High Low Linear Energy Transfer Research Articles

Cumulative Dose from Recurrent CT Scans: Exploring the DNA Damage Response in Human Non-Transformed Cells.

Recurrent computed tomography (CT) examination has become a common diagnostic procedure for several diseases and injuries. Though each singular CT scan exposes individuals at low doses of low linear energy transfer (LET) radiation, the cumulative dose received from recurrent CT scans poses an increasing concern for potential health risks. Here, we evaluated the biological effects of recurrent CT scans on the DNA damage response (DDR) in human fibroblasts and retinal pigment epithelial cells maintained in culture for five months and subjected to four CT scans, one every four weeks. DDR kinetics and eventual accumulation of persistent-radiation-induced foci (P-RIF) were assessed by combined immunofluorescence for γH2AX and 53BP1, i.e., γH2AX/53BP1 foci. We found that CT scan repetitions significantly increased both the number and size of γH2AX/53BP1 foci. In particular, after the third CT scan, we observed the appearance of giant foci that might result from the overlapping of individual small foci and that do not associate with irreversible growth arrest, as shown by DNA replication in the foci-carrying cells. Whether these giant foci represent coalescence of unrepaired DNA damage as reported following single exposition to high doses of high LET radiation is still unclear. However, morphologically, these giant foci resemble the recently described compartmentalization of damaged DNA that should facilitate the repair of DNA double-strand breaks but also increase the risk of chromosomal translocations. Overall, these results indicate that for a correct evaluation of the damage following recurrent CT examinations, it is necessary to consider the size and composition of the foci in addition to their number.

Cumulative Dose from Recurrent CT Scans: Exploring the DNA Damage Response in Human Non-Transformed Cells

Recurrent computed tomography (CT) examination has become a common diagnostic procedure for several diseases and injuries. Though each singular CT scan exposes individuals at low doses of low linear energy transfer (LET) radiation, the cumulative dose received from recurrent CT scans poses an increasing concern for potential health risks. Here, we evaluated the biological effects of recurrent CT scans on the DNA damage response (DDR) in human fibroblasts and retinal pigment epithelial cells maintained in culture for five months and subjected to four CT scans, one every four weeks. DDR kinetics and eventual accumulation of persistent-radiation-induced foci (P-RIF) were assessed by combined immunofluorescence for γH2AX and 53BP1, i.e., γH2AX/53BP1 foci. We found that CT scan repetitions significantly increased both the number and size of γH2AX/53BP1 foci. In particular, after the third CT scan, we observed the appearance of giant foci that might result from the overlapping of individual small foci and that do not associate with irreversible growth arrest, as shown by DNA replication in the foci-carrying cells. Whether these giant foci represent coalescence of unrepaired DNA damage as reported following single exposition to high doses of high LET radiation is still unclear. However, morphologically, these giant foci resemble the recently described compartmentalization of damaged DNA that should facilitate the repair of DNA double-strand breaks but also increase the risk of chromosomal translocations. Overall, these results indicate that for a correct evaluation of the damage following recurrent CT examinations, it is necessary to consider the size and composition of the foci in addition to their number.

Mechanistic insights from high resolution DNA damage analysis to understand mixed radiation exposure

Cells exposed to densely ionising high and scattered low linear energy transfer (LET) radiation (50 % dose of each) react more strongly than to the same dose of each separately. The relationship between DNA double strand break location inside the nucleus and chromatin structure was evaluated, using high-resolution transmission electron microscopy (TEM) in breast cancer MDA-MB-231 cells at 30 min post 5 Gy. Additionally, response to high and/or low LET radiation was assessed using single (1 ×1.5 Gy) versus fractionated dose delivery (5 ×0.3 Gy). By TEM analysis, the highest total number of γH2AX nanobeads were found in cells irradiated with alpha radiation just prior to gamma radiation (called mixed beam), followed by alpha, then gamma radiation. γH2AX foci induced by mixed beam radiation tended to be surrounded by open chromatin (lighter TEM regions), yet foci containing the highest number of beads, i.e. larger foci representing complex damage, remained in the heterochromatic areas. The γH2AX large focus area was also greater in mixed beam-treated cells when analysed by immunofluorescence. Fractionated mixed beams given daily induced the strongest reduction in cell viability and colony formation in MDA-MB-231 and osteosarcoma U2OS cells compared to the other radiation qualities, as well as versus acute exposure. This may partially be explained by recurring low LET oxidative DNA damage by every fraction together with a delay in recompaction of chromatin after high LET, demonstrated by low levels of heterochromatin marker H3K9me3 at 2 h after the last mixed beam fraction in MDA-MB-231. In conclusion, early differences in response to complex DNA damage may lead to a stronger cell kill induced by fractionated exposure, which suggest a therapeutic potential of combined high and low LET irradiation.

