Protein SH Groups Research Articles

Активність ензимів поліольного шляху у нирках щурів за умов різної забезпеченості раціону протеїном і сахарозою

The study examined the activity of enzymes in the polyol pathway and the status of free radical processes in the kidneys of rats subjected to different dietary protein and sucrose levels. It was found that the kidneys of animals consuming a highsucrose diet, irrespective of protein content, exhibited the highest increase in the activity of polyol pathway enzymes, namely aldose reductase and sorbitol dehydrogenase. Consumption of a high-sucrose diet enhances the generation of O2 •- in the kidneys by almost twofold, and hydroxyl radical by over fourfold compared to control indicators. This is accompanied by a twofold increase in the content of TBARS-active products and a threefold increase in the content of carbonyl derivatives of proteins, against the background of a decrease in the content of free thiol groups in proteins. The pronounced generation of hydroxyl radicals, accumulation of TBARS-active products, and reduction in the content of free SH-groups in proteins are characteristic of animals that consumed a high-sucrose diet in the context of dietary protein deficiency. It has been demonstrated that excessive sucrose consumption is a critical factor, influencing the activity of enzymes in the polyol pathway and the intensity of free radical processes. The obtained results may be considered as predisposing factors for disruptions in the structural and functional organization of the kidneys under conditions of nutrient imbalance.

Thiol Homeostasis in The Crystalline Eye and Influence on It Irradiation by Decimeter Electromagnetic Radiation (Experiments on Rats)

Electromagnetic radiation (EMR) in the microwave range, even at non-thermal intensity, causes significant biochemical and physiological changes in living organisms, which are supposed to be associated with their possible oxidative effect. This work is devoted to the study of the mechanism of realization of the EMR effect in the eye lens at the level of redox state elements, based on the fact that this organ is the most suitable model: it functions semiautonomously and has a well-organized system of antioxidant protection. The transparency of the lens is maintained by preserving the redox balance, in which the homeostasis of thiol compounds of protein and non-protein nature plays an important role. Our experiments were performed on rats using 460 MHz EMR for exposure at non-thermal intensities (power flux density between 10 and 30 µW/cm2). It has been shown that chronic exposure to EMR for up to two weeks caused changes in the redox state of the lens, which manifested in changes in the level of lipid peroxidation processes and the content of thiols of various natures. The substructures of the lens (cortical and nuclear regions) reacted to EMR exposure in different ways. Depending on the EMR intensity, pro- and antioxidant characters were revealed in their reactions. The dynamics of the oxidative reaction of lens substructures were also different under high- and low-intensity exposure. The character of the kinetics of changes in the products of oxidative reactions (malondialdehyde and lipid hydroperoxides) and reducing agents (non-protein and protein SH groups) in the lens of the irradiated organism suggested the role of the enzymatic thiolation-dethiolation system to preserve the redox balance in the substructures of the lens. In addition, the results on changes (kinetics) in the content of various protein SH-groups, i.e., hidden inside the protein molecule and exposed on its surface, during EMR exposure, as well as the data available in the literature, allow us to put forward suggestions about the supramolecular mechanism of homeostasis regulation, in particular, thiol homeostasis regulation in such high-protein structures as the lens, which can be realized by aggregation-disaggregation of protein molecules (crystallins in the case of the lens). Our results can serve as a basis for developing a new non-invasive approach to cataract prevention using low-intensity microwave radiation.

Crystalline lens: redox changes in response to the action of non-ionizing electromagnetic radiation

Electromagnetic radiation (EMR) in the microwave range, even at non-thermal intensity, causes significant biochemical and physiological changes in living organisms, which are supposed to be associated with its possible oxidative effect. This work is devoted to the study of the mechanism of realization of the EMR effect in the eye lens at the level of redox state elements, based on the fact that this organ is the most suitable model: it functions semi-autonomously and has a well-organized system of antioxidant protection. The transparency of the lens is maintained by preserving the redox balance, in which the homeostasis of thiol compounds of protein and non-protein nature plays an important role. Our experiments were performed on rats using 460 MHz EMR for exposure at non-thermal intensities (power flux density between 10 and 30 µW/cm2). It has been shown that chronic exposure to EMR for up to two weeks caused changes in the redox state of the lens, which manifested in changes in the level of lipid peroxidation processes and the content of thiols of various natures. The substructures of the lens (cortical and nuclear regions) reacted to EMR exposure in different ways. Depending on the EMR intensity, pro- and antioxidant characters were revealed in their reactions. The dynamics of the oxidative reaction of lens substructures were also different under high- and low-intensity exposure. The character of the kinetics of changes in the products of oxidative reactions (malondialdehyde and lipid hydroperoxides) and reducing agents (non-protein and protein SH groups) in the lens of the irradiated organism suggested the role of the enzymatic thiolation-dethiolation system to preserve the redox balance in the substructures of the lens. In addition, the results on changes (kinetics) in the content of various protein SH-groups, i.e., hidden inside the protein molecule and exposed on its surface, during EMR exposure, as well as the data available in the literature, allow us to put forward suggestions about the supramolecular mechanism of homeostasis regulation, in particular, thiol homeostasis regulation in such high-protein structures as the lens, which can be realized by aggregation-disaggregation of protein molecules (crystallins in the case of the lens). Our results can serve as a basis for developing a new non-invasive approach to cataract prevention using low-intensity microwave radiation.

