Gy Exposure Research Articles

A non-linear detection of phospho-histone H2AX in EA.hy926 endothelial cells following low-dose X-irradiation is modulated by reactive oxygen species

BackgroundA discontinuous dose response relationship is a major characteristic of the anti-inflammatory effects of low-dose X-irradiation therapy. Although recent data indicate an involvement of a variety of molecular mechanisms in these characteristics, the impact of reactive oxygen species (ROS) production to give rise or contribute to these phenomena in endothelial cells (EC) remains elusive.Material and methodsHUVEC derived immortalized EA.hy926 cells were stimulated by tumor necrosis factor-α (TNF-α, 20 ng/ml) 4 h before irradiation with doses ranging from 0.3 to 1 Gy. To analyse DNA repair capacity, phospho-histone H2AX foci were assayed at 1 h, 4 h and 24 h after irradiation. ROS production and superoxide dismutase (SOD) activity were analysed by fluorometric 2′,7′-dichlorodihydrofluorescein-diacetate (H2DCFDA) and colorimetric assays. A functional impact of ROS on γH2AX production was analysed by treatment with the scavenger N-acetyl-L-cysteine (NAC).ResultsIrrespective of stimulation by TNF-α, EA.hy926 cells revealed a linear dose response characteristic of γH2AX foci detection at 1 h and 4 h after irradiation. By contrast, we observed a discontinuity in residual γH2AX foci detection at 24 h after irradiation with locally elevated values following a 0.5 Gy exposure that was abolished by inhibition of ROS by NAC. Moreover, SOD protein expression was significantly decreased at doses of 0.5 Gy and 0.7 Gy concomitant with a reduced SOD activity.ConclusionThese data implicate a non-linear regulation of ROS production and SOD activity in EA.hy926 EC following irradiation with doses < 1 Gy that may contribute to a discontinuous dose-response relationship of residual γH2AX foci detection.

Radiation Effects on Human Heredity

In experimental organisms such as fruit flies and mice, increased frequencies in germ cell mutations have been detected following exposure to ionizing radiation. In contrast, there has been no clear evidence for radiation-induced germ cell mutations in humans that lead to birth defects, chromosome aberrations, Mendelian disorders, etc. This situation exists partly because no sensitive and practical genetic marker is available for human studies and also because the number of people exposed to large doses of radiation and subsequently having offspring was small until childhood cancer survivors became an important study population. In addition, the genome of apparently normal individuals seems to contain large numbers of alterations, including dozens to hundreds of nonfunctional alleles. With the number of mutational events in protein-coding genes estimated as less than one per genome after 1 gray (Gy) exposure, it is unsurprising that genetic effects from radiation have not yet been detected conclusively in humans.

Distinctive microRNA expression signatures in proton-irradiated mice

Proton particles comprise the most abundant ionizing radiation (IR) in outer space. These high energy particles are known to cause frequent double- and single-stranded DNA lesions that can lead to cancer and tumor formation. Understanding the mechanism of cellular response to proton-derived IR is vital for determining health risks to astronauts during space missions. Our understanding of the consequences of these high energy charged particles on microRNA (miRNA) regulation is still in infancy. miRNAs are non-coding, single-stranded RNAs of ~22 nucleotides that constitute a novel class of gene regulators. They regulate diverse biological processes, and each miRNA can control hundreds of gene targets. To investigate the effect of proton radiation on these master regulators, we examined the miRNA expression in selected mice organs that had been exposed to whole-body proton irradiation (2 Gy), and compared this to control mice (0 Gy exposure). RNA was isolated from three tissues (testis, brain, and liver) from treated and control mice and subjected to high-throughput small RNA sequencing. Bioinformatics analysis of small RNA sequencing data revealed dysregulation of (p < 0.05; 20 up- and 10 down-regulated) 14 mouse testis, 8 liver, and 8 brain miRNAs. The statistically significant and unique miRNA expression pattern found among three different proton-treated mouse tissues indicates a tissue-specific response to proton radiation. In addition to known miRNAs, sequencing revealed differential expression of 11 miRNAs in proton-irradiated mice that have not been previously reported in association with radiation exposure and cancer. The dysregulation of miRNAs on exposure to proton radiation suggest a possible mechanism of proton particles involvement in the onset of cell tumorgenesis. In summary, we have established that specific miRNAs are vulnerable to proton radiation, that such differential expression profile may depend upon the tissue, and that there are more miRNAs affected by proton radiation than have been previously observed.

