[RADIOPROTECTIVE ACTIVITY OF POLYMETOXY-LATED FLAVONOIDS OF CITRUS EXTRACT].

The aim of the study was to establish the radioprotective activity of citrus polymetoxylated flavonoids extract (CPMFE) on the X-irradiated rats. The experiments were carried out on white Wistar rats. Animals were irradiated with X rays in doses of 5 Gy and 7 Gy. The control group consisted the sham-irradiated rats. Part of animals of each group were treated with intramusculary injections of CPMFE (dose 30 mg/kg) during 7 days; blood was taken from the tail vein (0.5 ml) for detection of lipoperoxides (LOO.) content. On the 3rd day after irradiation 3 animals from each group were sacrificed (under ether anesthesia) and blood samples were taken for the study of antioxidant status. The activity of antioxidant enzymes (catalase (CAT) and superoxidedismutase (SOD)) was determined by the spectrophotometric method; the content of LOO.in the blood was determined by electron paramagnetic resonance (EPR) mrthod. In group of irradiated rats a sharp dose-dependent inactivation of blood antioxidant enzymes (SOD, CAT) and intensification of the lipid peroxidation were detected. The direct and feedback mechanism in the regulation of CAT and SOD activity, ensuring the implementation of antioxidant protection in the body was revealed. Under irradiation with 7Gy rapid death of animals (on 3-d day after irradiation the mortality of animals was 70%, and on the 5th day all died) were detected. During irradiation with dose 5 Gy the survival of animals increased (on the 8-th day after irradiation - 50% survival rate). CPMFE in dose-dependent manner supported the reduce the intensity of lipid peroxidation processes - at relatively low doses of radiation (5Gy) during the first 3 days the content of LOO.in the blood decreased insignificantly compared with indices in untreated animals, whereas with an increase in the dose of irradiation (7Gy) a statistically significant antiradical effect of CPMFE (a statistically significant decrease in the LOO. content) was detected. Under the effect of CPMFE in the blood of rats irradiated with a dose of 5 Gy and 7 Gy, the activity of CAT and SOD, not statistically significant tends to increase (more significant with a dose of 7 Gy). CPMFE did not affect the cumulative survival of animals irradiated with a dose of 5 Gy, but reduced the mortality of rats by 20% (on the 3rd day of irradiation), and contributed to an increase in the life expectancy of animals by 2 times (up to 7 days) in the case of dose 7 Gr. Based on the analysis of the research results, it can be assumed that under conditions of radiation damage, exogenous antioxidants synergistically with a dose-dependently activated endogenous non-enzymatic antioxidant system of the body (especially at 7Gy) contribute to the effective suppression of chain reactions of peroxidation, reduction of mortality and increase in life expectancy of animals.

Ciprofloxacin Therapy Results in Mitigation of ATP Loss after Irradiation Combined with Wound Trauma: Preservation of Pyruvate Dehydrogenase and Inhibition of Pyruvate Dehydrogenase Kinase 1.

