[Methyltetrazolium bromide (MTT) was applied for bioassay of environmental radioactivity in vivo].

The MTT-assay is a colorimetric assay that measures the activity of enzymes that reduce MTT (a yellow tetrazolium bromide) in living cells. The MTT-test has been traditionally applied for the analysis of drug cytotoxicity in vitro. In our study MTT-assay was first applied for the investigation in vivo of the mechanisms of non-targeted effects of radiation and development of stress in multicellular crustaceans Daphnia magna. MTT test was based on the analysis of variation in the optical density of the irradiated Daphnia, which is proportional to the amount of formazan formed as a result of restoring MT with the help of dehydrogenases. So this indicator measures the effectiveness of the toxic effect of gamma-radiation. It describes the change in the balance of normal and damaged cells, suppression of the total dehydrogenase activity and other factors that are responsible for the metabolism of a multicellular organism. Daphnia were exposed to acute 60Co gamma-rays. According to our data, the effectiveness of toxicity was significantly raised in the two groups exposed to 100 and 1000 mGy of gamma-rays. Given the results of in vitro studies, our data therefore indicate that the compromised viability of irradiated Daphnia may be attributed to the cytotoxic effects within the dose-range of 100 and 1000 mGy. The results obtained in this study show that Daphnia represent a very useful experimental model, which allows a very efficient and quick analysis of many aspects of non-targeted effects of ionising radiation.

[Influence of ionizing radiation on the parameters of oxidative stress markers in the rat liver and thymus].

Influence of ionizing radiation on the parameters of oxidative stress markers in the liver and thymus of the rats exposed to gamma-radiation 60Co at a doze of 4 Gy was investigated. The animals were decapitated on the 1, 4, 7, 10, 14, 22 and 30th day after irradiation and cell suspensions from the liver and thymus were obtained. After centrifugation, the content of MDA, the spontaneous and NADH-induced synthesis of superoxide anion radical of oxygen, the content of total and free iron were determined in the cellular sediment and centrifugate containing intercellular fluid. It is shown that the content of MDA and the levels of spontaneous and NADH-induced synthesis of superoxide anion radical of oxygen increases in intercellular fluid and thymus and liver cells on the 1st day after radiation exposure. In the liver, these parameters are normalized by the 4th day and do not significantly differ from the control level in the period of time following radiation exposure. In thymus, as compared with liver, the level of oxidative stress parameters increases by the 4th day after radiation and remains at the raised level within 22 days after irradiation exposure. It is shown that the content of free iron in thymus cells of irradiated animals increases 3.6 times by the 4th day and reliably exceeds the control level within the next 22 days. Radiation does not lead to any changes in the content of free iron in liver cells. Different levels of the free iron content can serve the reason for various sensitivity of oxidative stress markers in thymus and liver cells to radiation exposure.

[DNA damage in thymocytes of mice under combined acute whole body exposure to cadmium ions and gamma-radiation].

The effect of the combined acute whole body exposure to cadmium chloride (0.5 mg Cd2+ per kg body weight of animals) and gamma-radiation (1 Gy) on the DNA damage induction in thymocytes and thymic cellularity of mice was studied. It has been shown that CdCl2 solution injection 0.5 h before irradiation reduces the quantity of single-strand DNA breaks and alkali-labile sites in thymocytes 48 h after injection compared to gamma-radiation action only. The observed effect is accompanied by a sharp decrease of the thymic cellularity compared with the separate effects of both cadmium ions and irradiation, which masks the overall genotoxic effect of combined exposure and gives an illusion of cadmiumL ions radioprotective action. Cadmium chloride injection 24 h before irradiation leads to a significant additive increase in the single-strand DNA breaks and alkali-labile sites number as compared to the separate effects of cadmium ions and irradiation alone. At the same time the decrease in the percentage of DNA tightly bound to proteins (DNA-protein cross-links) was noted in comparison with the action of gamma-radiation only. Statistically significant changes in thymic cellularity compared with separate effects of cadmium ions and irradiation were not found. Thus, our research has shown that under a combined action of cadmium ions and gamma-radiation on thymocytes in mice at the applied doses and exposure schemes the additive effects, rather than antagonism or radioprotective effects are observed.

[Influence of ionizing radiation, application of iron ions and their chelate complexes on the oxidative status of blood serum of rats].

