Emotional Stress Induces Adaptive Response in Rat Lymphocytes to Subsequent Ionizing Radiation Exposure.

We studied the molecular mechanisms of cross-adaptation to ionizing radiation (1 Gy) of lymphocytes isolated from rats subjected to emotional stress. The effects of chronic (CES; various types of stress exposure) and acute (AES; forced swimming) emotional stress in rats on indicators of oxidative stress, cell death, and levels of NRF2 and NOX4 proteins involved in the development of the adaptive response were analyzed in isolated lymphocytes. It was found that stress induced an adaptive response in rat lymphocytes and triggered processes similar to the adaptive response induced by low doses of ionizing radiation: an increase in the level of oxidized DNA and cell death, as well as an increase in the content of NOX4 and NRF2 proteins. In animals subjected to emotional stress, suppressed DNA oxidation in response to irradiation, reduced levels of protective factor NRF2, as well as lymphocyte death were observed.

[Antioxidant status of blood plasma of acutely psychotic patients and its correlation with Nrf2 activation]. / Antioksidantnyi status plazmy krovi patsientov s ostrym psikhozom i ego svyaz' s aktivatsiei transkriptsionnogo faktora Nrf2.

OBJECTIVE: To study the correlation between the blood plasma antioxidant profile and the transcriptional activity of the Nrf2 gene in acute psychosis in patients with schizophrenia and alcoholism. MATERIAL AND METHODS: The study included 40 patients with the first episode of the paranoid form of schizophrenia, 33 patients with schizophrenic psychosis who had previously received therapy, 22 patients with first-time acute alcohol psychosis, and 25 healthy volunteers. The level of Nrf2 in peripheral blood mononuclear cells was estimated by flow cytometry, and the antioxidant profile of blood plasma was estimated with chemiluminometry. RESULTS: The total and «thiol¼ antioxidant capacity were reduced in patients with initially diagnosed schizophrenic psychosis and alcoholic psychosis. In patients after treatment, the total antioxidant capacity was higher compared to previously untreated patients. The level of Nrf2 protein in mononuclear cells in patients with the first psychotic episode was significantly lower than in patients with alcoholism and lower than in the control group. In patients with alcoholic psychosis, Nrf2 level was correlated with both the total antioxidant capacity due to uric acid and the «thiol¼ antioxidant capacity; in patients with psychosis in schizophrenia, Nrf2 level was correlated only with the «thiol¼ antioxidant capacity. CONCLUSIONS: The correlation between the total and «thiol¼ antioxidant capacity and the level of Nrf2 in mononuclear cells of patients with alcohol delirium indicates the undamaged state of the regulation. The absence of a correlation between the total antioxidant capacity and the level of Nrf2 in patients with schizophrenia indicates a disturbance of the activation of the Nrf2 pathway due to, possibly, a part associated with the participation of uric acid.

[Antioxidant status in patients with paranoid schizophrenia and Alzheimer disease]. / Antioksidantnyi status pri paranoidnoi shizofrenii i bolezni Al'tsgeimera.

OBJECTIVE: To study the antioxidant profile of blood plasma in patients with paranoid schizophrenia and Alzheimer disease (AD). MATERIAL AND METHODS: Thirty-three patients with paranoid schizophrenia and 18 patients with AD were included in the study. Patients with schizophrenia were stratified into two subgroups by response to therapy. The indicators of the antioxidant profile were determined using methods based on chemiluminometry and spectrofluorimetry. RESULTS: Systemic oxidative stress due to insufficiency of low molecular weight plasma antioxidants is not determined neither in AD nor in treatment resistant schizophrenia. At the same time, a «thiol¼ oxidative stress, which indirectly indicates a deficiency of the glutathione system, is present in both groups. In patients with paranoid schizophrenia responsive to treatment, systemic oxidative stress is more pronounced and «thiol¼ oxidative stress is less significant. Among the antipsychotics studied, haloperidol, zuclopenthixol, risperidone and ziprasidone do not exhibit antioxidant properties, but periciazine, clozapine and especially chlorpromazine exhibit strong antioxidant properties, but they unlikely affect the antioxidant potential of blood plasma. CONCLUSIONS: The glutathione part of the antioxidant system is mostly affected, but systemic oxidative stress is not significant in patients with treatment resistant paranoid schizophrenia and AD. Oxidative disorders are more pronounced in treatment responsive paranoid schizophrenia.

