It has been shown that nicotinamide coenzymes (NADPH, NADH, NADP+, NAD+) are capable of generating superoxide anions (O2─●) in an alkaline environment. The superoxide-generating activity of coenzymes is associated with high pH values and is sensitive to SOD. However, nicotinamide itself, being a functional part of coenzyme molecules, does not have this property. Polarographic studies have shown that in the presence of coenzymes, molecular oxygen is consumed from the buffer, namely oxygen activation occurs due to the formation of O2-●. Based on the results obtained and in accordance with the literature, our observations suggest that the formation of adducts of nicotinamide, which is part of the coenzyme molecule, and hydroxyl anions (OH−) may lead to the formation of O2─●. Under mild conditions in the organism, the studied coenzymes, while performing their main functions, are expected to generate superoxide, meaning that they can be signaling molecules.

Nicotinamide coenzymes can generate superoxide radicalsin alkaline environment. Their formation was registered by reduction of nitroblue tetrazolium (NBT) present in the buffer with formation of diformasan. Inhibition of diformazan formation occurs when superoxide dimutase (SOD) is added to the system, thus confirming generation of O─●. The highest superoxide generating activity was observed with NADPH. In the case of NADPH and NADH, the rate of superoxide generation was significantly lower (by approximately 50%). No O─● was detected when NAD was used under the same conditions and in the same time; however, 4 h later, diformasan was detected in the same sample. The superoxide generating activity decreased in the following order: NADPH > NADH ? NADP > NAD. Other compounds tested (adenosine, ADP and ATP) did not generate superoxide radicals even after prolonged incubation. In a cell, where a local changes in the pH of the environment are possible, nicotinamide coenzymes can be potential sources of O─● and thus participate in cell signaling. A change in pH can initiate this process.

This study has determined a survival rate of mice irradiated with accelerated carbon ions (450 MeV/nucleon) in the Bragg peak or X-ray at a dose of 6.5 Gy, using the comet assay. Levels of DNA damage (%TDNA) in blood leukocytes from mice were measured 1 day before, 1-23 days after exposure to carbon ions and 1-28 days after exposure to X-ray radiation at the same dose. According to survival and % TDNA parameters, it was found that a damaging effect of carbon ions is greater than that of X-rays and substantial variations in % TDNA, which occur in individual animals, could appear to cause individual differences in the development of genome instability in the long term. It is assumed that a higher % TDNA in leukocytes after carbon ion exposure compared to that of X-rays, a wide range of variations and asynchronous changes in individuals in the post-irradiation period are associated with the induction of clustered DNA damages and mitochondrial dysfunction, and are also due to genetic and epigenetic factors. The results obtained point to the need to assess the state of blood leukocytes in animals with a heterogeneous genetic background using the comet assay before irradiation in order to form a group with similar %TDNA values. The revealed differences in individual laboratory animals require further study in order to improve animal models in the light of the development of personalized biomedicine.

Показано, что при активации кислорода в модельных химических и биологических системах, связанных с образованием супероксидных анион-радикалов (О2●─), происходит снижение содержания молекулярного кислорода в буфере. Рассмотрены следующие химические и ферментативные реакции: автоокисление адреналина в щелочной среде, система феназинметасульфат/НАДН, а также ксантин-ксантиноксидазная реакция. В процессе химического превращения субстрата в каждой из этих реакций наблюдалось потребление О2 из буфера, что приводило к созданию временных гипоксических условий. Анион-радикалы О2●─ идентифицировались с помощью нитросинего тетразолия и супероксиддисмутазы. Впервые было установлено образование О2●─ в реакции «глюкоза-глюкозооксидаза», которую не называют супероксидгенерирующей. Каталаза в исследуемых реакциях выявляла пероксид водорода (Н2О2) - продукт диспропорционирования О2●─, и в буфере появлялся молекулярный кислород. Таким образом, показаны следующие превращения кислорода в исследуемых реакциях: О2 → О2●─ → Н2О2 → О2, которые могут происходить и во внутриклеточных системах при участии соответствующих оксидоредуктаз и антиоксидантных ферментов.

