Quantitative modeling of carcinogenesis induced by single beams or mixtures of space radiations using targeted and non-targeted effects.

Ionizing radiations encountered by astronauts on deep space missions produce biological damage by two main mechanisms: (1) Targeted effects (TE) due to direct traversals of cells by ionizing tracks. (2) Non-targeted effects (NTE) caused by release of signals from directly hit cells. The combination of these mechanisms generates non-linear dose response shapes, which need to be modeled quantitatively to predict health risks from space exploration. Here we used a TE + NTE model to analyze data on APC(1638N/+) mouse tumorigenesis induced by space-relevant doses of protons, 4He, 12C, 16O, 28Si or 56Fe ions, or γ rays. A customized weighted Negative Binomial distribution was used to describe the radiation type- and dose-dependent data variability. This approach allowed detailed quantification of dose-response shapes, NTE- and TE-related model parameters, and radiation quality metrics (relative biological effectiveness, RBE, and radiation effects ratio, RER, relative to γ rays) for each radiation type. Based on the modeled responses for each radiation type, we predicted the tumor yield for a Mars-mission-relevant mixture of these radiations, using the recently-developed incremental effect additivity (IEA) synergy theory. The proposed modeling approach can enhance current knowledge about quantification of space radiation quality effects, dose response shapes, and ultimately the health risks for astronauts.

Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy.

BACKGROUND: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. MATERIAL AND METHODS: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). RESULTS: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. CONCLUSION: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

Relative Role of Physical Mechanisms on Complex Biodamage Induced by Carbon Irradiation.

The effective use of swift ion beams in cancer treatment (known as hadrontherapy) as well as appropriate protection in manned space missions rely on the accurate understanding of the energy delivery to cells that damages their genetic information. The key ingredient characterizing the response of a medium to the perturbation induced by charged particles is its electronic excitation spectrum. By using linear-response time-dependent density functional theory, we obtained the energy and momentum transfer excitation spectrum (the energy-loss function, ELF) of liquid water (the main constituent of biological tissues), which was in excellent agreement with experimental data. The inelastic scattering cross sections obtained from this ELF, together with the elastic scattering cross sections derived by considering the condensed phase nature of the medium, were used to perform accurate Monte Carlo simulations of the energy deposited by swift carbon ions in liquid water and carried away by the generated secondary electrons, producing inelastic events such as ionization, excitation, and dissociative electron attachment (DEA). The latter are strongly correlated with cellular death, which is scored in sensitive volumes with the size of two DNA convolutions. The sizes of the clusters of damaging events for a wide range of carbon-ion energies, from those relevant to hadrontherapy up to those for cosmic radiation, predict with unprecedented statistical accuracy the nature and relative magnitude of the main inelastic processes contributing to radiation biodamage, confirming that ionization accounts for the vast majority of complex damage. DEA, typically regarded as a very relevant biodamage mechanism, surprisingly plays a minor role in carbon-ion induced clusters of harmful events.

Design and dosimetry of a facility to study health effects following exposures to fission neutrons at low dose rates for long durations.

PURPOSE: During extended missions into deep space, astronauts will be exposed to a complex radiation field that includes high linear energy transfer (LET) radiation from high energy, heavy ions (HZE particles) at low dose rates of about 0.5 mGy/d for long durations. About 20% of the dose is delivered by ions with LET greater than 10 keV/µm. There are sparse empirical data in any species for carcinogenic effects from whole-body exposures to external sources of mixed or high LET radiation at this level of dose rates. For the induction of solid tumors, acute exposures to HZE ions have been shown to be substantially more effective per unit dose than low LET exposures associated with photons. To determine the health effects of high LET radiation at space-relevant dose rates on experimental animals, we developed a vivarium in which rodents could be irradiated with Californium (252Cf) neutrons for protracted periods of time. MATERIALS AND METHODS: The neutron source is a panoramic irradiator containing 252Cf located in a concrete shielded vault with a footprint of 53 m2. The vault can accommodate sufficient caging to simultaneously irradiate 900 mice and 60 rats for durations up to 400 d at a dose rate of 1 mGy/d and is approved for extended animal husbandry. RESULTS: The mixed field fluence is a combination of neutrons and photons emitted directly from the source and scattered particles from the concrete walls and floor. Mixed field dosimetry was performed using a miniature GM counter and CaF2:Dy thermoluminescent dosimeters (TLD) for photons and tissue-equivalent proportional counters (TEPC) for neutrons. TEPC data provided macroscopic dose rates as well as measurements of radiation quality based on lineal energy, y, and LET. The instantaneous dose rate from the source decreases with a half-life of 2.6 years. The exposure time is adjusted weekly to yield a total dose 1 mGy/d. The photon contribution is 20% of the total dose. The uncertainty in the delivered dose is estimated to be ±20% taking into account spatial variations in the room and random position of mice in each cage. The dose averaged LET for the charged particle recoil nuclei is 68 keV/µ. CONCLUSIONS: We have developed a facility to perform high LET studies in mice and rats at space relevant dose rates and career-relevant doses using neutrons emitted from the spontaneous fission of 252Cf.