Changes in the parameters of pro-oxidant–antioxidant balance in the brain of rats with stepwise incomplete cerebral ischemia

Objective: to evaluate the activity of oxidative stress in rats with stepwise incomplete cerebral ischemia (SICI). Material and methods. Experiments were performed on 24 male outbred rats weighing 260±20 g, allocated into 3 subgroups (6 animals in each) based on time of ligation of both common carotid arteries (CCA). The control group comprised of sham-operated rats of the same sex and similar weights (n=6). To identify the pro-oxidant–antioxidant state of the brain based on its homogenates, the activity of lipid peroxidation processes, the content of thiobarbituric acid reactive substances (TBARS), the concentration of reduced glutathione (GSH), total thiol groups (TSH), and the activity of glutathione peroxidase were determined. Results. SICI with ligation interval of both CCAs of 1 day and 3 days led to a significant decrease in the total SH groups of proteins and glutathione by 30% (p=0.038) and 46% (p=0.044), respectively, TBARS concentration by 29% (p=0.038) and 31% (p=0.043), respectively. SICI with the maximum interval between CCA ligations was manifested by less pronounced changes in the pro-oxidant–antioxidant state of the brain. Conclusion. In SICI with ligation of both CCAs 7 days apart, at which histological changes were the least pronounced, changes in the pro-oxidant–antioxidant balance were insignificant. The most pronounced disorders of the pro-oxidant–antioxidant balance in the brain were observed in the subgroup with the minimum interval between CCA ligations, which implied the highest activity of oxidative stress.

ПОКАЗНИКИ СИСТЕМИ АНТИОКСИДАНТНОГО ЗАХИСТУ У ПЛАЗМІ КРОВІ ХВОРИХ НА ОСТЕОАРТРИТ ПІСЛЯ SARS-CoV-2-ІНФЕКЦІЇ

An outbreak of pneumonia that began in December 2019 in Wuhan (China) caused the spread of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). After an increase in the number of positive cases of infection in China, on January 30, 2020, the World Health Organization declared the virus epidemic a public health emergency. The 2019 coronavirus disease (Coronavirus disease 2019, COVID-19) has a devastating effect on the health of people in many countries around the world. It is known that the SARS-CoV-2 virus, in addition to damaging the lung parenchyma, can damage other organ systems and complicate the condition of patients with comorbid diseases, in particular, with osteoarthritis. Oxidative stress plays an important role in the formation and progression of osteoarthritis. Its development is associated with a violation of the oxidative-antioxidant balance. The aim of the work was to determine the indicators of the antioxidant system in the blood plasma of patients with OA after SARS-CoV-2 infection. All study participants were further divided into three experimental groups: Group I – conditionally healthy people, Group II – patients with OA of the knee joints of the II-III degree, and Group III – patients with OA of the knee joints of the II–III degree who suffered a mild or moderately severe form COVID-19 6–9 months ago. Superoxide dismutase and catalase activity and the content of sulfhydryl groups were determined by generally accepted biochemical methods. Processing of research results was carried out using generally accepted methods of variational statistics. It was found that the activity of anti-radical enzymes (superoxide dismutase and catalase) and the content of compounds containing SH-groups (of protein and non-protein nature) are reduced in the blood plasma of osteoarthritis patients who contracted COVID-19. The obtained results indicate a shift of the oxidant-antioxidant balance in the pro-oxidant direction. These disorders are more profound compared to patients with osteoarthritis who did not suffer from coronavirus infection. The results obtained by us indicate that patients with osteoarthritis may experience a more severe course of osteoarthritis and develop complications after infection with COVID-19.