Ionizing radiation exposure combined with wound injury increases animal mortalities than ionizing radiation exposure alone. Ciprofloxacin (CIP) is in the fluroquinolone family of synthetic antibiotic that are available from the strategic national stockpile for emergency use and is known to inhibit bacterial sepsis. The purpose of this study was to evaluate the efficacy of ciprofloxacin as a countermeasure to combined injury mortality and determine the signaling proteins involved in energy machinery. B6D2F1/J female mice were randomly assigned to receive either 9.75 Gy irradiation with Co-60 gamma rays followed by skin wounding (combined injury; CI) or sham procedure (sham). Either ciprofloxacin (90 mg/kg/day) or vehicle (VEH) (water) was administered orally to these mice 2 h after wounding and thereafter daily for 10 days. Determination of tissue adenosine triphosphate (ATP) was conducted, and immunoblotting for signaling proteins involved in ATP machinery was performed. Combined injury resulted in 60% survival after 10 days compared to 100% survival in the sham group. Furthermore, combined injury caused significant reductions of ATP concentrations in ileum, pancreas, brain, spleen, kidney and lung (-25% to -95%) compared to the sham group. Ciprofloxacin administration after combined injury resulted in 100% survival and inhibited reductions in ileum and kidney ATP production. Ileum protein levels of heat-shock protein 70 kDa (HSP-70, a chaperone protein involved in ATP synthesis) and pyruvate dehydrogenase (PDH, an enzyme complex crucial to conversion of pyruvate to acetyl CoA for entrance into TCA cycle) were significantly lower in the CI group (vs. sham group). Using immunoprecipitation and immunoblotting, HSP-70-PDH complex was found to be present in the ileum tissue of CI mice treated with ciprofloxacin. Furthermore, phosphorylation of serine residues of PDH resulting in inactivating PDH enzymatic activity, which occurred after combined injury, was inhibited with ciprofloxacin treatment, thus enabling PDH to increase ATP production. Increased ileum levels of pyruvate dehydrogenase kinase 1 protein (PDK1, an enzyme responsible for PDH phosphorylation) after combined injury were also prevented by ciprofloxacin treatment. Taken together, these data suggest that ciprofloxacin oral administration after combined injury had a role in sustained ileum ATP levels, and may have acted through preservation of PDH by HSP-70 and inhibition of PDK1. These molecular changes in the ileum are simply one of a host of mechanisms working in concert with one another by which ciprofloxacin treatment mitigates body weight loss and drastically enhances subsequent survival after combined injury. To this end, our findings indicate that oral treatment of ciprofloxacin is a valuable therapeutic treatment after irradiation with combined injury and warrants further analyses to elucidate the precise mechanisms involved.

Inhibiting glycogen synthase kinase-3 mitigates the hematopoietic acute radiation syndrome in mice.

Exposure to a nuclear accident or radiological attack can cause death from acute radiation syndrome (ARS), which results from radiation injury to vital organs such as the hematopoietic system. However, the U.S. Food and Drug Administration (FDA) has not approved any medical countermeasures for this specific purpose. With growing concern over nuclear terrorism, there is an urgent need to develop small molecule deliverables that mitigate mortality from ARS. One emerging modulator of hematopoietic stem/progenitor cell (HSPC) activity is glycogen synthase kinase-3 (GSK-3). The inhibition of GSK-3 has been shown to augment hematopoietic repopulation in mouse models of bone marrow transplantation. In this study, we performed an in vitro screen using irradiated bone marrow mononuclear cells (BM-MNCs) to test the effects of four GSK-3 inhibitors: CHIR99021; 6-Bromoindirubin-3'-oxime (BIO); SB415286; and SB216763. This screen showed that SB216763 significantly increased the frequency of c-Kit(+) Lin(-) Sca1(+) (KLS) cells and hematopoietic colony-forming cells in irradiated BM-MNCs. Importantly, administration of a single dose of SB216763 to C57BL/6J mice by subcutaneous injection 24 h after total-body irradiation significantly improved hematopoietic recovery and mitigated hematopoietic ARS. Collectively, our results demonstrate that the GSK-3 inhibitor SB216763 is an effective medical countermeasure against acute radiation injury of the hematopoietic system.

Flagellin administration protects gut mucosal tissue from irradiation-induced apoptosis via MKP-7 activity.

BACKGROUND AND AIMS: Radiotherapy for neoplastic disease is associated with significant adverse enteric effects associated with excessive cell death. Ionising radiation induces cell death by a mechanism that is dependent on JNK (c-jun N-terminal kinase) pathway signalling. Additionally, it is known that cells exposed to extracellular bacterial products such as flagellin, pleiotropically activate a number of innate immune pathways, including that of JNK. The JNK pathway controls its own activity by inducing the transcription of mitogen-activated protein kinase phosphatase-7 (MKP-7) which directly targets phosphorylated JNK, thus functioning as a negative feedback loop. Previously, it has been shown that flagellin limits ionising radiation-induced mortality in mice, but the cellular mechanism of protection remained unknown. METHODS: Wild-type C57BL/6 or tlr5(-/-) C57BL/6 were injected with flagellin 2 h before exposure to irradiation, and their intestines were examined for apoptosis. Candidate proteins mediating cytoprotection from irradiation were identified by expression profiling. One of these candidates, MKP-7, was cloned and packaged into adenovirus particles, used to infect cultured cells, and examined for the extent to which its activity reduced cellular apoptosis by flow cytometry or immunoblot analysis. RESULTS: Flagellin pretreatment protected mice from radiation-induced intestinal mucosal injury and apoptosis via a Toll-like receptor 5 (TLR5)-dependent mechanism. Expression profiling of flagellin-treated mice showed upregulation of MKP-7, an inducible repressor of the JNK pathway. MKP-7 expression reached a maximum at 2 h after flagellin treatment, coinciding with suppression of phosphorylated JNK and JNK pathway inhibition. Furthermore, constitutive MKP-7 expression protected cultured cells from radiation-induced apoptosis. CONCLUSIONS: Flagellin is a promising adjuvant for suppressing ionising radiation-induced injury. MKP-7 activity exhibits cytoprotective effects, and is thus a candidate cellular molecule for limiting the damaging effect of radiotherapy on the gastreointestinal system.

