Experimental investigation at CATANA facility of n-10B and p-11B reactions for the enhancement of proton therapy.

The aim of the NEPTUNE (Nuclear process-driven Enhancement of Proton Therapy UNravEled) project is to investigate in detail both the physical and radiobiological phenomena that could justify an increase of the proton-induced cytogenetic effects in cells irradiated in presence of an agent containing natural boron. In this work, a double-stage silicon telescope coupled to different boron converters was irradiated at the CATANA proton therapy facility (INFN-LNS) for studying the proton boron fusion and the neutron boron capture reactions by discriminating secondary particles from primary protons. Different boron targets were developed by depositing boric acid, enriched with a higher than 99% content of 10B or 11B, on a 50 µm thick PolyMethilMetacrylate (PMMA) substrate. The 10B target allows to evaluate the contribution of lithium and alpha particles produced by the boron neutron capture reaction triggered by secondary thermal neutrons, while the 11B target is exploited for studying the effect of the p + 11B → 3α nuclear reaction directly triggered by primary protons. Experimental results clearly show the presence of alpha particles from both the reactions. The silicon telescope is capable of discriminating, by means of the so-called "scatter plots", the contribution of alpha particles originated by thermal neutrons on 10B with respect to the ones produced by protons impinging on 11B. Although a reliable quantitative study of the alpha production rate has not been achieved yet, this work demonstrates that low energy and, therefore, high-LET particles from both the reactions can be measured.

Radiobiological quantities in proton-therapy: Estimation and validation using Geant4-based Monte Carlo simulations.

PURPOSE: The Geant4 Monte Carlo simulation toolkit was used to reproduce radiobiological parameters measured by irradiating three different cancerous cell lines with monochromatic and clinical proton beams. METHODS: The experimental set-up adopted for irradiations was fully simulated with a dedicated open-source Geant4 application. Cells survival fractions was calculated coupling the Geant4 simulations with two analytical radiobiological models: one based on the LEM (Local Effect Model) approach and the other on a semi-empirical parameterisation. Results was evaluated and compared with experimental data. RESULTS AND CONCLUSIONS: The results demonstrated the Geant4 ability to reproduce radiobiological quantities for different cell lines.

Raman spectroscopy for the evaluation of the radiobiological sensitivity of normal human breast cells at different time points after irradiation by a clinical proton beam.

Among different radiotherapy techniques, proton irradiation is an established and effective method for treatment of several types of cancer, because less healthy tissue is exposed with respect to conventional radiotherapy by photons/electrons. Recently, proton therapy has been proposed for the treatment of breast cancer. In vitro studies of proton irradiated normal human breast cells can provide information about cellular radioresponse, particularly as far as healthy tissue is concerned. In this paper, a study of the effects at different time points, following proton irradiation at different doses, of human normal MCF10A breast cells is performed by Raman spectroscopy. The aim of this investigation is to detect the unwanted effects of proton treatment and to investigate the possibility of monitoring them and of making an assessment of the cellular sensitivity by means of such a technique. The obtained results seem to indicate a rather significant sensitivity of MCF10A cells to proton irradiation. In fact, even at doses as low as 0.5 Gy, biological effects are clearly detectable in Raman spectra. In particular, ratiometric analysis of the Raman spectra measured from the nucleoplasm compartment showed that DNA/RNA damage increases with time, suggesting that most cells are unable to repair DNA/RNA broken bonds. The results obtained by the Raman spectroscopy analysis exhibit a similar trend with regard to dose to those obtained by commonly used radiobiological assays (i.e. MTT, clonogenic assay, senescence, apoptosis and necrosis). The results of this study strongly suggest the possibility that the Raman technique can be used to identify molecular markers predicting radiation response.

X-ray irradiation effects on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells investigated by Raman micro-spectroscopy.

Raman micro-spectroscopy was performed in vitro on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). The acquired spectra were analyzed by principal component analysis (PCA) and interval-PCA (i-PCA) methods. Biochemical changes occurring in the different regions of single cells as a consequence of the radiation exposure were observed in cells fixed immediately after the irradiation. The most relevant effects arose from the analysis of the spectra from the cell nucleus region. The observed changes were discussed in terms of the modifications in the cell cycle, resulting in an increase in the DNA-related signal, a protein rearrangement and changes in lipid and carbohydrates profiles within the nucleus. Potential markers of an apoptotic process in cell population irradiated with 6 and 8-Gy X-ray doses could have been singled out. No significant effects were found in spectra from cells fixed 24 h after the irradiation, thus suggesting the occurrence of repairing processes of the X-ray induced damage.

First experimental proof of Proton Boron Capture Therapy (PBCT) to enhance protontherapy effectiveness.

Protontherapy is hadrontherapy's fastest-growing modality and a pillar in the battle against cancer. Hadrontherapy's superiority lies in its inverted depth-dose profile, hence tumour-confined irradiation. Protons, however, lack distinct radiobiological advantages over photons or electrons. Higher LET (Linear Energy Transfer) 12C-ions can overcome cancer radioresistance: DNA lesion complexity increases with LET, resulting in efficient cell killing, i.e. higher Relative Biological Effectiveness (RBE). However, economic and radiobiological issues hamper 12C-ion clinical amenability. Thus, enhancing proton RBE is desirable. To this end, we exploited the p + 11B → 3α reaction to generate high-LET alpha particles with a clinical proton beam. To maximize the reaction rate, we used sodium borocaptate (BSH) with natural boron content. Boron-Neutron Capture Therapy (BNCT) uses 10B-enriched BSH for neutron irradiation-triggered alpha particles. We recorded significantly increased cellular lethality and chromosome aberration complexity. A strategy combining protontherapy's ballistic precision with the higher RBE promised by BNCT and 12C-ion therapy is thus demonstrated.