Intracellular Location and Function of Nuclear Factor of Erythroid Origin 2 (Nrf2) in Modeling Oxidative Stress <i>in vitro</i>

INTRODUCTION: Nuclear factor E2-related factor 2 (Nrf2) is a member of capncollar (CNC) family of subfamily of leucine zipper transcription factors that regulates cell protection against toxic substances and oxidants.
 AIM: To determine location, mechanism of activation and role of Nrf2 in conditions of oxidative stress in vitro.
 MATERIALS AND METHODS: The study was performed on human colon adenocarcinoma cell line (Caco-2). Oxidative stress (OS) was modeled by adding hydrogen peroxide (Н2О2) at concentrations of 0.1 М100 М to the nutritive medium and incubation for 24 and 72 hours. In assessment of Nrf2 function, its inhibitor ― AEM1 ― was added to cells at a concentration of 5 М. The extent of OS development was determined using photometric methods by the concentration of protein SH-groups and carbonyl derivatives of protein, and the activity of superoxide dismutase (SOD). Viability of cells was assessed by the results of cytotoxic test (MTT assay), the amount of Nrf2 in the cytoplasm and nucleus was determined by heterogenous ELISA method.
 RESULTS: Incubation of Caco-2 cells with Н2О2 resulted in decrease in the level of protein SH-groups and increase in the concentration of carbonyl derivatives of protein. In incubation with H2O2 at concentrations of 0.1 М10 М for 24 hours and 10 М for 72 hours, the activity of SOD increased. At concentrations of Н2О2 of 50 М and 100 М (24 hour and 72 hour), SOD activity and viability of cells decreased. Exposure to Н2О2 led to translocation of Nrf2 from the cytoplasm into nucleus. Direct correlation dependence was revealed between concentration of protein SH-groups and the amount of Nrf2 in the cytoplasm in incubation with H2O2 for 24 hour (r = 0.44, р = 0.03), 72 hour (r = 0.34, р = 0.05). The amount of Nrf2 in the nucleus positively correlated with SOD activity in the cytoplasm on exposure to H2O2 for 24 hour (r = 0.77, р = 0.0001) and 72 hour (r = 0.36, р = 0.06). In inhibition of Nrf2 in conditions of exposure to H2O2, the viability of cells decreased to a larger extent.
 CONCLUSION: Hydrogen peroxide induces the nuclear translocation of Nrf2, which promotes activation of antioxidant enzyme SOD and preserves viability of cells of OS conditions in vitro.

DISTURBANCE OF PRO-OXIDANT-ANTIOXIDANT BALANCE IN RATS WITH CEREBRAL ISCHEMIA

Excess of reactive oxygen can lead to membrane damage, accumulation of lipid, protein, and nucleic acid oxidation products, deficiency of reduced pyridine nucleotides and phospholipids of mitochondrial membranes, and then to electrolyte imbalance, mitochondrial swelling, uncoupling of oxidation and phosphorylation processes, and ischemic neuronal death. Thus, the study of oxidative stress and antioxidant system activity is relevant.
 The aim of the study is to examine the changes in the pro-oxidant-antioxidant balance in rats with ischemic brain damage of different degrees of severity (subtotal and total cerebral ischemia).
 Materials and Methods. The experiments were performed on 30 male outbred white rats weighing 260±20 g in compliance with the requirements of the Directive of the European Parliament and the Council of the European Union No. 2010/63/EU of September 22, 2010 on the protection of animals used for scientific purposes.
 Results. A more significant decrease in the content of total SH-groups of proteins and glutathione (by 58 (51; 64) % (p<0.05)), and GSH concentration (by 29 (19; 35) % (p<0.05)) was observed under 24-hour subtotal brain ischemia (SBI) compared with 1-hour SBI. Changes in the glutathione peroxidase activity were multidirectional: in 1-hour SBI, the activity increased by 12 (9; 18) % (p<0.05compared to the control level, and in 24-hour SBI, it decreased by 74 (67; 81) % (p<0.05). In 1-hour SBI, the content of total SH-groups of proteins and glutathione was higher by 60 (54; 65) % (p<0.05), and GSH concentration was higher by 42 (39; 56) % (p<0.05) compared with 1-hour total brain ischemia (TBI). The content of products that react with thiobarbituric acid increased by 59 (51; 63) % (p<0.05). In 24-hout SBI, the content of total SH-groups of proteins and glutathione was higher by 36 (29; 45) % (p<0.05), and GSH concentration was higher by 63 (59; 75) % (p<0.05) compared with 24-hour TBI. The content of products that react with thiobarbituric acid increased by 83 (78; 91) % (p<0.05). The glutathione peroxidase activity in TBI was equal to zero.
 Conclusions. Thus, the most pronounced disturbances in the pro-oxidant-antioxidant balance were observed in 24-hour TBI. Similar, but less pronounced disturbances were observed in 24-hour SBI.

