Consequences of ionizing radiation-induced damage in human neural stem cells

Cranial irradiation remains a frontline treatment for brain cancer, but also leads to normal tissue damage. Although low-dose irradiation (≤ 10 Gy) causes minimal histopathologic change, it can elicit variable degrees of cognitive dysfunction that are associated with the depletion of neural stem cells. To decipher the mechanisms underlying radiation-induced stem cell dysfunction, human neural stem cells (hNSCs) subjected to clinically relevant irradiation (0–5 Gy) were analyzed for survival parameters, cell-cycle alterations, DNA damage and repair, and oxidative stress. hNSCs showed a marked sensitivity to low-dose irradiation that was in part due to elevated apoptosis and the inhibition of cell-cycle progression that manifested as a G2/M checkpoint delay. Efficient removal of DNA double-strand breaks was indicated by the disappearance of γ-H2AX nuclear foci. A dose-responsive and persistent increase in oxidative and nitrosative stress was found in irradiated hNSCs, possibly the result of a higher metabolic activity in the fraction of surviving cells. These data highlight the marked sensitivity of hNSCs to low-dose irradiation and suggest that long-lasting perturbations in the CNS microenvironment due to radiation-induced oxidative stress can compromise the functionality of neural stem cells.     

DNA damage responses at low radiation doses

Increased cell killing after exposure to low acute doses of X rays (0-0.5 Gy) has been demonstrated in cells of a number of human tumor cell lines. The mechanisms underlying this effect have been assumed to be related to a threshold dose above which DNA repair efficiency or fidelity increases. We have used cells of two radioresistant human tumor cell lines, one that shows increased sensitivity to low radiation doses (T98G) and one that does not (U373), to investigate the DNA damage response at low doses in detail and to establish whether there is a discontinuous dose response or threshold in activation of any important mediators of this response. In the two cell lines studied, we found a sensitive, linear dose response in early signaling and transduction pathways between doses of 0.1 and 2 Gy with no evidence of a threshold dose. We demonstrate that ATM-dependent signaling events to downstream targets including TP53, CHK1 and CHK2 occur after doses as low as 0.2 Gy and that these events promote an effective damage response. Using chemical inhibition of specific DNA repair enzymes, we show that inhibition of DNA-PK-dependent end joining has relatively little effect at low (<1 Gy) doses in hyper-radiosensitive cells and that at these doses the influence of RAD51-mediated repair events may increase, based on high levels of RAD51/BRCA2 repair foci. These data do not support a threshold model for activation of DNA repair in hyper-radiosensitive cells but do suggest that the balance of repair enzyme activity may change at low doses.     

Low doses of radiation decrease the level of spontaneous and gamma-induced chromosomal mutagenesis in bone marrow cells of mice in vivo

Low doses of ionizing radiation are known to induce adaptive response (AR), which is characterized in most cases by temporary nature, though the possibility of long-term persistence of AR is not ruled out. In this investigation we studied the effect of low doses of gamma-radiation on both high-dose radiation-induced and spontaneous level of cytogenetic damage throughout the life of mice. SHK male mice 2 months old were used. Priming doses of 0.1 and 0.2 Gy (0.125 Gy/min, gamma-radiation from 60Co) were used. A challenging dose of 1.5 Gy (1 Gy/min) was used in the experiments using a routine AR experimental design. The frequency of micronucleated polychromatic erythrocytes in bone marrow cells of primed, primed and challenged, and control groups was assessed at various times of animal life span. It was shown that: a) single low-dose gamma-irradiation induces a cytogenetic AR which can be revealed at 1, 3, 6, 9, 12 months after priming; b) single low-dose gamma-irradiation decreases the cytogenetic damage to a level below the spontaneous rate at the end of lifetime (20 months) of animals; c) ability to induce adaptive response does not depend on the age of animals at the moment of priming irradiation. In conclusion, the mechanisms underlying AR not only protect from chromosome damage induced by high-dose irradiation but also may play a role in spontaneous mutagenesis during aging of animals.     

