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Breast cancer (BC) is the most common cancer in women, highly heterogeneous at both the clinical and molecular level. Radiation therapy (RT) represents an efficient modality to treat localized tumor in BC care, although the choice of a unique treatment plan for all BC patients, including RT, may not be the best option. Technological advances in RT are evolving with the use of charged particle beams (i.e. protons) which, due to a more localized delivery of the radiation dose, reduce the dose administered to the heart compared with conventional RT. However, few data regarding proton-induced molecular changes are currently available. The aim of this study was to investigate and describe the production of immunological molecules and gene expression profiles induced by proton irradiation. We performed Luminex assay and cDNA microarray analyses to study the biological processes activated following irradiation with proton beams, both in the non-tumorigenic MCF10A cell line and in two tumorigenic BC cell lines, MCF7 and MDA-MB-231. The immunological signatures were dose dependent in MCF10A and MCF7 cell lines, whereas MDA-MB-231 cells show a strong pro-inflammatory profile regardless of the dose delivered. Clonogenic assay revealed different surviving fractions according to the breast cell lines analyzed. We found the involvement of genes related to cell response to proton irradiation and reported specific cell line- and dose-dependent gene signatures, able to drive cell fate after radiation exposure. Our data could represent a useful tool to better understand the molecular mechanisms elicited by proton irradiation and to predict treatment outcome

Background: Intra operative radiation therapy (IORT) is a cancer treatment modality combining surgery and radiation therapy that permits the delivery of a large single dose of radiation at the time of the tumour resection. During the last years, IORT has become a widespread technique in clinical routine. This was possible thanks to the recent development of small dedicated, and hence very flexible, electron accelerators suitable for a surgical operation environment. However, the electron beams produced by these dedicated linacs are different with respect to those produced by linacs used in conventional radiation therapy in terms of dose rate (dose delivered/time of irradiation), dose per pulse (dose delivered per emitted beam pulse) and energy spectrum. In particular the very high dose rate and dose per pulse (i.e., respectively about 20 and 100 times greater than those of conventional radiation therapy) pose some radiobiological questions that have not yet been fully investigated. The aim of this work was to evaluate the relative biological effectiveness (RBE) of the IORT beams through the experimental construction of dose-response curves for cell survival. Methods: Since breast cancer represents the elective malignancy for treatment by IORT, clonogenic survival was assayed in the MCF-7 cell line from breast tissue malignant adenocarcinoma as a function of dose for different dose rates (5, 25 and 75 Gy/min) and doses per pulse (4.2 and 7.4 cGy/pulse). Irradiations were performed at the IORT accelerator Novac7 (Hitesys SpA, Italy) delivering electrons at the nominal energy of 7 MeV. For comparison, irradiations were also carried out with a conventional radiotherapy accelerator (ClinacR DHX, Varian), providing a 6 MeV pulsed electron beam and 6 MV X-rays (dose rate and dose-per-pulse 2 Gy/min and 0.1 cGy/pulse respectively) as reference irradiations. Results: The IORT beam effectiveness was found almost the same with varying dose rate (5, 25 and 75 Gy/min) and dose per pulse (4.2 and 7.4 cGy/pulse). No significant difference was also found between IORT and conventional electron beams, while 6 MV X-rays (beam used in the conventional treatment of breast cancer) proved to be more effective. Conclusions: Data show that cell killing effectiveness of the electron beams from the IORT accelerator does not differ significantly from that of an electron beam used in conventional radiotherapy. This suggests that changes in the dose rate and dose per pulse, up to the values of 75 Gy/min and 7.4 cGy/pulse, do not affect the radiobiological characteristics of the beam. In the light of our results, new experimental data are needed to better compare the RBE of a IORT breast cancer treatment (5–9 MeV electron beams, 21 Gy at the 90% isodose level in single fraction) and the RBE of a conventional breast cancer treatment (6 MV X-rays, 2 Gy per fraction, 30 fractions).

An automated analysis system was developed that is able to scan nuclear track detectors CR-39. The system consists of an optical microscope, a CCD camera, a frame grabber and a personal computer. On the latter a software written in Labview language (version 7.1) is implemented to control the motion of the microscope stage, the autofocus function and to measure the geometric characteristics of particle tracks. The system is able to detect and measure couples of partially overlapped tracks. As a preliminary application of this system, we estimated the projectile total charge-changing cross section for a 1 GeV/n iron ion beam impacting a polyethylene target.

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 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.

Purpose The biological optimization of proton therapy can be achieved only through a detailed evaluation of relative biological effectiveness (RBE) variations along the full range of the Bragg curve. The clinically used RBE value of 1.1 represents a broad average, which disregards the steep rise of linear energy transfer (LET) at the distal end of the spread-out Bragg peak (SOBP). With particular attention to the key endpoint of cell survival, our work presents a comparative investigation of cell killing RBE variations along monoenergetic (pristine) and modulated (SOBP) beams using human normal and radioresistant cells with the aim to investigate the RBE dependence on LET and intrinsic radiosensitvity. Methods and Materials Human fibroblasts (AG01522) and glioma (U87) cells were irradiated at 6 depth positions along pristine and modulated 62-MeV proton beams at the INFN-LNS (Catania, Italy). Cell killing RBE variations were measured using standard clonogenic assays and were further validated using Monte Carlo simulations and the local effect model (LEM). Results We observed significant cell killing RBE variations along the proton beam path, particularly in the distal region showing strong dose dependence. Experimental RBE values were in excellent agreement with the LEM predicted values, indicating dose-averaged LET as a suitable predictor of proton biological effectiveness. Data were also used to validate a parameterized RBE model. Conclusions The predicted biological dose delivered to a tumor region, based on the variable RBE inferred from the data, varies significantly with respect to the clinically used constant RBE of 1.1. The significant RBE increase at the distal end suggests also a potential to enhance optimization of treatment modalities such as LET painting of hypoxic tumors. The study highlights the limitation of adoption of a constant RBE for proton therapy and suggests approaches for fast implementation of RBE models in treatment planning.

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.

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.

Uncertainties surround the radiobiological consequences of exposure to charged particles, despite the increasing use of accelerated ion beams for cancer treatment (hadrontherapy). In particular, little is known about the long-term effects on normal tissue at the beam entrance or in the distal part of the Spread-Out Bragg Peak (SOBP). Moreover, although the relative biological effectiveness (RBE) of particle radiation has been traditionally related to the radiation linear energy transfer (LET), it has become increasingly evident that radiation-induced cell death, as well as long term radiation effects, is not adequately described by this parameter. Hence, exploring the effectiveness of various ion beams at or around the Bragg peak of monoenergetic ion beams can prove useful to gain insights into the role played by parameters other than the particle LET in determining the outcome of particle radiation exposures. In this context, the upgrade of the Tandem irradiation facility at Naples University here described, has allowed us to perform a series of preliminary radiobiological measurements using proton and carbon ion beams. The facility is currently used to irradiate normal and cancer cell lines with ion beams such as oxygen and fluorine.