Alexey G. Kruglov

Reactive oxygen species are involved in the stimulation of the mitochondrial permeability transition by dihydrolipoate

Dihydrolipoic acid (DHLA) has been found to stimulate the Ca(2+)-induced mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) [Biochem. Mol. Biol. Int. 44 (1998) 127] which could be due to its prooxidant properties. We therefore investigated whether DHLA stimulated superoxide anion (O(2)(.-)) generation in RLM and in bovine heart submitochondrial particles (SMP). In RLM DHLA caused a concentration-dependent O(2)(.-) generation assayed by lucigenin chemiluminiscence. The stimulation was seen with the lowest concentrations of DHLA (5 microM) with pyruvate as the respiratory substrate, with 2-oxoglutarate or especially succinate the stimulation was less pronounced. Stimulation of O(2)(.-) production by DHLA was also observed in bovine heart SMP using an electron spin-trapping technique. Radical scavengers (butylhydroxytoluene and TEMPO) decreased O(2)(.-) generation induced by DHLA and inhibited MPT. Slight reduction of the mitochondrial membrane potential by a small amount of a protonophorous uncoupling agent also delayed the DHLA-induced MPT. These data indicate that the stimulation of MPT by DHLA is due to DHLA-derived prooxidants, i.e. stimulated production of O(2)(.-) and possibly other free radicals.

Novel Mycotoxin from Acremonium exuviarum Is a Powerful Inhibitor of the Mitochondrial Respiratory Chain Complex III

A novel mycotoxin named acrebol, consisting of two closely similar peptaibols (1726 and 1740 Da), was isolated from an indoor strain of the mitosporic ascomycete fungus Acremonium exuviarum. This paper describes the unique mitochondrial toxicity of acrebol, not earlier described for any peptaibol. Acrebol inhibited complex III of the respiratory chain of isolated rat liver mitochondria (1 mg of protein mL(-1)) with an IC(50) of approximately 80 ng mL(-1) (50 nM) after a short preincubation, and 350 ng mL(-1) caused immediate and complete inhibition. Acrebol thus is a complex III inhibitor almost as potent as antimycin A and myxothiazol but completely different in structure. Similarly to myxothiazol but in contrast to antimycin A, acrebol decreased the level of mitochondrial superoxide anion detectable by chemiluminescent probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one. Unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria, did not induce disturbance of the ionic homeostasis or the osmotic balance of mitochondria, and did not release apoptogenic proteins like cytochrome c from the intermembrane space of mitochondria. In boar spermatozoa, acrebol inhibited the respiratory chain and caused ATP depletion by activation of the oligomycin-sensitive F(0)F(1)-ATPase, which resulted in the inhibition of the progressive movement. In mouse insulinoma MIN-6 cells, whose energy supply solely depends on oxidative phosphorylation, acrebol induced necrosis-like death. The pathophysiological relevance of these findings is discussed.

Combined Vitamins B12b and C Induce the Glutathione Depletion and the Death of Epidermoid Human Larynx Carcinoma Cells HEp-2

The combination of hydroxocobalamin (vitamin B12b) and ascorbicacid (vitamin C) can cause the death of tumor cells at the concentrationsof the components at which they are nontoxic when administeredseparately. This cytotoxic action on epidermoid human larynx carcinomacells HEp-2 in vitro is shown to be due to the hydrogen peroxidegenerated by the combination of vitamins B12b and C. The drop inthe glutathione level preceding cell death was found to be the result ofcombined action of the vitamins. It is supposed that the induction of celldeath by combined action of vitamins B12b and C is connected to the damageof the cell redox system.

External mitochondrial NADH-dependent reductase of redox cyclers: VDAC1 or Cyb5R3?

