Involvement of poly (ADP-ribose) in the radiation response of mammalian cells

The evidence implicating poly (ADP-ribose) in the radiation response of mammalian cells is reviewed. It is concluded that the apparently conflicting results using inhibitors of ADP-ribosyl transferase (ADPRT) can be explained by a working hypothesis. This hypothesis maintains that poly (ADP-ribose) is required for repair of radiation damage (presumably to facilitate ligation). In most cells the synthesis of poly (ADP-ribose) is not rate limiting for repair and therefore, an almost complete inhibition of ADPRT activity is required to potentiate the radiation response. In radiation-sensitive cells (e.g. resting lymphocytes, L5178Y-S cells) with a deficient poly (ADP-ribose) metabolism, its synthesis can become rate limiting for repair. In such cells even a partial inhibition of ADPRT activity may enhance radiation-induced cell killing. It is suggested that if such differences exist between normal and cancer cells, they can be utilized to improve the therapeutic ratio of radiotherapy.     

Poly(ADP-ribose) metabolism in X-irradiated Chinese hamster cells: its relation to repair of potentially lethal damage

Nicotinamide-adenine dinucleotide (NAD+) is the substrate used by cells in poly(ADP-ribose) synthesis. X-irradiation of log-phase Chinese hamster cells caused a rapid decrease in NAD+ levels which was linearly dependent on radiation dose. The activity of ADP-ribosyl transferase ( ADPRT ) also increased linearly with radiation dose. The decrease of NAD+ was slower, and the increase in ADPRT activity was less pronounced, in a radiation sensitive line, V79- AL162 /S-10. An inhibitor of ADPRT , m-aminobenzamide, largely prevented the depletion of cellular NAD+ and reduced the rate at which ADPRT activity disappeared during post-irradiation incubation. Post-irradiation treatment with hypertonic buffer or with medium containing D2O--which inhibit repair of radiation-induced potentially lethal damage--enhanced the depletion of NAD+ and prevented the reduction in ADPRT activity following irradiation. The characteristics of the effects of treatment with hypertonic buffer on NAD+ metabolism were qualitatively similar to the effects that such treatment has on radiation-induced cell killing. These results suggest that poly(ADP-ribose) synthesis after irradiation plays a role in the repair of potentially lethal damage.     

The nuclear ceramide/diacylglycerol balance depends on the physiological state of thyroid cells and changes during UV-C radiation-induced apoptosis

Intranuclear lipid metabolism modifications in relation to cell proliferation and/or apoptosis were demonstrated in hepatocytes. The aim of this study was to establish whether nuclear lipid metabolites influence cell function in different experimental models using a rat thyroid cell line (FRTL-5) treated with UV-C radiation. After UV-C irradiation cells proliferate and undergo apoptosis in the presence of thyrotropin, are quiescent and resistant to radiation-induced apoptosis in its absence and finally are proapoptotic for nutrition withdrawal. In nuclei purified from proliferating cells, irradiation stimulates neutral-sphingomyelinase activity and inhibits sphingomyelin-synthase, phosphatidylcholine-specific phospholipase C and phosphatidylinositol-specific phospholipase C activity with a consequent increase in the ceramide/diacylglycerol ratio. This effect is marked in proapoptotic cell nuclei and low in quiescent cell nuclei. In conclusion, UV-C radiation induces apoptosis, modifying nuclear lipid metabolism in relation to the physiological state of cells.     

Effect of forskolin and quinacrine on leukocyte functions under the effect of low-dose radiation

The adenylate cyclase and phospholipase A2 incorporation in the functional responses as well as lipid peroxidation processes and glutathione system homeostasis of animal leukocytes to small doses of ionizing radiation (1-100 mGy) have been estimated. The cells were irradiated by introduction of radioactive isotope 14C-leucine into the incubation medium. It is established that the ionizing radiation has different effects on the modification of cellular functions by the agents, which change adenylate cyclase and phospholipase A2 activity. Neutralization of stimulative irradiation effect on chemokinesis of polymorphonuclear leukocytes by quinacrine (the inhibitor of phospholipase A2) indicates for certain, that metabolism of eicosanoids takes immediate part in the cell response to ionizing radiation. Apparently, adenylate cyclase has no influence on this process, where at indicates the lack of influence of forskolin (the stimulator of adenylate cyclase) on the spontaneous motility, and on the radiation action on this leukocyte function. Rosette forming ability of lymphocytes is regulated by both enzymes because it is modified both by the inhibitor of phospholipase A2, and by the adenylate cyclase stimulant. In this case it is impossible to exclude the action of ionizing radiation both through the adenylate cyclase cascade, and through the eicosanoid metabolism. In all the concentration range the radionuclides do not affect the studied biochemical indexes of the cell, but change the action of the modifiers.     