Influence of ionizing radiation, ions of iron and their chelate complexes on the oxidative status of blood serum of rats has been investigated. Animals were irradiated by gamma-rays 60Co at a dose of 4 Gy. Ions of iron and iron chelates with nitrilotriacetic acid and citric acid were introduced into animals intra-abdominally at a doze of 10 mg of iron on 1 kg of body weight. The oxidative status of blood serum was determined according to the estimated content of oxidizing peroxide equivalents which oxidize ferrous iron in ferric iron with the subsequent estimation of ferric iron by means of xylenol orange. We also estimated the total content of iron in blood serum using ferrozine as an indicator. The oxidative status was defined 24 and 96 hours after irradiation and 2 hours after introduction of iron ions and their chelates. The research conducted has shown that the concentration of oxidizing peroxide equivalents in serum and the total iron concentration increase 1.47 times and 1.63 times correspondingly 24 hours after irradiation. The increase in the content of oxidizing peroxide equivalents and iron owing to Fenton's reaction can lead to the appearance of OH* radical and raise the level of damage of nuclear and membrane structures in irradiated cells. 2 hours after introduction of iron ions and their chelates, the content of oxidizing peroxide equivalents increased in the blood serum of irradiated and non-irradiated rats, and the maximum effect was observed when introducing ferrous iron and its chelate with citric acid.

[Antioxidant and prooxidant properties of the ascorbic acid, dihydroquercetine and mexidol in the radical reactions induced by the ionizing radiation and chemical reagents].

Antioxidant and prooxidant properties of dihydroquercetine, mexidol and an ascorbic acid in reactions with participation of radicals OH* and O2(-)*, induced by gamma-irradiation, iron-catalyzed decomposition of hydrogen peroxide and oxidation of reduced NADH by phenazine metosulfate are investigafed. The efficiency of scavenging of radicals OH* estimated by the results of the analysis of deoxyribose degradation, and the efficiency of scavenging of superoxide anion-radicals O2(-)* is estimated by the results of the analysis of occurrence the reduced nitrotetrazolium blue. The concentrations of analyzed compounds, scavenging on 50% (C50%) formation of radicals OH* and O2(-)* are certain. It is shown, that an ascorbic acid, dihydroquercetine and mexidol decrease the generating of superoxide anion-radicals O2(-)* in the gamma-irradiated solutions of sodium format and at oxidation of reduced NADH by phenazine metosulfate scavanged of superoxide anion-radicals O2(-)*. In the gamma-irradiated saline solutions an ascorbic acid, dihydroquercetine and mexidol protected deoxyribose from oxidizing action of hydroxyl radicals OH*. However at presence Fe(3+), EDTA and hydrogen peroxide addition of an ascorbic acid (0.1 mmol/l) increased generating of hydroxyl radicals OH* and in 2.8 times raised the maintenance of products of deoxyribose oxidation, reacting with thiobarbituric acid. Prooxidant action of an ascorbic acid is observed as well in absence of hydrogen peroxide. Obtained data testify that in various modelling systems reagents, in particular ions of iron, and the formed active intermediate products render significant influence on scavenging efficiency of investigated compounds.

[Action of mixed gamma-neutron radiation with various dose rates on the number of cells in thymus and chromosomes of mice bone marrow and human lymphocytes].

The irradiation with mixed gamma-neutron radiation was carried out at the pulse nuclear reactor on fast neutrons BARS-6 in a regimen of one pulse (100 micros) and in a regimen of continuous irradiation during 60 minutes. Was shown, that the irradiation of mice with pulse radiation was 1.3-1.8 times more effective in the induction of the chromosome aberrations in bone marrow cells in comparison with the continuous regimen of irradiation. At the same time, other biological tests (yield of chromosome aberrations in human lymphocytes, decreasing the number of cells in thymus) demonstrated that pulsed and continuous regimens have almost equal biological effectiveness.

[Radioprotective and antistressful properties of nitric oxide production modulators].

The radioprotective and antistressful activities of L-arginine and the "Pronumol" preparation, in which L-arginine is contained in the complex of proteins with nucleic acids, were studied. In mice repeated peroral intake of L-arginine and "Pronumol" partially prevented radiation-induced and stress-induced lipid peroxidation and DNA degradation in thymus, increased hemopoietic stem cell survival, and prevented an increase in chromosome aberration frequency in bone marrow cells of irradiated mice. When repeatedly administered per os before irradiation, "Pronumol" increased survival of intestinal stem cells in irradiated mice and prevented thymus cell devastation induced by radiation and stress.

[Comparative efficiency of injurious action of radiation and stress on thymus and lipid peroxidation]. / Sravnitel'naia éffektivnost' povrezhdaiushchego deistviia oblucheniia i stressa na timus i perekisnoe okisleniia lipidov.

