Antioxidative and Radiation Modulating Properties of Guanosine-5′-Monophosphate

Employing enhanced chemiluminescence in luminol-p-iodophenol peroxidase system and coumarine-3-carboxylic acid, it was shown that guanosine-5′-monophosphate (GMP) appreciably reduces formation of H₂O₂ and hydroxyl radicals induced by x-ray irradiation. Using immunoenzyme assay, we revealed that GMP lowered 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) formation in DNA in vitro after irradiation. The results of survival test have shown that mice being injected intraperitoneally with GMP after irradiation with a dose of 7 Gy had better survival rate than the control mice. GMP reduced leucopoenia and thrombocytopenia in irradiated mice. Obtained results give premises that GMP may be promising therapeutic agent for treatment of radiation injuries.     

Immune effects of low-dose radiation: short-term induction of thymocyte apoptosis and long-term augmentation of T-cell-dependent immune responses

We and others have shown that low-dose X or gamma irradiation of mice leads to an increase in their survival after a subsequent lethal high-dose irradiation. The greatest increase in radioresistance appears at a fixed window of dose and time, e.g. 8 weeks after 5-10 cGy or 2 weeks after 50 cGy preirradiation. We show that low-dose irradiation induces thymocyte apoptosis with a maximal level at 6 h postirradiation that returns to background levels after 24 h. At the same time, we observed no morphological alteration of splenocytes and no early modification of the intensity of T-cell-dependent immune responses as measured by plaque-forming cell (PFC) counts. Nevertheless, we found that PFCs were increased 2 weeks after 50 cGy irradiation, which is the same time at which mice expressed the optimal increase in survival after a second lethal irradiation. We also examined thymocyte apoptosis and spleen PFCs in mice subjected to other stress-inducing pretreatments. Our results emphasize the existence of a lag time between the time of low-dose irradiation in vivo and the appearance of radioresistance. A mechanism that interconnects an environmental stimulus with the response of the animal is proposed based on the evidence presented here and reported in the literature.     

Radioprotective properties of oxolinic acid during long-term exposure to radiation

The antioxidant activity of oxolinic acid was studied in mongrel mice during the prolonged (6-8 hours) exposure to ionizing radiation. The drug administered before irradiation was shown to increase (by 7 to 33 per cent) the survival rate of mice. The effect was maximum after the subcutaneous injection. The results obtained on the effect of oxolinic acid on DNA synthesis by bone marrow karyocytes are submitted.     

Postirradiation changes in the cGMP system of the mouse thymus and liver

Changes have been revealed in the function of cyclic GMP system of thymus and liver of irradiated (8 Gy) mice. In the thymus the cGMP level increased during the first 60 min following irradiation. In the liver the concentration of cGMP exhibited two peaks: 30 min and 24 hr after irradiation. The changes observed in the cGMP level are connected with the increased guanylate cyclase activity of thymocytes and liver of irradiated mice and, less likely, with changes in the activity of cGMP phosphodiesterase of these tissues.     

Inhibitory mechanisms of metallothionein on platelet aggregation in in vitro and platelet plug formation in in vivo experiments

Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that contains heavy metals such as cadmium and zinc. The biological function of MT in platelets is not yet understood. Therefore, the aim of this study was to systematically examine the inhibitory mechanisms of metallothionein in platelet aggregation. In this study, metallothionein concentration-dependently (1-8 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 microM) inhibited phosphoinositide breakdown in [3H]-inositol-labeled platelets, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane A2 formation stimulated by collagen. In addition, metallothionein (4 and 8 microM) significantly increased the formation of cyclic GMP but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (100 nM). This phosphorylation was markedly inhibited by metallothionein (4 and 8 microM) in phosphorus-32-labeled platelets. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Metallothionein (6 microg/g) significantly prolonged the latency period for inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of metallothionein may involve the following pathways: (1) metallothionein may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization; (ii) Metallothionein also activated the formation of cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. The results strongly indicate that metallothionein provides protection against thromboembolism.     

Inhibition of cyclooxygenase 2 in mice increases production of g-csf and induces radioprotection

Meloxicam, a selective inhibitor of cyclooxygenase 2, was tested to determine its ability to modulate hematopoiesis and to influence survival of mid-lethally gamma-irradiated mice. A single dose of meloxicam (20 mg/kg) administered to mice intraperitoneally 1 h before irradiation was shown to enhance serum levels of granulocyte colony-stimulating factor (G-CSF) during the first 24 h after irradiation, to elevate numbers of granulocytic precursor cells in bone marrow and granulocyte counts in peripheral blood on day 10 after irradiation, and to increase 30-day survival of these mice. The results provide new evidence for the protective ability of meloxicam administration to mice irradiated with mid-lethal doses and contribute to the understanding of the mechanisms of this meloxicam action by drawing attention to the possible role of increased endogenous G-CSF production.     