[The functional activity of neutrophils in paranoid schizophrenia and Alzheimer's disease]. / Funktsional'naya aktivnost' neitrofilov pri paranoidnoi shizofrenii i bolezni Al'tsgeimera.

OBJECTIVE: To evaluate the radical-producing function of neutrophils in paranoid schizophrenia and Alzheimer's disease. MATERIAL AND METHODS: The study included 40 patients with paranoid schizophrenia and 22 with Alzheimer's disease. To assess the functional activity of neutrophils, whole blood samples were analyzed using the chemiluminescent method with two-step cell stimulation. Indicators of radical-producing activity of neutrophils in patients were compared to those in age-matched healthy people. RESULTS: The quantitative indicators of radical-producing activity of neutrophils in the paranoid schizophrenia group and in the Alzheimer's disease group correspond to reference intervals of healthy donors, however almost one third of patients with paranoid schizophrenia had an altered shape of the chemiluminescent curve. CONCLUSION: The neutrophil immune response might be involved in the pathogenesis of paranoid schizophrenia only in some cases.

[Functional activity of leucocytes in seminal fluid of patients with pathospermia].

OBJECTIVE: Assessment of leukocyte activity as the main source of ROS in seminal fluid of patients with normospermia and pathospermia using an original protocol based on the kinetic chemiluminescence method and adapted for semen analysis. MATERIALS AND METHODS: A prospective study was attended by 95 men of reproductive age who applied to the Research Center of Medical Genetics (Moscow) for semen analysis. The material for the study were samples of native ejaculate. Chemiluminescent measurements were performed on a Lum-1200 chemiluminometer (DISoft, Russia) using the original method. RESULTS: Both in amplitude of basal and stimulated response, between the "normozoospermia", "pathozoospermia" and "pathozoospermia + leucospermia" groups, significant differences were obtained in the level of ROS production by leukocytes (p<0.05): the median level of basal chemiluminescence, normalized on the count of leukocytes was 0.13, 0.71 and 1.78, respectively; the median level of stimulated chemiluminescence normalized to the number of leukocytes was 0.62, 2.14, 5.94, respectively. The level of stimulated response did not exceed 0.5 arb. units in normozoospermic samples. In pathospermic groups, the level of stimulated response was low in about a third of semen samples, it was moderate in one third of patients, and high in one third of patients. CONCLUSIONS: A protocol previously developed for blood analysis was adapted to analyze the total ROS level produced by leukocytes in seminal fluid. In the groups of "pathozoospermia", "pathozoospermia + leucospermia", the level of basal ROS production by leukocytes was about 5 and 15 times higher than in the "normozoospermia" group, the level of stimulated ROS production was 3.5 and 9.5 times; this indicates oxidative stress, including with a normal number of leukocytes.

[Antioxidative activity and clinical efficiency of dietary supplements Nefradoz for treatment of recurrent calcium oxalate urolithiasis].