Protection against radiation oxidative damage in mice by Triphala

Protection against whole body gamma-irradiation (WBI) of Swiss mice orally fed with Triphala (TPL), an Ayurvedic formulation, in terms of mortality of irradiated animals as well as DNA damage at cellular level has been investigated. It was found that radiation induced mortality was reduced by 60% in mice fed with TPL (1g/kg body weight/day) orally for 7 days prior to WBI at 7.5 Gy followed by post-irradiation feeding for 7 days. An increase in xanthine oxidoreductase activity and decrease in superoxide dismutase activity was observed in the intestine of mice exposed to WBI, which, however, reverted back to those levels of sham-irradiated controls, when animals were fed with TPL for 7 days prior to irradiation. These data have suggested the prevention of oxidative damage caused by whole body radiation exposure after feeding of animals with TPL. To further understand the mechanisms involved, the magnitude of DNA damage was studied by single cell gel electrophoresis (SCGE) in blood leukocytes and splenocytes obtained from either control animals or those fed with TPL for 7 days followed by irradiation. Compared to irradiated animals without administering TPL, the mean tail length was reduced about three-fold in blood leukocytes of animals fed with TPL prior to irradiation. Although, similar protection was observed in splenocytes of TPL fed animals, the magnitude of prevention of DNA damage was significantly higher than that observed in leukocytes. It has been concluded that TPL protected whole body irradiated mice and TPL induced protection was mediated through inhibition of oxidative damage in cells and organs. TPL seems to have potential to develop into a novel herbal radio-protector for practical applications.     

Bimodal changes in germinability of pea seeds under the influence of low doses of gamma-radiation

Pea seeds (cv. 'Nemchinovskii-85', harvest of 2002, 80%-germination percentage) were exposed to gamma-radiation with doses ranging from 19 cGy to 100 Gy. One week after the irradiation with doses of 19 cGy and 3 Gy. the germination percentage decreased to 58 and 45%, respectively; at doses of 7 and 10 Gy it was 73 and 70% respectively. At greater doses (25, 50, and 100 Gy), germination percentage decreased in proportion. Anomalous changes in seed germination percentage (as a function of irradiation dose) were caused by the redistribution of irradiated seeds between fractions I and II. The measurements of room temperature phosphorescence in air-dry seeds and the phosphorescence of endogenous porphyrines of imbibing seeds showen that the germination decrease after the irradiation with low doses (19 cGy and 3 Gy) was caused by the increase in the number of weak seeds of fraction II, which had high rates of water uptake and suffered from hypoxia under seed coat. Some of these seeds suffocated from hypoxia, and other seeds produced seedlings with morphological defects (such seeds were considered incapable of germination). During storage of seeds irradiated at doses 19 cGy-10 Gy, the recovery of germination percentage (after its initial decrease) was caused by the decrease in seed number in fraction II. The subsequent germination decrease was caused by seed death. The higher was the irradiation dose, the faster were changes in germination percentage during storage of irradiated seeds. Bimodal changes in pea seed germination with the increase of y-irradiation dose has apparently the same origin as the changes in seed germination during accelerated ageing.     

A model of interactions between radiation-induced oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation triggers oxidative stress, which can have a variety of subtle and profound biological effects. Here we focus on mathematical modeling of potential synergistic interactions between radiation damage to DNA and oxidative stress-induced damage to proteins involved in DNA repair/replication. When sensitive sites on these proteins are attacked by radiation-induced radicals, correct repair of dangerous DNA lesions such as double strand breaks (DSBs) can be compromised. In contrast, if oxidation of important proteins is prevented by strong antioxidant defenses, DNA repair may function more efficiently. These processes probably occur to some extent even at low doses of radiation/oxidative stress, but they are easiest to investigate at high doses, where both DNA and protein damage are extensive. As an example, we use data on survival of Deinococcus radiodurans after high doses (thousands of Gy) of acute and chronic irradiation. Our model of radiogenic oxidative stress is consistent with these data and can potentially be generalized to other organisms and lower radiation doses.     