Baicalein protects mice against radiation-induced DNA damages and genotoxicity

Baicalein is the major flavonoid extracted from the root of Scutellaria baicaleins. This flavonoid is used extensively in Chinese herbal medicine. In the present study baicalein is evaluated for its radioprotective properties. Human blood cells when exposed to the γ-radiation ex vivo in presence of baicalein underwent the reduced DNA damage compared to the control. Baicalein administration prior to the whole-body γ-radiation (4 Gy) exposure of mice resulted in protecting the damage to the DNA as measured in their blood cells by alkaline comet assay. Mice when exposed to the radiation (whole body; 1.7 Gy) resulted in damage to the bone marrow as measured by micronucleated reticulocyte (MNRET) formation. Baicalein pre-treatment reduces the radiation induced damage to the bone marrow cells, as there was decrease in the percentage MNRET formation. These findings indicate radio-protecting ability of baicalein.

Irradiation Induced Injury Reduces Energy Metabolism in Small Intestine of Tibet Minipigs

BackgroundThe radiation-induced energy metabolism dysfunction related to injury and radiation doses is largely elusive. The purpose of this study is to investigate the early response of energy metabolism in small intestinal tissue and its correlation with pathologic lesion after total body X-ray irradiation (TBI) in Tibet minipigs.Methods and Results30 Tibet minipigs were assigned into 6 groups including 5 experimental groups and one control group with 6 animals each group. The minipigs in these experimental groups were subjected to a TBI of 2, 5, 8, 11, and 14 Gy, respectively. Small intestine tissues were collected at 24 h following X-ray exposure and analyzed by histology and high performance liquid chromatography (HPLC). DNA contents in this tissue were also examined. Irradiation causes pathologic lesions and mitochondrial abnormalities. The Deoxyribonucleic acid (DNA) content-corrected and uncorrected adenosine-triphosphate (ATP) and total adenine nucleotides (TAN) were significantly reduced in a dose-dependent manner by 2–8 Gy exposure, and no further reduction was observed over 8 Gy.ConclusionTBI induced injury is highly dependent on the irradiation dosage in small intestine and inversely correlates with the energy metabolism, with its reduction potentially indicating the severity of injury.

Late Infusion of Cloned Marrow Fibroblasts Stimulates Endogenous Recovery from Radiation-Induced Lung Injury

In the current study, we used a canine model of radiation-induced lung injury to test the effect of a single i.v. infusion of 10×106/kg of marrow fibroblasts on the progression of damage following 15 Gy exposure to the right lung. The fibroblasts, designated DS1 cells, are a cloned population of immortalized cells isolated from a primary culture of marrow stromal cells. DS1 cells were infused at week 5 post-irradiation when lung damage was evident by imaging with high-resolution computed tomography (CT). At 13 weeks post-irradiation we found that 4 out of 5 dogs receiving DS1 cells had significantly improved pulmonary function compared to 0 out of 5 control dogs (p = 0.047, Fisher’s Exact). Pulmonary function was measured as the single breath diffusion capacity-hematocrit (DLCO-Hct), the total inspiratory capacity (IC), and the total lung capacity (TLC), which differed significantly between control and DS1-treated dogs; p = 0.002, p = 0.005, and p = 0.004, respectively. The DS1-treated dogs also had less pneumonitis detected by CT imaging and an increased number of TTF-1 (thyroid transcription factor 1, NKX2-1) positive cells in the bronchioli and alveoli compared to control dogs. Endothelial-like progenitor cells (ELC) of host origin, detected by colony assays, were found in peripheral blood after DS1 cell infusion. ELC numbers peaked one day after infusion, and were not detectable by 7 days. These data suggest that infusion of marrow fibroblasts stimulates mobilization of ELC, which is associated with a reduction in otherwise progressive radiation-induced lung injury. We hypothesize that these two observations are related, specifically that circulating ELC contribute to increased angiogenesis, which facilitates endogenous lung repair.