Role of radiation-induced granulocyte colony-stimulating factor in recovery from whole body gamma-irradiation

The purpose of this study was to further elucidate the radioprotective role of granulocyte colony-stimulating factor (G-CSF) induced in response to irradiation. The induction of G-CSF and interleukin-6 (IL-6) in response to radiation exposure was evaluated in mice. The level of cytokine in serum was determined by multiplex Luminex. The role of G-CSF on survival and tissue injury after total body gamma-irradiation was evaluated by administration of neutralizing antibody to G-CSF before radiation exposure. An isotype control was used for comparison and survival was monitored for 30 d after irradiation. Jejunum samples were used for immunohistochemistry. Ionizing radiation exposure induced significant levels of the hematopoietic cytokines G-CSF and IL-6, in mice receiving 9.2 Gy radiation. Maximal levels of G-CSF were observed in peripheral blood of mice 8h after irradiation. IL-6 levels were maximum at 12h after irradiation. Administration of G-CSF antibody significantly enhanced mortality in irradiated mice. G-CSF antibody-treated mice had higher numbers of CD68(+) cells and apoptotic cells in intestinal villi. Our results confirm that radiation exposure induces elevations of circulating G-CSF and IL-6. Neutralizing antibody to G-CSF exacerbates the deleterious effects of radiation, indicating that G-CSF induced in response to irradiation plays an important role in recovery.     

8-Bromo-cyclic GMP inhibits the calcium channel current in embryonic chick ventricular myocytes

Superfusion with 8-bromo-cyclic GMP or intracellular injection of cyclic GMP inhibits calcium-dependent slow action potentials in embryonic chick or guinea pig ventricular cells, suggesting that cyclic GMP inhibits calcium currents. Recently, cyclic GMP has been shown to reduce cyclic AMP-stimulated calcium currents in voltage-clamped ventricular myocytes. Since earlier results in intact cells had suggested that cyclic GMP might inhibit basal (i.e., unstimulated by cyclic AMP) calcium currents, we directly investigated the effect of 8-bromo-cyclic GMP on basal calcium channel currents (using barium as the charge carrier) in voltage-clamped ventricular myocytes isolated from embryonic chick hearts. Superfusion with 1 mM 8-bromo-cyclic GMP (without prior cyclic AMP elevation) progressively decreased peak calcium channel currents (-68% at 15 min after the onset of drug exposure). In contrast, the currents were unchanged during 15 min superfusion with control solution, or 1 mM 8-bromo-GMP (the noncyclic inactive analog of 8-bromo-cyclic GMP). The present results in voltage-clamped embryonic chick heart cells indicate that cyclic GMP can inhibit basal calcium channel currents, apparently through a cyclic AMP-independent mechanism.     

Steroidogenic effect of atrial natriuretic factor in isolated mouse Leydig cells is mediated by cyclic GMP.

The effects of different atrial natriuretic peptides on cyclic GMP formation and steroidogenesis have been studied in Percoll-purified mouse Leydig cells. Rat atrial peptides rANP (rat atrial natriuretic peptide), rAP-I (rat atriopeptin I) and rAP-II (rat atriopeptin II), in the presence of a phosphodiesterase inhibitor, stimulated cyclic GMP formation in a concentration-dependent manner. In the presence of saturating concentrations of the peptides, a 400-600 fold stimulation of cyclic GMP accumulation was observed. Among the peptides, rAP-II appeared to be the most potent. ED50 values (concentration causing half-maximal effect) for rAP-II, rANP and rAP-I were 1 X 10(-9) M, 2 X 10(-9) M and 2 X 10(-8) M respectively. A parallel stimulation of cyclic GMP formation and testosterone production by the cells was observed after incubation of the cells with various concentrations of rAP-II. In the presence of a saturating concentration of rAP-II (2 X 10(-8) M), maximum stimulation of intracellular cyclic GMP content was obtained within 5 min of incubation. Testosterone production by mouse Leydig cells could be stimulated by 8-bromo cyclic GMP in a concentration-related manner. At a 10 mM concentration of the cyclic nucleotide, steroidogenesis was stimulated to a similar extent as that obtained with a saturating concentration of human chorionic gonadotrophin (5 ng/ml). On the basis of these results we conclude that cyclic GMP acts as a second messenger in atrial-peptide-stimulated steroidogenesis in mouse Leydig cells. The steroidogenic effect of atrial peptides appears to be species-specific, since none of these peptides stimulated testosterone production by purified Leydig cells of rats, though in these cells a 40-60-fold stimulation of cyclic GMP formation in response to each of the three peptides was observed. However, 8-bromo cyclic GMP could stimulate testosterone production in rat Leydig cells. Therefore we conclude that the lack of steroidogenic response in rat Leydig cells to atrial-natriuretic-factor-stimulation results from an insufficient formation of cyclic GMP in these cells. This species difference would appear to result from a lower guanylate cyclase activity in rat Leydig cells.     