AIM: to evaluate antioxidant properties of dietary supplements Nefradoz, as well as its role in the prevention of calcium oxalate stones. MATERIALS AND METHODS: Our study consisted of two parts, clinical and experimental. In the clinical trial a total of 80 patients with recurrent calcium oxalate urolithiasis of various localization were included. All patients were divided into 2 groups. In treatment group (n=45) patients received Nefradoz 1 capsule 2 times a day. In control group (n=35) no treatment was prescribed. In all patients a biomarkers level (bikunin, osteopontin, nephrocalcin) in urine was determined by ELISA. In addition, Litos-test was performed. Follow-up duration was 6 months. In the experimental part of the study, the antioxidant properties of Nefradoz were evaluated using chemiluminescence method. RESULTS: The antioxidant activity of resveratrol, which is contained in 300 mg Nefradoz (1 capsule), was 23.66+/-1.18, which corresponds to the moderate activity. After 6 months of therapy a level of bikunin and osteopontin did not significantly changed, which indicates a minimal risk of stone recurrence. According to the Litos-test, the activity of stone formation in Nefradoz group significantly decreased to minimal values in 42 out 45 patients (p<0.05). In the control group an excretion of biomarkers was minimal, which significantly increased the risk of stone formation. CONCLUSION: Dietary supplement NefraDoz can be used for prevention of calcium oxalate urolithiasis in case of increased stone formation according to the Litos-test. The antispasmodic, uroseptic and diuretic effects allow to maintain the effective level of biomarkers which is required for a recurrence-free course of urolithiasis compared to the control. The antioxidant activity, primarily determined by the resveratrol, can sufficiently compensate for oxidative stress and prevent the development of calcium oxalate stone. Further studies are needed to confirm our results.

[Role of neutrophil dysfunction in the pathogenesis of systemic lupus erythematosus]. / Rol' disfunktsii neitrofilov v patogeneze sistemnoi krasnoi volchanki.

Neutrophil dysfunction plays a considerable role.in systemic lupus erythematosus (SLE) The protective function of neutrophils is carried out through various mechanisms: isolation of granular antimicrobial peptides (gAMP), microbial phagocytosis with subsequent degradation via reactive oxygen species inside the phagolysosomes; as well as bactericidal action due to the release of networks from chromatin and gAMP, also called neutrophil extracellular traps (NECTs). The development of neutropenia in SLE has multiple causes, including the formation of antibodies directly to leukocytes; that of neutralizing autoantibodies to the growth factors of neutrophils and cells - myeloid precursors; bone marrow suppression; involvement of neutrophils in the processes of apoptosis and NETosis. Neutrophils in SLE are characterized by reduced phagocytic ability and pathological oxidative activity. In SLE, there is a decrease in the ability to remove the products of neutrophil apoptosis, which is correlated with disease activity. SLE patients are noted to have a higher expression level of the genes specific for low-density granulocytes, an abnormal immature neutrophil population. The impaired processes of formation of NECTs and removal NETosis products play a substantial role in the pathogenesis of SLE. It is shown that the abnormal formation of NECTs also causes endothelial injury and increases the risk of thromboses. The design of novel drugs that act on the specific parts of the formation of NECTs or contribute to their removal from the extracellular environment can propel therapy for SLE and other autoimmune diseases to new heights. There is evidence for further investigations of neutrophil-mediated pathogenetic processes in SLE in order to identify potential therapeutic targets and to understand the mechanisms of action of drugs used in clinical practice.

A New Chemiluminescent Method for Evaluation of the Functional Activity of Neutrophils in Patients with Type 2 Diabetes Mellitus.

Functional activity of neutrophils was evaluated by the chemiluminescent method with successive double stimulation by soluble stimuli with different mechanisms of action: phorbol-12-myristate-13-acetate (PMA) and phormyl-methionyl-leucyl-phenilalanine (fMLP). The study was carried out in 26 patients receiving oral sugar-reducing therapy. In addition to the functional activity of neutrophils, the levels of TBA reactive products, inflammation markers, blood clotting values, and biochemical parameters were measured. The results showed mainly reduction of the granulocytic component of the immune system in the patients.

Kinetic Chemiluminescence as a Method for Oxidative Stress Evaluation in Examinations of Patients with Type 2 Diabetes Mellitus.