Nonmonotonous metabolic response of mammalian cells and tissues to ionizing radiation

Changes in the activity of ornithindecarboxylase in various tissues and in the amount of catecholamine in rat hypothalamus by the action of acute and chronic ionizing radiation were studied. A nonmonotonous relationship between the metabolic parameters of animal tissues and cells and the radiation dose was revealed. It was assumed that the nonmonotonous character of the dose-response dependence results from the nonmonotonous time course of the metabolic response to irradiation. It was also assumed that living systems have the property of responding to stress agents by nonmonotonous changes in metabolism. In the case of acute irradiation, this response manifests itself as oscillations of metabolic parameters about the control. The oscillations occur with a particular amplitude and periods, which vary with radiation dose, and damp out with time. As a result, in a fixed time interval, the dose-response curve may be nonmonotonous. Reverse dose-response relationships are also possible. In the case of chronic irradiation, the metabolic and functional parameters oscillate throughout irradiation time, and a modification of the response occurs. A prolong exposure to ionizing radiation causes strong changes in the metabolism of lipids of cell membranes, organelles and chromatin, as well as in the functional properties of some mammalian cells and tissues. The necessity of constructing quantitative models for explaining the nonmonotonous dose-response dependence is discussed.     

Abnormal myo-inositol and phospholipid metabolism in cultured fibroblasts from patients with ataxia telangiectasia

Ataxia telangiectasia (AT) is a complex autosomal recessive disorder that has been associated with a wide range of physiological defects including an increased sensitivity to ionizing radiation and abnormal checkpoints in the cell cycle. The mutated gene product, ATM, has a domain possessing homology to phosphatidylinositol-3-kinase and has been shown to possess protein kinase activity. In this study, we have investigated how AT affects myo-inositol metabolism and phospholipid synthesis using cultured human fibroblasts. In six fibroblast lines from patients with AT, myo-inositol accumulation over a 3-h period was decreased compared to normal fibroblasts. The uptake and incorporation of myo-inositol into phosphoinositides over a 24-h period, as well as the free myo-inositol content was also lower in some but not all of the AT fibroblast lines. A consistent finding was that the proportion of 32P in total labeled phospholipid that was incorporated into phosphatidylglycerol was greater in AT than normal fibroblasts, whereas the fraction of radioactivity in phosphatidic acid was decreased. Turnover studies revealed that AT cells exhibit a less active phospholipid metabolism as compared to normal cells. In summary, these studies demonstrate that two manifestations of the AT defect are alterations in myo-inositol metabolism and phospholipid synthesis. These abnormalities could have an effect on cellular signaling pathways and membrane production, as well as on the sensitivity of the cells to ionizing radiation and proliferative responses.     

Radiation-Induced Glycogen Accumulation Detected by Single Cell Raman Spectroscopy Is Associated with Radioresistance that Can Be Reversed by Metformin

Altered cellular metabolism is a hallmark of tumor cells and contributes to a host of properties associated with resistance to radiotherapy. Detection of radiation-induced biochemical changes can reveal unique metabolic pathways affecting radiosensitivity that may serve as attractive therapeutic targets. Using clinically relevant doses of radiation, we performed label-free single cell Raman spectroscopy on a series of human cancer cell lines and detected radiation-induced accumulation of intracellular glycogen. The increase in glycogen post-irradiation was highest in lung (H460) and breast (MCF7) tumor cells compared to prostate (LNCaP) tumor cells. In response to radiation, the appearance of this glycogen signature correlated with radiation resistance. Moreover, the buildup of glycogen was linked to the phosphorylation of GSK-3β, a canonical modulator of cell survival following radiation exposure and a key regulator of glycogen metabolism. When MCF7 cells were irradiated in the presence of the anti-diabetic drug metformin, there was a significant decrease in the amount of radiation-induced glycogen. The suppression of glycogen by metformin following radiation was associated with increased radiosensitivity. In contrast to MCF7 cells, metformin had minimal effects on both the level of glycogen in H460 cells following radiation and radiosensitivity. Our data demonstrate a novel approach of spectral monitoring by Raman spectroscopy to assess changes in the levels of intracellular glycogen as a potential marker and resistance mechanism to radiation therapy.     

Histogenetic,metabolic, and immunologic aspects of the effect of infrared laser radiation on injured skeletal muscles from irradiated and nonirradiated rats

Using biochemical, histological, morphometric and cytogenetic methods, it was shown that low-intensive infrared laser radiation (total dose 3.6 J/cm2), applied to the injured rat skeletal muscles, stimulated metabolism and regeneration more efficiently in the muscles locally exposed to 20 Gy X-rays compared to the unexposed muscles. The laser irradiation promoted postradiative recovery in bone marrow cells, but did not provide normalization in thymus lymphocyte activity.     