Effects of low dose and low dose rate low linear energy transfer radiation on animals - review of recent studies relevant for carcinogenesis.

Purpose: Carcinogenic effects of radiation are often assumed to be universally understood, more often than, for example, carcinogenic effects of many different chemicals. This in turn leads to an assumption that any dose of radiation, delivered at any dose rate, poses a serious health challenge. This remains an issue of dispute and low dose radiation research is focused on understanding whether these exposures contribute to cancer incidence. This review is focused on the low linear energy transfer (low LET) radiation exposures for which the data is the most abundant in recent years. Materials and methods: Review of the literature between 2008 and today, highlighting some of the most diverse studies in low dose research. Results: Low dose and low dose rate, low LET ionizing radiation animal studies suggest that the effects of exposure very much depend on animal genotype and health status.Conclusions: Only the integration of all of the data from different models and studies will lead to a fuller understanding of low dose radiation effects. Therefore, we hope to see an increase in international archival efforts and exchange of raw data information opening the possibilities for new types of meta analyses.

A Computer Modeling Study of Water Radiolysis at High Dose Rates. Relevance to FLASH Radiotherapy.

"FLASH radiotherapy" is a new method of radiation treatment by which large doses of radiation are delivered at high dose rates to tumors almost instantaneously (a few milliseconds), paradoxically sparing healthy tissue while preserving anti-tumor activity. To date, no definitive mechanism has been proposed to explain the different responses of the tumor and normal tissue to radiation. As a first step, and given that living cells and tissues consist mainly of water, we studied the effects of high dose rates on the transient yields (G values) of the radical and molecular species formed in the radiolysis of deaerated/aerated water by irradiating protons, using Monte Carlo simulations. Our simulation model consisted of two steps: 1. The random irradiation of a right circular cylindrical volume of water, embedded in nonirradiated bulk water, with single and instantaneous pulses of N 300-MeV incident protons ("linear energy transfer" or LET ∼ 0.3 keV/µm) traveling along the axis of the cylinder; and 2. The development of these N proton tracks, which were initially contained in the irradiated cylinder, throughout the solution over time. The effect of dose rate was studied by varying N, which was calibrated in terms of dose rate. For this, experimental data on the yield G(Fe3+) of the super-Fricke dosimeter as a function of dose rate up to ∼1010 Gy/s were used. Confirming previous experimental and theoretical studies, significant changes in product yields were found to occur with increasing dose rate, with lower radical and higher molecular yields, which result from an increase in the radical density in the bulk of the solution. Using the kinetics of the decay of hydrated electrons, a critical time (τc), which corresponds to the "onset" of dose-rate effects, was determined for each value of N. For the cylindrical irradiation model, τc was inversely proportional to the dose rate. Moreover, the comparison with experiments with pulsed electrons underlined the importance of the geometry of the irradiation volume for the estimation of τc. Finally, in the case of aerated water radiolysis, we calculated the yield of oxygen consumption and estimated the corresponding concentration of consumed (depleted) oxygen as a function of time and dose rate. It was shown that this concentration increases substantially with increasing dose rate in the time window ∼1 ns-10 µs, with a very pronounced maximum around 0.2 µs. For high-dose-rate irradiations (>109 Gy/s), a large part of the available oxygen (∼0.25 mM for an air-saturated solution) was found to be consumed. This result, which was obtained on a purely water radiation chemistry basis, strongly supports the hypothesis that the normal tissue-sparing effect of FLASH stems from temporary hypoxia due to oxygen depletion induced by high-dose-rate irradiation.

