Elena M. Mil

We investigated the antiradical properties of antioxidants: carnitinate 2-ethyl-6-methyl-3-hydroxypyridine (CAR-3-HP) and carboxychromate-2-ethyl-6-methyl-3-hydroxypyridine (CCR-3-HP), and also assessed the mitochondrial response to stress (hypoxia) by analyzing the intensity of lipid peroxidation, membrane fatty acid composition, and activation of autophagy markers LC3 and Beclin-1 using spectrofluorimetry, chromatography, and immunoblotting, respectively. The studied antioxidants exhibited high antiradical activity, which was reflected in their ability to inhibit lipid peroxidation. Five-day administration of antioxidants to mice at a dose of 2 × 10⁻⁹ mol/kg prevented the activation of lipid peroxidation in liver mitochondrial membranes under conditions of acute hypobaric hypoxia. The drugs also prevented a decrease in C₁₈ and C₂₀ fatty acid levels, and also activated autophagy. In both cases, CAR-3-HP was more effective than CCR-3-HP. This drug increased the lifespan of mice almost twice as effectively under conditions of various types of hypoxias.

Oxidative stress occurs when there is an excess of reactive oxygen species (ROS) in the cell, primarily produced by mitochondria. Excess ROS trigger membrane lipid peroxidation (LPO), cause mitochondrial swelling, and release proapoptotic proteins into the cytoplasm, which can lead to apoptosis. It is assumed that antioxidants that reduce excessive ROS formation by mitochondria can increase the body’s resistance to stress factors. We investigated the effects of hypoxia and the antioxidant Ambiol (2-methyl-4-dimethylaminomethylbenzimidazole-5-ol dihydrochloride) on the functional characteristics of mitochondria, which were assessed by measuring lipid peroxidation intensity using spectrofluorimetry, mitochondrial membranes fatty acid composition using chromatography, mitochondrial morphology using atomic force microscopy, and respiration rate using polarography. Injecting mice with Ambiol at a dose of 10⁻⁶ mol/kg for 5 days prevented the stress-induced activation of lipid peroxidation, a decrease in the unsaturation index of C₁₈ and C₂₀ fatty acids in mitochondrial membranes, and swelling of these organelles. The drug also increased the efficiency of oxidative phosphorylation during the oxidation of NAD-dependent substrates. Furthermore, Ambiol increased the lifespan of mice by 3.0–4.0 times under various types of hypoxia. Ambiol’s ability to maintain initial (control) levels of C₁₈ and C₂₀ unsaturated fatty acids appears to protect against stress-induced mitochondrial dysfunction.

Currently, poly- and monophenol antioxidants should be considered not only as inhibitors that interact with free radicals, but also take into account that they are biologically active substances that affect specific targets in cells and can induce the activity of certain genes or stimulate various signaling pathways. The phenols can directly influence different points of the apoptotic process, and/or the expression of regulatory proteins. In our present study the effect of two antioxidants, sterically hindered monophenols sodium anphene (ANa) and potassium phenosan (PhK), on cell apoptosis of splenocytes was studied by fluorescence and confocal microscopy. PhK has already been introduced into medical practice in the Russian Federation because it proved effective as an anticonvulsant and was useful in treating neonatal hypoxia. The study of ANa continues; it may be a promising anticancer drug for some types of tumors. The fluorescent and confocal microscopy methods demonstrate that ANa in combination with H₂O₂ enhances apoptosis in suspension of Lewis carcinoma cells and to a lesser extent in splenocyte culture. We also discovered that autofluorescence of FAD and immunofluorescence of NADPH enzymatic complexes (with the AV-FITC fluorophore) in splenocytes of normal cells increases symbatically. The autofluorescence of FAD in splenocytes of Lewis carcinoma cells significantly exceeded that of splenocytes of healthy animals. The exact distinctive result was obtained when using potassium phenozan. It turned out that PhK prevents the development of apoptosis in mouse splenocyte cell culture (F1(CBA×C57B)). The combined use of ANa and PhK had no effect on splenocyte apoptosis. We show that fluorescence and confocal microscopy allow observing and quantifying the apoptotic effect of ANa and hydrogen peroxide, and make it possible to visualize metabolic changes in the cell, increased FAD fluorescence in tumor cells and NADPH -oxidase complexes in splenocytes. The data obtained indicate the possibility of using ANa in combination with hydrogen peroxide as an antitumor drug acting on certain types of cells. The different effects of sterically hindered monophenols ANa and PhK on the level of the anti-apoptotic protein Bcl-2 in the cell were established. ANa acts to lower Bcl-2 levels, signaling apoptosis, while PhK prevents the development of apoptosis and induces repair processes.