Exposure to radiation, as well as holding under conditions of limited mobility during 24 h, induced decrease in thymus cell number, increase in number of DNA breaks. The content the products of lipid peroxidation reactive with thiobarbituric acid in blood serum of mice decreased as well. The stress effect is comparable with radiation doses in the range of 50-60 cGy.

[Effect of preliminary adaptive irradiation on the concentration of lipid peroxidation products in blood serum and degradation of DNA in thymus of irradiated mice]. / Vliianie predvaritel'nogo adaptiruiushchego oblucheniia na soderzhanie produktov perekisnogo okisleniia lipidov v syvorotke krovi i povrezhdeniie DNK v timuse obluchennykh myshei.

The animals were irradiated within adaptive dose of 0.1 Gy and 5 hours later with a challenge dose of 2 Gy. The adaptive dose reduced the effects induced by the challenge dose of 2 Gy: the increased content of the products of lipid peroxidation reactive to thiobarbituric acid in blood serum, and the increased number of breaks in thymus DNA of irradiated mice.

[The molecular, cellular and systemic mechanisms of the radioprotective action of multivitamin antioxidant complexes]. / Molekuliarnye, kletochnye i sistemnye mekhanizmy radioprotektornogo deistviia polivitaminnykh antioksidantnykh kompleksov.

Exposure to radiation, as well as holding under conditions of limited mobility during 24 h induced decrease of thymus cell number in mice. Usage of water-dispersed forms of beta-carotene with addition of vitamins E and C protected thymus against cellular devastation induced by irradiation and stress, and increased the efficiency of DNA repair in spleen of irradiated mice.

[The inhibition by the tumor necrosis factor of the death and DNA fragmentation of lymphoid cells in irradiated rats]. / Ingibirovanie faktorom nekroza opukholi gibeli i fragmentatsii DNK limfoidnykh kletok obluchennykh krys.

The influence of a tumor necrosis factor, administered 16 h before irradiation of rats, on the radiation response of thymus and bone marrow cells has been investigated. Three and 6 h after irradiation the following indices were analyzed: the number of apoptotic cells in the thymus; the accumulation of polydeoxyribonucleotides and the appearance of single-strand breaks in DNA of bone marrow and thymus cells; and the electrophoretic properties of thymocyte DNA. The injection of a tumor necrosis factor reduced the number of polydeoxyribonucleotides, inhibited internucleosome DNA fragmentation, and did not influence the formation of single-strand breaks in DNA.

The effects of radiation and alkylating agents on chromatin degradation in normal and tumour lymphoid cells.

The dynamic of chromatin degradation was studied in thymocytes and LS/BL tumour cells. In permeabilised LS/BL cells, the rate of DNA degradation induced by endogenous calcium and magnesium-dependent endonuclease was approx. 25 times slower than in thymocytes. In LS/BL cells irradiation does not induce chromatin degradation. The alkylating agent TS 160 induced chromatin degradation in both LS/BL lymphosarcoma cells and thymocytes.

[The effect of irradiation and alkylating agents on chromatin breakdown in normal and malignant lymphoid cells]. / Vliianie oblucheniia i alkiliruiushchikh agentov na raspad khromatina v normal'nykh i malignizirovannykh limfoidnykh kletkakh.

Regularities of chromatin degradation in thymocytes and LS/BL tumor cells have been investigated. It has been shown that the rate of DNA degradation by Ca/Mg-dependent endonuclease in LS/BL tumor cells is 25 times lower than that in thymocytes, and radiation does not induce chromatin degradation. The alkylating agent TS 160 causes chromatin degradation in both LS/BL cells and thymocytes. In contrast to radiation TS 160 inhibits the endogenous chromatin degradation by Ca/Mg-dependent endonuclease in thymocytes.

[Electrophoretic analysis of the internucleosome fragmentation of DNA in irradiated thymocytes of rats]. / Elektroforeticheskii analiz mezhnukleosomnoi fragmentatsii DNK v obluchennykh timotsitakh krys.

Total DNA and DNA of chromatin degradation products obtained from rat thymocytes 6 h after irradiation with a dose of 10 Gy were separated electrophoretically. Relative shares of mononucleosomes and their oligomers were determined. Experimental distributions of DNA fragments differ from those calculated on the basis of the assumption of a random breakage of bonds between the nucleosomes.

