Впервые в исследовании in vivo на низших ракообразных рода Daphnia использован МТТ-тест, традиционно применяемый in vitro для тестирования лекарственных препаратов на цитотоксичность. В данной работе метод применен для изучения механизмов низкодозовых радиационных воздействий. МТТ-тест основан на анализе изменения показателя оптической плотности суспензии облученных гомогенизированных дафний, величина которого пропорциональна количеству формазана, образованного при восстановлении МТТ дегидрогеназами. Этот показатель определяет эффективность цитотоксического действия -излучения, характеризуя изменение соотношения в организме нормальных и поврежденных клеток, а также подавление общей дегидрогеназной активности. Дафний облучали -квантами 60. Показано значимое цитотоксическое действие -излучения в дозах 100 и 1000 мГр. При этом между группами, дозы облучения которых отличались в 10 раз, значимого отличия в проявлении цитотоксичности -квантов не выявлено. Сделано предположение, что снижение выживаемости, обнаруженное ранее при облучении дафний в дозах 100 и 1000 мГр, может быть связано с цитотоксическим эффектом излучения в клетках многоклеточного организма. Показана возможность использования дафний и МТТ-теста для целей биотестирования in vivo низкодозового радиационного воздействия.

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.

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, 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.

The effect of combined action of cadmium chloride at a dose of 0.5 mg/kg of body weight and γ-radiation of 1 Gy on induction of DNA damage in thymocytes and the total number of cells in the thymus of mice was studied. We found that injection of CdCl 2 0.5 h prior to irradiation decreased the number of single-strand DNA breaks and the number of alkali-labile sites in thymocytes 48 h after irradiation as compared to the γ-radiation effect only. This effect was associated with a strong decrease in the total number of thymocytes in this organ as compared to the action of cadmium ions and γ radiation separately. This masked the general genotoxic effect of combined treatment and created an illusion of a radioprotective effect of cadmium ions. Injection of cadmium chloride into mice 24 h prior to irradiation was followed by an additive increase in the number of the single-strand DNA breaks and the number of alkali-labile sites in thymocytes as compared to the respective controls such as the separate effects of cadmium ions and irradiation. We revealed a simultaneous decrease in the part of DNA tightly bound to proteins, i.e., DNA-protein cross-links as compared to the effect of γ-radiation only. We did not observe any statistically significant changes in the total number of thymocytes as compared to the separate effects of cadmium ions and irradiation. Thus, our data show that exposure of murine thymocytes to combined action of cadmium ions and γ-radiation at the doses and with the methods of treatment used induced additive effects but not antagonistic effects or protection against radiation.

Изучено влияние острого сочетанного воздействия хлорида кадмия (0.5 мг Cd2+ на кг массы тела животных) и -излучения (1 Гр) на индукцию повреждений ДНК в тимоцитах и общую численность клеток в тимусе мышей. Показано, что через 48 ч после инъекции раствора CdCl2 за 0.5 ч перед облучением происходит уменьшение количества однонитевых разрывов ДНК и щелочнолабильных сайтов в тимоцитах в сравнении с действием только -излучения. Наблюдаемый эффект сопровождается резким снижением общего числа тимоцитов в органе по сравнению с раздельным воздействием как ионов кадмия, так и -излучения, что маскирует общий генотоксический эффект сочетанного воздействия и создает иллюзию радиозащитного действия ионов кадмия. При инъекции мышам хлорида кадмия за 24 ч до облучения было отмечено аддитивное увеличение количества однонитевых разрывов ДНК и щелочнолабильных сайтов в тимоцитах по сравнению с соответствующими контролями: раздельным эффектом воздействия ионов кадмия и облучения. Одновременно отмечалось снижение доли ДНК, прочно связанной с белками (ДНК-белковые сшивки), по сравнению с вариантом действия только -излучения. Статистически достоверного изменения общего числа клеток в тимусе по сравнению с вариантами раздельного воздействия ионов кадмия и облучения не зарегистрировано. Таким образом, наши исследования показали, что при сочетанном действии ионов кадмия и -излучения на тимоциты мышей в использованных дозах и схемах воздействия отмечаются аддитивные эффекты, но не эффекты антагонизма или защиты от излучения.

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.

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.

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.

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.

In the present work we report that a prior injection of Pb, Cd Or Zn salt solutions in SHK male mice decreases the effect followed gamma-irradiation on thymus lymphocyte DNA structure and level of lipid peroxidation. It is assumed that the observed phenomenon is caused by activation of protective mechanisms of cells, expression of the genes of antioxidant proteins such as the metallothioneins, etc. Indeed the measurement of malondialdehyde (MDA) in blood plasma showed that the injection of metal salt solutions at median lethal doses a half hour before gamma-irradiation (1 Gy) causes the decrease of the MDA contents at 48 h after irradiation on 100% (Zn), 70% (Cd) and 20% (Pb). However we found that combined exposure of the mice also results to significant decrease of the thymus lymphocytes total number of as compared to the irradiation without metals. The elimination of the cells with high level of DNA lesions and existence at least a subset of cells which would survive the current oxidative stress (gamma-irradiation) possibly represents one path-way of the survival of individual organism facing stress. In turn the observed decrease of the lesion levels may be reflection of the cell number change.

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.

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.

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 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 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.