High and low dose responses of transcriptional biomarkers in ex vivo X-irradiated human blood

Purpose: Modifications of gene expression following ionizing radiation (IR) exposure of cells in vitro and in vivo are well documented. However, little is known about the dose-responses of transcriptionally responsive genes, especially at low doses. In this study, we investigated these dose-responses and assessed inter-individual variability.Materials and methods: High dose (0.5–4 Gy) and low dose (5–100 mGy) gene expression responses at 2 h and 24 h using 13 biomarkers transcriptionally regulated through the DNA damage response by the tumor suppressor p53 were investigated. Inter-individual variation was also examined.Results: High dose-response curves were best constructed using a polynomial fit while the low dose-response curves used a linear fit with linear R2 values of 0.841–0.985. Individual variation was evident in the high and low dose ranges. The FDXR, DDB2 high dose gene combination produced a mean dose estimate of 0.7 Gy for 1 Gy irradiated ‘unknown’ samples (95% CIs of 0.3–1.1 Gy) and 1.4 Gy for 2 Gy exposure (95% CIs of 0.6–2.1 Gy). The FDXR, DDB2, CCNG1 low dose gene combination estimated 98 mGy (95% CIs of 27–169 mGy) for 100 mGy exposure.Conclusions: These findings identify genes that fulfill some of the requirements of a good exposure biomarker even at low doses, such as sensitivity, reproducibility and simple proportionality with dose.

Effects of radiation and vitamin C treatment on metronidazole genotoxicity in mice

The impact of exposure to low dose radiation (LDR) on human health is not clear. Besides, cross adaptation or sensitization with pharmaceutical agents may modify the risk of LDR. In the present study, we analyzed the interaction of radiation and metronidazole (MTZ) in inducing chromosome aberration (CA) and micronucleus (MN) in the bone marrow cells of Balb/C mice in vivo. Further, we evaluated the efficacy of vitamin C to reduce MTZ induced genotoxicity. We found that 10, 20 and 40mg/kg of MTZ induced dose dependent increase in the frequency of CA (r=0.9923, P<0.01) as well as MN (r=0.9823, P<0.05) in polychromatic erythrocytes. However, MTZ did not affect the ratio of polychromatic erythrocytes to normochromatic erythrocytes indicating lack of cytotoxicity. Supplementation with vitamin C prior to MTZ treatment significantly reduced the frequency of CA (P<0.001) as well as MN (P<0.001). Radiation (0.5Gy) exposure prior to MTZ treatment produced a less than additive (for CA) to additive (for MN) effects. However, radiation exposure following MTZ treatment produced additive (for CA) and synergistic (for MN) effects. Further, vitamin C pre-treatment also reduced the genotoxicity indices following the combined treatment of MTZ and radiation. Our findings suggest that MTZ may sensitize bone marrow cells to radiation exposure and enhances genotoxicity. We recommend more studies on the interaction of LDR and marketed pharmaceuticals to minimize possible harmful outcomes through appropriate precautionary measures.

Mushroom beta glucan: Potential candidate for post irradiation protection

The in vivo radioprotective effect of a beta-glucan (BG) isolated from the mushroom Ganoderma lucidum, against radiation (RT) induced damage was investigated taking mouse survival, hematology, liver GSH (Reduced glutathione), liver Malondialdehyde (MDA) and bone marrow chromosomal aberrations as end points. Young adult swiss albino mice were whole body exposed to gamma radiation. For mouse survival study, BG was administered orally (250μg/kg body wt or 500μg/kg body wt) 15min before or 5min after 8Gy exposure. For other parameters BG was given orally 5min after 4Gy exposure. The radioprotective effect of BG was compared with that of clinically used radioprotective drug amifostine (WR-2721), at 300mg/kg body wt administered intraperitoneally, 30min before irradiation. BG (500μg/kg body wt) produced (66%) mouse survival at 30 days given post irradiation, and 83% survived at 30 days with 300mg/kg body wt of amifostine administered before RT while RT alone produced 100% mortality. BG is not toxic at the radioprotective dose. Significant reduction in number of aberrant cells and different types of aberration was observed in both BG and amifostine administered groups compared to radiation alone treated group. BG seems to have potential for use in protection against unplanned radiation exposures.

