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Hadron therapy by proton beams represents an advanced anti-cancer strategy due to its highly localized dose deposition allowing a greater sparing of normal tissue and/or organs at risk compared to photon/electron radiotherapy. However, it is not clear to what extent non-targeted effects such as transcriptional modulations produced along the beamline may diffuse and impact the surrounding tissue. In this work, we analyze the transcriptome of proton-irradiated mouse skin and choose two biomarker genes to trace their modulation at different distances from the beam's target and at different doses and times from irradiation to understand to what extent and how far it may propagate, using RNA-Seq and quantitative RT-PCR. In parallel, assessment of lipids alteration is performed by FTIR spectroscopy as a measure of tissue damage. Despite the observed high individual variability of expression, we can show evidence of transcriptional modulation of two biomarker genes at considerable distance from the beam's target where a simulation system predicts a significantly lower adsorbed dose. The results are compatible with a model involving diffusion of transcripts or regulatory molecules from high dose irradiated cells to distant tissue's portions adsorbing a much lower fraction of radiation.

Raman micro-spectroscopy and different approaches for multivariate analysis were used for an investigation of subcellular regions of X-ray exposed single SH-SY5Y human neuroblastoma cells. Nucleus and cytoplasm regions of single cells were investigated after X-rays irradiation (0, 2, 4, 6 and 8 Gy). Cells fixed immediately after irradiation and 24h irradiation were considered. Principal component analysis (PCA) and interval-PCA (i-PCA) were used for analyzing the spectra in order to highlight the changes due to the different treatments. Biochemical changes occurring in the nucleus and cytoplasm regions of single cells upon X-ray irradiation were observed. The analysis of Raman spectra allowed us to detect modifications in the contribution from proteins, nucleic acids, lipids, and carbohydrates of cells, induced at different extent on the two cell regions. The biochemical changes occurring in these cells were also discussed by using an alternative approach, namely the analysis of difference spectra, obtained by subtracting the cytoplasm-related spectrum from the corresponding one detected at the nucleus. The proposed approach enabled us to evidence some features not outlined in previous investigations. The results showed that it is possible to study in a selective way the effects of ionizing radiation on different neuroblastoma cell spatial regions. An increase of the signal related to the nucleobases, a decrease of DNA and/or RNA backbone contribution, a protein rearrangement with changes in the secondary structure, and an increase in lipid saturation were observed. These results indicated that the development of accurate data analysis methods enabling to take into account the complexity of the Raman spectra of cells and tissues and the high number of spectra needed to consider the intrinsic variability of biological samples.

The ability of Fourier transform infrared (FTIR) spectroscopy in analyzing cells at a molecular level was exploited for investigating the biochemical changes induced in protein, nucleic acid, lipid, and carbohydrate content of cells after irradiation by graded X-ray doses. Infrared spectra from in vitro SH-SY5Y neuroblastoma cells following exposure to X-rays (0, 2, 4, 6, 8, 10 Gy) were analyzed using a ratiometric approach by evaluating the ratios between the absorbance of significant peaks. The spectroscopic investigation was performed on cells fixed immediately (t0 cells) and 24 h (t24 cells) after irradiation to study both the initial radiation-induced damage and the effect of the ensuing cellular repair processes. The analysis of infrared spectra allowed us to detect changes in proteins, lipids, and nucleic acids attributable to X-ray exposure. The ratiometric analysis was able to quantify changes for the protein, lipid, and DNA components and to suggest the occurrence of apoptosis processes. The ratiometric study of Amide I band indicated also that the secondary structure of proteins was significantly modified. The comparison between the results from t0 and t24 cells indicated the occurrence of cellular recovery processes. The adopted approach can provide a very direct way to monitor changes for specific cellular components and can represent a valuable tool for developing innovative strategies to monitor cancer radiotherapy outcome.