Інтенсивність вільнорадикальних процесів у скелетних м’язах щурів за умов різної забезпеченості раціону нутрієнтами

The intensity of free-radical processes in the skeletal muscles of rats at different dietary supply with protein and sucrose was studied. It has been established that the most pronounced intensification of free radical processes in the mitochondria of skeletal muscles is found in animals kept on a low-protein/ high-sucrose diet. In particular, the generation of superoxide anion-radical increases more than 5,3-fold and the generation of hydroxyl radical more than 3,2-fold compared with the control, more than a 14,3-fold increase in the carbonyl derivatives levels, and also decreases by three times in the free protein SH-groups levels against the background of a 1,7-fold decrease in catalase activity. It has been shown that excessive consumption of sucrose is a critical factor influencing the intensity of free radical processes in skeletal muscle mitochondria. The detected changes can be considered as prerequisites for skeletal muscle dysfunction under the conditions of nutrient imbalance.

Status of prooxidant and antioxidant systems in the sperm and seminal plasma of breeding boars of large white breed and SS23 synthetic line.

The indicators of pro- and antioxidant systems in sperm and sperm plasma of breeding boars of Large White breed and SS23 synthetic line were studied. Measurements of antioxidant enzyme activity, determination of lipid peroxidation (LPO) product content, and antioxidant factor were performed. Lipid peroxidation in the semen of healthy breeding boars was characterized by a stable level of activity, which is necessary to ensure normal reproductive functions. Additionally, there was a high content of low molecular weight thiols and proteins. The concentration of SH-groups in spermatozoa was higher (P≤0.05) compared to sperm plasma. The number of total, protein, and free SH-groups in the semen of boars of the synthetic line was higher (P<0.05) in relation to animals of the Large White breed. Low catalase (CAT) activity in the sperm was compensated by glutathione peroxidase (GPX). The content of ceruloplasmin (CP) in the sperm of boars was almost twice as high as that of sperm plasma. In spermatozoa, high content of reduced glutathione (GTH) was recorded, which was more than 3 times higher than in the seminal fluid. The main antioxidants of spermatozoa were superoxide dismutase (SOD), CP, SH-groups of proteins, and reduced content of GTH. We revealed that CAT is a key enzyme that neutralizes excess hydrogen peroxide in boar semen. In contrast, in sperm, hydrogen peroxide was inactivated mainly by GPX. Further research on the mechanisms of action of reactive oxygen species on boar semen will help to develop effective methods for sperm storage and successful fertilization of oocytes.

INTENSITY OF FREE RADICAL PROCESSES IN THE POSTNUCLEAR FRACTION OF RAT KIDNEYS UNDER INFLUENCE OF LASER IRRADIATION AND ω-3 POLYUNSATURATED FATTY ACIDS

Introduction. The effect of laser irradiation on the body can lead to local tissue damage, which will be accompanied by changes in biochemical processes that occur in the kidneys. The direction of these changes is completely unclear, so the study of free radical oxidation of lipids and proteins as the main markers of oxidative stress in the body and their correction by ω-3 polyunsaturated fatty acids (PUFA) remains relevant. Aim. To evaluate the intensity of lipid peroxidation and oxidative modification of proteins in the postnuclear fraction of rat kidneys under the action of laser irradiation and additional administration of ω-3 PUFA. Methods. The studies used white outbred rats, which were irradiated daily for 4 minutes with a laser diode with a wavelength of 650 nm, power of 50 mW in the abdominal cavity at a distance of 10 cm from the skin surface. The state of lipid peroxidation (LPO) was determined by the level of primary, secondary and final products. The intensity of oxidative modification of proteins (OMP) was evaluated by the level of carbonyl derivatives and protein SH-groups. To correct the prooxidant state, the animals were additionally injected with ω-3 PUFA. Results. Low-intensity laser irradiation has a destructive effect on the cell membranes of the kidneys, which is expressed by an increase in primary, secondary and final products of LPO in phospholipid extracts and an increase in the intensity of OMP. The introduction of ω-3 PUFAs reduces free radical processes in the kidneys of irradiated rats, but this effect depends on the scheme of their introduction. ω-3 PUFAs do not show antioxidant effect under the conditions of their introduction after laser irradiation. Daily administration of ω-3 PUFA two hours before irradiation shows a slight antioxidant effect only in the initial stages of irradiation. Preliminary seven-day administration of ω-3 PUFA before laser irradiation is the most effective, as it helps to reduce free radical processes. Conclusion. It is established that ω-3 PUFAs are able to have a corrective effect on the action of low-intensity laser irradiation, which depends on the scheme of their introduction. The highest antioxidant effect is observed in groups of animals to which ω-3 PUFA was previously administered before the action of laser irradiation.