Pre-Adipocytes Differentiate Into Megakaryocytes (MK) by a Paracrine Thrombopoietin (TPO) Loop

Abstract 4742The regulation of megakaryopoiesis and thrombopoiesis are incompletely understood. Better understanding of the underlying mechanisms would be of biological import, but may also lead to novel approaches for generating of platelets for clinical application. One cell line that undergoes terminal differentiation into megakaryocytes is pre-adipocytes. These cells represent a potential candidate cell for ex vivo generation of megakaryocytes. Here we demonstrate that pre-adipocytes differentiate into MKs and platelets by using an endogenous paracrine loop involving TPO, the primary cytokine involved in megakaryopoiesis, and its receptor c-mpl. We previously reported that pre-adipocytes differentiate into MKs and form platelets when the culture medium is switched from maintenance medium to MK lineage induction (MKLI) medium. Neither media include TPO. Based on these observations, the present study tested the hypothesis that pre-adipocytes might differentiate into MKs and platelets by endogenous TPO and c-mpl expression of sufficient magnitude to drive megakaryopoiesis. We used primary mouse pre-adipocytes (CD45-, CD117-, Sca1+, CD29+, CD73+, CD90+, CD105+, CD106+) from subcutaneous adipose tissues and also the mouse pre-adipocyte line 3T3-L1 cells. The TPO levels, as assessed by ELISA, in the supernatant from 106 pre-adipocytes cultured in 2 ml MKLI medium without exogenous TPO (TPO-) were unmeasurable level on Day 0, 29±14 pg/ml on Day 7 and 8±2 pg/ml on Day 12. Similar results were obtained in the supernatant from 3T3-L1 during MK differentiation. We did not observe measureable TPO in supernatants from pre-adipocytes and 3T3-L1 cells cultured in maintenance medium on Days 0, 7 and 12. We then compared MK differentiation from pre-adipocytes in MKLI media in the absence (TPO-) or addition (final concentration, 50 ng/ml; TPO+) of TPO. The frequency of CD41-postive pre-adipocytes in culture after 6 days was 58±11% for TPO+ and 70±7% for TPO- (p>0.05), consistent with endogenous TPO being sufficient under these circumstances to stimulate MK differentiation of pre-adipocytes. DNA ploidy and c-mpl expression assessed by immunohistochemistry were also similar with or without added exogenous TPO. We next examined the number of CD41-expressing large-sized (>15 μm) cells beginning with 1.2 × 104 preadipocytes in the absence or presence of the c-mpl inhibitor rat anti-mouse c-mpl inhibitor AMM2 (final concentration, 10 μg/ml). In the absence of AMM2, 10-fold more CD41-positive, large cells were seen (6.8±1.7×103) than in its presence (0.6±0.2×103, p<0.05). These observations support that pre-adipocytes differentiate into MKs using endogenous TPO stimulation via c-mpl. We next examined the ability to release platelets from these treated pre-adipocytes in each experimental condition (pre-adipocytes and 3T3-L1 cells, each ± exogenous TPO). Infusion of 2 × 105 carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled MKs into irradiated thrombocytopenic mice (7 days post-2.0Gy exposure) led to a time-dependent appearance of CFSE+/CD41+ platelet-sized particles with a peak at 4 hours after the infusion reaching ∼2.5% of the total circulating platelet population (∼30 platelets/infused MK) under all tested experimental conditions. For platelet function, blood samples from each of these mice were perfused over a collagen-coated chip for 10 minutes, and the incorporation of CFSE+ particles into thrombi on the chip was determined (Total Thrombus-formation Analyzing System). A similar degree of platelet incorporation was observed under all experimental conditions and each was with an efficiency similar to that seen when CFSE+ control platelets were infused. These findings demonstrate that pre-adipocytes differentiate into MKs and subsequent platelets by an endogenous TPO release and a paracrine loop involving c-mpl. We propose that such pre-adipocytes could be used as a model of a continuously replicating cell line that upon switching media simultaneous expresses TPO and its receptor c-mpl to establish a paracrine loop leading to terminal differentiation into functional MKs. The basis of why this media switch induces this change needs to be clarified to further develop this pre-adipocyte strategy as a donor-independent source for platelet transfusion as well as for studying mechanism underlying megakaryopoiesis and thrombopoiesis. Disclosures:Matsubara:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees. Murata:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees.