The Inflection Point of Single Event Transient in SiGe HBT at a Cryogenic Temperature

Basing our findings on our previous pulsed laser testing results, we have experimentally demonstrated that there is an inflection point of a single event transient (SET) in the silicon-germanium heterojunction bipolar transistors (SiGe HBTs) with a decreasing temperature from +20 °C to −180 °C. Additionally, the changes in the parasitic resistivity of the carrier collection pathway due to incomplete ionization could play a key role. In this paper, we found that the incident-heavy ion’s parameters could also have an important impact on the SET inflection point by introducing the ion track structures generated by Geant4 simulation to the TCAD transient simulation. Heavy ion with a low linear energy transfer (LET) will not trigger the ion shunt effect of SiGe HBT and the inflection point will not occur until −200 °C. For high LET ions’ incidence, the high-density electron-hole pairs (EHPs) could significantly affect the parasitic resistivity on the pathway and lead to an earlier inflection point. The present results and methods could provide a new reference for the effective evaluation of single-event effects in bipolar transistors and circuits at cryogenic temperatures and provide new evidence of the SiGe technology’s potential for applications in extreme cryogenic environments.

Interaction of curcumin with glioblastoma cells via high and low linear energy transfer radiation therapy inducing radiosensitization effects

Glioblastoma is a deadly cancer tumor in the brain and has a survival rate of about 15 months. Despite the high mortality rate, temozolomide has proven to increase the survival rate of patients when combined with radiotherapy. However, its effects may be limited because some patients develop therapeutic resistance. Curcumin has proven to be a cancer treatment due to its broad anticancer spectrum, high efficiency and low toxic level. Additionally, curcumin significantly enhanced radiation efficacy under high and low Linear Energy Transfer (LET) radiation conditions in vitro. In combination with radiation, curcumin increased the cell population in the sub-G1 phase and the reactive oxygen species (ROS) level, ultimately increasing GBM cellular apoptosis. The radiosensitizing effects of curcumin are much higher in neutron (high LET)-irradiated cell lines than in γ (low LET)-irradiated cell lines. Curcumin plus neutron combination significantly inhibited cell invasion compared with that of single treatment or curcumin combined γ-ray treatment. Curcumin enhances the radiosensitivity of Glioblastoma (GBM), suggesting it may have clinical utility in combination cancer treatment with neutron high-LET radiation.

Radiation cancer risk at different dose rates: new dose-rate effectiveness factors derived from revised A-bomb radiation dosimetry data and non-tumor doses.

The dose rate of atomic bomb (A-bomb) radiation to the survivors has still remained unclear, although the dose–response data of A-bomb cancers has been taken as a standard in estimating the cancer risk of radiation and the dose and dose-rate effectiveness factor (DDREF). Since the applicability of the currently used DDREF of 2 derived from A-bomb data is limited in a narrow dose-rate range, 0.25-75 Gy/min as estimated from analysis of DS86 dosimetry data in the present study, a non-tumor dose (Dnt) was applied in an attempt to gain a more universal dose-rate effectiveness factor (DREF), where Dnt is an empirical parameter defined as the highest dose at which no statistically significant tumor increase is observed above the control level and its magnitude depends on the dose rate. The new DREF values were expressed as a function of the dose rate at four exposure categories, i.e. partial body low LET, whole body low linear energy transfer (LET), partial body high LET and whole body high LET and provided a value of 14 for environmental level radiation at a dose rate of 10−9 Gy/min for whole body low LET.

