Post-irradiation Time Research Articles

Investigation of relative metabolic changes in the organs and plasma of rats exposed to X-ray radiation using HR-MAS (1)H NMR and solution (1)H NMR.

Excess exposure to ionizing radiation generates reactive oxygen species and increases the cellular inflammatory response by modifying various metabolic pathways. However, an investigation of metabolic perturbations and organ-specific responses based on the amount of radiation during the acute phase has not been conducted. In this study, high-resolution magic-angle-spinning (HR-MAS) NMR and solution NMR-based metabolic profiling were used to investigate dose-dependent metabolic changes in multiple organs and tissues--including the jejunum, spleen, liver, and plasma--of rats exposed to X-ray radiation. The organs, tissues, and blood samples were obtained 24, 48, and 72 h after exposure to low-dose (2 Gy) and high-dose (6 Gy) X-ray radiation and subjected to metabolite profiling and multivariate analyses. The results showed the time course of the metabolic responses, and many significant changes were detected in the high-dose compared with the low-dose group. Metabolites with antioxidant properties showed acute responses in the jejunum and spleen after radiation exposure. The levels of metabolites related to lipid and protein metabolism were decreased in the jejunum. In addition, amino acid levels increased consistently at all post-irradiation time points as a consequence of activated protein breakdown. Consistent with these changes, plasma levels of tricarboxylic acid cycle intermediate metabolites decreased. The liver did not appear to undergo remarkable metabolic changes after radiation exposure. These results may provide insight into the major metabolic perturbations and mechanisms of the biological systems in response to pathophysiological damage caused by X-ray radiation.

Transient delay of radiation-induced apoptosis by phorbol acetate

The mechanisms of interference of a model tumour promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) with radiation-induced apoptosis in human peripheral lymphocytes have been investigated. The cells were treated with TPA under various conditions and thereafter exposed to a single lethal dose of gamma radiation. Morphological and biochemical changes characteristic of apoptosis were followed up to 72 h of post-irradiation time. Acute exposure to low concentration of TPA resulted in delay in the onset of radiation-induced apoptosis (determined as morphological changes and rate of mitochondrial demise) by 24-48 h as compared to the irradiated, sham TPA-treated cells. The time course of this delay correlated well with confinement of the p53 protein to the cytoplasm and increase in bcl-2 levels at the nuclear periphery of irradiated cells. Our results indicate that confinement of p53 in the cytoplasm is one of the potential mechanisms by which TPA interferes with the process of radiation-induced apoptosis in human lymphocytes.

A Gamma-Knife-Enabled Mouse Model of Cerebral Single-Hemisphere Delayed Radiation Necrosis.

PurposeTo develop a Gamma Knife-based mouse model of late time-to-onset, cerebral radiation necrosis (RN) with serial evaluation by magnetic resonance imaging (MRI) and histology.Methods and MaterialsMice were irradiated with the Leksell Gamma Knife® (GK) PerfexionTM (Elekta AB; Stockholm, Sweden) with total single-hemispheric radiation doses (TRD) of 45- to 60-Gy, delivered in one to three fractions. RN was measured using T2-weighted MR images, while confirmation of tissue damage was assessed histologically by hematoxylin & eosin, trichrome, and PTAH staining.ResultsMRI measurements demonstrate that TRD is a more important determinant of both time-to-onset and progression of RN than fractionation. The development of RN is significantly slower in mice irradiated with 45-Gy than 50- or 60-Gy, where RN development is similar. Irradiated mouse brains demonstrate all of the pathologic features observed clinically in patients with confirmed RN. A semi-quantitative (0 to 3) histologic grading system, capturing both the extent and severity of injury, is described and illustrated. Tissue damage, as assessed by a histologic score, correlates well with total necrotic volume measured by MRI (correlation coefficient = 0.948, with p<0.0001), and with post-irradiation time (correlation coefficient = 0.508, with p<0.0001).ConclusionsFollowing GK irradiation, mice develop late time-to-onset cerebral RN histology mirroring clinical observations. MR imaging provides reliable quantification of the necrotic volume that correlates well with histologic score. This mouse model of RN will provide a platform for mechanism of action studies, the identification of imaging biomarkers of RN, and the development of clinical studies for improved mitigation and neuroprotection.