Potentials of intravenous laser blood irradiation to correct peroxidation and antioxidant activity as a result of surgical distress

Purpose: to reveal protective effects of intravenous low-level laser blood irradiation (ILBI) during surgical interventions at the gallbladder as well as to study the state of antioxidant activity, lipid peroxidation and endogenous intoxication.Material and methods. In the perioperative period, 40 patients from the main group (I) had traditional treatment which included pharmacological preparations and sessions of intravenous laser blood irradiation. 45 patients from the control group (II) had only traditional (pharmacotherapeutic) treatment. In both groups during surgery, the anesthetic protection consisted of classical neuroleptanalgesia. The age of patients in both groups did not differ significantly. Intravenous laser blood irradiation was done with helium-neon laser “Atoll” 632 nm (Fokon Ltd, St-Petersburg, Russia). During surgery, three irradiation sessions were performed at radiation power of 20 mW and exposure 15 minutes at various stages of surgery: 30 minutes before induction of anesthesia, at the traumatic stage and 30 minutes before the expected finish of the surgery. Ceruloplasmin and transferrin levels in the blood plasma were assessed with the electron paramagnetic resonance technique. The ratio of ceruloplasmin/transferrin which characterizes the level of antioxidant activity was also established. The level of endogenous intoxication was assessed using the previously published technique. The quantitative characteristics of endogenous intoxication by this technique is the value of optical density (D280) and total optical density of components related to high molecular weight and low molecular weight fractions.Results. ILBI sessions in patients neutralize changes in the level of ceruloplasmin and transferrin in blood. The data obtained also confirm ILBI protective effects at protein SH-groups or activation of the restoration of protein SH-groups that were damaged by oxidation as a result of surgical aggression and trauma.Conclusion. The protective effect of intravenous laser blood irradiation in addition to the standard preoperative therapy optimizes pharmacological anesthetic protection during surgery under the existing traditional schemes of combined general anesthesia.

Identification of a metallothionein gene and the role of biological thiols in stress induced by short-term Cd exposure in Ostrinia nubilalis

Cadmium (Cd) is a non-essential metal that is highly toxic to all living forms, characterized by an extremely high affinity for thiol (SH) groups. The aim of this work was to identify and experimentally verify metallothionein gene and to analyze the role of biological thiols in stress induced by short-term Cd exposure in Ostrinia nubilalis, one of the most important corn pests. The coding region of a metallothionein (MT) gene in O. nubilalis was identified, encoding protein, OnMT1, which contains 46 amino acids, including 12 cysteine residues, and has no aromatic amino acids. Phylogenetic analysis revealed that OnMT1 clustered together with metallothionein from Bombyx mori. Structural bioinformatics analysis strongly suggests that OnMT1 is a metallothionein with affinity for multiple transition metals. Further, in order to elucidate the role of biological thiols, O. nubilalis L5 larvae were exposed to increasing Cd concentrations in diet (6.85, 41.71, 77.35 mg kg-1) during a 48 h period, after which Cd concentration in larvae was measured (3.50, 12.02, 47.37 mg kg-1, respectively). Due to short-term Cd exposure, concentration of free protein SH groups and relative expression of OnMT1 and thioredoxin (Trx) genes was elevated, while the reduced glutathione content remained unchanged. The presented results provide evidence that OnMT1 plays a role in Cd detoxification and homeostasis, and confirm the importance of biological thiols, especially OnMT1 and Trx, in the early response of O. nubilalis to Cd poisoning, indicating interaction between Cd and thiol-linked redox reactions. Insects provide valuable insight into molecular adaptations to metals.