Guanosine and inosine display antioxidant activity, protect DNA in vitro from oxidative damage induced by reactive oxygen species, and serve as radioprotectors in mice

The effect of ribonucleosides on 8-oxoguanine formation in salmon sperm DNA dissolved in 1 mM phosphate buffer, pH 6.8, upon exposure to gamma rays was examined by ELISA using monoclonal antibodies against 8-oxoguanine. Nucleosides (1 mM) decreased the radiation-induced yield of 8-oxoguanine in the order Guo > Ino > Ado > Thd > Urd > Cyd. Guanosine and inosine considerably reduced deamination of cytosine in the DNA solutions upon heating for 24 h at 80 degrees C. The action of nucleosides on the heat-induced generation of reactive oxygen species in the phosphate buffer was studied. The concentration of hydrogen peroxide was measured by enhanced chemiluminescence in a peroxidase-luminol-p-iodophenol system; the hydroxyl radical formation was measured fluorometrically by the use of coumarin-3-carboxylic acid. Guanosine and inosine considerably decreased the heat-induced production of both hydrogen peroxide and OH radicals. Guanosine and inosine increased survival of mice after a lethal dose of radiation. They especially enhanced the survival of animals when were administered shortly after irradiation. The results indicate that guanosine and inosine, natural antioxidants, prevent oxidative damage to DNA, decrease the generation of ROS, and protect mice against gamma-radiation-induced death.     

Dose- and time-dependence of radiation-induced nitric oxide formation in mice as quantified with electron paramagnetic resonance.

In vivo nitric oxide (NO) formation was quantified in mice after exposure to high-dose whole-body X-ray irradiation. NO produced and accumulated in the livers of irradiated mice was determined using NO trapping method with iron-dithiocarbamate complex combined with electron paramagnetic resonance (EPR) spectroscopy. When mice were irradiated with 50 Gy X-ray, NO formation peaked in approximately 3 h after the irradiation was terminated. Dose-dependence study indicated that NO formation measured 5 h after irradiation was leveled off at the dose higher than 50 Gy. Administration of NO synthase inhibitor, N(G)-monomethyl L-arginine (L-NMMA) shortly after irradiation completely abolished the NO signal, indicating that radiation-induced NO is produced through L-arginine-dependent NO synthase pathways. These results suggest that irradiation of X-ray initiates inflammation processes, resulting in delayed NO synthase expression and NO formation.     

The induction of the migration of bone marrow stem elements to the periphery by the cytokines of cortisone-resistant thymocytes]

We present evidence about the functional activity of factors produced by cortisol-resistant thymocytes. Experiments in vivo have shown that the administration of the supernatant prepared from cortisol-resistant thymocytes leads to a strong stimulation of endogenous colony formation, significantly prolongs survival of sublethally irradiated mice, increases the rate of restoration of the number of thymocytes in the thymus after sublethal irradiation, and contributes to the recovery of the humoral immune response of nude mice to T-dependent antigens. The results obtained suggest that along with other cytokines, cortisol-resistant thymocytes spontaneously produce a chemotactic factor inducing migration of stem cells from the bone marrow to the periphery.     