It is shown that activation of oxygen in model chemical and biological systems associated with the formation of superoxide anion radicals ( $${\text{O}}_{2}^{{\centerdot - }}$$ ) leads to a decrease in the content of molecular oxygen in the buffer. The following chemical and enzymatic reactions were considered: autooxidation of adrenaline in an alkaline medium, phenazine methosulfate/NADH, as well as the xanthine–xanthine oxidase reaction. It was found that when the chemical transformation of the substrate occurs in each of these reactions O2 is consumed from the buffer, leading to temporary hypoxic conditions. Nitro blue tetrazolium and superoxide dismutase were used to determine the production of anion radicals $${\text{O}}_{2}^{{\centerdot - }}$$ . It was discovered for the first time that $${\text{O}}_{2}^{{\centerdot - }}$$ is produced in the glucose–glucose oxidase reaction, which is known as a reaction that does not generate superoxide. Catalase in reactions reacted with hydrogen peroxide, the product of $${\text{O}}_{2}^{{\centerdot - }}$$ disproportionation, and molecular oxygen was detected under study buffer. Thus, the following oxygen conversions in the studied reactions are shown: О2 → $${\text{O}}_{2}^{{\centerdot - }}$$ → Н2О2 → О2. They may also occur in intracellular systems with the involvement of appropriate oxidoreductases and antioxidant enzymes.

This review is focused on literature data and our own research of the nontrivial quinoid pathway for the oxidation of adrenaline. All catecholamines can be oxidized similarly with formation of corresponding aminochromes. This process is simulated in vitro in an alkaline medium and is known as the adrenaline autoxidation chain reaction, whose products are adrenochrome and radical compounds, superoxide anions ( $${\text{O}}_{2}^{{ - {\kern 1pt} \centerdot }}$$ ), and other. This reaction was previously used to determine the activity of superoxide dismutase as a model of superoxide generation. We have proposed various new methodical approaches that allow the determination of the enzyme activity and reveal the anti/prooxidant properties of various compounds and materials. This pathway of conversion of one of the catecholamines (dopamine) is currently described as a “preclinical model of Parkinson’s disease.” In this regard, we have proposed the reaction of adrenaline autoxidation to be used in search for substances that can inhibit the process of quinoid oxidation, that is, to identify potential neuroprotective agents. Experimental and theoretical studies of this reaction expand the understanding of the mechanisms of free radical processes that occur in the body.

The superoxide-generating reaction of adrenaline autoxidation in an alkaline medium, used in vitro to identify the antioxidant properties of various compounds, simulates the complex multistep process of quinoid oxidation of catecholamines (CA) in the body. Sulfur-containing cysteine (Cys) and reduced glutathione (GSH), as well as oxidized glutathione (GSSG), have been shown to inhibit this process. The studied substances were considered as inhibitors of quinoid oxidation and are evaluated as antioxidants. The IC50 values for Cys and GSH were close to 7.5 mM. Inhibition by GSSG was weaker; represented approximately 50-70% of Cys and GSH. Other sulfur-containing compounds that differ in chemical structure, the amino acids taurine and methionine were ineffective. The interest in this model and the search for effective compounds acting on this reaction is associated with one of the mechanisms of the etiopathogenesis of Parkinson's disease (PD) discussed in the literature, which occurs when the biochemical transformations of dopamine CA and its quinoid oxidation process are violated. Cys, GSH and GSSG in the model system inhibit quinoid oxidation of adrenaline, as a result of which the formation of superoxide (O2 ·-) is also inhibited. Experiments with the superoxide-generating enzymatic reaction xanthine xanthioxidase, the chemistry of which is different and not related to formation of quinoid metabolites, showed that the studied substances did not inhibit O2 ·- formation in this model. Thus, it was established that the biologically active sulfur-containing compounds Cys, GSH and GSSG are specific inhibitors of quinoid oxidation of CA, and are likely to be able to play the role of a neuroprotector. It is proposed to use these compounds in the treatment and prevention of PD by activating their biosynthesis in the body.

A fluorescent biosensor is synthesized and described. The biosensor consists of polyelectrolyte microcapsules with glucose oxidase (GOx) entrapped in the cavities and an oxygen-sensitive fluorescent indicator Ru(dpp) immobilized in shells, where Ru(dpp) is tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride. The theoretical activity of the encapsulated GOx and the effect storage time and medium composition have on the stability of sensor microcapsules are determined from polarographic measurements. No change in the activity of the encapsulated enzyme and or its loss to the storage medium are detected over the test period. The dispersion medium (water or a phosphate buffer) are shown to have no effect on the activity of microcapsules with immobilized GOx. The described optical sensor could be used as an alternative to electrochemical sensors for in vitro determination of glucose in the clinically important range of concentrations (up to 10 mmol/L).