Previous works showed that spatially resolved Raman spectra of cytoplasm and nucleus region of single cells exposed to X-rays evidence different features. The present work aims to introduce a new approach to profit from these differences to deeper investigate X-ray irradiation effects on single SH-SY5Y human neuroblastoma cells. For this aim, Raman micro-spectroscopy was performed in vitro on single cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). Spectra from nucleus and cytoplasm regions were selectively acquired. The examination by interval Principal Component Analysis (i-PCA) of the difference spectra obtained by subtracting each cytoplasm-related spectrum from the corresponding one detected at the nucleus enabled us to reveal the subtle modifications of Raman features specific of different spatial cell regions. They were discussed in terms of effects induced by X-ray irradiation on DNA/RNA, lipids, and proteins. The proposed approach enabled us to evidence some features not outlined in previous investigations.

A micro-Raman spectroscopy investigation has been performed in vitro on single human mammary epithelial cells after irradiation by graded x-ray doses. The analysis by principal component analysis (PCA) and interval-PCA (i-PCA) methods has allowed us to point out the small differences in the Raman spectra induced by irradiation. This experimental approach has enabled us to delineate radiation-induced changes in protein, nucleic acid, lipid, and carbohydrate content. In particular, the dose dependence of PCA and i-PCA components has been analyzed. Our results have confirmed that micro-Raman spectroscopy coupled to properly chosen data analysis methods is a very sensitive technique to detect early molecular changes at the single-cell level following exposure to ionizing radiation. This would help in developing innovative approaches to monitor radiation cancer radiotherapy outcome so as to reduce the overall radiation dose and minimize damage to the surrounding healthy cells, both aspects being of great importance in the field of radiation therapy.

The polykaryon-forming unit (PFU) cell survival assay is based on the postirradiation flow cytometric analysis of the DNA content accumulated in high-ploidy cells (polykaryons) induced by the cytokinesis inhibitor cytochalasin B and can provide a meaningful measure of cell radiosensitivity. In this assay, cell survival is defined as the ability to form a polykaryon of a given ploidy after irradiation. The slope of the polykaryon dose response has been shown to be highly correlated with the initial slope of the clonogenic survival curves after gamma irradiation, which implies a common subset of lethal lesions. We reported previously on an apoptotic mode of cell death in the polykaryon system and on the heritability of small variations in polykaryon radioresponse. We now show that exposure of PFUs to a given dose of alpha particles results in a greater reduction in the proportion of cells able to reach at least 16C when compared to the same dose of low-LET radiation. This reduction is less than that observed in the low-dose (alpha term) region of the clonogenic curve. On the basis of published LET-dependent spectra of radiation-induced DNA damage, we suggest that this behavior reflects a differential expression of lethal damage that can be probed by varying the LET of the radiation and that base damages contributing additional complexity to clustered DNA lesions may be more deleterious in PFUs than in clonogens.

We report results for chromosomal aberrations in human peripheral blood lymphocytes after they were exposed to high-energy iron ions with or without shielding at the HIMAC, AGS and NSRL accelerators. Isolated lymphocytes were exposed to iron ions with energies between 200 and 5000 MeV/nucleon in the 0.1-1-Gy dose range. Shielding materials consisted of polyethylene, lucite (PMMA), carbon, aluminum and lead, with mass thickness ranging from 2 to 30 g/cm(2). After exposure, lymphocytes were stimulated to grow in vitro, and chromosomes were prematurely condensed using a phosphatase inhibitor (calyculin A). Aberrations were scored using FISH painting. The yield of total interchromosomal exchanges (including dicentrics, translocations and complex rearrangements) increased linearly with dose or fluence in the range studied. Shielding decreased the effectiveness per unit dose of iron ions. The highest RBE value was measured with the 1 GeV/nucleon iron-ion beam at NSRL. However, the RBE for the induction of aberrations apparently is not well correlated with the mean LET. When shielding thickness was increased, the frequency of aberrations per particle incident on the shield increased for the 500 MeV/nucleon ions and decreased for the 1 GeV/nucleon ions. Maximum variation at equal mass thickness was obtained with light materials (polyethylene, carbon or PMMA). Variations in the yield of chromosomal aberrations per iron particle incident on the shield follow variations in the dose per incident particle behind the shield but can be modified by the different RBE of the mixed radiation field produced by nuclear fragmentation. The results suggest that shielding design models should be benchmarked using both physics and biological data.