Role of splenic stroma in the action of bacterial lipopolysaccharides on radiation mortality: a study in mice carrying the Slj allele

Slj/+ mice display a slight macrocytic anaemia due to a defect in their haemopoietic organ stroma. They have a deficient endogenous spleen colony (CFU-end) formation following sublethal doses of gamma-radiation compared with their normal +/+ littermates, which is likely to be due to the low pre-irradiation CFU-S content of the Slj/+ spleen. CFU-S in these congenic mice do not differ in their sensitivity to gamma-irradiation or stem cell-activating factor. While injection of +/+ mice with 10 micrograms of lipopolysaccharide-W (LPS) one day prior to irradiation led to a substantial increase in their survival, the survival of Slj/+ mice was only slightly increased. Irradiation induced a similar dose-related reduction in the numbers of CFU-S in the spleen and femora of LPS-injected Slj/+ mice compared to similarly treated +/+ mice when measured directly after irradiation. At Day 9 after irradiation, injection of LPS led to a significantly higher CFU-end formation and higher numbers of CFU-S and nucleated cells in the Slj/+ spleens compared to LPS-injected +/+ mice. No such differences in the radioprotective effect of LPS were observed in the +/+ and Slj/+ mice with respect to the splenic and femoral 59Fe-incorporation and the femoral CFU-S numbers at Day 9. These data strongly suggest a contribution by immigrating CFU-S to the CFU-S numbers and endogenous colony formation in at least the Slj/+ spleen after LPS injection and subsequent sublethal irradiation. The observations also imply that the splenic organ stroma may play a mediatory role in the radioprotective action of LPS. In addition, the data represent an extreme example of a lack of correlation between animal survival and haemopoietic parameters. Caution should be taken when applying endogenous colony counts as a means of screening potential anti-radiation drugs.     

Evaluation of the cataractogenic effect of 9 GeV protons

A study was made of the regularities of formation of lenticular opacity in mice exposed to 9 GeV protons and 60Co-gamma-rays. The RBE coefficients, calculated by the nonparametric method, were found to depend upon dose and time after irradiation. It was shown that after small radiation doses (0.25 to 0.50 Gy) the RBE coefficients increased from 1 to 8 with increasing period of observation. With higher doses (up to 5.0 Gy) the RBE coefficient increase in time was less pronounced.     

Role of splenic stroma in the action of bacterial lipopolysaccharides on radiation mortality: a study in mice carrying the Slj allele

Abstract. Sl_j/+ mice display a slight macrocytic anaemia due to a defect in their haemopoietic organ stroma. They have a deficient endogenous spleen colony (CFU-end) formation following sublethal doses of gamma-radiation compared with their normal +/+ littermates, which is likely to be due to the low pre-irradiation CFU-S content of the Sl_j/+ spleen. CFU-S in these congenic mice do not differ in their sensitivity to gamma-irradiation or stem cell-activating factor. While injection of +/+ mice with 10 μg of lipopolysaccharide-W (LPS) one day prior to irradiation led to a substantial increase in their survival, the survival of Sl_j/+ mice was only slightly increased. Irradiation induced a similar dose-related reduction in the numbers of CFU-S in the spleen and femora of LPS-injected Sl_j/+ mice compared to similarly treated +/+ mice when measured directly after irradiation. At Day 9 after irradiation, injection of LPS led to a significantly higher CFU-end formation and higher numbers of CFU-S and nucleated cells in the Sl_j/+ spleens compared to LPS-injected +/+ mice. No such differences in the radioprotective effect of LPS were observed in the +/+ and Sl_j/+ mice with respect to the splenic and femoral ⁵⁹Fe-incorporation and the femoral CFU-S numbers at Day 9. These data strongly suggest a contribution by immigrating CFU-S to the CFU-S numbers and endogenous colony formation in at least the Sl_j/+ spleen after LPS injection and subsequent sublethal irradiation. The observations also imply that the splenic organ stroma may play a mediatory role in the radioprotective action of LPS. In addition, the data represent an extreme example of a lack of correlation between animal survival and haemopoietic parameters. Caution should be taken when applying endogenous colony counts as a means of screening potential anti-radiation drugs.     

Protective effects of 5,6,7,8-tetrahydroneopterin against X-ray radiation injury in mice

The protective effects of 5,6,7,8-tetrahydroneopterin (NH4) against radiation injury in mice were studied. (C57BL/6xA/J)F1 (B6A) mice received a single whole-body irradiation dose of 200, 400, 700 or 800 cGy of X-rays. NH4 (30 mg/kg body weight) or phosphate-buffered saline (PBS) was injected intraperitoneally into irradiated mice 10 min before and after the irradiation and again after 6 h. All mice which received the 800 cGy radiation+PBS died between 8 and 11 days after the treatment. In contrast, those which also received NH4 demonstrated a significantly prolonged survival time and 40% lived more than 5 months. Total numbers of thymocytes and spleen cells on day 5 post-irradiation were dramatically reduced in line with the radiation dose. The survival was significantly enhanced by NH4 in treated mice. The proliferation of spleen cells in mice stimulated by concanavalin A (Con A) or lipopolysaccharide (LPS) was also greater in NH4 treated mice. The immune response of survivors 5 months after 800 cGy+NH4 treatments, against Con A, LPS, allogenic mouse, and sheep red blood cells had essentially recovered to the levels of normal mice. These results indicate that NH4 had an important role in modifying radiation injury.     

