Changes of reduced glutathion, glutathion reductase, and glutathione peroxidase after radiation in guinea pigs

In this series of experiments the protective action of reduced glutathion due to ionizing radiation has been studied. In the experimental group 18 guinea pigs were exposed to successive radiations of 150 rad 3 or 4 days apart. Total dose given amounted to 750 rad which is the LD50 for guinea pigs. Blood samples were taken 30 min after each exposure. The control series were sham radiated but otherwise treated identically. The cells of the removed blood samples were separated by centrifugation and were subjected to the reduced glutathion stability test. GSSGR, GPer, and LDH enzyme activities were also measured of which the latter served as a marked enzyme. It was found that LDH did not show any alteration after radiation. The reduced glutathion stability test showed a consistent but minor reduction (P greater than 0.05), in the experimental group. GSSGR enzyme activity on the other hand was reduced significantly (from 176.48 +/- 11.32 to 41.34 +/- 1.17 IU/ml of packed erythrocytes, P less than 0.001) in the same group. GPer activity showed a consistent but minor elevation during the early phase of the experimental group. It was later increased significantly beginning after 600 rad total radiation on the fourth session (P less than 0.050).     

Alterations in monoamine oxidase activity of the mouse brain and liver after mixed neutron-gamma irradiation

Monoamine oxidase (MAO) plays an important role in the metabolism of neuro-transmitter biogenic amines. Its activity was determined in mouse brain and liver after exposure to different kinds of ionizing radiation and after pretreatment with a radioprotective agent. After a lethal dose of mixed neutron-gamma irradiation the MAO activity decreased in the brain and increased in the liver. In contrast, after a lethal dose of 60Co-gamma irradiation enzyme activity was considerably increased in the brain while in the liver it increased like after mixed neutron-gamma irradiation. AET (S2-aminoethyl-isothiuronium-Br X HBr), when administered in a radio-protective dose, inhibited MAO activity in the brain, while it increased in the liver. Even more marked changes of enzyme activity were observed in both brain and liver after AET pretreatment and mixed neutron-gamma irradiation. On the basis of the results it is suggested that different kinds of ionizing radiation lead to different types of lipid peroxidation in the lipid environment surrounding MAO, an event leading to altered enzyme activity. AET itself inhibited MAO in the brain and increased the activity in the liver but did not prevent the alterations caused by ionizing radiation in enzyme activity.     

CHANGED ACTIVITIES OF BRAIN ENZYMES INVOLVED IN NEUROTRANSMITTER METABOLISM IN RATS EXPOSED TO DIFFERENT QUALITIES OF IONIZING RADIATION

—The effect of different qualities of ionizing radiation on the activity of brain enzymes involved in the metabolism of neurotransmitters in specific regions of the brain of rats was investigated. Groups of Sprague-Dawley adult male rats were exposed to approx. 18,000 rads of radiation either rich in neutrons or rich in gamma rays. It was found that, when the animals were exposed to radiation rich in neutrons, monoamine oxidase (MAO) activity was markedly decreased in all brain areas studied. In contrast, a very marked increase in the activity of this enzyme was observed when the animals received the same dose of radiation rich in gamma rays. Relatively minor changes were observed in the activity of choline acetyl transferase (ChAc). Acetylcholinesterase (AChE) activity did not change appreciably.     

Reversible alterations in cultured pulmonary artery endothelial cell monolayer morphology and albumin permeability induced by ionizing radiation

The effects of ionizing irradiation (0, 600, 1,500, or 3,000 rads) on the permeability of pulmonary endothelial monolayers to albumin were studied. Pulmonary endothelial cells were grown to confluence on gelatin-coated polycarbonate filters, placed in serum-free medium, and exposed to a 60Co source. The monolayers were placed in modified flux chambers 24 hours after irradiation; 125I-albumin was added to the upper well, and both the upper and lower wells were serially sampled over 4 hours. The amount of albumin transferred from the upper well/hour over the period of steady-state clearance (90-240 min after addition of 125I-albumin) was 2.8 +/- 0.2% in control monolayers and was increased in monolayers exposed to 1,500 or 3,000 rads (increase of 63 +/- 10% and 61 +/- 10%, respectively, P less than 0.01). No increase was found in monolayers exposed to 600 rads. The increases in endothelial albumin transfer rates were associated with morphologic evidence of monolayer disruption and endothelial injury which paralleled the changes in albumin permeability. Dose-dependent alterations in endothelial actin filament organization were also found. Incubation of the monolayers exposed to 3,000 rads with medium supplemented with 10% fetal calf serum for 24 hours resulted in normalization of albumin permeability, improvement in morphologic appearance of the monolayers, and reorganization of the actin filament structure. These studies demonstrate that ionizing radiation is an active principle in the reversible disorganization of cultured pulmonary endothelial cell monolayers without the need of other cell types or serum components.     