Investigation of an improved MAA-based polymer gel for thermal neutron dosimetry

The aims of this study are to increase the melting point of a normoxic Methacrylic acid-based polymer gel as well as investigation of its responses to thermal neutron and gamma irradiation. New polymer gel was named MAGIC-A (MAGIC and agarose). The R 2-dose sensitivity of MAGIC-A polymer gel for gamma irradiation was 0.381 ± 0.010 Gy−1 s−1 at 24 h post irradiation time which decreased to 0.180 ± 0.004 Gy−1 s−1 for thermal neutron irradiation. Results of this research confirmed the applicability of the MAGIC-A polymer gels for dosimetry of thermal neutrons as well as gamma irradiation.

EGR1 regulates radiation-induced apoptosis in head and neck squamous cell carcinoma.

The transcription factor, early growth response1 (EGR1) belongs to the early growth response family. EGR1 regulates the transactivation of genes involved in growth inhibition and apoptosis by ionizing radiation. The aims of the present study were to evaluate the expression of EGR1, and its relationship to prognosis, in patients with advanced laryngeal and hypopharyngeal squamous cell carcinoma (LHSCC) receiving chemoradiation therapy, and to observe the effect of EGR1 on the apoptosis of head and neck squamous cell carcinoma (HNSCC) cells treated with ionizing radiation. Expression of the EGR1 protein in tissue samples from patients with LHSCC was detected by immunohistochemistry. A high expression of the EGR1 protein was observed in 37 (67.3%) of the 55LHSCC tissue samples examined. A high EGR1 protein expression in patients with LHSCC who were treated with chemoradiation was significantly associated with improved larynx-preservation survival (p=0.04). The 5-year disease-specific survival rate with larynx preservation was 59% in patients with a high EGR1 protein expression vs. 30% in those with a low EGR1 protein expression. In the human HNSCC cell line, PCI50, EGR1 mRNA expression was induced at 30-60min, and EGR1 protein expression was induced at 60-120min, after exposure to a 5Gy dose of ionizing radiation. To evaluate the impact of EGR1 on radiation-induced apoptosis, we used small‑interfering RNA to knock down endogenous EGR1 gene expression. Cleaved caspase3, cleaved caspase7, and cleaved PARP were decreased, while XIAP was increased, in EGR1-knockdown PCI50 cells compared to negative control PCI50 cells, at all observed post-irradiation time points. These findings suggested that EGR1 knockdown inhibits radiation-induced apoptosis. In conclusion, EGR1 may be associated with larynx-preservation survival, through the regulation of radiation-induced apoptosis in patients with LHSCC treated with chemoradiation. Although further investigations are required to support the present study, EGR1 serves as a favorable biomarker of radiosensitivity in the treatment of LHSCC.

Curcumin Stimulates the Antioxidant Mechanisms in Mouse Skin Exposed to Fractionated γ-Irradiation.

Fractionated irradiation is one of the important radiotherapy regimens to treat different types of neoplasia. Despite of the immense therapeutic gains accrued by delivering fractionated irradiation to tumors, the radiation burden on skin increases significantly. Low doses of irradiation to skin adversely affect its molecular and metabolic status. The use of antioxidant/s may help to alleviate the radiation-induced changes in the skin and allow delivering a higher dose of radiation to attain better therapeutic gains. Curcumin is an antioxidant and a free radical scavenging dietary supplement, commonly used as a flavoring agent in curries. Therefore, the effect of 100 mg/kg body weight curcumin was studied on the antioxidant status of mice skin exposed to a total dose of 10, 20 and 40 Gy γ-radiation below the rib cage delivered as a single fraction of 2 Gy per day for 5, 10 or 20 days. Skin biopsies from both the curcumin treated or untreated irradiated groups were collected for the biochemical estimations at various post-irradiation times. The irradiation of animals caused a dose dependent decline in the glutathione concentration, glutathione peroxidase, and superoxide dismutase activities and increased the lipid peroxidation in the irradiated skin. Curcumin treatment before irradiation resulted in a significant rise in the glutathione concentration and activities of both the glutathione peroxidase and superoxide dismutase enzymes in mouse skin, whereas lipid peroxidation declined significantly. The present study indicates that curcumin treatment increased the antioxidant status of mouse exposed to different doses of fractionated γ-radiation.

Particle size analysis of PAGAT gel dosimetry

This work represents the response of PAGAT gel dosimeter using UV-Visible Spectrophotometry. The particle size and optimal wavelength of the gel sample were analyzed from the obtained spectrum. In addition, the compressibility was estimated using Ultrasonic Interferometer. The results showed that the particle size of the PAGAT varied appreciably with respect to the dose applied but did not vary significantly with the post irradiation time.