Differences in Linear Energy Transfer Affect Cell-killing and Radiosensitizing Effects of Spread-out Carbon-ion Beams.

BACKGROUND/AIM: The cell-killing and radiosensitizing effects of carbon-ion (C-ion) beams with low linear energy transfer (LET) are underexplored. We aimed to demonstrate the cell-killing effects of 60Co gamma rays and C-ion beams at various LET values and the radiosensitizing effect of C-ion beams at various LET and cisplatin levels. MATERIALS AND METHODS: Human uterine cervical cancer cells were irradiated with 60Co gamma rays and C-ion beams at different levels of LET, with and without cisplatin treatment. RESULTS: Low-LET C-ion beams had a superior cell-killing effect compared to 60Co gamma rays. Survival curves under low-LET C-ion beams were more similar to that of 60Co gamma rays than that of high-LET C-ion beams. Cisplatin significantly reduced cell survival after 1, 2, and 3 Gy C-ion beam irradiations at LET values of 13/30/70 keV/µm, 13/30 keV/µm, and 13 keV/µm, respectively. CONCLUSION: Low-LET C-ion beams combined with cisplatin have higher radiosensitizing effects than high-LET C-ion beams.

A radiobiological study of carbon ions of different linear energy transfer in resistant human malignant cell lines.

PURPOSE: Analysis of elimination of four human radioresistant malignant cell lines to mono-energetic and non mono-energetic incoming carbon ion beams, characterized by different linear energy transfer (LET) qualities is performed. Comparisons with protons from the middle of the therapeutic spread out Bragg peak (SOBP) and reference γ-rays are also included. MATERIALS AND METHODS: HTB140 cells were irradiated at five positions, with different LET, along the 62 MeV carbon pristine Bragg peak. To provide reliable reproducibility of irradiations at INFN-LNS, as the carbon Bragg peak is very narrow, precise positioning of samples for desired LET value is complicated. The peak was slightly widened using two ripple filters. After defining irradiation position and LET at the peak itself where cell killing is almost the highest, irradiation position with the same LET value was found within somewhat broadened peak. HTB140, MCF-7, HTB177 and CRL5876 cells were irradiated at the two described positions. Additionally, irradiations in the middle of 62 MeV proton SOBP and reference γ-rays were performed. Doses ranged from 0.5 to 16 Gy. Cell survival and corresponding radiobiological parameters were assessed seven days after irradiations. RESULTS: When moving irradiation position along the carbon Bragg curve, LET rises from 85 to 747 keV/µm, while surviving fraction at 2 Gy (SF2) for HTB140 cells, falls from 0.72 to 0.57 further rising to 0.73 on the distal fall-off part of the curve. Improved cell radiosensitivity is seen for the doses below 4 Gy. Relative biological effectiveness (RBE) increases from 4.56 to 7.69 and drops to 4.23. Almost the highest cell killing LET, being ∼200 keV/µm, is used to irradiate HTB140, MCF-7, HTB177 and CRL5876 cells within the pristine and slightly broadened Bragg peak. After irradiations with protons of the mid SOBP, carbon ions of the pristine and slightly widened Bragg peak RBE ranges for HTB140 cells from 2.08, 4.81 to 7.06, for MCF-7 from 1.70, 3.28 to 4.17, for HTB177 from 1.98, 4.18 to 5.08 and for CRL5876 from 1.33, 2.57 to 3.51. CONCLUSIONS: Significant elimination of HTB140 cells is observed along the carbon Bragg curve. The highest one is achieved by LET that is at the level of already reported. For the same LET, mono-energetic carbon ions provide higher cell elimination than the non mono-energetic. For all cell lines, both carbon ion beams, more the monoenergetic one, express stronger killing rate than protons and especially γ-rays.

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.

The effect of hypoxia on the induction of strand breaks in plasmid DNA by alpha-, beta- and Auger electron-emitters 223Ra, 188Re, 99mTc and DNA-binding 99mTc-labeled pyrene.