Prevention of recurrence of endometrial polyps in women of reproductive age

The objective: the optimization of management of women in reproductive age after removal of endometrial polyps based on the study of the antioxidant status of the organism.Materials and methods. We examined 150 women in reproductive age with endometrial polyps, 65 (43.3%) patients of them were diagnosed with glandular polyp, 85 (56.7%) – with glandular fibrous polyps. 110 patients after polypectomy were divided into three clinical groups: the first group (32 (29.1%) women) did not receive anti-relapse therapy, the second group (36 (32.7%) persons) used dydrogesterone, the third group (42 (38.2%) women) – received complex drug anti-recurrence therapy (thioctic acid preparation, ascorbic acid, α-tocopherol acetate, indole-3-carbinol). In all women, the antioxidant indicators in blood and the presence of recurrent polyps were determined in 12 months after the treatment. Results. Before polypectomy in patients with endometrial polyps, a decrease of indicators of antiradical activity by 28.52%, restored forms of non-protein SH-groups – by 16.46%, protein SH-groups – by 25.31%, ascorbic acid – by 27.04%, α-tocopherol – by 21.62%, the value of the integral indicator of antioxidant protection – by 31.01% were found compared to the persons in the control group, as well as an increase in the concentration of malonic dealdehyde by 37.23%, non-protein SS groups – by 43.48%, protein SS-groups - by 29.43% and oxidized forms of ascorbic acid – by 29.68%.In 12 months after polypectomy, recurrence of the pathology was in 21.9% of patients of the first group, in 16.7% – of the second group, and no recurrence was detected in women in the third group.Conclusions. In patients with endometrial polyps there are a decrease in level of components of antioxidant protection in the organism and the expressed intensification of processes of free radical and peroxide oxidation of lipids. The use of a comprehensive approach that combines the polypectomy with the use of antioxidant drugs in the postoperative period, in such patients allows to prevent a recurrence of the pathology.

Mechanisms of the Cytotoxic Action of Organotin Compounds

This review analyzed the literature data on the in vitro preclinical study of the cytotoxic properties of organotin compounds, as well as the main mechanisms of their action. The latter consist in interacting with SH groups of proteins, initiating oxidative stress, binding to DNA, interacting with receptors, as well as activate apoptosis by increasing the expression of caspases, proapoptotic proteins, and decreasing antiapoptotic proteins. Organotin compounds, depending on the donor ligand, exhibit specifi c cytotoxicity towards certain tumor cell lines. The high cytotoxic potential indicates the possibility of further development in vivo and research of organotin compounds as candidates for the creation of drugs for anticancer and antimetastatic therapy.

Therapeutic irradiation in the management of gynecological cancer and predictability of radiation-induced complications

Background. Today, applying radiotherapy (RT) in management of both primary and secondary vaginal cancer (SVC) take pride of place in the spectrum of specialized treatments for cancer patients. Secondary vaginal tumors are more common (6% to 33%) in cervical cancer (CC) patients, while in uterine cancer (UC) occur in 8–10% of cases treated either surgically, or by means of radiotherapy, otherwise via a combination approach. Therefore, RT is administered in about 80% of primary vaginal cancer and particularly SVC patients. When using even the most advanced radiotherapy equipment implying the cutting-edge technologies, there is a risk of radiation-induced complications in healthy organs and tissues that fall under the irradiation area. Keeping in mind the key radiobiological paradigms makes it possible not only to predict the probability of tumor resorption upon radiation exposure, but also to assess the biological effectiveness of absorbed dose, as well as the risk of late radiation complications.&#x0D; Purpose – to enhance the effectiveness and assess the toxicity of SVC RT via ascertaining BT (brachytherapy) most suitable techniques depending on the type of ionizing radiation and exploring predictability of radiation-induced complications in terms of biomolecular cell properties.&#x0D; Materials and methods. Clinical study was performed at the National Cancer Institute Clinic (Radiation Oncology Department), using a high-energy BT unit with a HDR 192Ir source. The SVC patients (n = 106) were treated according to the developed BT methods. They had been pretreated for the CC (n = 65) with squamous cell carcinoma histologically diagnosed in the most cases or UC (n = 41) with histologically prevalent adenocarcinoma of a variable grade. Patients had a locally advanced pelvic tumor process with tumor staging II–III, T2-3N0-1M0. Along with clinical study the radiobiological research was conducted to count the apoptotic cells in both intact and irradiated peripheral blood lymphocytes (PBL), as well as the level of SH-groups of plasma proteins and peptides in gynecological cancer patients and healthy donors to predict the risk of radiation-induced complications.&#x0D; Results and discussion. Given the delayed effect of RT, the treatment effective- ness was analyzed immediately after RT session and also 3 months upon completion of the conservative therapy. Thus, positive tumor response upon three months of observation over time was registered in 67.9 ± 5.2% of patients in study group I, in 72.5 ± 6.9% in study group II, and in 51.3 ± 6.8% in comparison group. Hence the values in study groups were higher than in comparison group by 16.6% and 21.2% respectively. All patients tolerated BT satisfactorily. Neither general nor local rectum or bladder severe (above grade II) toxicities were noted both during treatment and over the next 3 months upon its completion in all the patients regardless of study group. Results obtained in radiobiological studies correlated with clinical and literature data.&#x0D; Conclusions. Decrease in manifestations of RT toxicity, namely of the ear- ly radiation reactions from interfacing critical organs was established in groups I and II vs comparison group I, especially in group I where a high- energy 192Ir source was used in the SBD irradiation mode of 3 Gy twice a week. The obtained results of the experimental study suggest that the content of SH-groups in blood plasma and the level of PBL apoptosis can be considered as additional predictive measures of radiosensitivity of non-malignant cells from the irradiated tumor environment.