It was reported that VDAC1 possesses an NADH oxidoreductase activity and plays an important role in the activation of xenobiotics in the outer mitochondrial membrane. In the present work, we evaluated the participation of VDAC1 and Cyb5R3 in the NADH-dependent activation of various redox cyclers in mitochondria. We show that external NADH oxidoreductase caused the redox cycling of menadione ≫ lucigenin>nitrofurantoin. Paraquat was predominantly activated by internal mitochondria oxidoreductases. An increase in the ionic strength stimulated and suppressed the redox cycling of negatively and positively charged acceptors, as was expected for the Cyb5R3-mediated reduction. Antibodies against Cyb5R3 but not VDAC substantially inhibited the NADH-related oxidoreductase activities. The specific VDAC blockers G3139 and erastin, separately or in combination, in concentrations sufficient for the inhibition of substrate transport, exhibited minimal effects on the redox cycler-dependent NADH oxidation, ROS generation, and reduction of exogenous cytochrome c. In contrast, Cyb5R3 inhibitors (6-propyl-2-thiouracil, p-chloromercuriobenzoate, quercetin, mersalyl, and ebselen) showed similar patterns of inhibition of ROS generation and cytochrome c reduction. The analysis of the spectra of the endogenous cytochromes b5 and c in the presence of nitrofurantoin and the inhibitors of VDAC and Cyb5R3 demonstrated that the redox cycler can transfer electrons from Cyb5R3 to endogenous cytochrome c. This caused the oxidation of outer membrane-bound cytochrome b5, which is in redox balance with Cyb5R3. The data obtained argue against VDAC1 and in favor of Cyb5R3 involvement in the activation of redox cyclers in the outer mitochondrial membrane.

Sulfur-containing compounds quench 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one chemiluminescence: Discrimination between true antioxidants and quenchers using xanthine oxidase.

The probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) is widely used for studying the superoxide anion production and the efficiency of antioxidants in biological systems. Here we report that a number of sulfur-containing compounds applied in biochemical and cytological studies are able to suppress MCLA-derived chemiluminescence (MDCL) independent of their capability to scavenge superoxide anion. The most effective MDCL quenchers appeared to be the substances with thiocarbamoyl and thiocarbonyl groups coupled to cyclic molecules and several thiol- and disulfide-containing compounds. The analysis of MDCL kinetics in a xanthine oxidase system allows one to rapidly discriminate between true antioxidants and the quenchers of chemiluminescence.

Cereulide produced by Bacillus cereus increases the fitness of the producer organism in low-potassium environments

Cereulide, produced by certain Bacillus cereus strains, is a lipophilic cyclic peptide of 1152 Da that binds K(+) ions with high specificity and affinity. It is toxic to humans, but its role for the producer organism is not known. We report here that cereulide operates for B. cereus to scavenge potassium when the environment is growth limiting for this ion. Cereulide-producing B. cereus showed higher maximal growth rates (µ(max)) than cereulide non-producing B. cereus in K(+)-deficient medium (K(+) concentration ~1 mM). The cereulide-producing strains grew faster in K(+)-deficient than in K(+)-rich medium with or without added cereulide. Cereulide non-producing B. cereus neither increased µ(max) in K(+)-deficient medium compared with K(+)-rich medium, nor benefited from added cereulide. Cereulide-producing strains outcompeted GFP-labelled Bacillus thuringiensis in potassium-deficient (K(+) concentration ~1 mM) but not in potassium-rich (K(+) concentration ~30 mM) medium. Exposure to 2 µM cereulide in potassium-free medium lacking an energy source caused, within seconds, a major efflux of cellular K(+) from B. cereus not producing cereulide as well as from Bacillus subtilis. Cereulide depleted the cereulide non-producing B. cereus and B. subtilis cells of a major part of their K(+) stores, but did not affect cereulide-producing B. cereus strains. Externally added 6-10 µM cereulide triggered the generation of biofilms and pellicles by B. cereus. The results indicate that both endogenous and externally accessible cereulide supports the fitness of cereulide-producing B. cereus in environments where the potassium concentration is low.

Role of mitochondrial thiols of different localization in the generation of reactive oxygen species

The role of thiols of the outer and the inner membranes of mitochondria in the regulation of generation of reactive oxygen species (ROS) has been studied. It was found that N-ethylmaleimide (NEM), which penetrates through the mitochondrial membrane and binds thiols to form thioesters, at concentrations from 20 to 250 μM activates the production of superoxide anion and hydrogen peroxide during the oxidation of the substrates of complexes I and II of the respiratory chain. 5′,5′-Dithiobis-(2-nitrobenzoate) (DTNB), which does not penetrate into mitochondria and binds thiols to form disulfides, weakly activates hydrogen peroxide production during the oxidation of NAD-dependent substrates and inhibits the ROS production upon succinate oxidation. DTNB is particularly effective in inhibiting the menadione-induced formation of ROS. The differences in the ROS formation by these reagents are explained by the fact that they influence different thiol-containing proteins and enzymes. As distinct from NEM, which inhibits complex I of the respiratory chain, DTNB has no effect on the respiratory chain of mitochondria but can bind the SH-groups of NADH-quinone oxidoreductase, which is localized in the outer mitochondrial membrane and participates in the redox cycle of menadione. It was also shown that the ability to inhibit the ADP-stimulated respiration, a feature inherent in both reagents, does not significantly contribute to ROS production.