INTRODUCTION: Radiation-induced DNA damage occurs from direct and indirect effects. The induction is influenced by the physical characteristics of the radionuclide, especially its linear energy transfer. Hypoxia reduces the effect of irradiation treatment in tumor cells and leads to poor patient outcomes. High linear energy transfer emitters can overcome this obstacle. Our aim is to demonstrate the influence of hypoxia on the interaction of different radiation qualities with isolated DNA. METHODS: PuC19 Plasmid DNA was irradiated with 223Ra, 188Re, 99mTc and 99mTc-labeled pyrene with and without DMSO under hypoxia or normoxic conditions. DNA damages in form of single-(SSB) and double-strand breaks (DSB) were analyzed by gel electrophoresis. RESULTS: Radiation doses up to 200 Gy of 223Ra, 188Re and 99mTc led to maximal yields of 80% SSB and 30%, 28% and 32% DSB, respectively. Hypoxia had minor effects on damages from 223Ra, but caused a small enhancement in DSB for 188Re and 99mTc. DMSO prevented DSB completely and reduced SSB from the "free" radionuclides to comparable levels. DNA-binding 99mTc-labeled pyrene induced less SSB and DSB compared to [99mTc]TcO4-. However, the incubation with DMSO could prevent the SSB and DSB induction only to a minor extent. CONCLUSIONS: Hypoxia does not limit DNA damage induced by 223Ra, 188Re, 99mTc and 99mTc-labeled pyrene. Dose-dependent radiation effects were comparable for alpha-emitters and both high- and low-energy electron emitters. The radioprotection by DMSO was not influenced by hypoxia. The results indicate the contribution of mainly indirect radiation effects for 99mTc, 188Re and 223Ra. 99mTc-labeled pyrene caused direct DNA damages and Auger-electrons from 99mTc-labeled pyrene are more effective than high-energy electrons or alpha particles. ADVANCES IN KNOWLEDGE: Without the consideration of DNA repair mechanisms, oxygen has no direct influence in radiation-induced DNA damages by different radiation qualities. IMPLICATIONS FOR PATIENT CARE: The short-time stimulation with oxygen during patient radiation could have minor influence compared to constant oxygen flooding to overcome hypoxic barriers.

Influence of a perpendicular magnetic field on biological effectiveness of carbon-ion beams.

Purpose: Our previous study revealed that the application of a magnetic field longitudinal to a carbon-ion beam of 0.1 ≤ B//≤ 0.6 T enhances the biological effectiveness of the radiation. The purpose of this study is to experimentally verify whether the application of a magnetic field perpendicular to the beam also alters the biological effectiveness. Methods and materials: Most experimental conditions other than the magnetic field direction were the same as those used in the previous study to allow comparison of their results. Human cancer and normal cells were exposed to low (12 keV/µm) and high (50 keV/µm) linear energy transfer (LET) carbon-ion beams under the perpendicular magnetic fields of B⊥ = 0, 0.15, 0.3, or 0.6 T generated by a dipole magnet. The effects of the magnetic fields on the biological effectiveness were evaluated by clonogenic cell survival. Doses that would result in the survival of 10%, D10s, were determined for the exposures and analyzed using Student's t-tests. Results: For both cancer and normal cells treated by low- and high-LET carbon-ion beams, the D10s measured in the presence of the perpendicular magnetic fields of B⊥ ≥ 0.15 T were not statistically different (p ≫ .05) from the D10s measured in the absence of the magnetic fields, B⊥ = 0 T. Conclusions: Exposure of human cancer and normal cells to the perpendicular magnetic fields of B⊥ ≤ 0.6 T did not alter significantly the biological effectiveness of the carbon-ion beams, unlike the exposure to longitudinal magnetic fields of the same strength. Although the mechanisms underlying the observed results still require further exploration, these findings indicate that the influence of the magnetic field on biological effectiveness of the carbon-ion beam depends on the applied field direction with respect to the beam.

The radiobiological effects of He, C and Ne ions as a function of LET on various glioblastoma cell lines.