Setting Reaction of Dental Resin-Modified Glass Ionomer Restoratives as a Function of Curing Depth and Postirradiation Time

Specular reflectance Fourier transform infrared (SR-FTIR) spectroscopy was used to study the setting reaction of dental resin-modified glass ionomer (RMGI) restoratives as a function of curing depth and postirradiation time. Two light-cure and one tri-cure RMGI materials were selected and used according to the manufacturers’ instructions. Samples were prepared by filling the mixed materials into custom-made molds and then light-irradiating using a dental curing light. The degree of conversion and the extent of acid-base reaction of the materials at different depths (0, 1, 2, and 4 mm) and postirradiation times (10 min, 1 day, and 7 days) were determined using SR-FTIR spectroscopy in conjunction with the Kramers-Kronig (K-K) transformation. The setting reaction was also investigated using microhardness measurements. The results showed that the depth of cure increased over time by the continuous acid-base reaction rather than photopolymerization or chemical polymerization. Microhardness tests seemed less suitable for studying the setting reaction as a function of postirradiation time, probably due to softening from the humidity. Analysis using specular reflectance in conjunction with the K-K algorithm was an easy and effective method for monitoring the setting reaction of dental RMGI materials.

Unraveling early defects in the in utero irradiated mouse neocortex

Event Abstract Back to Event Unraveling early defects in the in utero irradiated mouse neocortex Tine Verreet1, 2*, Mieke Verslegers2, Roel Quintens2, Sarah Baatout2, Lieve Mooons1 and Mohammed A. Benotmane2 1 KU Leuven, Belgium 2 SCK-CEN, Belgium Epidemiological studies demonstrated an increased mental disability and microcephaly in prenatally exposed atomic bomb survivors. We recapitulated these human findings in the mouse by exposing E11 embryos to X-rays. We now aimed at characterising underlying mechanisms using immunohistochemistry on E11 brain sections at different times post irradiation (PI). At 1 and 2 h PI in the developing cortex, a widespread and dose-dependent increase in DNA damage was accompanied by a transient reduction in mitotically active cells. At these early time points, apoptosis was induced in the frontal region of the telencephalon. At 6 h PI, apoptotic cells were found throughout the brain, while at 24 h PI, apoptosis was restricted to the preplate constituting the differentiated neurons. This highly specific spatiotemporal spread of apoptosis has not been reported yet in the irradiated brain and warrants additional experiments. Furthermore, we could detect a reduction in the number of radial glia at 6 h PI, whereas post-mitotic migrating neurons were found in the proliferative zone of the cortex. Together, this suggests that the loss of radial glia might be related to a decrease in mitotic divisions, an increase in apoptosis or an accelerated differentiation. Notably, all of these mechanisms have previously been implicated in the etiology of microcephaly. Which of these is primarily responsible for the observed reduction in brain size after prenatal irradiation, is currently under investigation. Acknowledgements This work was financially supported by the EU FP7 project CEREBRAD (GA n° 295552). Keywords: Apoptosis, Brain Development, differentiation and proliferation, Microcephaly, Radiation exposure Conference: 11th National Congress of the Belgian Society for Neuroscience, Mons, Belgium, 22 May - 22 May, 2015. Presentation Type: Oral or Poster presentation Topic: Neuroscience Citation: Verreet T, Verslegers M, Quintens R, Baatout S, Mooons L and Benotmane MA (2015). Unraveling early defects in the in utero irradiated mouse neocortex. Front. Neurosci. Conference Abstract: 11th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2015.89.00020 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 May 2015; Published Online: 05 May 2015. * Correspondence: Mrs. Tine Verreet, KU Leuven, Leuven, Belgium, tverreet@sckcen.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Tine Verreet Mieke Verslegers Roel Quintens Sarah Baatout Lieve Mooons Mohammed A Benotmane Google Tine Verreet Mieke Verslegers Roel Quintens Sarah Baatout Lieve Mooons Mohammed A Benotmane Google Scholar Tine Verreet Mieke Verslegers Roel Quintens Sarah Baatout Lieve Mooons Mohammed A Benotmane PubMed Tine Verreet Mieke Verslegers Roel Quintens Sarah Baatout Lieve Mooons Mohammed A Benotmane Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Does nitrogen gas bubbled through a low density polymer gel dosimeter solution affect the polymerization process?