We propose a new approach to evaluation of oxidative stress based on kinetic chemiluminescence: measurement of antioxidant and pro-oxidant activities of the plasma. The study included 50 patients with type 2 diabetes mellitus receiving peroral hypoglycemic therapy. In addition to the above parameters, the levels of TBA-reactive products, inflammation markers, clotting parameters, and biochemical values were studied. The new method provides information on oxidative stress in patients with type 2 diabetes mellitus irrespective of the clinical and laboratory values. The use of this method in complex with the clinical, laboratory, and instrumental studies allows comprehensive evaluation of patient's status for the diagnosis and choice of therapy.

[Changes in Kinetics of Chemiluminescence of Plasma as a Measure of Systemic Oxidative Stress in Humans].

Oxidative stress is a pathogenetic factor of many diseases. The control of its level is important for early diagnosis and therapy adjustment. In this work, antioxidant status was estimated in blood plasma. In the system of 2,2'-azo-bis(2-amidinopropane)dihydrochloride-luminol a set of chemiluminescence kinetic curve parameters is proposed for oxidative stress level estimation (the latent period τ(lat) and the increasing of analytical signal ΔI(CL)). Uric acid and albumin were shown as the main components that responsible for changes in chemiluminescence kinetic curve of plasma. Serum albumin undergoes oxidative modification in dose-depend manner under the action of UV irradiation, it causes the enhancement of antioxidant properties. Changes in plasma chemiluminescence kinetics are proposed as a measure of oxidative stress in human body.

Cytochrome c-Cardiolipin Complex in a Nonpolar Environment.

Programmed cell death (apoptosis) plays an important role in the life of multicellular organisms and in the development of socially significant human diseases. Cytochrome c-cardiolipin complex (Cyt-CL) is formed at the very beginning of a cascade of apoptotic reactions. Nevertheless, the structure of the complex and the mechanism of its participation in lipid peroxidation in mitochondrial membranes are not yet understood. In previous work (Vladimirov, Y. A., et al. (2011) Crystallography, 56, 712-719), it was shown that the Cyt-CL complex precipitates in concentrated water solution, the sediment containing orderly nanospheres formed by cytochrome c molecules with changed conformation and surrounded by a cardiolipin monolayer, and they are essentially hydrophobic. In this work, we obtained chloroform and hexane solutions of Cyt-CL with lipid/protein ratio of 77 ± 11. The conditions are described under which the solutions were obtained. Study of the properties of Cyt-CL solutions in hydrophobic media will reveal their structure and the mechanism of their catalytic activity inside the lipid layer of biological membranes.

Molecular mechanisms of apoptosis. structure of cytochrome c-cardiolipin complex.

One of the functions of cytochrome c in living cells is the initiation of apoptosis by catalyzing lipid peroxidation in the inner mitochondrial membrane, which involves cytochrome c bound with acidic lipids, especially cardiolipin. In this paper the results of studies of cytochrome c-cardiolipin complex structure carried out by different authors mainly on unilamellar cardiolipin-containing phospholipid liposomes are critically analyzed. The principal conclusion from the published papers is that cytochrome c-cardiolipin complex is formed by attachment of a cytochrome c molecule to the membrane surface via electrostatic interactions and the subsequent penetration of one of the fatty-acid cardiolipin chains into the protein globule, this being associated with hydrophobic interactions that break the >Fe·×·S(Met80) coordinate bond and giving rise to appearance of cytochrome c peroxidase activity. Nevertheless, according to data obtained in our laboratory, cytochrome c and cardiolipin form spherical nanoparticles in which protein is surrounded by a monolayer of cardiolipin molecules. Under the action of cooperative forces, the protein in the globule expands greatly in volume, its conformation is modified, and the protein becomes a peroxidase. In extended membranes, such as giant monolayer liposomes, and very likely in biological membranes, the formation of nanospheres of cytochrome c-cardiolipin complex causes fusion of membrane sections and dramatic chaotization of the whole membrane structure. The subsequent disintegration of the outer mitochondrial membrane is accompanied by cytochrome c release from the mitochondria and triggering of a cascade of programmed cell death reactions.

[Antioxidants as aromatic amino acid oxidation products].