The effects of the charged ion species 4He, 12C and 20Ne on glioblastoma multiforme (GBM) T98G, U87 and LN18 cell lines were compared with the effects of 200 kVp X-rays (1.7 keV/µm). These cell lines have different genetic profiles. Individual GBM relative biological effectiveness (RBE) was estimated in two ways: the RBE10 at 10% survival fraction and the RBE2Gy after 2 Gy doses. The linear quadratic model radiosensitivity parameters α and ß and the α/ß ratio of each ion type were determined as a function of LET. Mono-energetic 4He, 12C and 20Ne ions were generated by the Heavy Ion Medical Accelerator at the National Institute of Radiological Sciences in Chiba, Japan. Colony-formation assays were used to evaluate the survival fractions. The LET of the various ions used ranged from 2.3 to 100 keV/µm (covering the depth-dose plateau region to clinically relevant LET at the Bragg peak). For U87 and LN18, the RBE10 increased with LET and peaked at 85 keV/µm, whereas T98G peaked at 100 keV/µm. All three GBM α parameters peaked at 100 keV/µm. There is a statistically significant difference between the three GBM RBE10 values, except at 100 keV/µm (P < 0.01), and a statistically significant difference between the α values of the GBM cell lines, except at 85 and 100 keV/µm. The biological response varied depending on the GBM cell lines and on the ions used.

Enhancement of biological effectiveness of carbon-ion beams by applying a longitudinal magnetic field.

Purpose: A magnetic field longitudinal to an ion beam will potentially affect the biological effectiveness of the radiation. The purpose of this study is to experimentally verify the significance of such effects. Methods and materials: Human cancer and normal cell lines were exposed to low (12 keV/µm) and high (50 keV/µm) linear energy transfer (LET) carbon-ion beams under the longitudinal magnetic fields of B// = 0, 0.1, 0.2, 0.3, or 0.6 T generated by a solenoid magnet. The effects of the magnetic fields on the biological effectiveness were evaluated by clonogenic cell survival. Doses that would result in a survival fraction of 10% (D10s) were determined for each cell line and magnetic field. Results: For cancer cells exposed to the low (high)-LET beams, D10 decreased from 5.2 (3.1) Gy at 0 T to 4.3 (2.4) Gy at 0.1 T, while no further decrease in D10 was observed for higher magnetic fields. For normal cells, decreases in D10 of comparable magnitudes were observed by applying the magnetic fields. Conclusions: Significant decreases in D10, i.e. significant enhancements of the biological effectiveness, were observed in both cancer and normal cells by applying longitudinal magnetic fields of B// ≥ 0.1 T. These effects were enhanced with LET. Further studies are required to figure out the mechanism underlying the observed results.

Relating Linear Energy Transfer to the Formation and Resolution of DNA Repair Foci After Irradiation with Equal Doses of X-ray Photons, Plateau, or Bragg-Peak Protons.

Proton beam therapy is increasingly applied for the treatment of human cancer, as it promises to reduce normal tissue damage. However, little is known about the relationship between linear energy transfer (LET), the type of DNA damage, and cellular repair mechanisms, particularly for cells irradiated with protons. We irradiated cultured cells delivering equal doses of X-ray photons, Bragg-peak protons, or plateau protons and used this set-up to quantitate initial DNA damage (mainly DNA double strand breaks (DSBs)), and to analyze kinetics of repair by detecting γH2A.X or 53BP1 using immunofluorescence. The results obtained validate the reliability of our set-up in delivering equal radiation doses under all conditions employed. Although the initial numbers of γH2A.X and 53BP1 foci scored were similar under the different irradiation conditions, it was notable that the maximum foci level was reached at 60 min after irradiation with Bragg-peak protons, as compared to 30 min for plateau protons and photons. Interestingly, Bragg-peak protons induced larger and irregularly shaped γH2A.X and 53BP1 foci. Additionally, the resolution of these foci was delayed. These results suggest that Bragg-peak protons induce DNA damage of increased complexity which is difficult to process by the cellular repair apparatus.

Monte Carlo Simulation of the Oxygen Effect in DNA Damage Induction by Ionizing Radiation.