The Role of P-Glycoprotein in Decreasing Cell Membranes Permeability during Oxidative Stress.

P-Glycoprotein (P-gp) is one of the most clinically significant representatives of the ABC transporter superfamily due to its participation in the transport of biotic components and xenobiotics across the plasma membrane. It is known that various chemicals, environmental factors, and pathological processes can affect P-gp activity and expression. In this study, we investigated the role of P-gp in limiting the cell membrane permeability during oxidative stress. Human adenocarcinoma colon cells (Caco-2) overexpressing P-gp were cultured for 72 h in the medium containing hydrogen peroxide (0.1-50 µM). The transport of the P-gp substrate fexofenadine was evaluated in a special Transwell system. The amounts of P-gp and Nrf2 transcription factor were analyzed by the enzyme-linked immunosorbent assay. The concentration of SH-groups in proteins and the contents of lipid peroxidation products and protein carbonyl derivatives were determined spectrophotometrically. Hydrogen peroxide at a concentration of 0.1-5 µM did not significantly affect the studied parameters, while incubation with 10 µM H2O2 decreased in the level of SH groups in cell lysates and increased in the amount of Nrf2 in the cell lysates. Nrf2, in its turn, mediated an increase in the content and activity of the P-gp transporter, thus limiting the increasing permeability of the cell membrane. Hydrogen peroxide at a concentration of 50 µM promoted oxidative stress, which was manifested as a decrease in the content of SH-groups, increase in the concentration of lipid peroxidation products and protein carbonyl derivatives, and decrease in the P-gp level, which led to a significantly increased permeability of the plasma membrane. These results show that the transport and protective roles of P-gp, in particular, reduction of the cell membrane permeability, are affected by the intensity of oxidative stress and can be manifested only if the extent of membrane damage is insignificant.

Participation of nitrogen oxide and its metabolites in the genesis of hyperimmune inflammation in COVID-19.

Despite the success in the tactics of treating COVID-19, there are many unexplored issues related to the development and progression of the process in the lungs, brain, and other organs, as well as the role of individual elements, in particular, nitric oxide (NO), and in the pathogenesis of organ damage. Based on the analyzed literature data, we considered a possible pathophysiological mechanism of action of NO and its derivatives in COVID-19. It can be noted that hyperimmune systemic inflammation and "cytokine storm" are enhanced by the production of NO, products of its oxidation ("nitrosative stress"). It is noted in the work that as a result of the oxidation of NO, a large amount of the toxic compound peroxynitrite is formed, which is a powerful proinflammatory agent. Its presence significantly damages the endothelium of the vascular walls and also oxidizes lipids, hemoglobin, myoglobin, and cytochrome, binds SH-groups of proteins, and damages DNA in the target cells. This is confirmed by the picture of the vessels of the lungs on computed tomography and the data of biochemical studies. In case of peroxynitrite overproduction, inhibition of the synthesis of NO and its metabolic products seems to be justified. Another aspect considered in this work is the mechanism of damage by the virus to the central and peripheral nervous system, which remains poorly understood but may be important in understanding the consequences, as well as predicting brain functions in persons who have undergone COVID-19. According to the analyzed literature, it can be concluded that brain damage is possible due to the direct effect of the virus on the peripheral nerves and central structures, and indirectly through the effect on the endothelium of cerebral vessels. Disturbances in the central nervous regulation of immune responses may be associated with the insufficient function of the acetylcholine anti-inflammatory system. It is proposed to further study several approaches to influence various links of NO exchange, which are of interest for theoretical and practical medicine.