The protective effect of alpha-lipoic acid against oxidative damage in rabbit conjunctiva and cornea exposed to ultraviolet radiation.

PURPOSE: The purpose of this study was to determine the protective effect of alpha-lipoic acid against oxidative damage in rabbit conjunctiva and cornea exposed to ultraviolet radiation. METHODS: 20 rabbits weighing 2,500- 3,000 g were used, and we divided them into 4 groups with 5 randomly selected rabbits. The rabbits were exposed to 2 J/cm(2)/h of ultraviolet A radiation (UVA) in the range of 320-405 nm for 12 h per day within 90 days. The control group did not undergo any procedure, the UVA group was only exposed to UVA radiation. The PUVA group was treated with 8-methoxypsoralen and UVA. The alpha-lipoic acid group was administered 8-methoxypsoralen + UVA + alpha-lipoic acid. At the end of 90 days, the rabbits were killed by decapitation, and the eyes were enucleated. Both eyes of each rabbit were used for biochemical evaluation. Conjunctival and corneal free malondialdehyde (MDA), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) levels were compared among the groups. RESULTS: Conjunctival free MDA levels were lower in the alpha-lipoic acid group compared with the UVA and PUVA groups (p < 0.05, p < 0.001, respectively). Both conjunctival SOD levels (p < 0.05, p < 0.01, respectively) and conjunctival GSH-PX levels (p < 0.01, p < 0.001, respectively) were higher in the alpha-lipoic acid group compared with other groups. Corneal free MDA levels were lower in the alpha-lipoic acid group compared with the UVA and PUVA groups (p < 0.01, p < 0.001, respectively). Both corneal SOD levels (p < 0.01, p < 0.01, respectively) and corneal GSH-PX levels (p < 0.01, p < 0.01, respectively) were higher in the alpha-lipoic acid group compared with the other groups. CONCLUSION: alpha-Lipoic acid which is considered as potent antioxidant protects the eye from the damaging effect of ultraviolet exposure.

Effects of oral Ginkgo biloba supplementation on cataract formation and oxidative stress occurring in lenses of rats exposed to total cranium radiotherapy.

PURPOSE: To determine the antioxidant role of Ginkgo biloba (GB) in preventing radiation-induced cataracts in the lens after total-cranium irradiation of rats with a single radiation dose of 5 Gy. METHODS: Sprague-Dawley rats were randomly divided into three groups. Group 1 received neither GB nor irradiation (control group). Group 2 was exposed to total-cranium irradiation of 5 Gy in a single dose [radiation therapy (RT) Group], and group 3 received total cranium irradiation from a cobalt-60 teletherapy unit, plus 40 mg/kg per day GB (RT+GB group). At the end of the tenth day, the rats were killed and their eyes were enucleated to measure the antioxidant enzymes, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the lipid peroxidation level [malondialdehyde (MDA)]. RESULTS: Irradiation significantly increased both the MDA level and the activity of GSH-Px, and significantly decreased the activity of SOD in the rat lenses. GB supplementation significantly increased the activities of SOD and GSH-Px enzymes and significantly decreased the MDA level. Total cranium irradiation of 5 Gy in a single dose promoted cataract formation, and GB supplementation protected the lenses from radiation-induced cataracts. CONCLUSIONS: We suggest that Ginkgo biloba is an antioxidant that protects the rat lens from radiation-induced cataracts.