DNA damage induced by ionizing radiation exposure is enhanced in the presence of oxygen (the "oxygen effect"). Despite its practical importance in radiotherapy, the oxygen effect has largely been excluded from models that predict DNA damage from radiation tracks. A Monte Carlo-based algorithm was developed in MATLAB software to predict DNA damage from physical and chemical tracks through a cell nucleus simulated in Geant4-DNA, taking into account the effects of cellular oxygenation (pO2) on DNA radical chemistry processes. An initial spatial distribution of DNA base and sugar radicals was determined by spatially clustering direct events (that deposited at least 10.79 eV) and hydroxyl radical (•OH) interactions. The oxygen effect was modeled by increasing the efficiency with which sugar radicals from direct-type effects were converted to strand breaks from 0.6 to 1, the efficiency with which sugar radicals from the indirect effect were converted to strand breaks from 0.28 to 1 and the efficiency of base-to-sugar radical transfer from •OH-mediated base radicals from 0 to 0.03 with increasing pO2 from 0 to 760 mmHg. The DNA damage induction algorithm was applied to tracks from electrons, protons and alphas with LET values from 0.2 to 150 keV/µm under different pO2 conditions. The oxygen enhancement ratio for double-strand break induction was 3.0 for low-LET radiation up to approximately 15 keV/µm, after which it gradually decreased to a value of 1.3 at 150 keV/µm. These values were consistent with a range of experimental data published in the literature. The DNA damage yields were verified using experimental data in the literature and results from other theoretical models. The spatial clustering approach developed in this work has low memory requirements and may be suitable for particle tracking simulations with a large number of cells.

Linear Energy Transfer Modulates Radiation-Induced NF-kappa B Activation and Expression of its Downstream Target Genes.

Nuclear factor kappaB (NF-κB) is a central transcription factor in the immune system and modulates cell survival in response to radiotherapy. Activation of NF-κB was shown to be an early step in the cellular response to ultraviolet A (UVA) and ionizing radiation exposure in human cells. NF-κB activation by the genotoxic stress-dependent sub-pathway after exposure to different radiation qualities had been evaluated to a very limited extent. In addition, the resulting gene expression profile, which shapes the cellular and tissue response, is unknown. Therefore, in this study the activation of NF-κB after exposure to low- and high-linear energy transfer (LET) radiation and the expression of its target genes were analyzed in human embryonic kidney (HEK) cells. The activation of NF-κB via canonical and genotoxic stress-induced pathways was visualized by the cell line HEK-pNF-κB-d2EGFP/Neo L2 carrying the destabilized enhanced green fluorescent protein (d2EGFP) as reporter. The NF-κB-dependent d2EGFP expression after irradiation with X rays and heavy ions was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after irradiation with X rays (significant NF-κB activation for doses >4 Gy) and heavy ions (significant NF-κB activation at doses as low as 1 Gy), it was expected that radiation quality (LET) played an important role in the cellular radiation response. In addition, the relative biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival were compared for heavy ions having a broad LET range (∼0.3-9,674 keV/µm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real-time reverse transcriptase quantitative PCR (RT-qPCR). The maximal RBE for NF-κB activation and cell killing occurred at an LET value of 80 and 175 keV/µm, respectively. There was a dose-dependent increase in expression of NF-κB target genes NF-κB1A and CXCL8. A qPCR array of 84 NF-κB target genes revealed that TNF and a set of CXCL genes (CXCL1, CXCL2, CXCL8, CXCL10), CCL2, VCAM1, CD83, NF-κB1, NF-κB2 and NF-κBIA were strongly upregulated after exposure to X rays and neon ions (LET 92 keV/µm). After heavy-ion irradiations, it was noted that the expression of NF-κB target genes such as chemokines and CD83 was highest at an LET value that coincided with the LET resulting in maximal NF-κB activation, whereas expression of the NF-κB inhibitory gene NFKBIA was induced transiently by all radiation qualities investigated. Taken together, these findings clearly demonstrate that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ∼50-200 keV/µm. The upregulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, CXCL8/IL-8 and TNF) could be important for cell-cell communication among hit as well as nonhit cells (bystander effect).

A single low dose of Fe ions can cause long-term biological responses in NL20 human bronchial epithelial cells.