Role of phospholipase A2 and epoxygenase in inhibition of respiration burst in neutrophils by low intensity radiation of extremely high frequency

The role of some components of the phospholipid metabolism in the activation of neutrophil respiratory burst and its inhibition by electromagnetic radiation (EMR) of extremely high frequencies (EHF) was studied. It was shown that EHF EMR has effect on cells with a high sensitivity to the inhibitor of phospholipase A2 4-bromophenacyl bromide. However, againsts the background of the inhibitor, the effect of EHF EMR was not observed on cells with either high or low sensitivity to the inhibitor. EHF EMR was also inefficient with cells pretreated with proadifen, an inhibitor of epoxygenase (cytochrome P-450). The results obtained suggest that the effect of EHF EMR manifests itself in cells with a high activity of phospholipase A2 and is realized with the participation of epoxygenase metabolites of arachidonic acid.     

The action of helium-neon laser radiation on the growth of Mycobacterium tuberculosis

The growth properties of M. tuberculosis subjected to the action of helium-neon laser radiation was studied. Laser radiation was shown to change the quantitative and qualitative composition of mycobacterial population. Disturbances in the viability of mycobacteria appear as a consequence of changes in the morphological structure of mycobacterial cells. The maximum effect of helium-neon laser radiation was achieved after the irradiation of M. tuberculosis culture on days 2-3 after inoculation. These results made it possible to suggest that the effect of helium-neon laser radiation was most pronounced in cells at the stage of mitosis (the logarithmic stage of growth) with the highest degree of metabolism.     

Long-term consequences of radiation-induced bystander effects depend on radiation quality and dose and correlate with oxidative stress

Widespread evidence indicates that exposure of cell populations to ionizing radiation results in significant biological changes in both the irradiated and nonirradiated bystander cells in the population. We investigated the role of radiation quality, or linear energy transfer (LET), and radiation dose in the propagation of stressful effects in the progeny of bystander cells. Confluent normal human cell cultures were exposed to low or high doses of 1GeV/u iron ions (LET ～ 151 keV/μm), 600 MeV/u silicon ions (LET ～ 51 keV/μm), or 1 GeV protons (LET ～ 0.2 keV/μm). Within minutes after irradiation, the cells were trypsinized and co-cultured with nonirradiated cells for 5 h. During this time, irradiated and nonirradiated cells were grown on either side of an insert with 3-μm pores. Nonirradiated cells were then harvested and allowed to grow for 20 generations. Relative to controls, the progeny of bystander cells that were co-cultured with cells irradiated with iron or silicon ions, but not protons, exhibited reduced cloning efficiency and harbored higher levels of chromosomal damage, protein oxidation and lipid peroxidation. This correlated with decreased activity of antioxidant enzymes, inactivation of the redox-sensitive metabolic enzyme aconitase, and altered translation of proteins encoded by mitochondrial DNA. Together, the results demonstrate that the long-term consequences of the induced nontargeted effects greatly depend on the quality and dose of the radiation and involve persistent oxidative stress due to induced perturbations in oxidative metabolism. They are relevant to estimates of health risks from exposures to space radiation and the emergence of second malignancies after radiotherapy.     

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.     

Effect of Increased Glucose Levels on Na+/K+-Pump Activity in Cultured Neuroblastoma Cells

Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo-inositol metabolism and Na+/K+-pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+-pump activity, myo-inositol metabolism, and myo-inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo-inositol metabolism and partially restored Na+/K+-pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo-inositol metabolism and these abnormalities are associated with a reduction in Na+/K+-pump activity.     

Ionizing Radiation Impairs T Cell Activation by Affecting Metabolic Reprogramming

Ionizing radiation has a variety of acute and long-lasting adverse effects on the immune system. Whereas measureable effects of radiation on immune cell cytotoxicity and population change have been well studied in human and animal models, little is known about the functional alterations of the surviving immune cells after ionizing radiation. The objective of this study was to delineate the effects of radiation on T cell function by studying the alterations of T cell receptor activation and metabolic changes in activated T cells isolated from previously irradiated animals. Using a global metabolomics profiling approach, for the first time we demonstrate that ionizing radiation impairs metabolic reprogramming of T cell activation, which leads to substantial decreases in the efficiency of key metabolic processes required for activation, such as glucose uptake, glycolysis, and energy metabolism. In-depth understanding of how radiation impacts T cell function highlighting modulation of metabolism during activation is not only a novel approach to investigate the pivotal processes in the shift of T cell homeostasis after radiation, it also may lead to new targets for therapeutic manipulation in the combination of radiotherapy and immune therapy. Given that appreciable effects were observed with as low as 10 cGy, our results also have implications for low dose environmental exposures.     