The accumulation of UV photolysis products of amino acids tyrosine and tryptophan, which possess an antioxidant activity, has been studied by the method of luminol-activated chemiluminescence. The amount of antioxidant products was judged by the value of the total antioxidant potential of a UV-irradiated solution, the measure of which was the distance between the peaks of the chemiluminescence curve in the system 2,2'-azo-bis(2-amidinopropane)hydrochloride + luminol in a UV-irradiated and an unirradiated samples (induction period, tau(i)). Simultaneously, the absorption and fluorescence spectra of unirradiared and UV-irradiated amino acid solutions were recorded. It was shown that, upon the exposure of a tryptophan solution to radiation, the accumulation of the fluorescent product N-formyl kynurenine (lambda(em) = 325 nm, lambda(max) = 440 nm) occures, and the curve of its accumulation was similar to the curve of growth of tau(i) photoproducts produced during UV-radiation. When a tyrosine solution was irradiated, the main fluorescent product was dityrosine (lambda(em) = 310 nm, lambda(max) = 415 nm). Nevertheless, the dose dependencies of the formation of dityrosine, and the total antioxidant potential (tau(i)) were completely different. It was found that another product of tyrosine UV-photolysis, dioxyphenylalanine, possessed a pronounced antioxidant activity. It was concluded that the main antioxidants produced under UV-irradiation of tryptophan is formyl kynurenine, and under the irradiation of tyrosine, dioxyphenylalanine.

Determination of superoxide dismutase and SOD-mimetic activities by a chemical system: Co2/H2O2/lucigenin.

The bright chemiluminescence has been observed in the system: Co(2+)/hydrogen peroxide/lucigenin. The chemiluminescence intensity was directly proportional to either cobalt, hydrogen peroxide, or lucigenin concentrations. A procedure of determination of superoxide dismutase (SOD) activity by the chemiluminescence method in the cobalt-hydrogen peroxide-lucigenin system at pH 8.5 is suggested. A linear dependence was established between a relative chemiluminescence intensity and SOD concentration in the range of SOD concentrations between 0 and 4.5 nM, c (1/2) = 0.8 nM. The determination of SOD activity was performed in several tissue samples (rat plasma, erythrocyte hemolysate, and liver mitochondria). A technique of tissue sample preparation with the use of thermal inactivation of interfering proteins at 60 °C was used. The method was successfully applied for comparison of the efficiency of SOD mimetics.

Dihydroquercetin (taxifolin) and other flavonoids as inhibitors of free radical formation at key stages of apoptosis.

Formation of free radicals in mitochondria plays a key role in the development of apoptosis, which includes formation of superoxide by the respiratory chain, formation of radicals by cytochrome c-cardiolipin complex in the presence of hydrogen peroxide or lipids, and chain lipid peroxidation resulting in cytochrome c release from mitochondria and initiation of the apoptotic cascade. In this work the effect of taxifolin (dihydroquercetin) and some other antioxidants on these three radical-producing reactions was studied. Peroxidase activity of the complex of cytochrome c with dioleyl cardiolipin estimated by chemiluminescence with luminol decreased by 50% with quercetin, taxifolin, rutin, Trolox, and ionol at concentrations 0.7, 0.7, 0.8, 3, and 10 microM, respectively. The lipid radical production detected by coumarin C-525-activated chemiluminescence decreased under the action of rutin and taxifolin in a dose-dependent manner, so that a 50% inhibition of chemiluminescence was observed at the antioxidant concentrations of 3.7 and 10 microM, respectively. Thus, these two radical-producing reactions responsible for apoptosis onset are inhibited by antioxidants at rather low concentrations. Experiments performed on liver slices and mash showed that taxifolin, quercetin, naringenin, and Trolox have low inhibitory effect on the lucigenin-dependent chemiluminescence in the tissue only at concentrations higher than 100 microM.

Free radicals and cell chemiluminescence.