Space radiation cancer risk may be a potential obstacle for long-duration spaceflight. Among all types of cancer space radiation may induce, lung cancer has been estimated to be the largest potential risk. Although previous animal study has shown that Fe ions, the most important contributor to the total dose equivalent of space radiation, induced a higher incidence of lung tumorigenesis per dose than X-rays, the underlying mechanisms at cellular level remained unclear. Therefore, in the present study, we investigated long-term biological changes in NL20 human bronchial epithelial cells after exposure to Fe ion or X-ray irradiation. We found that compared with sham control, the progeny of NL20 cells irradiated with 0.1 Gy of Fe ions showed slightly increased micronucleus formation, significantly decreased cell proliferation, disturbed cell cycle distribution, and obviously elevated intracellular ROS levels accompanied by reduced SOD1 and SOD2 expression, but the progeny of NL20 cells irradiated with 0.9 Gy of X-rays did not show any significant changes. More importantly, Fe ion exposure caused much greater soft-agar colony formation than X-rays did in the progeny of irradiated NL20 cells, clearly suggesting higher cell transformation potential of Fe ions compared with X-rays. These data may shed the light on the potential lung tumorigenesis risk from Fe ion exposure. In addition, ATM inhibition by Ku55933 reversed some of the changes in the progeny of Fe ion-irradiated cells but not others such as soft-agar colony formation, suggesting complex processes from DNA damage to carcinogenesis. These data indicate that even a single low dose of Fe ions can induce long-term biological responses such as cell transformation, etc., suggesting unignorable health risk from space radiation to astronauts.

Oxygen Enhancement Ratio in Radiation-Induced Initial DSBs by an Optimized Flow Cytometry-based Gamma-H2AX Analysis in A549 Human Cancer Cells.

High-linear energy transfer (LET) heavy ions cause higher therapeutic effects than low-LET radiation due to lower dependency on oxygen concentration in tumor cell killing. The lethality after irradiation largely depends on DNA double-strand breaks (DSBs), however the detailed LET dependency for DSB induction under oxic and hypoxic conditions has not been reported. Therefore, we evaluated the oxygen enhancement ratio (OER) of heavy ion-induced DSB induction using a highly-optimized flow cytometry-based method of γ-H2AX detection. Non-small cell lung cancer (NSCLC) A549 cells were exposed to X-ray, carbon-ion and iron-ion radiations under oxic or hypoxic condition. As a DSB marker, the γ-H2AX signal was measured 1 h postirradiation and analyzed by flow cytometry. DSB slope values were calculated as DSB induction per Gy. Our method was able to detect high-LET radiation-induced DSBs even from clustered DNA damage sites. We also showed a decrease in OER value in an LET-dependent manner regardless of radiation type. In summary, we demonstrated a simple, quick and highly-optimized flow cytometry-based method of DSB analysis that detects DSBs induced by heavy-ion radiation for hypoxic and nonhypoxic cancer cells. Our study may provide a useful biological basis for heavy-ion radiotherapy.

Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving.

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

Radiation damage to neuronal cells: Simulating the energy deposition and water radiolysis in a small neural network.

Radiation damage to the central nervous system (CNS) has been an on-going challenge for the last decades primarily due to the issues of brain radiotherapy and radiation protection for astronauts during space travel. Although recent findings revealed a number of molecular mechanisms associated with radiation-induced impairments in behaviour and cognition, some uncertainties exist in the initial neuronal cell injury leading to the further development of CNS malfunction. The present study is focused on the investigation of early biological damage induced by ionizing radiations in a sample neural network by means of modelling physico-chemical processes occurring in the medium after exposure. For this purpose, the stochastic simulation of incident particle tracks and water radiation chemistry was performed in realistic neuron phantoms constructed using experimental data on cell morphology. The applied simulation technique is based on using Monte-Carlo processes of the Geant4-DNA toolkit. The calculations were made for proton, 12C, and 56Fe particles of different energy within a relatively wide range of linear energy transfer values from a few to hundreds of keV/µm. The results indicate that the neuron morphology is an important factor determining the accumulation of microscopic radiation dose and water radiolysis products in neurons. The estimation of the radiolytic yields in neuronal cells suggests that the observed enhancement in the levels of reactive oxygen species may potentially lead to oxidative damage to neuronal components disrupting the normal communication between cells of the neural network.