Gadolinium delivery agents for neutron capture therapy

<p indent=0mm>Neutron capture therapy (NCT) is a binary form of radiation therapy that combines neutron irradiation with a tumor targeting agent labeled with a stable isotope having a high thermal neutron capture cross section and yielding charged particle reaction products after neutron capture. At present, NCT has largely, but not exclusively, focused on the use of boron-10 as the target nuclide. Gadolinium neutron capture therapy (Gd-NCT) is an alternative and supplementary method of boron neutron capture therapy (BNCT). Gd-157, which has the largest cross-section (254000 barn) of thermal neutrons of all stable isotopes, is the most effective isotope in terms of neutron capture. The Gd neutron capture reaction, ¹⁵⁷Gd(n, γ)¹⁵⁸Gd, provokes complicated nuclear decay transitions that generate low linear energy transfer (LET) gamma photons and the emission of high LET conversion electrons and Auger electrons. Therefore, Gd is considered to be highly suitable for the absorption of thermal neutrons in the NCT reaction. Another advantage of Gd-NCT is Gd³⁺ has a longer retention time and can thus be tracked by means of magnetic resonance imaging (MRI) methods. Currently, Gd(III) based MRI contrast agents are most commonly used in clinical practice. However, due to lack of appropriate tumor-selective Gd agents, the Gd-NCT concept has not yet been clinically tested. For a successful neutron capture therapy, a target nuclide delivery system with high therapeutic efficiency and low adverse effects is crucial. Three major classes Gd delivery agents have been developed so far, small molecule chelates of Gd, nano- delivery agents, and Gd-B dual delivery agents. In the first trial of Gd-NCT, commercially available MRI contrast agents composed of Gd-chelating compounds were used as Gd delivery agents. However, due to the insufficient number of tumor cell nuclei incorporating Gd, efficacy of small molecule chelates of Gd as Gd-NCT delivery agents is generally low. In recent years, with the rapid development of nanotechnology, nanomaterials have been widely employed as carriers for cancer theranostics. A variety of Gd-based nanomaterials have been developed for pre-clinical trials of NCT. Majority of them are constructed by incorporating commercially available MRI contrast agents in biocompatible nanomaterials (liposomes, albumin, and micelles), or employing Gd based nanoparticles with their unique physiochemical properties. While nano-Gd delivery agents have proved to be a promising strategy in Gd-NCT, it is also possible to integrate of both Gd and B in one delivery agent. The design of many Gd-NCT agents to date has focused on combined BNCT and Gd-NCT agents in order to maximise cytotoxicity from the combined effects or to use the B component for NCT and the Gd component as a MRI imaging agent. This paper highlights the recent advances in Gd selective and efficient delivery methods and the preclinical outcome of Gd-NCT, both advantages and disadvantages of various Gd delivery agents were addressed. Finally, we offered our perspectives on the challenges and future development of Gd delivery agents for Gd-NCT.

Cellular Response to Proton Irradiation: A Simulation Study with TOPAS-nBio.

The cellular response to ionizing radiation continues to be of significant research interest in cancer radiotherapy, and DNA is recognized as the critical target for most of the biologic effects of radiation. Incident particles can cause initial DNA damages through physical and chemical interactions within a short time scale. Initial DNA damages can undergo repair via different pathways available at different stages of the cell cycle. The misrepair of DNA damage results in genomic rearrangement and causes mutations and chromosome aberrations, which are drivers of cell death. This work presents an integrated study of simulating cell response after proton irradiation with energies of 0.5-500 MeV (LET of 60-0.2 keV/µm). A model of a whole nucleus with fractal DNA geometry was implemented in TOPAS-nBio for initial DNA damage simulations. The default physics and chemistry models in TOPAS-nBio were used to describe interactions of primary particles, secondary particles, and radiolysis products within the nucleus. The initial DNA double-strand break (DSB) yield was found to increase from 6.5 DSB/Gy/Gbp at low-linear energy transfer (LET) of 0.2 keV/µm to 21.2 DSB/Gy/Gbp at high LET of 60 keV/µm. A mechanistic repair model was applied to predict the characteristics of DNA damage repair and dose response of chromosome aberrations. It was found that more than 95% of the DSBs are repaired within the first 24 h and the misrepaired DSB fraction increases rapidly with LET and reaches 15.8% at 60 keV/µm with an estimated chromosome aberration detection threshold of 3 Mbp. The dicentric and acentric fragment yields and the dose response of micronuclei formation after proton irradiation were calculated and compared with experimental results.