The effect of N-acetyl serotonin on ultraviolet-radiation-induced cataracts in rats.

AIMS: To investigate the effects of ultraviolet radiation (UVR) on cataract development in rat lenses and whether or not N-acetyl serotonin has an effect on changes in these lenses. MATERIAL AND METHODS: The study was performed using 5 groups of Sprague-Dawley albino rats, with each group consisting of 15 rats. The 5th group being the control group did not receive any applications, whilst the other 4 groups received a daily dose of 0.2 J/cm(2)/day UVR (305 nm wavelength) for 60 days. A dose of 4 mg/kg/0.1 ml N-acetyl serotonin was injected intraperitoneally to group 1 and group 2 every day and on alternate days, respectively. Group 3 received an intraperitoneal injection of 0.1 ml phosphate buffer solution every day, whilst group 4 received no injection. On the 60th day, an intracardiac withdrawal of blood was performed, after the rats had been anesthetized with ether. Following the withdrawal of blood, the rats were killed using a high dose of ether and their eyes were enucleated. The lens fresh weights, plasma malondialdehyde (P-MDA), erythrocyte glutathione peroxidase (E-GSHPx), erythrocyte glutathione reductase, blood reduced glutathione (B-RGSH), erythrocyte catalase (E-CAT), lenticular malondialdehyde, lenticular superoxide dismutase (L-SOD), lenticular glutathione peroxidase (L-GSHPx) and lenticular glutathione (L-GSH) levels were all assessed. RESULTS: The lens fresh weights were determined to be lower in group 1 and in the control group in comparison with the other groups (p < 0.01). Whilst the P-MDA level was found to be lower (p < 0.001), the E-GSHPx level was higher (p < 0.01) in the control group than in the other groups. The E-GSHPx level was higher in groups 1 and 2 than in groups 3 and 4 (p < 0.01). The B-RGSH level was higher in the control group than in the other groups (p < 0.001). The E-CAT level was higher in both the control group and group 1 than in groups 2, 3 and 4 (p < 0.01), whilst it was higher in group 2 when compared to groups 3 and 4 (p < 0.01). The L-SOD levels were found to be higher in the control group and group 1 than in groups 2, 3 and 4 (p < 0.001). Whilst the L-GSHPx levels were determined to be higher only in the control group (p < 0.001), the L-GSH levels were higher in the control group and group 1 than in the other groups (p < 0.001). CONCLUSIONS: In recent years, the depletion of the atmospheric ozone layer has resulted in the penetration of more UVR to the earth, which causes various effects on different tissues of organisms. N-acetyl serotonin, a melatonin precursor, may well be effective in the prevention of the negative effects induced by the UVR upon the lens tissue, in which case the capability of melatonin to capture free radicals as well as its antioxidative properties should be taken into consideration.

Radioprotective effect of silymarin against radiation induced hepatotoxicity.

The radioprotective effect of silymarin using different modes of treatment against radiation (3 or 6 Gy) induced hepatotoxicity 1, 3 and 7 days post-irradiation was studied. Whole-body gamma-irradiation revealed an increase in serum alkaline phosphatase (AP) activity as well as liver glutathione reductase (GR) and glutathione peroxidase (GSH-PX) activities on the first post-exposure day with respect to the control value. However, 3 days after radiation exposure, these parameters showed a significant decrease below the control level which persisted till the end of the experimental time except for serum AP activity that showed another increase on the seventh post-exposure day at 3 Gy dose of radiation. A gradual increase in serum alanine and aspartate aminotransferase (ALT&AST) as well as gamma glutamyl transpeptidase activities were observed due to irradiation throughout the experimental time. Administration of silymarin as single (70 mg kg (-1)), fractionated (490 mg kg (-1)) oral doses or as intravenous (i.v.) injection (50 mg kg (-1)), caused significant protection. Intravenous treatment showed the most pronounced protection. The protective effect of silymarin was attributed to its antioxidant and free radicals scavenging properties.

