojtczak L. (1993) Acta Biochim. Polon., 40, 539-544], the present results suggest that cytoplasmic Ca2+ plays a crucial role in the Crabtree effect.

Microtubule-active drugs suppress the closure of the permeability transition pore in tumour mitochondria.

We report the effects of anticancer drugs, inhibitors of microtubule organisation, on the mitochondrial permeability transition pore (PTP) in Ehrlich ascites tumour cells. Taxol (5-20 microM) and colchicine (100-500 microM) prevented closing of the cyclosporin A-sensitive PTP. No taxol or colchicine effects on oxidative phosphorylation were observed in the range of concentrations used. We suggest that either membrane-bound tubulin per se can be part of PTP and/or the attachment of mitochondria to the microtubular network is essential for PTP regulation. The taxol inhibition of PTP closure, mediated through interaction with the cytoskeleton, sheds new light on the cytotoxic properties of this anticancer drug.

Effect of glucose and deoxyglucose on cytoplasmic concentration of free Ca2+ in Ehrlich ascites tumour: studies on single cells.

Concentration of free cytoplasmic Ca2+ ([Ca2+]i) in Ehrlich ascites tumour cells loaded with fura-2 was measured in single cells applying a video imaging system. In resting cells [Ca2+]i amounted to 60-340 nM and was increased after addition of 10 mM D-glucose or D-2-deoxyglucose by 80-200 nM. This increase occurred within 30-60 s following addition of the sugars and lasted for several minutes. Pretreatment of the cells with thapsigargin resulted in a much smaller [Ca2+]i increase after addition of glucose or deoxyglucose and, vice versa, thapsigargin added after the sugars mobilized less Ca2+ than when added before. A possible relation of the [Ca2+]i rise evoked by glucose and deoxyglucose to the Crabtree effect is discussed.

Combined vitamins Bl2b and C induce the glutathione depletion and the death of epidermoid human larynx carcinoma cells HEp-2.

The combination of hydroxocobalamin (vitamin B12b) and ascorbic acid (vitamin C) can cause the death of tumor cells at the concentrations of the components at which they are nontoxic when administered separately. This cytotoxic action on epidermoid human larynx carcinoma cells HEp-2 in vitro is shown to be due to the hydrogen peroxide generated by the combination of vitamins B12b and C. The drop in the glutathione level preceding cell death was found to be the result of combined action of the vitamins. It is supposed that the induction of cell death by combined action of vitamins B12b and C is connected to the damage of the cell redox system.

The high calcium ion uptake capacity of Ehrlich ascites tumour cell mitochondria is due to inhibition of the permeability transition and phospholipase A2 activity by magnesium.

Tumour cells frequently have a high Ca2+ threshold for the mitochondrial permeability transition which occurs when a large pore in the inner membrane is opened. We studied whether this was due to the known high content of Mg2+ in Ehrlich ascites tumour cell mitochondria or to the increased expression of the protooncogene bcl-2. The latter was found not to be the case. Mg2+ potently inhibited the permeability transition and the binding of Ca2+ to the inner membrane. Also, phospholipase A2 activity was reduced by Mg2+. It is concluded that the high Ca2+ threshold is due to the high Mg2+ content in these tumour mitochondria.

[The effect of fluorocitrate on oxygen consumption and Ca2+ transport in the mitochondria of liver cells]. / Vliianie ftortsitrata na potreblenie kisloroda i transport Ca2+ v mitokhondriiakh kletok pecheni.

The effect of fluorocitrate on oxidative reactions and energy production systems of rat liver mitochondria has been studied. It was shown that oxidation of endogenous substrates and malate with pyruvate as well as the phosphorylation of the added ADP were inhibited by fluorocitrate. Inhibition of oxygen consumption by fluorocitrate induced the efflux of Ca2+ ions from mitochondria and a decrease in the Ca(2+)-accumulating capacity. The effect of fluorocitrate on Ca2+ transport in mitochondria is due to activation of the Ca-efflux pathway in those sensitive to ruthenium red.

[Cytotoxic effect of energy supply system inhibitors on cells from Ehrlich ascite carcinoma]. / Tsitotoksicheskoe deistvie ingibitorov sistem énergoobespecheniia na kletki astsitnoi kartsinomy Erlikha.

It was shown that inhibitors of oxidative phosphorylation (cyanide, rotenone, and oligomycin) and very low concentrations of exogenous prooxidants exerted a pronounced cytotoxic effect on Ehrlich ascites carcinoma cells. We propose that cell injury by reactive oxygen forms is the cause of the cytotoxic effect of the studied inhibitors. It was shown via flow cytometry that inhibitors of oxidative phosphorylation and exogenous prooxidants block cell progress in the cell cycle and induce appearance of cells with reduced DNA content.

[Biological role and mechanisms of realization of the Crabtree effect in rapidly proliferating cells. The role of Ca2+ ions]. / Biologicheskoe znachenie i mekhanizmy realizatsii éffekta Krebtri v bystro proliferiruiushchikh kletkakh. Rol' ionov Ca2+.

Characteristic differences in energy supply systems in normal differentiated cells versus rapidly proliferating cells including tumor cells are summarized. Previously suggested mechanisms of the Crabtree effect (inhibition of respiration by glycolysis metabolites) are specifically evaluated. The effect cannot be explained by the competition of glycolysis systems with oxidative phosphorylation for ADP and Pi. A novel mechanism of the Crabtree effect is suggested based on regulatory (inhibitory) effect of Ca2+ as the second messenger on oxidative phosphorylation in tumor cells and other rapidly proliferating cells. The Crabtree effect can be one of the main mechanisms which switch cellular energy metabolism form oxidation to anaerobic glycolysis, the latter being more beneficial for reductive biosynthetic reactions and rapid growth of the cells.