Bystander-type effects mediated by long-lived inflammatory signaling in irradiated bone marrow

Radiation-induced bystander and abscopal effects, in which DNA damage is produced in nonirradiated cells as a consequence of communication with irradiated cells, indicate mechanisms of inducing damage and cell death additional to the conventional model of deposition of energy in the cell nucleus at the time of irradiation. In this study we show that signals generated in vivo in the bone marrow of mice irradiated with 4 Gy γ rays 18 h to 15 months previously are able to induce DNA damage and apoptosis in nonirradiated bone marrow cells but that comparable signals are not detected at earlier times postirradiation or at doses below 100 mGy. Bone marrow cells of both CBA/Ca and C57BL/6 genotypes exhibit responses to signals produced by either irradiated CBA/Ca or C57BL/6 mice, and the responses are mediated by the cytokines FasL and TNF-α converging on a COX-2-dependent pathway. The findings are consistent with indirect inflammatory signaling induced as a response to the initial radiation damage rather than to direct signaling between irradiated and nonirradiated cells. The findings also demonstrate the importance of studying tissue responses when considering the mechanisms underlying the consequences of radiation exposures.     

小剂量^16O^8＋离子预辐射减轻大剂量辐射所致小鼠睾丸组织损伤

The testes of the B6C3F1 hybrid strain mice were irradiated with 0.05 Gy of 16O8+ ion as the pre-exposure dose (D1), and were then irradiated with 2 Gy of 16O8+ ion as challenging radiation dose (D2) at 4 h after per-exposure. Testicular morphology was observed by light microscope at 35th day after radiation. The results showed that irradiation of mouse testes with 2 Gy of 16O8+ ion significantly impaired, mainly reduction of tubule diameter and decrease or loss of germ cells in various developing stages, especially spermatogenic elements. Pre-exposure to a low-dose (0.05 Gy) of 16O8+ ion significantly alleviated above mentioned damage on testicular morphology induced by subsequent a high-dose (2 Gy) radiation.     

Dose- and time-dependent changes in gene expression in human glioma cells after low radiation doses

We have used DNA microarrays to identify changes in gene expression in cells of the radioresistant human glioma cell lines T98G and U373 after low radiation doses (0.2-2 Gy). Using Bayesian linear models, we have identified a set of genes that respond to low doses of radiation; furthermore, a hypothesis-driven approach to data analysis has allowed us to identify groups of genes with defined non-linear dose responses. Specifically, one of the cell lines we have examined (T98G) shows increased radiosensitivity at low doses (low-dose hyper-radiosensitivity, HRS); thus we have also assessed sets of genes whose dose response mirrors this survival pattern. We have also investigated a time course for induction of genes over the period when the DNA damage response is expected to occur. We have validated these data using quantitative PCR and also compared genes up-regulated in array data to genes present in the polysomal RNA fraction after irradiation. Several of the radioresponsive genes that we describe code for proteins that may have an impact on the outcome of irradiation in these cells, including RAS homologues and kinases involved in checkpoint signaling, so understanding their differential regulation may suggest new ways of altering radioresistance. From a clinical perspective these data may also suggest novel targets that are specifically up-regulated in gliomas during radiotherapy treatments.     

Mitochondrial reactive oxygen species-mediated genomic instability in low-dose irradiated human cells through nuclear retention of cyclin D1

Mitochondria are associated with various radiation responses, including adaptive responses, mitophagy, the bystander effect, genomic instability, and apoptosis. We recently identified a unique radiation response in the mitochondria of human cells exposed to low-dose long-term fractionated radiation (FR). Such repeated radiation exposure inflicts chronic oxidative stresses on irradiated cells via the continuous release of mitochondrial reactive oxygen species (ROS) and decrease in cellular levels of the antioxidant glutathione. ROS-induced oxidative mitochondrial DNA (mtDNA) damage generates mutations upon DNA replication. Therefore, mtDNA mutation and dysfunction can be used as markers to assess the effects of low-dose radiation. In this study, we present an overview of the link between mitochondrial ROS and cell cycle perturbation associated with the genomic instability of low-dose irradiated cells. Excess mitochondrial ROS perturb AKT/cyclin D1 cell cycle signaling via oxidative inactivation of protein phosphatase 2A after low-dose long-term FR. The resulting abnormal nuclear accumulation of cyclin D1 induces genomic instability in low-dose irradiated cells.     