Effects of expression level of DNA repair-related genes involved in the NHEJ pathway on radiation-induced cognitive impairment

Cranial radiation therapy can induce cognitive decline. Impairments of hippocampal neurogenesis are thought to be a paramountly important mechanism underlying radiation-induced cognitive dysfunction. In the mature nervous system, DNA double-strand breaks (DSBs) are mainly repaired by non-homologous end-joining (NHEJ) pathways. It has been demonstrated that NHEJ deficiencies are associated with impaired neurogenesis. In our study, rats were randomly divided into five groups to be irradiated by single doses of 0 (control), 0 (anesthesia control), 2, 10, and 20 Gy, respectively. The cognitive function of the irradiated rats was measured by open field, Morris water maze and passive avoidance tests. Real-time PCR was also used to detect the expression level of DNA DSB repair-related genes involved in the NHEJ pathway, such as XRCC4, XRCC5and XRCC6, in the hippocampus. The influence of different radiation doses on cognitive function in rats was investigated. From the results of the behavior tests, we found that rats receiving 20 Gy irradiation revealed poorer learning and memory, while no significant loss of learning and memory existed in rats receiving irradiation from 0–10 Gy. The real-time PCR and Western blot results showed no significant difference in the expression level of DNA repair-related genes between the 10 and 20 Gy groups, which may help to explain the behavioral results, i.e. DNA damage caused by 0–10 Gy exposure was appropriately repaired, however, damage induced by 20 Gy exceeded the body's maximum DSB repair ability. Ionizing radiation-induced cognitive impairments depend on the radiation dose, and more directly on the body's own ability to repair DNA DSBs via the NHEJ pathway.

Effect of Low-dose Radiation on Cognitive Function of Rats at Different Ages

Purpose/Objective(s): It is known that radiation can induce cognitive dysfunction, but we still know little about the threshold of radiation dose. It turns out that 0.1 Gy radiation can modulate the expression of gene pathways involved in cognitive function. However, it isn’t clear whether 0.1 Gy whole brain irradiation can induce cognitive impairments. In this study, the relationship between 0.1 Gy exposure and cognitive function was investigated in rats at different ages. Materials/Methods: Thirty-two 1-month-old male Sprague-Dawley rats and thirty two six-month-old male Sprague-Dawley rats were divided randomly into 0.1Gy irradiated group and anesthesia control group. The rats were anesthetized, and then the whole brains were irradiated using 4MeV electron beams delivered by a linear accelerator. Behavioral tests were performed 3 months after irradiation in the order of open field, Morris water maze and passive avoidance test. The tests were used to evaluate the anxiety level, hippocampal dependent spatial learning and memory, and nonspatial learning and memory respectively. In Morris water maze, the place navigation test was conducted on the 1st to 4th day; while on the 5th day, the spatial probe test was done. Results: In the place navigation test of Morris water maze, six-month-old rats exhibited age dependent impairments in latency to find the hidden platform compared with the one-month-old rats. But radiation did not affect the six-month-old rats’ spatial learning and memory. Irradiated group (one month old) had significantly longer latency time than control group. Radiation did not have the effect on the anxiety level in the open field test, and there was no significant difference between groups in the latency of the passive avoidance. Conclusions: In present study, the hippocampal dependent spatial learning and memory capacity of the one-month-old rats was impaired by 0.1Gy whole brain irradiation, but it did not have the same effect on the six-month-old rats. These findings strongly suggest that low dose radiation is sensitive to induce cognitive impairments in younger rats. Author Disclosure: R. Sun: E. Research Grant; NSFC 81172128, NSFC 81102077, NSFC 30870740, Project of Postgraduate’s Innovation of Jiangsu Province cx10b-055z. L.Y. Zhang: E. Research Grant; NSFC 81172128, NSFC 81102077, NSFC 30870740, Project of Postgraduate’s Innovation of Jiangsu Province cx10b-055z. L.S. Chen: E. Research Grant; NSFC 81172128, NSFC 81102077, NSFC 30870740, Project of Postgraduate’s Innovation of Jiangsu Province cx10b-055z. Y. Tian: E. Research Grant; NSFC 81172128, NSFC 81102077, NSFC 30870740, Project of Postgraduate’s Innovation of Jiangsu Province cx10b-055z.