Effects of acute low doses of Gamma-radiation on erythrocytes membrane

It is believed that any dose of ionizing radiation may damage cells and that the mutated cells could develop into cancer cells. Additionally, results of research performed over the past century on the effects of low doses of ionizing radiation on biological organisms show beneficial health effects, called hormesis. Much less is known about the cellular response to low doses of ionizing radiation, such as those typical for medical diagnostic procedures, normal occupational exposures or cosmic-ray exposures at flight altitudes. Extrapolating from the effects observed at higher doses to predict changes in cells after low-dose exposure is problematic. We examined the biological effects of low doses (0.01–0.3 Gy) of γ-radiation on the membrane characteristics of erythrocytes of albino rats and carried out osmotic fragility tests and Fourier transform infrared spectroscopy (FTIR). Our results indicate that the lowest three doses in the investigated radiation range, i.e., 0.01, 0.025 and 0.05 Gy, resulted in positive effects on the erythrocyte membranes, while a dose of 0.1 Gy appeared to represent the limiting threshold dose of those positive effects. Doses higher than 0.1 Gy were associated with the denaturation of erythrocyte proteins.     

Effect of ionizing radiation on DNA synthesis in ataxia telangiectasia cells.

The effect of ionizing radiation on DNA synthesis in control and ataxia telangiectasia (AT) lymphoblastoid cell lines was determined. A dose dependent decrease in DNA synthesis was observed in control cells, and the rate and extent of thi decrease in synthesis increased with time after irradiation. No decrease in DNA synthesis was obtained in AT cells, immediately following irradiation, at doses up to 400 rads. At longer times postirradiation, inhibition of synthesis increased but the extent of inhibition was less in AT cell than controls at all doses used. An immediate depression of DNA synthesis was evident in control cells after a radiation dose of 200 rads reaching a maximum at 90 min postirradiation. Little or no decrease in DNA synthesis was evident in AT cells up to 60 min after the same radiation dose, but a decrease occurred between 60 and 90 min after irradiation. The rate of recovery of DNA synthesis to normal levels was more rapid in AT cells than in controls.     

REGULATION OF PROTEIN SYNTHESIS DURING POSTNATAL MATURATION OF THE BRAIN

—The activities of sRNA-aminoacyl synthetases (EC 6.1.1) were investigated in the cerebral white and grey matter of rabbits subjected during their prenatal life to a single X-ray dose of 150 rad. The results of investigations have shown that ionizing radiation acting during intrauterine development of the experimental animal brings about a distinct depression of all sRNA-aminoacyl synthetase activities in the newborn irradiated litter. During the postnatal development of these animals the activities of some of the synthetases further decreased and even at adulthood, where they are normally very low, their activities were below the control values. The activities of some other synthetases, after the initial depression, showed no further decrease and at adulthood had values comparable to controls. Our results indicate clearly that prenatal exposure to ionizing radiation also affects the step of protein biosynthesis which depends on the activity of sRNA-aminoacyl synthetases.     

SOD2-mediated adaptive responses induced by low-dose ionizing radiation via TNF signaling and amifostine