Background:On account of the lower electron density in the lung tissue, the dose distribution in the lung cannot be verified with the existing polymer gel dosimeters. Thus, the aims of this study are to make a low density polymer gel dosimeter and investigate the effect of nitrogen gas bubbles on the R2 responses and its homogeneity.Materials and Methods:Two different types of low density polymer gel dosimeters were prepared according to a composition proposed by De Deene, with some modifications. In the first type, no nitrogen gas was perfused through the gel solution and water. In the second type, to expel the dissolved oxygen, nitrogen gas was perfused through the water and gel solution. The post-irradiation times in the gels were 24 and 5 hours, respectively, with and without perfusion of nitrogen gas through the water and gel solution.Results:In the first type of gel, there was a linear correlation between the doses and R2 responses from 0 to 12 Gy. The fabricated gel had a higher dynamic range than the other low density polymer gel dosimeter; but its background R2 response was higher. In the second type, no difference in R2 response was seen in the dose ranges from 0 to 18 Gy. Both gels had a mass density between 0.35 and 0.45 g.cm-3 and CT values of about -650 to -750 Hounsfield units.Conclusion:It appeared that reactions between gelatin-free radicals and monomers, due to an increase in the gel temperature during rotation in the household mixer, led to a higher R2-background response. In the second type of gel, it seemed that the collapse of the nitrogen bubbles was the main factor that affected the R2-responses.

Enhancement of viability of radiosensitive (PBMC) and resistant (MDA-MB-231) clones in low-dose-rate cobalt-60 radiation therapy.

ObjectiveIn the present study, the authors investigated the in vitro behavior of radio-resistant breast adenocarcinoma (MDA-MB-231) cells line and radiosensitive peripheral blood mononuclear cells (PBMC), as a function of different radiation doses, dose rates and postirradiation time kinetics, with a view to the interest of clinical radiotherapy.Materials and MethodsThe cells were irradiated with Co-60, at 2 and 10 Gy and two different exposure rates, 339.56 cGy.min–1 and the other corresponding to one fourth of the standard dose rates, present over a 10-year period of cobalt therapy. Post-irradiation sampling was performed at pre-established kinetics of 24, 48 and 72 hours. The optical density response in viability assay was evaluated and a morphological analysis was performed.ResultsRadiosensitive PBMC showed decrease in viability at 2 Gy, and a more significant decrease at 10 Gy for both dose rates. MDAMB- 231 cells presented viability decrease only at higher dose and dose rate. The results showed MDA-MB-231 clone expansion at low dose rate after 48–72 hours post-radiation.ConclusionLow dose rate shows a possible potential clinical impact involving decrease in management of radio-resistant and radiosensitive tumor cell lines in cobalt therapy for breast cancer.

Low-dose γ-irradiation induces dual radio-adaptive responses depending on the post-irradiation time by altering microRNA expression profiles in normal human dermal fibroblasts

Exposure to high-dose ionizing radiation, including γ-radiation, induces severe skin disorders. However, the biological consequences and molecular mechanisms responsible for the response of human skin to low-dose γ-radiation (LDR) are largely unknown. In the present study, we demonstrate that LDR (0.1 Gy) induces distinct cellular responses in normal human dermal fibroblasts (NHDFs) depending on the post-irradiation time point. A MTT-based cell viability assay and propidium iodide staining-based cell cycle assay revealed that the viability and proportion of the cells in the G2/M phase were differed at 6 and 24 h post-irradiation. Reverse transcription quantitative PCR (RT-qPCR) revealed that LDR significantly upregulated the mRNA expression of collagen type I alpha 1 (COL1A1), but downregulated the mRNA expression of matrix metalloproteinase 1 (MMP1) at 24 h post-irradiation. MicroRNA (miRNA) microarray analysis further demonstrated that LDR induced changes in the expression profiles of specific miRNAs and that some of the deregulated miRNAs were specific to either the early or late radio-adaptive response. Our results suggest that LDR generates dual radio-adaptive responses depending on the post-irradiation time by altering specific miRNA expression profiles in NHDFs.

Melanin nanoparticles (MNPs) provide protection against whole-body ɣ-irradiation in mice via restoration of hematopoietic tissues.