Validation and investigation of reactive species yields of Geant4‐DNA chemistry models

Indirect biological damage due to reactive species produced in water radiolysis reactions is responsible for the majority of biological effect for low linear energy transfer (LET) radiation. Modeling water radiolysis and the subsequent interactions of reactive species, as well as track structures, is essential to model radiobiology on the microscale. Recently, chemistry models have been developed for Geant4-DNA to be used in combination with the comprehensive existing physics models. In the current work, the first detailed, independent, in silico validation of all species yields with published experimental observations and comparison with other radiobiological simulations is presented. Additionally, the effect of LET of protons and heavier ions on reactive species yield in the model was examined, as well as the completeness of the chemical reactions following the radiolysis within the time after physical interactions simulated in the model. Yields over time of reactive species were simulated for water radiolysis by incident electrons, protons, alpha particles, and ions with various LETs using Geant4 and RITRACKS simulation tools. Water dissociation and recombination was simulated using Geant4 to determine the completeness of chemical reactions at the end of the simulation. Yield validation was performed by comparing yields simulated using Geant4 with experimental observations and other simulations. Validation was performed for all species for low LET radiation and the solvated electron and hydroxyl radical for high LET ions. It was found that the Geant4-DNA chemistry yields were generally in good agreement with experimental observations and other simulations. However, the Geant4-DNA yields for the hydroxyl radical and hydrogen peroxide at the end of the chemistry stage were found to be respectively considerably higher and lower than the experimentally observed yields. Increasing the LET of incident hadrons increased the yield of secondary species and decreased the yield of primary species. The effect of LET on the yield of the hydroxyl radical at 100ns simulated with Geant4 was in good agreement with experimental measurements. Additionally, by the end of the simulation only 40% of dissociated water molecules had been recombined and the rate of recombination was slowing. The yields simulated using Geant4 are within reasonable agreement with experimental observations. Higher LET radiation corresponds with increased yields of secondary species and decreased yields of primary species. These trends combined with the LET having similar effects on the 100ns hydroxyl radical yield for Geant4 and experimental measurements indicate that Geant4 accurately models the effect of LET on radiolysis yields. The limited recombination within the modeled chemistry stage and the slowing rate of recombination at the end of the stage indicate potential long-range indirect biological damage.

DNA Repair Deficient Chinese Hamster Ovary Cells Exhibiting Differential Sensitivity to Charged Particle Radiation under Aerobic and Hypoxic Conditions

It has been well established that hypoxia significantly increases both cellular and tumor resistance to ionizing radiation. Hypoxia associated radiation resistance has been known for some time but there has been limited success in sensitizing cells to radiation under hypoxic conditions. These studies show that, when irradiated with low linear energy transfer (LET) gamma-rays, poly (ADP-ribose), polymerase (PARP), Fanconi Anemia (FANC), and mutant Chinese Hamster Ovary (CHO) cells respond similarly to the non-homologous end joining (NHEJ) and the homologous recombination (HR) repair mutant CHO cells. Comparable results were observed in cells exposed to 13 keV/μm carbon ions. However, when irradiated with higher LET spread out Bragg peak (SOBP) carbon ions, we observed a decrease in the oxygen enhancement ratio (OER) in all the DNA of repair mutant cell lines. Interestingly, PARP mutant cells were observed as having the largest decrease in OER. Finally, these studies show a significant increase in the relative biological effectiveness (RBE) of high LET SOBP carbon and iron ions in HR and PARP mutants. There was also an increase in the RBE of NHEJ mutants when irradiated to SOBP carbon and iron ions. However, this increase was lower than in other mutant cell lines. These findings indicate that high LET radiation produces unique types of DNA damage under hypoxic conditions and PARP and HR repair pathways play a role in repairing this damage.

Increased Hematopoietic Stem Cells/Hematopoietic Progenitor Cells Measured as Endogenous Spleen Colonies in Radiation-Induced Adaptive Response in Mice (Yonezawa Effect).

The existence of radiation-induced adaptive response (AR) was reported in varied biosystems. In mice, the first in vivo AR model was established using X-rays as both the priming and the challenge doses and rescue of bone marrow death as the end point. The underlying mechanism was due to the priming radiation-induced resistance in the blood-forming tissues. In a series of investigations, we further demonstrated the existence of AR using different types of ionizing radiation (IR) including low linear energy transfer (LET) X-rays and high LET heavy ion. In this article, we validated hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) measured as endogenous colony-forming units-spleen (CFU-S) under AR inducible and uninducible conditions using combination of different types of IR. We confirmed the consistency of increased CFU-S number change with the AR inducible condition. These findings suggest that AR in mice induced by different types of IR would share at least in part a common underlying mechanism, the priming IR-induced resistance in the blood-forming tissues, which would lead to a protective effect on the HSCs/HPCs and play an important role in rescuing the animals from bone marrow death. These findings provide a new insight into the mechanistic study on AR in vivo.