The superoxide-generating reaction of adrenaline autoxidation is widely used for determination of superoxide dismutase activity and pro/antioxidant properties of various materials. There are two variants of the spectrophotometric registration of the products of this reaction. The first one is based on registration of adrenochrome (a product of adrenaline autoxidation) at 347 nm; the second approach employs nitro blue tetrazolium (NBT) and registration of diformazan (a product of NBT reduction) at 560 nm. In the present work, recommendations for the standardization of the reaction rate in both variants have been given. The main approach consists in the use of a pharmaceutical form of 0.1% adrenaline hydrochloride solution. Although each of two adrenaline preparations available in the Russian market has some individual features in kinetic behavior of adrenaline autoxidation, they are applicable for the superoxide generating system. Performing measurements at 560 nm, the reaction rate can be regulated by lowering concentration of added adrenaline, whereas during spectrophotometric registration at 347 nm, this is not applicable. These features of the adrenaline autoxidation reaction may be attributed to the multistage process of adrenaline conversion to adrenochrome and also to coupled electron transfer from adrenaline and intermediate products of its oxidation to oxygen, carbon dioxide, and carbonate bicarbonate ions. This results in formation of corresponding radicals detectable by adding NBT.

The antioxidant properties of para-aminobenzoic acid, a substance from the group of vitamins, and its sodium salt has been found using the reaction of adrenaline autoxidation in an alkaline medium as a superoxide-generating model system. These compounds inhibited the accumulation of adrenochrome, which is a product of adrenaline oxidation, and the formation of superoxide anions, detected by their reaction with nitro blue tetrazolium. Approaches have been developed to produce a true solution of para-aminobenzoic acid and conditions were established to measure the antioxidant activity of para-aminobenzoic acid and its sodium salt. The antioxidant properties of these compounds indicate their possible participation in the redox reactions of the cell and can also be one reason that they are essential.

A new model system has been developed to study the influence of reactive oxygen species on isolated mammalian cells in conjunction with the comet assay. The glucose-glucose oxidase system was used as a hydrogen peroxide generating source. The level of DNA damage was assessed in the splenocytes and the cells of bone marrow of mouse and in human leukocytes both in untreated cells and in cells treated with hydrogen peroxide generated by glucose oxidase using the alkaline comet assay in vitro. Various options for the location of the enzyme in the slides have been studied: in the layer with the cells, in the layer above the cells, or in solution on the surface of the slides. The option where glucose oxidase was in the upper layer of 0.5% agarose over the layer of the cells was optimal. It provided separation of the enzyme from the cells and avoided obstruction to the hydrogen peroxide exposure. For the whole blood study, the content of endogenous glucose must be taken into account. This approach can be used to study the level of DNA damage induced in vitro and for the detection of DNA repair, thereby expanding the possibilities of the method, while the experiments are conducted under controlled conditions.

The physiologically active metal ions with fixed valence Ca2+ and Mg2+ were shown to accelerate epinephrine autoxidation at an alkaline pH, which proceeds via the known quinoid pathway and is accompanied by the generation of reactive oxygen species. A higher efficiency was observed for Ca2+ ions compared with Mg2+ ions. The activation of epinephrine autoxidation was evident from a decrease in the time of the initiation of the chain reaction to begin (i.e., the reaction lag) and an increase in the rate of both oxygen uptake and the formation of adrenochrome. Based on the observed effects, Ca2+ and Mg2+ cations were assumed to have the potential to play a role in the free radical processes that are associated with redox reactions in the cell and can also modulate the effect of epinephrine in the organism its oxidation via the quinoid pathway.