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.     

Gene expression profiles of normal human fibroblasts after exposure to ionizing radiation: a comparative study of low and high doses

Several types of cellular responses to ionizing radiation, such as the adaptive response or the bystander effect, suggest that low-dose radiation may possess characteristics that distinguish it from its high-dose counterpart. Accumulated evidence also implies that the biological effects of low-dose and high-dose ionizing radiation are not linearly distributed. We have investigated, for the first time, global gene expression changes induced by ionizing radiation at doses as low as 2 cGy and have compared this to expression changes at 4 Gy. We applied cDNA microarray analyses to G1-arrested normal human skin fibroblasts subjected to X irradiation. Our data suggest that both qualitative and quantitative differences exist between gene expression profiles induced by 2 cGy and 4 Gy. The predominant functional groups responding to low-dose radiation are those involved in cell-cell signaling, signal transduction, development and DNA damage responses. At high dose, the responding genes are involved in apoptosis and cell proliferation. Interestingly, several genes, such as cytoskeleton components ANLN and KRT15 and cell-cell signaling genes GRAP2 and GPR51, were found to respond to low-dose radiation but not to high-dose radiation. Pathways that are specifically activated by low-dose radiation were also evident. These quantitative and qualitative differences in gene expression changes may help explain the non-linear correlation of biological effects of ionizing radiation from low dose to high dose.     

Activation of denovo GSH synthesis pathway in mouse spleen after long term low-dose γ-ray irradiation

Abstract

Glutathione (GSH) is an important cellular antioxidant and has a critical role in maintaining the balance of cellular redox. In this study, we investigated the GSH biosynthesis genes involved in the elevation of endogenous GSH levels using an irradiation system with an irradiation dose rate of 1.78 mGy/h, which was about 40,000 times less than the dose rates used in other studies. The results showed that GSH levels were significantly increased in the low-dose (0.02 and 0.2 Gy) irradiated group compared to those in the non-irradiated group, but enzymatic antioxidants such as superoxide dismutase and catalase were not induced at any doses tested. The elevation in GSH was accompanied by elevated expression of glutamate–cysteine ligase modifier subunit, but no changes were observed in the expression of glutamate–cysteine ligase catalytic subunit and thioredoxin in de novo GSH synthesis. In the case of genes involved in the GSH regeneration cycle, the expression of glutathione reductase was not changed after irradiation, whereas glutathione peroxidase was only increased in the 0.2 Gy irradiated group. Collectively, our results suggest that the de novo pathway, rather than the regeneration cycle, may be mainly switched on in response to stimulation with long-term low-dose radiation in the spleen.     

Site-specific DNA damage at the GGG sequence by UVA involves acceleration of telomere shortening