Morphofunctional characteristics of murine kidneys in single and fractionated irradiation in air and hypoxia (8% of O2)]

The protective effect of hypoxic gas mixture containing 8% of oxygen (HGM-8) has been studied in long terms after local single and fractional X-ray irradiation of the kidney in mice. Some criteria of injury shown that hypoxia protects the kidneys against irradiation, changes in irradiation dose constituting 1.25-1.33. When passing from single irradiation to five-fold daily one, the protective effect of hypoxia does not fall significantly.     

Specific effect of attenuated strain of Francisella tularensis on the development of radiation induced lesions in experimental animals and effictiveness of antibacterial therapy for lesions induced by radiation combined with virulemt strain of the bacteria

For study of the effects of whole-body gamma-radiation (1 and 4 Gy) on the response of the body to administration of vaccines and virulent strains of tularemia 206 outbred white mice were used. The results of the study shown that the administration of attenuated bacterial cells in 5 days after exposure to radiation (1 and 4 Gy) caused more severe post-radiation effects and the increase in the number of died animals. The severity of the disease was less if mice were vaccinated in 26 days after irradiation (4 Gy). The treatment of tularemia in irradiated mice twith Riphampicin (daily peroral administration, 5 mg/mouse, duration of treatment--7 days) administered in 4 hours after infection was effective and caused high survival of affected mice. The results show effectiveness of the riphampicin treatment of tularemia in the animals exposed to sublethal dose of radiation.     

Cyclic GMP regulates cromakalim-induced relaxation in the rat aortic smooth muscle: role of cyclic GMP in K(ATP)-channels.

Recent studies have shown that nitric oxide (NO) modulates K+-channel activity which play an important role in controlling vascular tone. The formation of cyclic guanosine 3',5'-monophosphate (cyclic GMP) has also been recognized to be associated with the vasodilatory effect of NO. Both cyclic GMP and NO increase whole-cell K+-current by activating Ca2+-activated K+-channels (K(Ca)-channels). Here, we show evidence that activators of soluble guanylyl cyclase sodium nitroprusside or 3-morpholino-sydnonimine (SIN-1), and an analogue of cyclic GMP 8-bromo-cyclic GMP enhance the relaxation induced by cromakalim which is blocked by glibenclamide (a specific inhibitor of ATP-sensitive K+-channels [K(ATP)-channels]), and partially attenuated by methylene blue (an inhibitor of cyclic GMP formation). However, this is not due to the increase of cyclic GMP level by cromakalim itself because the relaxation induced by cromakalim is not associated with the changes of cyclic GMP level formed in the aortic smooth muscle. Thus, it is most likely that cyclic GMP also modulates activity of K(ATP)-channels, in addition to K(Ca)-channels, in the rat aorta.     

减毒沙门氏菌介导的血小板第四因子活性片段的放射保护作用研究

目的:观察减毒沙门氏菌携带的血小板第四因子活性片段PF417 70 的放射保护作用。方法:通过口服途经喂饲小鼠携带PF4活性片段的减毒沙门氏菌,在第 2次喂饲后小鼠接受 70 0cGy全身照射,然后观察PIRES2 EGFP PF417 70 在小鼠体内的表达,并观察小鼠的造血恢复情况。结果:在小鼠的肝脏、脾脏、肾脏、小肠、外周血及骨髓均能检测到GFP的表达和转基因的整合。与对照组比较,实验组小鼠的生存期明显延长,照射后第 7d和 1 4d骨髓有核细胞数、骨髓培养的CFU GM和HPP CFC数量明显增加 (P <0 0 5 )。结论:首次应用减毒沙门氏菌SL32 61为载体来介导PF4活性片段的生物学作用,并证实通过口服途径可以保护小鼠免受放射损伤,并促进放射损伤后小鼠的造血恢复。