Reduction of high-energy shock-wave-induced renal tubular injury by selenium.

In shock-wave-induced renal injury cavitation-generated free radicals play an important role. Using an in vitro model with Madin-Darby canine kidney (MDCK) cells, we investigated the influence of selenium, a free radical scavenger, in shock-wave-induced tubular cell injury. Suspensions of MDCK cells (33 x 10(6) cells/ml) were placed in small containers (volume 1.1 ml) for shock wave exposure. Two groups of 12 containers each were examined: (1) control (no medication), (2) selenium (0.4 microg/ml nutrient medium). Six containers in each group were exposed to shock waves (impulse rate 256, frequency 60 Hz, generator voltage 18 kV), while the other six containers in each group served as a control. After shock wave exposure, the concentration of cellular enzymes such as lactate dehydrogenase (LDH), N-acetyl-beta-glucosaminidase (NAG), glutamate oxaloacetate transaminase (GOT) and glutamate lactate dehydrogenase (GLDH) in the nutrient medium was examined. Following shock wave exposure there was a significant rise in LDH, NAG, GOT and GLDH concentrations. Selenium reduced this enzyme leakage significantly. Thus we conclude that selenium protects renal tubular cells against shock-wave-induced injury. Since selenium is an essential part of glutathione peroxidase, this effect seems to be mediated by a reduction in reactive oxygen species.

[The effect of the parenteral administration of alpha-ketoglutarate on the resistance of rats to ionizing radiation and on their cholinergic systems]. / Vplyv parenteral'noho vvedennia al'fa-ketohlutaratu na rezystentnist' shchuriv do ionizuiuchoho oprominennia ta kholinerhichnu systemu ïkh orhanizmu.

We have investigate the effect of sodium ketoglutarate intraperitoneal injection (20 mg/100 g body weight) made 0.5 hour before and 1, 2 and 3 hours after total X-ray treatment (259 mKl/kg) on the survival of rats. Simultaneously we have define changes in cholinesterase-acetylcholine system and content of adrenaline and noradrenaline in liver and pancreas tissues, small intestines mucous and in blood. All the data were taking at 1, 2, 5, 4, 24 and 72 hours after treatment and sodium ketoglutarate injection. We have found that sodium ketoglutarate injection made 0.5 hour before the treatment results in increasing of percentage death rate and injection made 1 and 2 hours after treatment results in increase in survival of rats. The effect of alpha-ketoglutarate injection made 1 hour after treatment in more pronounced. It is accompanied with increase of cholinergic status of organism on the catecholamine deficit background, decrease in the cholinesterase activity and increase of acetylcholine content in tissues of treated organism.

[Effect of polyunsaturated phosphatidylcholine on the activity of antioxidant and lipolytic enzymes in alimentary hypercholesterolemia in irradiated rats]. / Deistvie polinenasyshchennogo fosfatidylkholina na aktivnost' antioksidantnykh i lipoliticheskikh fermentov pri alimentarnoi giperkholesterinemii u obluchennykh krys.

A high-cholesterol diet containing 3.5% cholesterol, 0.2% methyl thiouracil and 20% of heated sunflower-seed oil caused an increase in the content of cholesterol, triacylglycerols, diene conjugates and in the activity of phospholipase A2 in rat liver and of serum total cholesterol and cholesterol of low density lipoproteins, and a decrease in the activity of hepatic cholesterol esterase, in the fractional activity of lecithin:cholesterol acetyl transferase (LCAT), in the activity of the enzymes involved in the lipolytic transformation of lipoproteins and in the serum levels of very low density lipoprotein cholesterol. The content of malonic dialdehyde in liver tissue and molar activity of LCAT in blood serum were increased after preirradiation (0.5 and 5.0 Gy) of rats kept on the cholesterol diet. The preparation of polyunsaturated phosphatidylcholine was shown to decrease the content of triacylglycerols and diene conjugates in liver tissue of rats with alimentary hypercholesterolemia. In rats irradiated in a dose of 0.5 Gy the phosphatidylcholine preparation decreased the content of cholesterol in liver tissue and blood serum, exhibited the normalizing effect on the content of cholesterol of high density lipoproteins, on the activity of superoxide dismutase and glutathione peroxidase; in a dose of 5.0 Gy this phosphatidylcholine preparation normalized the content of malonic dialdehyde, activity of the enzymes involved in lipolytic transformation of lipoproteins and increased the LCAT activity.