Telomere shortening is associated with cellular senescence. We investigated whether UVA, which contributes to photoaging, accelerates telomere shortening in human cultured cells. The terminal restriction fragment (TRF) from WI-38 fibroblasts irradiated with UVA (365-nm light) decreased with increasing irradiation dose. Furthermore, UVA irradiation dose-dependently increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in both WI-38 fibroblasts and HL-60 cells. To clarify the mechanism of the acceleration of telomere shortening, we investigated site-specific DNA damage induced by UVA irradiation in the presence of endogenous photosensitizers using (32)P 5'-end-labeled DNA fragments containing the telomeric oligonucleotide (TTAGGG)(4). UVA irradiation with riboflavin induced 8-oxodG formation in the DNA fragments containing telomeric sequence, and Fpg protein treatment led to chain cleavages at the central guanine of 5'-GGG-3' in telomere sequence. The amount of 8-oxodG formation in DNA fragment containing telomere sequence [5'-CGC(TTAGGG)(7)CGC-3'] was approximately 5 times more than that in DNA fragment containing nontelomere sequence [5'-CGC(TGTGAG)(7)CGC-3']. Catalase did not inhibit this oxidative DNA damage, indicating no or little participation of H(2)O(2) in DNA damage. These results indicate that the photoexcited endogenous photosensitizer specifically oxidizes the central guanine of 5'-GGG-3' in telomere sequence to produce 8-oxodG probably through an electron-transfer reaction. It is concluded that the site-specific damage in telomere sequence induced by UVA irradiation may participate in the increase of telomere shortening rate.     

Vicia faba chromosome damage induced by low doses of gamma radiation

The rate of radiation damage to chromosomes by low doses of gamma rays (0.01-0.30 Gy) was studied in the root tips ofVicia faba. As criteria of the effect of ionizing radiation, the frequency of sister chromatid exchanges (SCEs), incidence of chromosomal aberrations and the number of micronuclei were evaluated and compared in irradiated cells. The results obtained confirmed that the analysis of SCEs did not represent an efficient indicator of radiation damage to chromosomes. On the contrary, the formation of chromosomal aberrations and micronuclei was effectively stimulated by low radiation doses, there being linear dose-effect relationships in the low doses region used.     

X-ray-induced base sequence damage in primate alphoid DNA

Radiation-induced single-strand breaks were found throughout the 172 bp repeat units of African green monkey component alpha DNA. Two kinds of 3'-ends of 5'-32P-labeled restriction fragments were found, as previously described by others. After irradiation in vitro, the yield of single-strand breaks was 4 X 10(-5) breaks/nucleotide/Gy, as determined by analyses in DNA sequencing type gels. Protection from X-ray damage was found when the DNA received 150 Gy in the presence of 2-mercaptoethanol. The results demonstrate a very sensitive quantitative means to study the role of indirect effects of ionizing radiation on strand-break induction and protection at the base sequence level. Component alpha DNA was isolated from irradiated CV-1 cells and was analyzed for single-strand breaks. Under these conditions the frequency of breaks was less than the frequency obtained when purified DNA was irradiated. The methodology is presented because of its relevance to the study of DNA strand breakage in living cells.     

Radiosensitization by hypoxic pretreatment with misonidazole: an interaction of damage at the DNA level

Prolonged exposures to misonidazole (MISO) in vitro under hypoxic conditions result in radiosensitization which is characterized by a decrease in the size of the radiation survival curve shoulder for cells irradiated under hypoxic or aerobic conditions after drug removal. Although intracellular glutathione (GSH) was depleted during hypoxic exposures to MISO, this could not account for the dose-additive radiosensitization (decrease in shoulder size) since GSH depletion by diethylmaleate had no effect on the sensitivity of cells irradiated in air. The alkaline elution assay was used to measure DNA strand breaks and their repair after exposure to MISO, graded doses of X rays, and the combination of MISO pretreatment with X rays. The elution rate of DNA from irradiated cells increased linearly with X-ray dose, with and without MISO pretreatment. However, the DNA elution rates measured after MISO pretreatment were greater by a constant amount at all X-ray doses greater than 1 Gy. In terms of both cell survival and DNA elution rate, MISO-pretreated cells behaved as though they had received an extra 1.5 Gy. Although the initial damage after X rays was greater in MISO-pretreated cells, there was no effect of MISO pretreatment on the rate of repair of radiation-induced DNA strand breaks. The agreement between the differences in survival levels and DNA elution rates for irradiated control and MISO-pretreated cells and absence of an effect on DNA repair rates suggest that the pretreatment sensitization is due to an additive interaction of damage at the DNA level.