Strategies to Enhance Radiosensitivity to Heavy Ion Radiation Therapy.

Heavy ion radiation therapy has been increasingly used due to several advantages over low linear energy transfer (LET) photon therapy, but further improvement of its therapeutic efficacy would be necessary. In this review, we summarize effective radiosensitizers for heavy ion radiation therapy and mechanisms associated with the radiosensitization. High LET heavy ions induce more complex and clustered DNA damage than low LET radiation. Inhibition of homologous recombimation repair or nonhomologous end rejoining and dysfunctional cell cycle checkpoint have been reported to sensitize cancer cells to heavy ions. Radiosenstizing agents, including DNA damage response inhibitors, Hsp90 inhibitors, histone acetylase inhibitors, and nanomaterials have been found to enhance cell killing by heavy ion irradiation through disrupted DNA damage response, cell cycle arrest, or other cellular processes. The use of these radiosensitizers could be a promising strategy to enhance the efficacy of heavy ion radiation therapy.

Experimental characterization of the dominant multiple nodes charge collection mechanism in metal oxide-semiconductor transistors

We propose an experimental method to investigate the dominant multiple node charge collection mechanism. A transistor array-based test structure is used to distinguish charge collection owing to the drift-diffusion and parasitic bipolar amplification effect. Heavy ion experimental results confirm that drift-diffusion dominates multiple node charge collection at low linear energy transfer (LET). However, the parasitic bipolar amplification effect dominates it at high LET. We also propose simple equations to determine the critical LET which may change the dominant multiple node charge collection mechanism. The calculated LET value is consistent with the heavy ion experimental results.

Very low doses of heavy oxygen ion radiation induce premature ovarian failure.

Astronauts are exposed to charged particles during space travel, and charged particles are also used for cancer radiotherapy. Premature ovarian failure is a well-known side effect of conventional, low linear energy transfer (LET) cancer radiotherapy, but little is known about the effects of high LET charged particles on the ovary. We hypothesized that lower LET (16.5 keV/µm) oxygen particles would be less damaging to the ovary than we previously found for iron (LET = 179 keV/µm). Adult female mice were irradiated with 0, 5, 30 or 50 cGy oxygen ions or 50 cGy oxygen plus dietary supplementation with the antioxidant alpha lipoic acid (ALA). Six-hour after irradiation, percentages of ovarian follicles immunopositive for γH2AX, a marker of DNA double strand breaks, 4-HNE, a marker of oxidative lipid damage and BBC3 (PUMA), a proapoptotic BCL-2 family protein, were dose dependently increased in irradiated mice compared to controls. One week after irradiation, numbers of primordial, primary and secondary follicles per ovary were dose dependently decreased, with complete absence of follicles in the 50 cGy groups. The ED50 for primordial follicle destruction was 4.6 cGy for oxygen compared to 27.5 cGy for iron in our previous study. Serum FSH and LH concentrations were significantly elevated in 50 cGy groups at 8 week. Supplementation with ALA mitigated the early effects, but not the ultimate depletion of ovarian follicles. In conclusion, oxygen charged particles are even more potent inducers of ovarian follicle depletion than charged iron particles, raising concern for premature ovarian failure in astronauts exposed to both particles during space travel.

TAS-116, a Novel Hsp90 Inhibitor, Selectively Enhances Radiosensitivity of Human Cancer Cells to X-rays and Carbon Ion Radiation.