The aim was to evaluate the effect of modelled microgravity on radiation-induced chromosome aberrations (CAs). G(0) peripheral blood lymphocytes were exposed to 60 MeV protons or 250 kV(p) X-rays in the dose range 0 - 6 Gy, and allowed to repair DNA damage for 24 h under either normal gravity or microgravity modelled by the NASA-designed rotating-wall bioreactor. Cells were then stimulated to proliferate by phytohaemagglutinin (PHA) under normal gravity conditions and prematurely condensed chromosomes were harvested after 48 h. CAs were scored in chromosomes 1 and 2 by fluorescence in-situ hybridization. Proliferation gravisensitivity was examined by cell growth curves and by morphological evaluation of mitogen-induced activation. Cell replication rounds were monitored by bromodeoxyuridine labelling. Modelled microgravity markedly reduced PHA-mediated lymphocyte blastogenesis and cell growth. However, no significant differences between normal gravity and modelled microgravity were found in the dose - response curves for the induction of aberrant cells or total interchromosomal exchange frequency. Rotating-wall bioreactor-based microgravity reproduced space-related alterations of mitogen stimulation in human lymphocytes but did not affect the yield of CAs induced by low-linear energy transfer radiation.

The possible use of radioactivity dispersal devices by terrorist groups has been recently reported in the news. In this paper, we discuss the threat of terrorist attacks by plutonium, with particular attention to the dispersal of plutonium by explosion or fire. Doses resulting from inhalation of radioactive aerosol induced by a plutonium explosion or fire are simulated using a Gaussian plume model (the HOTSPOT code) for different meteorological conditions. Ground contamination and resuspension of dust are also considered in the simulations. Our simulations suggest that acute effects from a plutonium dispersal attack are very unlikely. For late stochastic effects, the explosion poses a greater hazard than fire. However, even in the worst-case scenario, the dispersed plutonium would cause relatively few excess cancers (around 80 in a city of 2 million inhabitants) after many years from the explosion, and these excess cancers would remain undetected against the background of cancer fatalities.

Accelerated proton beams have become increasingly common for treating cancer. The need for cost and size reduction of particle accelerating machines has led to the pioneering investigation of optical ion acceleration techniques based on laser-plasma interactions as a possible alternative. Laser-matter interaction can produce extremely pulsed particle bursts of ultra-high dose rates (≥ 109 Gy/s), largely exceeding those currently used in conventional proton therapy. Since biological effects of ionizing radiation are strongly affected by the spatio-temporal distribution of DNA-damaging events, the unprecedented physical features of such beams may modify cellular and tissue radiosensitivity to unexplored extents. Hence, clinical applications of laser-generated particles need thorough assessment of their radiobiological effectiveness. To date, the majority of studies have either used rodent cell lines or have focussed on cancer cell killing being local tumour control the main objective of radiotherapy. Conversely, very little data exist on sub-lethal cellular effects, of relevance to normal tissue integrity and secondary cancers, such as premature cellular senescence. Here, we discuss ultra-high dose rate radiobiology and present preliminary data obtained in normal human cells following irradiation by laser-accelerated protons at the LULI PICO2000 facility at Laser Lab Europe, France.