Application of chemiluminescence (CL) for study of free-radical reactions in human and animal cells and tissues is considered in this review. Historically, the study of intrinsic (ultraweak) luminescence gave place to the measurement of CL in the presence of chemical activators (CL probes) and physical activators (sensitizers) of luminescence, which made the method much more sensitive and specific. The methods of CL and EPR are direct methods of radical investigation, though the advantage of the CL method consists in the fact that CL intensity is directly proportional to a steady-state concentration of the radicals responsible for luminescence (first of all, lipid and oxygen radicals) irrespective the activity of these radicals. The mechanisms of CL reactions in the absence of activators and in the presence of luminol and lucigenin are considered. Examples of various applications of the CL method in medical, biological, and clinical investigations are given including those for estimation of the phagocytic activity of cells, antioxidant activity, determination of toxicity, and other purposes.

Chemiluminescence as a method for detection and study of free radicals in biological systems.

Chemiluminescence observed during LPO or reactions of nitric oxide and oxygen radicals and was named "ultraweak luminescence". In the presence of chemiluminescence activators (luminol, lucigenin, rhodamine G, or coumarine C-525) the appearance of radicals is associated with intensive fluorescence; the registration of this fluorescence is widely used in biomedical and clinical studies.

Mechanism of activation of cytochrome C peroxidase activity by cardiolipin.

In this work, the actions of bovine heart cardiolipin, synthetic tetraoleyl cardiolipin, and a nonspecific anionic detergent sodium dodecyl sulfate (SDS) on cytochrome c (Cyt c) peroxidase activity recorded by chemiluminescence in the presence of luminol and on the Fe...S(Met80) bond whose presence was estimated by a weak absorption band amplitude with peak at 695-700 nm (A(695)) were compared. A strict concurrency between Fe...S(Met80) breaking (A(695)) and cytochrome peroxidase activity enhancement was shown to exist at cardiolipin/Cyt c and SDS/Cyt c molar ratios of 0 : 1 to 50 : 1 (by chemiluminescence). Nevertheless, when A(695) completely disappeared, Cyt c peroxidase activity under the action of cardiolipin was 20 times more than that under the action of SDS, and at low ligand/protein molar ratios (=4), SDS failed to activate peroxidase activity while cardiolipin enhanced Cyt c peroxidase activity 16-20-fold. A(695) did not change on Cyt c binding with liposomes consisting of tetraoleyl cardiolipin and phosphatidylcholine (1 : 10 : 10), while peroxidase activity was enhanced by a factor of 8. Breaking of 70% of the Fe...S(Met80) bonds resulted in only threefold enhancement of peroxidase activity. Cardiolipin-activated Cyt c peroxidase activity was reduced by high ionic strength solution (1 M KCl). The aggregated data suggest that cardiolipin activating action is caused, first, by a nonspecific effect of Fe...S(Met80) breaking as the result of conformational changes in the protein globule caused by the protein surface electrostatic recharging by an anionic amphiphilic molecule, and second, by a specific acceleration of the peroxidation reaction which is most likely due to enhanced heme accessibility for H(2)O(2) as a result of the hydrophobic interaction between cardiolipin and cytochrome.

Cardiolipin activates cytochrome c peroxidase activity since it facilitates H(2)O(2) access to heme.

In this work, the effect of liposomes consisting of tetraoleyl cardiolipin and dioleyl phosphatidylcholine (1 : 1, mol/mol) on the rate of three more reactions of Cyt c heme with H2O2 was studied: (i) Cyt c (Fe2+) oxidation to Cyt c (Fe3+), (ii) Fe...S(Met80) bond breaking, and (iii) heme porphyrin ring decomposition. It was revealed that the rates of all those reactions increased greatly in the presence of liposomes containing cardiolipin and not of those consisting of only phosphatidylcholine, and approximately to the same extent as peroxidase activity. These data suggest that cardiolipin activates specifically Cyt c peroxidase activity not only because it promotes Fe...S(Met80) bond breaking but also facilitates H2O2 penetration to the reaction center.