Hsp90 inhibitors have been investigated as cancer therapeutics in monotherapy and to augment radiotherapy; however, serious adverse effects of early-generation Hsp90 inhibitors limited their development. TAS-116 is a novel Hsp90 inhibitor with lower adverse effects than other Hsp90 inhibitors, and here, we investigated the radiosensitizing effects of TAS-116 in low linear energy transfer (LET) X-ray and high LET carbon ion-irradiated human cancer cells and mouse tumor xenografts. TAS-116 decreased cell survival of both X-ray and carbon ion-irradiated human cancer cell lines (HeLa and H1299 cells), and similar to other Hsp90 inhibitors, it did not affect radiosensitivity of noncancerous human fibroblasts. TAS-116 increased the number of radiation-induced γ-H2AX foci and delayed the repair of DNA double-strand breaks (DSB). TAS-116 reduced the expression of proteins that mediate repair of DSBs by homologous recombination (RAD51) and nonhomologous end joining (Ku, DNA-PKcs), and suppressed formation of RAD51 foci and phosphorylation/activation of DNA-PKcs. TAS-116 also decreased expression of the cdc25 cell-cycle progression marker, markedly increasing G2-M arrest. Combined treatment of mouse tumor xenografts with carbon ions and TAS-116 showed promising delay in tumor growth compared with either individual treatment. These results demonstrate that TAS-116 radiosensitizes human cancer cells to both X-rays and carbon ions by inhibiting the two major DSB repair pathways, and these effects were accompanied by marked cell-cycle arrest. The promising results of combination TAS-116 + carbon ion radiotherapy of tumor xenografts justify further exploration of TAS-116 as an adjunct to radiotherapy using low or high LET radiation. Mol Cancer Ther; 16(1); 16-24. ©2016 AACR.

New Data and Modelling for Single Event Effects in the Stratospheric Radiation Environment

The upper atmosphere is a transition region between the neutron-dominated aviation environment and satellite environment where primary protons and ions dominate. We report high altitude balloon measurements and model results characterising this radiation environment for single event effects (SEE) in avionics. Our data, from the RaySure solid-state radiation monitor, reveal markedly different altitude profiles for low linear energy transfer (LET) and high LET energy depositions. We use models to show that the difference is caused by the influence of primary cosmic ray particles, which induce counts in RaySure via both direct and indirect ionization. Using the new Model of Atmospheric Ionizing Radiation Effects (MAIRE), we use particle fluxes and LET spectra to calculate single event upset (SEU) rates as a function of altitude from ground level to the edge of space at 100 km altitude. The results have implications for a variety of applications including high altitude space tourism flights, UAVs and missions to the Martian surface.

Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis.

Telomeres are nucleoprotein complexes comprised of tandem arrays of repetitive DNA sequence that serve to protect chromosomal termini from inappropriate degradation, as well as to prevent these natural DNA ends from being recognized as broken DNA (double-strand breaks) and triggering of inappropriate DNA damage responses. Preservation of telomere length requires telomerase, the specialized reverse transcriptase capable of maintaining telomere length via template-mediated addition of telomeric repeats onto the ends of newly synthesized chromosomes. Loss of either end-capping function or telomere length maintenance has been associated with genomic instability or senescence in a variety of settings; therefore, telomeres and telomerase have well-established connections to cancer and aging. It has long been recognized that oxidative stress promotes shortening of telomeres, and that telomerase activity is a radiation-inducible function. However, the effects of ionizing radiation (IR) exposure on telomeres per se are much less well understood and appreciated. To gain a deeper understanding of the roles, telomeres and telomerase play in the response of human cells to IRs of different qualities, we tracked changes in telomeric end-capping function, telomere length, and telomerase activity in panels of mammary epithelial and hematopoietic cell lines exposed to low linear energy transfer (LET) gamma(γ)-rays or high LET, high charge, high energy (HZE) particles, delivered either acutely or at low dose rates. In addition to demonstrating that dysfunctional telomeres contribute to IR-induced mutation frequencies and genome instability, we reveal non-canonical roles for telomerase, in that telomerase activity was required for IR-induced enrichment of mammary epithelial putative stem/progenitor cell populations, a finding also suggestive of cellular reprograming. Taken together, the results reported here establish the critical importance of telomeres and telomerase in the radiation response and, as such, have compelling implications not only for accelerated tumor repopulation following radiation therapy but also for carcinogenic potential following low dose exposures as well, including those of relevance to spaceflight-associated galactic cosmic radiations.