During radiotherapy, ionizing irradiation interacts with biological systems to produce free radicals, which attack various cellular components. The hematopoietic system is easily recognized to be radiosensitive and its damage may be severe. Melanin nanoparticles (MNPs) act as free radical scavengers prepared by polymerization of dopamine. In this study, a total of 110 male BALB/C mice were divided into five equal groups. Each group contained 22 mice. Mice of group A did not receive MNPs or irradiation (control group), group B was injected intraperitoneally (i.p.) with 50mg/kg MNPs. Mice of group C and D were exposed to a dose of 7Gy ɣ-irradiation and injected with the same dose of MNPs as in group B either 30min pre- or post-irradiation, and group E was exposed to a dose of 7Gy ɣ-irradiation only. The impact of MNPs on peripheral blood, spleen, and DNA damage induced by irradiation was evaluated by blood count, histopathology of the spleen, and comet assay for the DNA in the bone marrow at 1, 4, 8, and 12days post-irradiation. Results of group E compared with control group (A) showed a significant depression in complete blood count. Additionally, histopathological observation showed the absence of megakaryocytes with delayed time post-irradiation, deposition of eosinophilic protein of their spleen appeared, as well as a remarkable decrease in spleen size was observed. Moreover, ɣ-irradiation-induced DNA damage as can be inferred from a significant increase by about 5-10 folds in all comet parameters (% of DNA, tail length, tail moment, and olive moment) in the DNA of the bone marrow. In contrast, pre-post treatment with MNPs protected hematopoietic tissues against radiation damage, and therefore, enhanced the survival of mice with 40% in groups (C&D) compared with 10% to group (E) till 30days post-irradiation. In conclusion, these results demonstrated that synthetic MNPs provide significant radioprotection to the hematopoietic tissues.

Post-irradiation induction time in the radiolytic synthesis of UO2 nanoparticles in aqueous solutions

The radiolytic growth process of UO2 nanoparticles in aqueous solutions through electron beam irradiation was investigated. The growth of these nanoparticles was carried out in the aqueous solutions containing different volume fractions of propan-2-ol. Different volume fractions of ethylene glycol along with 10% propan-2-ol in aqueous solutions were used for the investigation of viscosity effect. The growth of UO2 nanoparticles was found to follow a stochastic mechanism. Moreover, the growth of these nanoparticles is always found to be associated with a post-irradiation induction time. This induction time increases with the decrease in the propan-2-ol content and the increase in solvent viscosity, whereas it decreases with the increase in temperature as well as precursor concentration. It is therefore established that the radiolytic formation of UO2 nanoparticles is accompanied by a post-irradiation induction time, which strongly depends on the solvent composition, viscosity, temperature and precursor concentration.

A GSK-3β Inhibitor Protects Against Radiation Necrosis in Mouse Brain

To quantify the effectiveness of SB415286, a specific inhibitor of GSK-3β, as a neuroprotectant against radiation-induced central nervous system (brain) necrosis in a mouse model. Cohorts of mice were treated with SB415286 or dimethyl sulfoxide (DMSO) prior to irradiation with a single 45-Gy fraction targeted to the left hemisphere (brain) using a gamma knife machine. The onset and progression of radiation necrosis (RN) were monitored longitudinally by noninvasive in vivo small-animal magnetic resonance imaging (MRI) beginning 13 weeks postirradiation. MRI-derived necrotic volumes for SB415286- and DMSO-treated mice were compared. MRI results were supported by correlative histology. Mice treated with SB415286 showed significant protection from radiation-induced necrosis, as determined by in vivo MRI with histologic validation. MRI-derived necrotic volumes were significantly smaller at all postirradiation time points in SB415286-treated animals. Although the irradiated hemispheres of the DMSO-treated mice demonstrated many of the classic histologic features of RN, including fibrinoid vascular necrosis, vascular telangiectasia, hemorrhage, and tissue loss, the irradiated hemispheres of the SB415286-treated mice consistently showed only minimal tissue damage. These studies confirmed that treatment with a GSK-3β inhibitor dramatically reduced delayed time-to-onset necrosis in irradiated brain. The unilateral cerebral hemispheric stereotactic radiation surgery mouse model in concert with longitudinal MRI monitoring provided a powerful platform for studying the onset and progression of RN and for developing and testing new neuroprotectants. Effectiveness of SB415286 as a neuroprotectant against necrosis motivates potential clinical trials of it or other GSK-3β inhibitors.

Treatment-interval associated effect of irradiation on locoregionally-relapsed ovarian cancer.