A Micro-Raman spectroscopy investigation has been performed on human mammary epithelial cells after irradiation by different X-ray doses. The analysis by interval­ Principal Component Analysis (iPCA) has allowed us to outline the subtle differences in the Raman spectra induced by irradiation. In particular, this experimental approach has enabled to delineate radiation-induced changes in structure, protein, nucleic acid, lipid, and carbohydrate content. This has further confirmed that micro-Raman spectroscopy coupled to properly chosen data analysis methods is a very sensitive technique to detect molecular changes even in single human cells following exposure to ionising radiation. This would help developing innovative approaches to monitor radiation treatment effects so as to reduce the overall radiation dose and minimize damage to the surrounding healthy cells, both aspects being of great importance in the field of radiation therapy. Keywords-Micro-Raman spectroscopy; X-ray induced damage cells; interval principal component analaysis

The use of charged particles significantly reduces the dose absorbed by normal cells due to the inverse dose-depth deposition profile. This is the physical pillar justifying hadrontherapy as the eligible treatment for deepseated tumours. However, a non-negligible amount of radiation is nevertheless absorbed in correspondence with the plateau region of the Bragg curve, which may result in the induction of sub-lethal effects. Very little experimental data exist on the induction of such effects. Moreover, reliable follow-up data on such adverse effects in hadrontherapy patients are limited since this type of treatment has been adopted relatively recently. A fortiori, the dependence of sub-lethal effects on unprecedented factors, such as the exceedingly high dose rates and/or the pulsed nature of beams originated by laser interaction with target materials, is unknown. This warrants investigation prior to a therapeutic use of such beams.

Micro-Raman technique can be particularly useful to investigate the chemical changes induced in structure, protein, nucleic acid, lipid, and carbohydrate contents of cells. The aim of this work is to inspect the possibility to employ Raman microspectroscopy to detect biochemical modifications in human mammary epithelial cells after exposure to different Xray doses. The samples consisted of cells cultured on polylysine-coated glass coverslips. After the exposition, control and treated cells were washed in phosphate-buffered saline (PBS) and then fixed in paraformaldehyde 3.7%. They were examined using a confocal micro-Raman system equipped with a He-Ne laser (λ = 632.8 nm; power on the sample= 3.5mW). Differences in the intensity ratio of specific Raman vibrational markers commonly assigned to phenylalanine and tyrosine amino acids (at 1000, 1030, 1618 cm -1 ), DNA bases (787, 1090, 1305 cm -1 ), and amide III (1237, and 1265 cm -1 ) with respect a reference peak (the one of lipids at 1450 cm -1 ) were evidenced between control and exposed cells. These differences may be indicative of damage in exposed cells as the fragmentation of individual amino acids and DNA bases, crosslink effects in molecular structure of DNA and protein conformational change that especially tend to "unwind" the protein due to the breaking of hydrogen bonds between peptide chains.

A Micro-Raman spectroscopic investigation has been performed on human mammary epithelial cells irradiated with different doses of X-ray. Results show that this experimental approach can detect radiation-induced changes in structure, protein, nucleic acid, lipid, and carbohydrate content. Micro- Raman spectroscopy appears to be a very sensitive technique to detect molecular changes even in single human cells following exposure to ionising radiation.

We measured residual cytogenetic damage in the progeny of human peripheral blood lymphocytes exposed to 1 GeV/ nucleon iron ions or gamma rays. Arm-specific DNA probes for chromosome 1 were used to detect aberrations as a function of dose in cells harvested 144 h after exposure. In addition, arm-specific mFISH was applied to samples exposed to a single dose of 2 Gy. These methods allowed the detection of interarm intrachanges (pericentric inversions) in addition to interchanges. The ratio of these types of aberrations (F ratio) has been proposed as a fingerprint of exposure to densely ionizing radiation. The fractions of aberrant cells in the progeny of cells exposed to iron ions were similar to those in the population exposed to gamma rays, possibly because many rearrangements induced by heavy ions ultimately lead to cell death. Simple inter- and intrachanges were also similar, but more complex rearrangements were found in cells that survived after exposure to iron ions. We did not find a significant difference in the ratio of simple interchanges to simple intrachanges for the two radiation types. However, iron ions induced a much higher frequency of events involving both inter- and intrachanges. We conclude that these complex rearrangements represent a hallmark of exposure to heavy ions and may be responsible of the decrease of the F ratio with increasing LET reported in the literature in some in vitro and in vivo experiments.