Manganese superoxide dismutase (SOD2)-mediated adaptive processes that protect against radiation-induced micronucleus formation can be induced in cells after a 2-Gy exposure by previously exposing them to either low-dose ionizing radiation (10cGy) or WR1065 (40μM), the active thiol form of amifostine. Although both adaptive processes culminate in elevated levels of SOD2 enzymatic activity, the underlying pathways differ in complexity, with the tumor necrosis factor α (TNFα) signaling pathway implicated in the low-dose radiation-induced response, but not in the thiol-induced pathway. The goal of this study was the characterization of the effects of TNFα receptors 1 and 2 (TNFR1, TNFR2) on the adaptive responses induced by low-dose irradiation or thiol exposure using micronucleus formation as an endpoint. BFS-1 wild-type cells with functional TNFR1 and 2 were exposed 24h before a 2-Gy dose of ionizing radiation to either 10cGy or a 40μM dose of WR1065. BFS2C-SH02 cells, defective in TNFR1, and BFS2C-SH22 cells, defective in both TNFR1 and TNFR2 and generated from BFS2C-SH02 cells by transfection with a murine TNFR2-targeting vector and confirmed to be TNFR2 defective by quantitative PCR, were also exposed under similar conditions for comparison. A 10-cGy dose of radiation induced a significant elevation in SOD2 activity in BFS-1 (P<0.001) and BFS2C-SH02 (P=0.005) but not BFS2C-SH22 cells (P=0.433), compared to their respective untreated controls. In contrast, WR1065 significantly induced elevations in SOD2 activity in all three cell lines (P=0.001, P=0.007, P=0.020, respectively). A significant reduction in the frequency of radiation-induced micronuclei was observed in each cell line when exposure to a 2-Gy challenge dose of radiation occurred during the period of maximal elevation in SOD2 activity. However, this adaptive effect was completely inhibited if the cells were transfected 24h before low-dose radiation or thiol exposure with SOD2 siRNA. Under the conditions tested, TNFR1 and 2 inhibition negatively affected the low-dose radiation-induced but not the thiol-induced adaptive responses observed to be mediated by elevations in SOD2 activity.     

Reduction of radiation-induced nitrative stress in leucocytes and kidney cells of rats upon administration of polyphenolic complex concentrates from red wine

The research has shown that exposure to ionizing radiation at the dose of 30 cGy leads to the activation of NO-synthase way of nitrogen oxide synthesis, as well as to the accumulation of its stable metabolites and 3'-nitrotyrosine modified proteins in rat peripheral blood leucocytes and the renal cortical layer. NO-synthase activity was preserved at the control value through the consumption of red wine naturalpolyphenolic complex concentrates by the irradiated animals. The content of proteins modified by tyrosine nitration decreased in the early period of post-radiation exposure due to the influence of the investigated concentrate. Thus the ability of red wine natural polyphenolic complex concentrates to prevent adverse changes in L-arginine/NO system and, therefore, inhibit the development of nitrative stress induced by low doses of ionizing radiation has been proved experimentally.     

Modifying action of Mn(II) chelate with ethyl ester of salicylydene tyrosine on some biophysical parameters of erythrocyte membranes of the irradiated organism

The presentation summarizes the results of studies on radioprotective activity of newly synthesized Mn(II) chelate with ethyl ester of salicylydene D,L-tyrosine, basing on the evaluation of its membrane-protective effects, revealed at different periods post the exposure of the organism to ionizing radiation. It was revealed that the administration of the compound into the animal organism before the radiation exposure (gamma-irradiation of 60Co at a dose level of 4.8 Gy) produced correcting action on structure-functional properties of erythrocyte membrane of irradiated rats: the excessive activation of lipid peroxidation of erythrocyte membranes was inhibited, the passive permeability of erythrocytes for K+ ions was normalized, the deformity and membrane potential of erythrocytes according to hydrogen-chlorine gradient were recovered.     

An investigation of the reaction kinetics of luciferase and the effect of ionizing radiation on the reaction rate

The bioluminescence produced by luciferase, a firefly enzyme, requires three substrates: luciferin, ATP and oxygen. We find that ionizing radiation, in the form of a proton beam from a cyclotron, will eliminate dissolved oxygen prior to any damage to other substrates or to the protein. The dose constant for removal of oxygen is 70 ± 20 Gy, a much smaller dose than required to cause damage to protein. Removal of oxygen, which is initially in excess, leads to a sigmoidal response of bioluminescence to radiation dose, consistent with a Michaelis–Menten relationship to substrate concentration. When excess oxygen is exhausted, the response becomes exponential. Following the irradiation, bioluminescence recovers due to a slow leak of oxygen into the solution. This may also explain previous observations on the response of bioluminescent bacteria to radiation. We have studied the dependence of the reaction rate on enzyme and substrate concentration and propose a model for the reaction pathway consistent with this data. The light output from unirradiated samples decreases significantly with time due to product inhibition. We observe that this inhibition rate changes dramatically immediately after a sample is exposed to the beam. This sudden change of the inhibition rate is unexplained but shows that enzyme regulatory function responds to ionizing radiation at a dose level less than 0.6 Gy.     