Molecular mechanisms of the effects of N-ethylmaleimide and 1,4-dithioerythritol on regulation of apoptosis in P19 cells under hypoxia

Relevance. Impairment of apoptosis regulation in P19 cells is correlated with generation of oxidative stress. Under hypoxia, changes in mitochondrial functions occur, which may exacerbate oxidative stress in the tumor cell. The aim of the study was to evaluate the effects of N-ethylmaleimide and 1,4-dithioerythritol on implementation and regulation of apoptosis in P19 cells under hypoxia in vitro . Materials and methods . P19 cells (mouse teratocarcinoma) cultured under hypoxia served as the material for the study. For redox status modulation, 5mM N-ethylmaleimide and 1,4-dithioerythritol in the final concentrations of 5 mM were used. The intracellular concentration of calcium ions, the transmembrane potential and the number of Annexin V, CD95 and CD120 positive cells were determined by flow cytometry. The levels of reduced, oxidized and protein-bound glutathione, protein SH groups, hydroxyl radical and protein carbonyl derivatives were measured by spectrophotometry. Results. The alteration in the redox status of the glutathione system under hypoxia, accompanied by oxidative modification of proteins (glutathionylation and carbonylation), influences the metabolism in the tumor cell on the whole. Under the effects of 1,4-dithioerythritol, an SH group protector, this alteration promotes formation of additional mechanisms to escape apoptosis, whereas under the effects of N-ethylmaleimide, an SH group blocker, it, on the contrary, promotes apoptosis activation. Conclusions . The changes in the redox homeostasis of the tumor cell and modulation of oxidative modification of proteins (glutathionylation and carbonylation) under hypoxia are one of the promising approaches to targeted regulation of cell death.

EFFECT OF N-ACETYLCYSTEINE ON APOPTOSIS OF P19 CANCER CELLS DURING HYPOXIA

Introduction. Hypoxia in tumor growth contributes to mitochondrial dysfunction and exacerbates oxidative stress in the immortalized cell. The objective of the study was to investigate the molecular mechanisms of the effects of N-acetylcysteine on redox regulation of tumor cell apoptosis under hypoxia.Material and Methods. P19 cells (mouse teratocarcinoma) cultured under hypoxia served as the material for the study. The redox status was modulated with N-acetylcysteine in the final concentration of 5 mM. The level of reactive oxygen species, concentration of calcium ions, transmembrane potential and the number of CD95-, CD120- and Annexin V-positive cells were determined by flow cytometry. The concentration of glutathione system components as well as the levels of protein SH groups and protein carbonyl derivatives were measured by spectrophotometry.Results. The use of N-acetylcysteine under hypoxic conditions was accompanied by the increased total glutathione concentration and protein SH groups levels, decreased levels of Са2+ ions, proteinbound glutathione and protein carbonyl derivatives, as well as the production of reactive oxygen species and more appropriate functioning of P19 cells mitochondria. N-acetylcysteine contributed to the development of additional resistance of P19 cells to apoptosis under hypoxia.Conclusion. The alteration in the state of the glutathione system under hypoxia influences the changes in tumor cell metabolism on the whole and promotes formation of additional mechanisms to escape apoptosis.

Вплив лазерного опромінення на ксантиноксидазну активність та генерацію супероксидного радикала в цитозольній фракції печінки щурів-пухлиноносіїв

The effect of tumor growth in the body and laser irradiation on the enzymatic activity of xanthine oxidase, in particular its D- and O-forms, and also the rate of generation of the superoxide radical (O2–) and the level of protein sulfhydryl groups in the liver rat cytosolic fraction has been investigated. It has been found that in the cytosolic fraction of rats with transplanted Guerin’s carcinoma decreases the enzymatic activity of the D-form of xanthine oxidase with a simultaneous increase in its O-form during the period of intensive (14 days, which corresponds to the logarithmic phase of on cogenesis) and the period of final tumor growth (21 days, which corresponds to the stationary phase of oncogenesis). The increase in the enzymatic activity of the O-form of xanthine oxidase was accompanied by an increase the rate of superoxide radical generation and a decrease in the level of protein SH-groups in the liver cytosolic fraction of tumor-bearing rats. Daily directed action of laser irradiation on the area of growth of Guerin’s carcinoma leads to less destructive changes in the liver. Thus, there is an increase in the enzymatic activity of the D-form of xanthine oxidase, a decrease the rate of superoxide radical formation and an increase the content of protein SH-groups in the cytosolic fraction of the liver of experimental animals compared with non-irradiated tumor-bearing rats.