Inactivation of bacteriophage lambda by combined X-ray and U.V.-light exposure.

Extracellular phage lambda has been successively exposed to X-rays and U.V. light. The plaque-forming ability of the irradiated phages was determined on host cells with different repair capacities. No change in sensitivity was found with a pre-treatment of one type of radiation to lethal damage inflicted by the other. This indicates that a prerequisite for an interaction of different types of radiation is either an active metabolism or repair process occurring during the two radiation exposures.     

Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity

DNA double-strand breaks (DSBs) can be processed by the Mre11-Rad50-Nbs1 (MRN) complex, which is essential to promote ataxia telangiectasia-mutated (ATM) activation. However, the molecular mechanisms linking MRN activity to ATM are not fully understood. Here, using Xenopus laevis egg extract we show that MRN-dependent processing of DSBs leads to the accumulation of short single-stranded DNA oligonucleotides (ssDNA oligos). The MRN complex isolated from the extract containing DSBs is bound to ssDNA oligos and stimulates ATM activity. Elimination of ssDNA oligos results in rapid extinction of ATM activity. Significantly, ssDNA oligos can be isolated from human cells damaged with ionizing radiation and injection of small synthetic ssDNA oligos into undamaged cells also induces ATM activation. These results suggest that MRN-dependent generation of ssDNA oligos, which constitute a unique signal of ongoing DSB repair not encountered in normal DNA metabolism, stimulates ATM activity.     

Cellular autofluorescence following ionizing radiation

Cells often autofluoresce in response to UV radiation excitation and this can reflect critical aspects of cellular metabolism. Here we report that many different human and murine cell types respond to ionizing radiation with a striking rise in autofluorescence that is dependent on dose and time. There was a highly reproducible fluorescent shift at various wavelengths, which was mirrored by an equally reproducible rise in the vital intracellular metabolic co-factors FAD and NADH. It appears that mitochondria, metabolism and Ca(2+) homeostasis are important for this to occur as cells without mitochondria or cells unable to alter calcium levels did not behave in this way. We believe these radiation-induced changes are of biological importance and that autofluorescence may even provide us with a tool to monitor radiation responses in the clinic.     

Influence of beta-radiation on human erythrocyte surface potential under eicosanoid metabolism inhibition

The aim of the research consisted in the study of influence of beta-radiation on response of erythrocyte surface potential to inhibitors of eicosanoid metabolism enzymes (cyclo-, lipoxygenase and phospholipase A2). It was shown, that inhibitors of phospholipase A2 (quinacrine, 10-100 microM), cyclooxygenase (aspirin, 10-100 microM) and cyclo- and lipoxygenase (BW755c, 1-100 microM) lowered electrophoretic mobility (EPM) of erythrocytes by 20-30%. An analogous effect can be exerted by beta-radiation. Nonradioactive leucine in the studied concentrations cannot simulate EPM erythrocytes. Response of cellular EPM to these inhibitors depended on their concentration in the incubation medium. Addition of 14C to the incubation medium changed response of EPM of cells to inhibitors of cyclo- and lipoxygenase but not to quinacrine. However beta-radiation fully abolished the stimulative action of nonspecific activator of phospholipase A2 (Ca-independent), H2O2, on cellular EPM. Under these conditions beta-radiation enhanced EPM response to aspirin only at concentration of 100 microM. The EPM response to BW755c is reduced by irradiation at all concentrations with the exception of equal-effective one (10 microM). Data obtained evidence for modification of eicosanoid metabolism by beta-radiation, probably, as a result of phospholipase A2 inhibition, as evident from elimination by radiation of stimulated action of hydrogen peroxide on EPM. The radiation action can also affect the cyclooxygenase lipoxygenase activity ratio, this activity being mediated by cellular membrane signaling systems.     

Induction of heme oxygenase-1 modulates cis-aconitase activity in lens epithelial cells

Heme oxygenase-1 is the heme catabolic enzyme induced in human dermal fibroblasts by environmental stress. We report an increase of heme oxygenase-1 message in lens epithelial cells after exposure to UVA radiation, followed by a 10-fold increase of protein expression. The size of message was larger than previously demonstrated for fibroblasts. The relationship between heme oxygenase-1 activation and iron metabolism was investigated by measurement of activities of both cytosolic and mitochondrial cis-aconitase enzymes. A 2-fold increase in mitochondrial cis-aconitase activity in UVA-exposed cells coincided with the time of maximal heme oxygenase-1 expression. We propose that modulation of cis-aconitase activity at the translational level by an increase of cellular iron is an important consequence of heme oxygenase-1 activation. This might be a novel aspect of the protective role of heme oxygenase-1 in modulating the response of cells challenged with oxidative stress.