Auditory Effects of Cranial Irradiation in a Mouse Model

Objective: 1) Determine whether radiation exposure in mice leads to measurable hearing losses similar to those seen in patients exposed to radiotherapy for acoustic neuromas, temporal bone malignancies, and nasopharyngeal carcinoma. 2) Assess for any associated pathologic changes in cochleas exposed to radiation. Method: Adult (11-13 weeks) 129Sv/J mice were exposed to radiation using an animal irradiator, ranging from a single exposure of 10 Gy to 100 Gy. Pre- and postradiation audiometric brainstem response (ABR) data were recorded to document hearing loss, with subsequent analysis of cochlear ultrastructure using transmission and scanning electron microscopy. Results: High-frequency hearing loss was noted in exposures exceeding 40 Gy. Anorexia was observed by day 6 post- irradiation in all mice exposed to doses ≥20 Gy. Normal hearing following 20 Gy exposure suggests that hearing losses seen after ≥40 Gy exposures is secondary to radiation effects rather than any confounding metabolic disturbance. Ultrastructural analysis revealed dramatically decreased neuron density in Rosenthal’s canal and loss of the associated extracellular matrix. Negligible changes were seen in the outer and inner hair cells, with overall preservation of the stria vascularis and cochlear vasculature. Age-matched controls failed to demonstrate similar histopathologic findings. Conclusion: This study demonstrates that the mouse is a viable model for the study of radiation-induced hearing loss. The initial impact of the radiation appears to focus on the eighth nerve cell body and the axonal projections to the organ of Corti. Future studies should incorporate fractionation of the radiation dose.

Changes in liver metabolic gene expression after radiation exposure

Increased exposure to radiation is one physiological stressor associated with spaceflight. While known to alter normal physiological function, how radiation affects metabolism of administered medications is unclear. Crew health could be affected if the actions of medications used in spaceflight deviated from expectations formed during terrestrial medication use. Three different doses of gamma radiation (50 mGy – 6.05 Gy) and a sham were administered to groups of 6 mice each, and after various intervals of recovery time, liver gene expression was measured with RT‐qPCR arrays for drug metabolism and DNA repair enzymes. Results indicated ~65 genes of the 190 tested were significantly affected by at least one of the radiation doses. Many of the affected genes are involved in the metabolism of drugs with hydrophobic or steroid‐like structures, maintenance of redox homeostasis and repair of DNA damage. Most affected genes returned to near control expression levels by 7 days post‐treatment. With 6 Gy exposure, metallothionein expression was 132‐fold more than control at the 4 hr time point, and fell at each later time point (11‐fold at 24 hrs, and 8‐fold at 7 days). In contrast, Cyp17a1 showed a 4‐fold elevation at 4 hrs after exposure and remained constant for 7 days.