In some cases, the catalytic processes involve the formation of self-organized supramolecular structures due to H-bonds and other non-covalent interactions. It has been suggested that the construction of self-assembled catalytic systems is a promising strategy to mimic enzyme catalysis at the model level. As a rule, the real catalysts are not the primary catalytic complexes, but rather, those that are formed during the catalytic process. In our earlier works, we have established that the effective catalysts M(II)_xL¹_y(L¹_ox)_z(L²)_n(H₂O)_m (M = Ni, Fe, L¹ = acac⁻, L² = activating electron-donating ligand) for the selective oxidation of ethylbenzene to α-phenyl ethyl hydroperoxide are the result of the transformation of primary (Ni(Fe)L¹)_x(L²)_y complexes during the oxidation of ethylbenzene. In addition, the mechanism of the transformation to active complexes is similar to the mechanism of action of NiFeARD (NiFe-acireductone dioxygenase). Based on kinetic and spectrophotometric data, we hypothesized that the high stability of effective catalytically active complexes may be associated with the formation of stable supramolecular structures due to intermolecular hydrogen bonds and possibly other non-covalent bonds. We confirmed this assumption using AFM. In this work, using AFM, we studied the possibility of forming supramolecular structures based on iron complexes with L²-crown ethers and quaternary ammonium salts, which are catalysts for the oxidation of ethylbenzene and are models of FeARD (Fe-acireductone dioxygenase). The formation of supramolecular structures based on complexes of natural Hemin with PhOH and L-histidine or Hemin with L-tyrosine and L-histidine, which are models of heme-dependent tyrosine hydroxylase and cytochrome P450-dependent monooxygenases (AFM method), may indicate the importance of outer-sphere regulatory interactions with the participation of Tyrosine and Histidine in the mechanism of action of these enzymes.

The possibility of using antioxidants-derivatives of 3-hydroxypyridine: N-acetylcysteinate 2-ethyl-6-methyl-3-hydroxypyridine and carnitinate 2-ethyl-6-methyl-3-hydroxypyridine as plant growth regulators investigated on the pea seedlings mitochondria (Pisum sativum L.) Flora 2 cultivar. The influence of water deficiency (WD) and 3-HP on the functional state of mitochondria was studied. Water deficiency activated lipid peroxidation and led to mitochondrial swelling. An increase in LPO intensity was associated with significant changes in the content of C₁₈ fatty acids The content of linoleic and linolenic acids in the mitochondrial membranes of seedlings decreased by 12.5 and 22.3%, respectively. There were also changes in the content of C₂₀ FA: the content of 20:2 ω6 and 20:1 ω9 decreased by almost 1.65 times and 1.5 times, respectively. The swelling of mitochondria, probably, resulted in a partial loss of cytochrome C from the intermembrane space of mitochondria into the cytosol, which led to a decrease in the electron transport rates at the end section of the respiratory chain. The treatment of pea seeds with antioxidants led to the prevention of lipid peroxidation, the prevention changes in the composition of FAs membranes and mitochondrial morphology and also restored the rate of electron transport at the end section of the respiratory chain of these organelles. By preventing lipid peroxidation, the drugs helped maintain the functional state of mitochondria under WD conditions, which probably increased the resistance of pea seedlings to stress.

The article analyzes the role of hydrogen bonds and supramolecular structures in enzyme catalysis and model systems. Hydrogen bonds play a crucial role in many enzymatic reactions. However, scientists have only recently attempted to harness the power of hydrogen bonds in homogeneous catalytic systems. One of the newest directions is associated with attempts to control the properties of catalysts by influencing the “second coordination sphere” of metal complexes. The role H-bonding, and the building of stable supramolecular nanostructures due to intermolecular H-bonds, based on catalytic active heteroligand iron (Fe) or nickel (Ni) complexes formed during hydrocarbon oxidations were assessed via the AFM (Atomic-force microscopy) method, which was proposed and applied by authors of this manuscript. Th is article also discusses the roles of hydrogen bonds and supramolecular structures in oxidation reactions catalyzed by heteroligand Ni and Fe complexes, which are not only effective homogeneous catalysts but also structural and functional models of Oxygenases.

For the first time AFM (atomic-force microscopy) was used to record significant changes in the geometric parameters of the image of erythrocytes in vitro under conditions of glycolytic starvation (ATP (Adenosine triphosphate) deficiency). The difference in the action of antioxidants, phenosan K, and Ihfan-10 on erythrocytes that we detected with AFM seems to be mainly due to their difference in hydrophobicity. We used the AFM method to research the self-organization of the components of the active center of P450 (Porphyrin-450) metalloenzymes that are part of a class of hemoproteins with functions of affinity to molecular oxygen O₂. Stable supramolecular nanostructures in the form of triangular prisms based on the iron porphyrin complex with amino acids due to self-assembly involving intermolecular hydrogen bonds were received. A possible scheme for the formation of such structures is proposed.

The role of ligands in the regulation of the catalytic activity of Ni-complexes (Ni(acac)₂) in green process-selective ethylbenzene oxidation with O₂ into α-phenyl ethyl hydroperoxide is considered in this article. The dual function of phenol (PhOH) included in the coordination sphere of the nickel complex as an antioxidant or catalyst depends on the ligand environment of the metal. The role of intermolecular H-bonds and supramolecular structures (AFM method) in the mechanisms of selective catalysis by nickel complexes in chemical and biological oxidation reactions is analyzed.