[The display of hormesis in the action of quinoid radiotoxins]. / Proiavlenie gormezisa v deistvii khinoidnykh radiotoksinov.

Two hours before irradiation (7.5 Gy) mice were injected with quinoid radiotoxins in concentrations close to those formed in mouse blood after gamma-irradiation with a dose of 10 cGy. This increased the survival rate (from 10-20% to 80%), prevented the radiation-induced weight loss, and increased catalase activity in the liver and blood of exposed animals.

[The effect of the prophylactic use of benzonal on the cytochrome P-450 content of the liver in irradiated rats]. / Vliianie profilakticheskogo primeneniia benzonala na soderzhanie tsitokhroma P-450 v pecheni obluchennykh krys.

The rats (100 mg/kg, once a day, per os, during 3 days) were administered suspension of benzonal in starch gel before irradiation of 12 Gy. Induced and uninduced rats were irradiated on the following day after stopping benzonal administration and were decapitated at 10, 12, 15 and 21 o'clock during the first and second day after irradiation and also on the fourth day (day of mass death of irradiated rats). It has been established that irradiation changes the dynamics of cytochrome P-450 concentration in microsomal fraction of rat liver. The essential decrease of the content of cytochrome on the second day after irradiation was accompanied by intensification of the process of its inactivation, but stoichiometry between the decrease of P-450 and the increase of cytochrome P-420 was not observed. The high inducing and stabilizing effects of benzonal on cytochrome P-450 and on the liver persisted. In comparison with irradiation the unfavourable effect of benzonal on immunocompetent organs (thymus, spleen) was not found.

[Pyridoxal phosphate as an agent for normalizing the monoamine oxidase activity of the brain in radiation sickness]. / Piridoksal'fosfat kak sredstvo normalizatsii monoaminoksidaznoi aktivnosti golovnogo mozga pri luchevoi bolezni.

A study was made of the influence of the coenzyme pyridoxal phosphate on the activity of mitochondrial monoaminoxidase (MAO) catalyzing oxidative deamination of serotonin at the exacerbation of acute radiation disease (on the 6th day after radiation exposure) in different parts of the brain (cerebral hemisphere, stem and cerebellum). Experiments were staged on rabbits, irradiated by x-ray at a dose of 4.5 Gy with a dose rate of 0.33 Gy/min. The peak of radiation disease was characterized by considerable changes in MAO activity resulting in catalysis of oxidative deamination of serotonin in different parts of the brain and in different mitochondrial subfractions. Pyridoxal phosphate produced a positive effect on monoaminoxidase activity, catalyzing serotonin deamination, and as a coenzyme it can be incorporated in a complex of drugs used for normalization of metabolism of mediators of the nervous system as well as for therapy of radiation injuries.

[Action of alpha-tocopherol on the enzymes of the electron transport chains in the liver microsomes of irradiated rats]. / Deistvie al'fa-tokoferola na fermenty élektron-transportnykh tsepei mikrosom pecheni obluchennykh krys.

Five days following single whole-body gamma irradiation of rats (8.5 Gy) the rate of NADPH and NADH oxidation, the activity of NADPH-cytochrome P-450 and NADH-cytochrome b5 reductases, and the content of cytochromes P-450 and b5 were found to decrease. The intragastric administration of alpha-tocopherol (100 mg/kg, two times a day) produced a normalizing effect.

Biogenic amine response to whole body irradiation. Prevention by APTH.

60Co whole body irradiation with 154.8 mC/kg (600 R) resulted in a mortality of 50 per cent within two weeks. Administration of 1-acetyl-3-phenylamidine thiocarbamide hydrochloride (APTH) 30 min before irradiation prevented this mortality. Irradiation eliminated neurosecretory material from the perikaryons of the supraoptic and paraventricular neurons, whereas APTH counteracted this action. APTH also increased the synthesis of bioamines (5-hydroxytryptamine, catecholamine and acetylcholine).