Recurrent ovarian cancer following chemotherapy is usually incurable, particularly when the tumor acquires a drug resistance. The present study aimed to define the effect of irradiation on locoregional recurrences and the impact of the factors on the efficacy. The study retrospectively reviewed the clinical records of 61 patients with epithelial ovarian cancer who received irradiation following repeated chemotherapy between 1997 and 2006. A positive-irradiation response was designated as complete response, partial response, minor response or no change (NC). Due to the possible synergistic effect of chemotherapy and irradiation, and the cross-resistance to chemotherapeutic drugs and radiation, the focus was on the treatment break between chemotherapy and radiation, and patients were classified into 3 categories: Category I, ≤1 month; II, 1-6 months; and III, >6 months. The effect of irradiation was analyzed in association with histology, treatment break, recurrent site, irradiation dose and chemosensitivity. The post-irradiation survival time was analyzed by the irradiation response and treatment category. The median biological-effective dose was 60.0 Gy (range, 15.6-72.0 Gy). The sites irradiated included nodal recurrence (36), abdominal (six) and pelvic cavity (five cases). Histologically, serous adenocarcinoma was the most common type of the disease (23 cases) compared to mucinous (four), endometrioid (three), and clear-cell types (six cases). The median survival times were 4.5 months in the radiation responders (13 cases) and 15.3 months in the non-responders (37) (P=0.004). The positive-irradiation response was significantly associated with the treatment break (P=0.026) and chemosensitivity (P=0.007). In conclusion, irradiation for recurrent ovarian cancer produced an improved survival benefit when applied to chemoresponsive, locoregional-recurrent tumors immediately following chemotherapy.

SU-E-T-571: Microdosimetric Characterization of Proton Biological Effectiveness

Purpose: The knowledge of cell repair kinetics is critical in order to understand relevant aspects of proton therapy such optimal fractionation. High LET radiation induces larger proportions of more complex DSBs that might not be resolved in standard interfractional times (24h). We propose a method to characterize the cell repair kinetic at different positions within the spread-out Bragg peak (i.e. as a function of proton LETd) and use this information to model normal tissue complication probabilities (NTCP) as a function of LETd Methods: Repair kinetics curves will be obtained using U251-glioblastoma cells plating over microscope slides that are immersed in culture-medium that will be exposed along the axis of a proton-beam (10cm in range, 2cm modulation) of 1Gy. Culture is immersed within a solid-water block to place the distal edge of the proton-beam within the slide. gamma-H2AX foci along the slide in the beam axis is imaged with a microscope at different positions in order to correlate number of gamma-H2AX foci with dose and LET, where dose is obtained from a strip of gafchromic film placed on the side of the slide. Each slide is fixed at different time points. Results: We will present the correlation between an increase of LET and the increase of the gamma-H2AX along the depth within the proton beam, at different post-irradiation times and doses. Different fractions of unrepaired damage at 24h after irradiation will be presented at each depth of the proton beam, with larger fractions at the distal edge of the beam, where normal tissue exist. Larger values of NTCP are therefore observed at the distal edge. Conclusion: Slower repair kinetics is observed at the distal edge of a proton beam, and this study shows a method to obtain such information and the correlation between increased LET and increased NTCP. This research is supported by the Department of Radiation Oncology at the Hospital of the University of Pennsylvania as a pilot project.

Liver metabolomics reveals increased oxidative stress and fibrogenic potential in gfrp transgenic mice in response to ionizing radiation.

Although radiation-induced tissue-specific injury is well documented, the underlying molecular changes resulting in organ dysfunction and the consequences thereof on overall metabolism and physiology have not been elucidated. We previously reported the generation and characterization of a transgenic mouse strain that ubiquitously overexpresses Gfrp (GTPH-1 feedback regulatory protein) and exhibits higher oxidative stress, which is a possible result of decreased tetrahydrobiopterin (BH4) bioavailability. In this study, we report genotype-dependent changes in the metabolic profiles of liver tissue after exposure to nonlethal doses of ionizing radiation. Using a combination of untargeted and targeted quantitative mass spectrometry, we report significant accumulation of metabolites associated with oxidative stress, as well as the dysregulation of lipid metabolism in transgenic mice after radiation exposure. The radiation stress seems to exacerbate lipid peroxidation and also results in higher expression of genes that facilitate liver fibrosis, in a manner that is dependent on the genetic background and post-irradiation time interval. These findings suggest the significance of Gfrp in regulating redox homeostasis in response to stress induced by ionizing radiation affecting overall physiology.