Nanoscopic exclusion between Rad51 and 53BP1 after ion irradiation in human HeLa cells

Many proteins involved in detection, signalling and repair of DNA double-strand breaks (DSB) accumulate in large number in the vicinity of DSB sites, forming so called foci. Emerging evidence suggests that these foci are sub-divided in structural or functional domains. We use stimulated emission depletion (STED) microscopy to investigate localization of mediator protein 53BP1 and recombination factor Rad51 after irradiation of cells with low linear energy transfer (LET) protons or high LET carbon ions. With a resolution better than 100 nm, STED microscopy and image analysis using a newly developed analyzing algorithm, the reduced product of the differences from the mean, allowed us to demonstrate that with both irradiation types Rad51 occupies spherical regions of about 200 nm diameter. These foci locate within larger 53BP1 accumulations in regions of local 53BP1 depletion, similar to what has been described for the localization of Brca1, CtIP and RPA. Furthermore, localization relative to 53BP1 and size of Rad51 foci was not different after irradiation with low and high LET radiation. As expected, 53BP1 foci induced by low LET irradiation mostly contained one Rad51 focal structure, while after high LET irradiation, most foci contained >1 Rad51 accumulation.

Hyperthermia-induced radiosensitization in CHO wild-type, NHEJ repair mutant and HR repair mutant following proton and carbon-ion exposure.

The DNA repair mechanisms involved in hyperthermia-induced radiosensitization with proton and carbon ion radiation exposure were investigated in the present study. In a previous study, Chinese hamster ovary (CHO) cells were exposed to low linear energy transfer (LET) photon radiation. These cells can be sensitized by hyperthermia as a result of inhibition of homologous recombination (HR) repair. The present study used wild-type, non-homologous end joining (NHEJ) and HR repair-deficient CHO cells to define the contributions of each repair pathway to cellular lethality following hyperthermia-induced hadron radiation sensitization. The cells were exposed to ionizing radiation, followed by hyperthermia treatment (42.5°C for 1 h). Hyperthermia-induced radiosensitization was determined by the colony formation assay and thermal enhancement ratio. HR repair-deficient cells exhibited no hyper-sensitization to X-rays, protons, or low and high LET carbon ions when combined with hyperthermia. Wild-type and NHEJ repair-deficient cells exhibited significant hyperthermia-induced sensitization to low LET photon and hadron radiation. Hyperthermia-induced sensitization to high LET carbon-ion radiation was less than at low LET radiation. Relative biological effectiveness (RBE) between radiation alone and radiation combined with hyperthermia cell groups was not significantly different in any of the cell lines, with the exception of wild-type cells exposed to high LET radiation, which exhibited a lower RBE in the combined group. The present study investigated additional cell lines to confirm the lower RBE observed in DNA repair-deficient cell lines. These findings suggested that hyperthermia-induced hyper-sensitization to hadron radiation is also dependent on inhibition of HR repair, as was observed with photon radiation in a previous study.

Space Environmental Factor Impacts upon Murine Colon Microbiota and Mucosal Homeostasis.

Astronaut intestinal health may be impacted by microgravity, radiation, and diet. The aim of this study was to characterize how high and low linear energy transfer (LET) radiation, microgravity, and elevated dietary iron affect colon microbiota (determined by 16S rDNA pyrosequencing) and colon function. Three independent experiments were conducted to achieve these goals: 1) fractionated low LET γ radiation (137Cs, 3 Gy, RAD), high Fe diet (IRON) (650 mg/kg diet), and a combination of low LET γ radiation and high Fe diet (IRON+RAD) in male Sprague-Dawley rats; 2) high LET 38Si particle exposure (0.050 Gy), 1/6 G partial weight bearing (PWB), and a combination of high LET38Si particle exposure and PWB in female BalbC/ByJ mice; and 3) 13 d spaceflight in female C57BL/6 mice. Low LET radiation, IRON and spaceflight increased Bacteroidetes and decreased Firmicutes. RAD and IRON+RAD increased Lactobacillales and lowered Clostridiales compared to the control (CON) and IRON treatments. Low LET radiation, IRON, and spaceflight did not significantly affect diversity or richness, or elevate pathogenic genera. Spaceflight increased Clostridiales and decreased Lactobacillales, and similar trends were observed in the experiment using a ground-based model of microgravity, suggesting altered gravity may affect colonic microbiota. Although we noted no differences in colon epithelial injury or inflammation, spaceflight elevated TGFβ gene expression. Microbiota and mucosal characterization in these models is a first step in understanding the impact of the space environment on intestinal health.