An important role of carbonate/bicarbonate ions has been recognized in the superoxide generating reaction of adrenaline autooxidation in an alkaline buffer (a model of quinoid adrenaline oxidation in the body). It is suggested that these ions are directly involved not only in formation of superoxide anion radical (О(2)(-)) but also other radicals derived from the carbonate/bicarbonate buffer. Using various buffers it was shown that the rate of accumulation of adrenochrome, the end product of adrenaline oxidation, and the rate of О(2)(-)· formation depend on concentration of carbonate/bicarbonate ions in the buffer and that these ions significantly accelerate adrenaline autooxidation thus demonstrating prooxidant properties. The detectable amount of diformazan, the product of nitro blue tetrazolium (NBT) reduction, was significantly higher than the amount of adrenochrome formed; taking into consideration the literature data on О(2)(-)· detection by NBT it is suggested that adrenaline autooxidation is accompanied by one-electron reduction not only of oxygen dissolved in the buffer and responsible for superoxide formation but possible carbon dioxide also dissolved in the buffer as well as carbonate/bicarbonate buffer components leading to formation of corresponding radicals. The plots of the dependence of the inhibition of adrenochrome and diformazan formation on the superoxide dismutase concentration have shown that not only superoxide radicals are formed during adrenaline autooxidation. Since carbonate/bicarbonate ions are known to be universally present in the living nature, their involvement in free radical processes proceeding in the organism is discussed.

In this paper the oil from seeds of Amaranthus cruentus L. (AmO) was shown to be an efficient modulator of the physical chemical properties of artificial lipid and rat hepatocyte plasma membranes. AmO improved the membrane stability, their stress resistance and the adsorption of neurotensin to plasma membranes with the distinct biphasic interactions being observed even after adrenalin stress exposure. The analysis of pro-/antioxidant balance in rat blood revealed a mild prooxidant activity after AmO intake, which was accompanied by accumulation of oxidative destruction products in plasma membranes. This prooxidant action of AmO was corroborated in vitro in an adrenalin autooxidation model. On the other hand, the observed improved resistance to adrenalin stress in AmO supplemented rats was associated with an antioxidant response in blood and plasma membrane studies. The AmO effects can be attributed to the modulation of the metabolic pathways involved into oxygen and free radical homeostasis.

Several parameters of the cytoplasmic enzymatic antioxidant system of the liver and brain of the rat have been investigated under conditions of immobilization stress and of an antioxidant preparation in the diet of animals. These included superoxide dismutase (SOD) and glutathione reductase (GR) activities and nonspecific NADPH oxidation. Only changes in the activity of SOD both in the liver and brain were revealed. In the liver of animals that receive no preparation, a decrease in the activity of SOD after 30-min immobilization and its restoration after a 360-min immobilization were observed. In the brain, the activity of SOD decreased only in preconditioned animals after 30 and 360 min of exposure to stress. In addition, the activity of SOD in the brain of preconditioned animals, both stressed and unstressed, was lower than in the corresponding groups of control animals. It is probable that, under the conditions of immobilization stress, the level of reactive oxygen species (ROS) and as a consequence the activity of SOD decrease. The intake of an antioxidant preparation under these conditions seems to be not correct.

The reaction of adrenaline autoxidation in an alkaline buffer with the formation of superoxide radicals and the product of its oxidation, adrenochrome, which models the quinoid pathway of adrenaline conversion in the body, is accompanied by oxygen consumption. This reaction is applicable for polarographic determination of the activity of superoxide dismutase and the antioxidant properties of biological and chemical compounds, it is based on evaluation of the latent period and the rate of oxygen consumption, which are measured in the presence of the compounds examined. It was assumed that the neuro- and cardiotoxicity of quinone products of adrenaline oxidation is related not only to their "own" properties and reactive oxygen species formed but also the hypoxia of those regions of the cell and tissue where the quinoid oxidation of adrenaline occurs.

The addition of nitro blue tetrazolium (NBT) into the reaction of adrenaline autooxidation allows direct identification of superoxide anion formation (O 2 −⊙ ) as well as demonstration of kinetics of their accumulation in this superoxide-generating system. The kinetics of adrenochrome and O 2 −⊙ formation has been compared under the same conditions. Three possible approaches to the use of the adrenaline autooxidation reaction for the determination of superoxide dismutase activity (SOD) and revealing antioxidant properties of various compounds are discussed. Two of these approaches have been described previously: the spectro-photometric method of registration of adrenochrome, an end product of adrenaline autooxidation, at 347 nm (Sirota, 1999) and the polarographic method, which measures oxygen consumption used for O 2 −⊙ formation (Sirota, 2011). Here, a novel approach to this problem is presented; it is based on spectrophotometric determination of O 2 −⊙ using NBT. The employment of this approach results in a significant decrease of the pH value of carbonate buffer from 10.5 to 9.7 and a 4-fold decrease in the amounts of added adrenaline, thus creating milder conditions for the revealing and investigation of antioxidant properties of materials being examined.