The case for a DNA-damaging action produced by radio-frequency (RF) signals remains controversial despite extensive research. With the advent of the Universal Mobile Telecommunication System (UNITS) the number of RF-radiation-exposed individuals is likely to escalate. Since the epigenetic effects of RF radiation are poorly understood and since the potential modifications of repair efficiency after exposure to known cytotoxic agents such as ionizing radiation have been investigated infrequently thus far, we studied the influence of UNITS exposure on the yield of chromosome aberrations induced by X rays. Human peripheral blood lymphocytes were exposed in vitro to a UNITS signal (frequency carrier of 1.95 GHz) for 24 h at 0.5 and 2.0 W/kg specific absorption rate (SAR) using a previously characterized waveguide system. The frequency of chromosome aberrations was measured on metaphase spreads from cells given 4 Gy of X rays immediately before RF radiation or sham exposures by fluorescence in situ hybridization. Unirradiated controls were RF-radiation- or sham-exposed. No significant variations due to the UNITS exposure were found in the fraction of aberrant cells. However, the frequency of exchanges per cell was affected by the SAR, showing a small but statistically significant increase of 0.11 exchange per cell compared to 0 W/kg SAR. We conclude that, although the 1.95 GHz signal (UNITS modulated) does not exacerbate the yield of aberrant cells caused by ionizing radiation, the overall burden of X-ray-induced chromosomal damage per cell in first-mitosis lymphocytes may be enhanced at 2.0 W/kg SAR. Hence the SAR may either influence the repair of X-ray-induced DNA breaks or alter the cell death pathways of the damage response.

Protons are the most abundant element in the galactic cosmic radiation, and the energy spectrum peaks around 1 GeV. Shielding of relativistic protons is therefore a key problem in the radiation protection strategy of crewmembers involved in long-term missions in deep space. Hydrogen ions were accelerated up to 1 GeV at the NASA Space Radiation Laboratory, Brookhaven National Laboratory, New York. The proton beam was also shielded with thick ( about 20 g/cm(2)) blocks of lucite (PMMA) or aluminium ( Al). We found that the dose rate was increased 40-60% by the shielding and decreased as a function of the distance along the axis. Simulations using the General Purpose Particle and Heavy-Ion Transport code System (PHITS) show that the dose increase is mostly caused by secondary protons emitted by the target. The modified radiation Weld after the shield has been characterized for its biological eVectiveness by measuring chromosomal aberrations in human peripheral blood lymphocytes exposed just behind the shield block, or to the direct beam, in the dose range 0.53 Gy. Notwithstanding the increased dose per incident proton, the fraction of aberrant cells at the same dose in the sample position was not significantly modified by the shield. The PHITS code simulations show that, albeit secondary protons are slower than incident nuclei, the LET spectrum is still contained in the low-LET range (< 10 keV/mu m), which explains the approximately unitary value measured for the relative biological effectiveness.

This review addresses the purported interplay between actual or simulated weightlessness and cellular response to ionizing radiation. Although weightlessness is known to alter several cellular functions and to affect signaling pathways implicated in cell proliferation, differentiation and death, its influence on cellular radiosensitivity has so far proven elusive. Renewed controversy as to whether reduced gravity enhances long-term radiation risk is fueled by recently published data that claim either overall enhancement of genomic damage or no increase of radiation-induced clastogenicity by modeled microgravity in irradiated human cells. In elucidating this crucial aspect of space radiation protection, ground-based experiments, such as those based on rotating-wall bioreactors, will increasingly be used and represent a more reproducible alternative to in-flight experiments. These low-shear vessels also make three-dimensional cellular co-cultures possible and thus allow to study the gravisensitivity of radioresponse in a context that better mimics cell-to-cell communication and hence in vivo cellular behavior.