Radiation-induced mitotic catastrophe in FANCD2 primary fibroblasts

Purpose: As the Fanconi anemia (FA) pathway is required for appropriate cell cycle progression through mitosis and the completion of cell division, the aim of the present study was to determine the destiny of FA cells after irradiation in vitro and to elucidate any difference in radiosensitivity between FA and control cells.Materials and methods: Analyses of phosphorylated histone H2AX (γ-H2AX) foci, micronuclei formation and cell cycle analysis were performed in unirradiated (0 min) and irradiated primary FA fibroblasts and in a control group at different post-irradiation times (30 min, 2 h, 5 h and 24 h).Results: The accumulation of γ-H2AX foci in irradiated FA fibroblasts was observed. At 24 h post-irradiation, 57% of FA cells were γ-H2AX foci-positive, significantly higher than in the control (p < 0.01). The cell cycle analysis has shown the transient G2/M arrest in irradiated FA fibroblasts. The portion of cells in the G2/M phase showed initial increase at 30 min post-irradiation and afterwards decreased over time reaching the pretreatment level 24 h after irradiation. Irradiated FA fibroblasts progressed to abnormal mitosis, as is shown by the production of cells with different nuclear morphologies from binucleated to multinucleated surrounded with micronuclei, and also by a high percentage of foci-positive micronuclei. The majority of radiation-induced micronuclei were γ-H2AX foci-positive, indicating that radiation-induced micronuclei contain fragments of damaged chromosomes. In contrast, in the control group, most of the micronuclei were classified as γ-H2AX foci-negative, which indicates that cells with unrepaired damage were blocked before entering mitosis.Conclusion: The results clearly indicate that mitotic catastrophe might be an important cell-death mechanism involved in the response of FA fibroblasts to ionizing radiation.

Primary and secondary clustering of DSB repair foci and repair kinetics compared for -rays, protons of different energies and high-LET 20Ne ions

Purpose: Ionizing radiations of different qualities (e.g. high-LET and low-LET) might differently interact with structurally and functionally distinct higher order chromatin domains (discussed in [ 1] and citations therein); this might be reflected by DNA double strand break (DSB) repair efficiency and the mechanism of how cancerogenous chromosomal translocations (CHT) form. Therefore, we compared the DSB repair kinetics and formation of γH2AX/p53BP1 repair clusters upon the action of γ-rays [ 2, 3], protons (15 and 30 MeV) [ 4], and 20Ne ions (preliminary data). Consequently, we discuss biological impacts of these clusters.Material and methods: Immunostaining methods in combination with high-resolution confocal microscopy, performed on 3D-fixed normal human skin fibroblasts [ 2– 4], were used to study initial distributions of γH2AX and p53BP1 repair foci and their changes during the post-irradiation (PI) time, with a special concern on foci clustering. Irradiations with γ-rays, protons of different energies (15 and 30 MeV), and high-LET 20Ne ions was performed in IBP ASCR Brno (CR), NPI AVCR Řež (CR) and JINR Dubna (Russia), respectively.Results: Upon irradiating cells with 20Ne ions, tracks of multiple clustered γH2AX and p53BP1 repair foci appeared immediately after the irradiation; these clusters, called here as the ‘primary clusters’, were rare in cells irradiated with γ-rays or protons (submitted). Though γH2AX/p53BP1 foci were positionally quite stable [ 2], ‘secondary clusters’ occasionally appeared after all kinds of irradiation during about 30 min PI. The formation of secondary clusters usually appeared due to the heterochromatin decondensation at the sites of heterochromatic DNA double-strand breaks (hcDSBs), followed by their protrusion into a limited space of nuclear subdomains of low density-chromatin (discussed in [ 1, 2, 5]).Conclusions: Primary clusters appear in cell nuclei immediately PI as the consequence of highly localized energy deposition, while secondary clusters develop during (and because of) DSB repair. Primary DSB clusters probably represent the main cause of chromosomal translocations induced with high-LET radiations while secondary clusters seem to be more important for low-LET γ-rays and protons. Secondary clusters of primary clusters (higher-order clusters) observed for 20Ne ions might explain frequent formation of complex translocations upon the action of high-LET radiations. Finally, we suggest [ 1, 2, 4] a model that describes the relationship between the higher order chromatin structure, DSB formation, repair and misrepair.