The RBE for skin tumors and hair follicle damage in the rat following irradiation with alpha particles and electrons

Rat skin was irradiated with cyclotron-accelerated alpha particles with doses ranging from 210 rads to 6850 rads and monoenergetic electrons with doses ranging from 810 rads to 12,300 rads. The beams were modified so that the depth-dose curves were approximately identical with penetrations of about 1.0 mm. Tumors were counted every 4 weeks for 80 weeks, and at death or sacrifice the hair follicle damage was assessed by using "whole mounts" of separated epithelium. The RBE values determined from a comparison of the dose response curves were: acute skin injury, RBE = 3.0+/-1.0; hair follicle survival, RBE = 2.1 +/- 0.7; hair follicle damage, RBE = 2.6 = 0.4; tumor induction, RBE = 2.9+/- 0.5. Within the experimental error, these values were independent of the dose. For both types of radiation, the tumor incidence increased approximately as the square of time and at low doses approximately as the 4th power of dose. The histological characteristics of the tumors and the correlation between the incidence of tumors and damaged hair follicles were independent of the type of radiation. The results were consistent with the hypothesis that structural damage to the hair follicles is a factor in the tumor induction process.     

Micronucleus frequency is increased in peripheral blood lymphocytes of nuclear power plant workers

Nuclear power plant workers are exposed to ionizing radiation at relatively low doses and for prolonged periods of time. To investigate the extent of genetic damage in these workers, a group of 133 nuclear power plant workers and 39 healthy controls were compared using the cytokinesis-block micronucleus assay. The frequency of micronuclei was significantly increased in peripheral lymphocytes of nuclear power plant workers (20.5 +/- 9.7% compared to 13.7 +/- 5.9%). A significant dose-response relationship was observed between micronucleus (MN) frequency and both the accumulated dose and the duration of employment (P < 0.01 for both variables after adjusting for age, gender and cigarette smoking) with an evident leveling off for exposures over 200 mSv. Accumulated dose and duration of employment were significantly correlated but exerted independent effects on MN frequency. For non-occupational parameters, age was significantly associated with the frequency of micronuclei, while gender was not. Smoking habit showed no overall effect, whereas increased chromosome damage was evident in smokers of more than 20 cigarettes per day. In conclusion, a dose-related association between MN frequency and exposure to ionizing radiation was evident in nuclear power plant workers, encouraging the application of the cytokinesis-block micronucleus assay in biomonitoring studies of human populations with prolonged exposure to ionizing radiation.     

Effect of cold exposure on liver and muscle cAMP content and cAMP phosphodiesterase activity

Adenosine 3',5'-cyclic monophosphate (cAMP) concentration and 3',5'-cyclic-nucleotide phosphodiesterase (PDE) activity were measured in skeletal muscle, heart, and liver of rats exposed to 1, 3, 5, and 7 days of cold. Cyclic nucleotide concentration increased in fast-twitch red muscle at the same time that PDE activity was decreasing. Nucleotide concentration and enzyme activity of slow-twitch red muscle were not altered by the cold exposure. The PDE activity of fast-twitch white muscle was elevated approximately 50% above control after 1 and 3 days of cold exposure. By the 5th day in the cold, white muscle PDE activity had returned to control levels and remained there through the 7th day of experimentation. cAMP concentration in hearts of cold-exposed rats was significantly (P less than 0.01) elevated above control at all time points measured. Myocardial PDE activity was elevated above control (P less than 0.05) at 1 and 3 days of cold exposure but returned to control levels by the 5th day in the cold. Hepatic cAMP and PDE activity were elevated above control at all time points analyzed. These data suggest that changes in cyclic nucleotide metabolism play a role in attaining homeostasis during acute cold exposure.     

Effects of radiation on host-tumor interactions using the multicellular tumor spheroid model

Summary Use of the multicellular tumor spheroid as a tumor model allows separate host or tumor treatment with ionizing radiation and examination of the effects on host-tumor immune interactions. Spheroids of EMT6/Ro, a BALB/c mammary tumor were implanted into the peritoneal cavity of syngeneic immunized mice, recovered, and dissociated into single cells. Cytolytic activity of mature spheroid associated cells and peritoneal cells was resistant to radiation doses as high as 1000 rads when irradiated directly prior to assay. Mice irradiated (200, 400, 700 rads) 24 h prior to spheroid injection had an increased number of tumor cells and decreased number of tumor infiltrating and peritoneal host cells upon spheroid recovery. This was paralleled by an increased colony forming efficiency per spheroid. Cytolytic activity of the spheroid associated cells against radiolabeled EMT6 cells was in many cases decreased with radiation although lysis was the same on a per cell basis. Cytolytic activity by peritoneal cells from these mice increased with dose as measured on a per cell basis. This activity from irradiated animals was carried out by a Thyl⁺ cell.     