Radio-protective response on the environmental pollutant induced oxidative stress

The purpose of this study was to investigate the protective effect of low dose radiation on TCE induced oxidative damage in rats. The oxidative damage of both liver and kidney was assessed by serum alkaline phosphatase (ALP), Gamma Glutamyl Trans-Peptidase (GGTP), Alanine and Aspartate Amino Transferase (ALT & AST) activities in addition concentrations of cholesterol, high density lipoprotein-cholesterol (HDL-c), low density lipoprotein-cholesterol (LDL-c), Triacyglycerols (TGs), urea and creatinine were analyzed. Liver lipid peroxidation (MDA), Nitric Oxide (NO), reduced glutathione (GSH) levels, and activities of both Super-Oxide Dismutase (SOD) and Glutathione Peroxidase (GSH-Px) were measured. Results: TCE administration increase serum ALP, GGTP, ALT, AST activities, cholesterol, triacylglycerols, LDL-c, urea and creatinine concentrations, besides liver MDA and NO, whereas it decreased SOD, GSH-Px activities, GSH level in liver, HDL-c in serum. Low dose of gamma rays (0.5 Gy) exposure significantly improved lipid peroxidation and oxidative injury induced by TCE. Conclusion: The study indicates that treatment with low dose of gamma rays ameliorate harmful effects induced by TCE taking in consideration the effect of gamma radiation as a stimulant of radical detoxification.

Mouse embryonic stem cells that survive γ‐rays exposure maintain pluripotent differentiation potential and genome stability

This study aimed to investigate the cell cycle, apoptosis, cytogenetics and differentiation capacity of mouse embryonic stem cells (mESCs) that survived a single dose of 2 or 5 Gy γ-rays during a period of up to 96 h of culture. After 2 Gy irradiation and 24 h culture, compared to control, a significant majority of cells was blocked at the G2/M phase and a massive apoptosis was recorded. Between 48 and 72 h post-irradiation, the parameters used to describe the cell cycle and apoptosis returned similar to those of control samples. When mESCs were irradiated with 5 Gy, a small fraction of cells, even after 96 h of culture, still presented clear evidences of a G2/M block and apoptosis. The cytogenetic analysis performed at 96 h showed that the structural stability of the aberrations did not change significantly when comparing control and 2 or 5 Gy-treated populations. However, the chromosomal damage observed in the progeny of the survived cells after 5 Gy exposure is significantly higher than that observed in control samples, although it is mostly of the stable and transmissible type. Ninety-six hours after irradiation, the survived mESCs maintained their undifferentiated status and capability to differentiate into the three germ layers. Overall, these results indicate a commitment of mESCs to maintain pluripotency and genome stability.

Evidence for formation of DNA repair centers and dose-response nonlinearity in human cells

The concept of DNA "repair centers" and the meaning of radiation-induced foci (RIF) in human cells have remained controversial. RIFs are characterized by the local recruitment of DNA damage sensing proteins such as p53 binding protein (53BP1). Here, we provide strong evidence for the existence of repair centers. We used live imaging and mathematical fitting of RIF kinetics to show that RIF induction rate increases with increasing radiation dose, whereas the rate at which RIFs disappear decreases. We show that multiple DNA double-strand breaks (DSBs) 1 to 2 μm apart can rapidly cluster into repair centers. Correcting mathematically for the dose dependence of induction/resolution rates, we observe an absolute RIF yield that is surprisingly much smaller at higher doses: 15RIF/Gy after 2 Gy exposure compared to approximately 64RIF/Gy after 0.1 Gy. Cumulative RIF counts from time lapse of 53BP1-GFP in human breast cells confirmed these results. The standard model currently in use applies a linear scale, extrapolating cancer risk from high doses to low doses of ionizing radiation. However, our discovery of DSB clustering over such large distances casts considerable doubts on the general assumption that risk to ionizing radiation is proportional to dose, and instead provides a mechanism that could more accurately address risk dose dependency of ionizing radiation.