[Effect of cycloheximide on the morphological and biochemical changes in chromatin in non-irradiated and irradiated rat thymocytes]. / Vliianie tsiklogeksimida na morfologicheskie i biokhimicheskie izmeneniia khromatina v neobluchennykh i obluchennykh timotsitov krys.

The level of chromatin degradation was studied and the method of electron-microscopy was used to estimate the changes in the ultrastructure of irradiated and nonirradiated thymocytes of rats treated with cycloheximide. The latter was found to decrease the degree of derangement of nuclear ultrastructure and the level of chromatin degradation in exposed animals and to increase the yield of these damages in thymocytes of nonirradiated animals. The electronmicroscopic determinations showed that the percentage of thymocytes with the impaired nucleus structure is twice as high as that of degraded chromatin. The causes of the quantitative disagreement between the morphological and biochemical indices of the interphase thymocyte death are discussed.

[Structural organization of chromatin as a factor determining the rate of its endonucleolysis in irradiated and nonirradiated thymocytes]. / Strukturnaia organizatsiia khromatina kak faktor, opredeliaiushchii skorost' ego éndonukleoliza v obluchennykh i neobluchennykh timotsitakh.

A study was made of chromatin endonucleolysis in hypotonic thymocytes incubated in digestive buffers containing different concentrations of potassium, magnesium, calcium, and mercaptoethanol. Inhibition of endonucleolysis by univalent cation during the first 20 min of incubation was followed by intensive chromatin degradation. A decrease in free potassium content retarded chromatin degradation and enhanced the inhibiting effect of the univalent cations. The regularities of changes in the rate of chromatin endonucleolysis in different digestive buffers were similar with both exposed and intact thymocytes.

[Thymidine excretion from thymocytes in irradiated and nonirradiated rats during endonucleolysis]. / Ekskretsiia timidina iz timotsitov obluchennykh i neobluchennykh krys v usloviiakh éndonukleoliza.

The concentration of free thymidine (dT), excreted from thymocytes of irradiated and nonirradiated rats, was determined after incubation of cells in various digestive buffers. The release of dT from thymocytes depended upon the rate of DNA fragmentation in conditions of chromatin endonucleolysis. The increase in the thymidine content, in conditions of chromatin endonucleolysis in buffers containing no calcium ions, was only noted in thymocytes of exposed rats: this was the consequence of chromatin DNA damages already available in these cells.

[Endonucleolysis of chromatin in thymocytes of irradiated and non-irradiated rats]. / Endonukleoliz khromatina v timotsitakh obluchennykh i neobluchennykh krys.

It was shown that in conditions optimal for Ca/Mg endonuclease, chromatin endonucleolysis in the nuclei and thymocytes occurs due to internucleosome fragmentation of DNA. Irradiation activates chromatin degradation in thymocytes washed by a buffer containing 0.25 M sucrose, 10 mM tris-HCl, pH 7.2, 3 mM MgCl2, and does not influence this process in thymocytes washed by 10 mM tris-HCl, pH 7.2, 3 mM MgCl2.

A comparison of chromatin degradation in irradiated normal and tumorous lymphoid cells.

Irradiation of mice with doses of 2 and 4 Gy induced extensive chromatin degradation in the thymocytes within 6 hours accompanied by an increase in polydeoxynucleotide (PDN) content (36 and 42 times, respectively). Fifteen hours after irradiation the PDN level was considerably lower, however, still being 4.7 and 14 times the control values after doses of 2 and 4 Gy. The PDN content in control LS/BL lymphosarcoma cells was similar as that in the thymocytes of non-irradiated mice. Unlike in the thymocytes, irradiation of lymphosarcoma cells did induce no statistically significant increase in the PDN level 6 and 15 hours after the irradiation, respectively. It has been reported previously (Matyásová et al. 1973) that chromatin of LS/BL cells degraded similarly as that in the irradiated thymocytes. The results of the present experiments thus provide additional evidence for changes of LS/BL cell properties due to long term cultivation. These cells, however, are still able to react by chromatin fragmentation to nitrogen mustard treatment.

[Degradation of DNA and chromatin in cells of mouse lymphoid organs after irradiation and PHA administration]. / Degradatsiia DNK i khromatina v kletkakh limfoidnykh organov myshei posle oblucheniia i vvedeniia FGA.

Phytohemagglutinin (PHA) administered to irradiated mice did not influence the postirradiation degradation of DNA and the yield of polydeoxynucleotides (PDN) in cells of thymus, spleen and bone marrow. The degree of degradation of DNA and chromatin was higher in the thymus as compared to other studied organs. A possible mechanism of the radiotherapeutic action of PHA is discussed.