Effects of mixed neutron-gamma total-body irradiation on physical activity performance of rhesus monkeys

Behavioral incapacitation for a physical activity task and its relationship to emesis and survival time following exposure to ionizing radiation were evaluated in 39 male rhesus monkeys (Macaca mulatta). Subjects were trained to perform a shock avoidance activity task for 6 hr on a 10-min work/5-min rest schedule in a nonmotorized physical activity wheel. Following stabilization of performance, each subject received a single, pulsed dose of mixed neutron-gamma, whole-body radiation (n/gamma = 3.0) ranging between 1274 and 4862 rad. Performance testing was started 45 sec after exposure. A dose-response function for early transient incapacitation (ETI) during the first 2 hr after irradiation was fitted, and the median effective dose (ED50) was calculated to be 1982 rad. More subjects experienced both incapacitation and emesis in this study than has been reported for other behavioral tasks in similar radiation fields. Analysis done on the relationship of dose to ETI, emesis, and survival time found (a) a significant relationship between the radiation dose and the number and duration of ETIs; (b) no correlation between emesis and dose, survival time, or ETI; (c) no relation between survival time and ETI at any dose; and (d) no significant difference in survival time for dose groups between 1766 +/- 9 (SEM) and 2308 +/- 23 rad.     

The rate of strand separation in alkali of DNA of irradiated Mammalian cells

When mammalian cells are treated with alkali of pH at about 12, the cells are lysed and the released DNA starts to uncoil. This process of DNA strand separation is accelerated if the cells have been exposed to ionizing radiation, and the effect is clearly detectable in the dose range 10-100 rads. The rate of strand separation is also influenced by temperature and ionic strength of the alkaline solution. The kinetics of DNA strand separation in alkali is studied for three conditions in terms of ionic strength and temperature, chosen in such a way that the effect of irradiation may conveniently be studied in the dose range 10 rads to 20 krads. The accelerating effect of ionizing radiation on DNA strand separation is probably due to DNA strand breakage and the technique described is thus a sensitive method of studying such damage to DNA. A model for the strand-separation process, based on the assumption that strand breakage causes the accelerating effect, is proposed and found to describe the experimental data adequately.     

Effects of pyridoxine supplementation on blood glucocorticoids level, aspartate aminotransferase activity and glucose tolerance pattern under acute hypoxic stress

Studies were conducted on male adult rabbits to find out the changes in blood glucocorticoid levels along with the changes in aspartate aminotransferase activity in blood and the role of pyridoxine on the glucose tolerance pattern under hypoxic stress. Hypoxic stress was produced by exposing the animals to a simulated altitude of 7,000 m for 6 h. In the first set of experiments 10 rabbits were used. Blood haemoglobin level, plasma and erythrocyte glucocorticoid levels and erythrocyte GOT activity were measured just before and after the exposure to hypoxia. Erythrocyte GOT activity was measured both without and with 50 mg of pyridoxal phosphate addition to the incubation mixture. Glucocorticoid levels in plasma increased by 11% whereas in erythrocytes the increase was 55% after hypoxia. Percent stimulation of erythrocyte GOT activity with pyridoxal phosphate before exposure to hypoxia was 180% but increased to 321% after exposure. In the second set of experiments another 10 rabbits were used. First they were exposed to hypoxia without pyridoxine hydrochloride feeding and then after 7 days with 3 mg of pyridoxine hydrochloride feeding. For glucose tolerance tests the animals were fed with 1 g of glucose immediately after the hypoxic exposures. Plasma reduced glutathione (GSH), LDH and ICDH activities increased and GOT activity was depressed after hypoxic stress, but when the animals were fed pyridoxine hydrochloride prior to the exposure the enzyme activities remained unaltered after hypoxic stress. Pyridoxine hydrochloride did not alter the pattern of glucose tolerance after hypoxic stress.