Endothelial cell damage induces a blood–alveolus barrier breakdown in the development of radiation‐induced lung injury

To investigate the role of endothelial cell damage in radiation-induced lung injury. A total of 60 male Sprague-Dawley rats were irradiated to the right hemithorax with a single dose of 0, 7.0 or 14.4 Gy. Serial studies were performed before and at 1, 7, 30 and 90 days after radiation, respectively. Pathological studies were carried out to detect changes in the lung after irradiation. Western blot studies were conducted to detect the expression of CD34 and of CD105 and vascular endothelial growth factor (VEGF). Compared to controls, the irradiated rat lung showed a dose-dependent and time-dependent decrease in the expression of CD34. The level of CD105 was significantly reduced by irradiation except at 90 days after radiation. The expression of VEGF increased 1 day after radiation, and then decreased from day 30 onwards, to be lower than the control group at 90 days. Pulmonary fibrosis was observed at 90 days after 14.4 Gy exposure; however, most of these phenomena were not observed in the 7.0 Gy group. These results support the notion that endothelial cells play an important role in radiation-induced lung injury, and may be critical to breakdown of the blood-alveolus barrier and microcirculation dysfunction related to radiation-induced inflammation and fibrosis.

Radiation-induced blood–brain barrier damages: An in vitro study

A radiation-induced blood–brain barrier (BBB) breakdown has been supposed to explain the acute radiation syndrome and the delayed brain radiation injury, but it has been clearly demonstrated only at high doses. In a previous study (Diserbo et al., 2002), we showed that non-lethal total body irradiation produced an early transient increase in BBB permeability in rats but the underlying mechanisms of radiation-induced BBB breakdown remain unclear. In the present work, the effects of ionizing radiation were studied on an in vitro BBB model. Gamma irradiation induced an increase in [ 14C]-sucrose BBB permeability that can be detected 72 h after exposure at doses up to 4 Gy. This increase was more important 8 days after irradiation and could be limited by dexamethasone treatment. An increase in fluorescein and FITC-dextrans (4 kDa/70 kDa) permeability was also observed, which can be related to a substantial opening of endothelial cell tight-junctions but without massive modification of tight-junction protein (ZO-1, ZO-2, claudin-5, occludin) immunolabeling even 8 days after 25 Gy exposure. Formation of actin stress fibers occurred in endothelial cells 8 days after 25 Gy exposure. A progressive decrease in cellular density associated with a simultaneous spreading of the endothelial cells was also observed after irradiation. Anti-γH2AX immunolabeling was used to investigate both DNA double-strand break induction and repair rates in endothelial cells. It revealed long-lasting DNA double-strand breaks after gamma irradiation. A better understanding and awareness of these phenomena are essential for designing appropriate pharmacotherapy in radiation-therapy and treatment of accidental overexposure.

Mitochondrial DNA alterations of peripheral lymphocytes in acute lymphoblastic leukemia patients undergoing total body irradiation therapy

BackgroundMitochondrial DNA (mtDNA) alterations, including mtDNA copy number and mtDNA 4977 bp common deletion (CD), are key indicators of irradiation-induced damage. The relationship between total body irradiation (TBI) treatment and mtDNA alterations in vivo, however, has not been postulated yet. The aim of this study is to analyze mtDNA alterations in irradiated human peripheral lymphocytes from acute lymphoblastic leukemia (ALL) patients as well as to take them as predictors for radiation toxicity.MethodsPeripheral blood lymphocytes were isolated from 26 ALL patients 24 hours after TBI preconditioning (4.5 and 9 Gy, respectively). Extracted DNA was analyzed by real-time PCR method.ResultsAverage 2.31 times mtDNA and 0.53 fold CD levels were observed after 4.5 Gy exposure compared to their basal levels. 9 Gy TBI produced a greater response of both mtDNA and CD levels than 4.5 Gy. Significant inverse correlation was found between mtDNA content and CD level at 4.5 and 9 Gy (P = 0.037 and 0.048). Moreover, mtDNA content of lymphocytes without irradiation was found to be correlated to age.ConclusionsmtDNA and CD content may be considered as predictive factors to radiation toxicity.