Rapid fluorescent visualization of multiple NAD(P)H oxidoreductases in homogenate, permeabilized cells, and tissue slices.

Intracellular NAD(P)H oxidoreductases are a class of diverse enzymes that are the key players in a number of vital processes. The method we present and validate here is based on the ability of many NAD(P)H oxidoreductases to reduce the superoxide probe lucigenin, which is structurally similar to flavins, to its highly fluorescent water-insoluble derivative dimethylbiacridene. Two modifications of the method are proposed: (i) an express method for tissue homogenate and permeabilized cells in suspensions and (ii) a standard procedure for cells in culture and acute thin tissue slices. The method allows one to assess, visualize, and localize, using fluorescent markers of cellular compartments, multiple NADH and NADPH oxidoreductase activities. The application of selective inhibitors (e.g., VAS2870, a NOX2 inhibitor; plumbagin, a NOX4 inhibitor) allows one to distinguish and compare specific NAD(P)H oxidoreductase activities in cells and tissues and to attribute them to known enzymes. The method is simple, rapid, and flexible. It can be easily adapted to a variety of tasks. It will be useful for investigations of the role of various NAD(P)H oxidoreductases in a number of physiological and pathophysiological processes.

Effect of Melatonin on Rat Heart Mitochondria in Acute Heart Failure in Aged Rats

Excessive generation of reactive oxygen species (ROS) in mitochondria and the opening of the nonselective mitochondrial permeability transition pore are important factors that promote cardiac pathologies and dysfunction. The hormone melatonin (MEL) is known to improve the functional state of mitochondria via an antioxidant effect. Here, the effect of MEL administration on heart mitochondria from aged rats with acute cardiac failure caused by isoprenaline hydrochloride (ISO) was studied. A histological analysis revealed that chronic intake of MEL diminished the age-dependent changes in the structure of muscle fibers of the left ventricle, muscle fiber swelling, and injury zones characteristic of acute cardiac failure caused by ISO. In acute heart failure, the respiratory control index (RCI) and the Ca2+ retention capacity in isolated rat heart mitochondria (RHM) were reduced by 30% and 40%, respectively, and mitochondrial swelling increased by 34%. MEL administration abolished the effect of ISO. MEL partially prevented ISO-induced changes at the subunit level of respiratory complexes III and V and drastically decreased the expression of complex I subunit NDUFB8 both in control RHM and in RHM treated with ISO, which led to the inhibition of ROS production. MEL prevents the mitochondrial dysfunction associated with heart failure caused by ISO. It was shown that the level of 2′,3′-cyclicnucleotide-3′-phosphodiasterase (CNPase), which is capable of protecting cells in aging, increased in acute heart failure. MEL also retained the CNPase content in RHM both in control experiments and after ISO-induced heart damage. We concluded that an increase in the CNPase level promotes cardioprotection.

Glutamate contributes to alcohol hepatotoxicity by enhancing oxidative stress in mitochondria

Chronic alcohol intoxication is associated with increased oxidative stress. However, the mechanisms by which ethanol triggers an increase in the production of reactive oxygen species (ROS) and the role of mitochondria in the development of oxidative stress has been insufficiently studied. The biochemical and proteomic data obtained in the present work suggest that one of the main causes of an increase in ROS generation is enhanced oxidation of glutamate in response to long-term alcohol exposure. In the course of glutamate oxidation, liver mitochondria from alcoholic rats generated more superoxide anion and H2O2 than in the presence of other substrates and more than control organelles. In mitochondria from alcoholic rats, rates of H2O2 production and NAD reduction in the presence of glutamate were almost twice higher than in the control. The proteomic study revealed a higher content of glutamate dehydrogenase in liver mitochondria of rats subjected to chronic alcohol exposure. Simultaneously, the content of mitochondrial catalase decreased compared to control. Each of these factors stimulates the production of ROS in addition to ROS generated by the respiratory chain complex I. The results are consistent with the conclusion that glutamate contributes to alcohol hepatotoxicity by enhancing oxidative stress in mitochondria.