Chromosome inter- and intrachanges detected by arm-specific DNA probes in the progeny of human lymphocytes exposed to energetic heavy ions.

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.

Effects of modulated microwave radiation at cellular telephone frequency (1.95 GHz) on X-ray-induced chromosome aberrations in human lymphocytes in vitro.

The case for a DNA-damaging action produced by radiofrequency (RF) signals remains controversial despite extensive research. With the advent of the Universal Mobile Telecommunication System (UMTS) 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 UMTS exposure on the yield of chromosome aberrations induced by X rays. Human peripheral blood lymphocytes were exposed in vitro to a UMTS 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 UMTS 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 (UMTS 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.

Accelerator-based tests of radiation shielding properties of materials used in human space infrastructures.

Shielding is the only practical countermeasure for the exposure to cosmic radiation during space travel. It is well known that light, hydrogenated materials, such as water and polyethylene, provide the best shielding against space radiation. Kevlar and Nextel are two materials of great interest for spacecraft shielding because of their known ability to protect human space infrastructures from meteoroids and debris. We measured the response to simulated heavy-ion cosmic radiation of these shielding materials and compared it to polyethylene, Lucite (PMMA), and aluminum. As proxy to galactic nuclei we used 1 GeV n iron or titanium ions. Both physics and biology tests were performed. The results show that Kevlar, which is rich in carbon atoms (about 50% in number), is an excellent space radiation shielding material. Physics tests show that its effectiveness is close (80-90%) to that of polyethylene, and biology data suggest that it can reduce the chromosomal damage more efficiently than PMMA. Nextel is less efficient as a radiation shield, and the expected reduction on dose is roughly half that provided by the same mass of polyethylene. Both Kevlar and Nextel are more effective than aluminum in the attenuation of heavy-ion dose.

Shielding of relativistic protons.

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/cm2) 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 field after the shield has been characterized for its biological effectiveness by measuring chromosomal aberrations in human peripheral blood lymphocytes exposed just behind the shield block, or to the direct beam, in the dose range 0.5-3 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/microm), which explains the approximately unitary value measured for the relative biological effectiveness.

Cytogenetic effects of high-energy iron ions: dependence on shielding thickness and material.

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/cm2. 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.

Modelled microgravity does not modify the yield of chromosome aberrations induced by high-energy protons in human lymphocytes.

The aim was to evaluate the effect of modelled microgravity on radiation-induced chromosome aberrations (CAs). G0 peripheral blood lymphocytes were exposed to 60 MeV protons or 250 kVp 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.

Fragmentation studies of relativistic iron ions using plastic nuclear track detectors.

We measured fluence and fragmentation of high-energy (1 or 5 A GeV) 56Fe ions accelerated at the Alternating Gradient Synchrotron or at the NASA Space Radiation Laboratory (Brookhaven National Laboratory, NY, USA) using solid-state CR-39 nuclear track detectors. Different targets (polyethylene, PMMA, C, Al, Pb) were used to produce a large spectrum of charged fragments. CR-39 plastics were exposed both in front and behind the shielding block (thickness ranging from 5 to 30 g/cm2) at a normal incidence and low fluence. The radiation dose deposited by surviving Fe ions and charged fragments was measured behind the shield using an ionization chamber. The distribution of the measured track size was exploited to distinguish the primary 56Fe ions tracks from the lighter fragments. Measurements of projectile's fluence in front of the shield were used to determine the dose per incident particle behind the block. Simultaneous measurements of primary 56Fe ion tracks in front and behind the shield were used to evaluate the fraction of surviving iron projectiles and the total charge-changing fragmentation cross-section. These physical measurements will be used to characterize the beam used in parallel biological experiments.

Correlation between the clonogenic initial slope and the response of polykaryon-forming units: the behavior of strains defective in XRCC5 and ATM and the heritability of small variations in radioresponse.

The polykaryon-forming unit (PFU) assay measures the survival of multiple cycles of DNA synthesis after exposure to ionizing radiation, and it is known that there is a strong correlation between the slope of the PFU dose-response curve and the clonogenic initial slope. This suggests that DNA lesions expressed in clonogens are also important in PFU. Cells having a mutation in XRCC5 (also known as Ku80; strain xrs-6) and ATM (strain AT5BIVA) were hypersensitive in the PFU assay and in clonogens, while a strain of xrs-6 cells transfected with hamster wild-type XRCC5 cDNA displayed wild-type resistance in both assays. These data suggest that the DNA double-strand break (DSB) is an important lesion in PFU, although the relative radioresistance of PFU compared to clonogens indicates differential DSB toxicity. We propose that this results from the absence of cytokinesis-related loss of DNA fragments. Small variations in the radioresponse of PFU were observed between CHO K1 cell substrains, such that the xrs parental substrain RR-CHOK1 (carrying wild-type XRCC5) was more sensitive than an independent K1 substrain (E-CHOK1). Somatic hybridization showed that this variation is heritable and that the resistant E phenotype is dominant. In RR-CHOK1 cells there was a biphasic PFU radioresponse, which suggests that there may be transient expression at a locus selectively affecting PFU sensitivity.

Apoptosis is a mode of cell death in the polykaryon-forming unit assay.

In the polykaryon-forming unit (PFU) assay, which defines cell survival as the ability to form a cytochalasin-induced polykaryon of predetermined ploidy, the mode of PFU deletion is not known. Incubation of L5178Y-S PFU in cytochalasin resulted in polyploidy (> or =32C) and most polykaryons (>75%) ultimately underwent apoptosis, detected using chromatin condensation and externalised phosphatidylserine. However, large polykaryons carrying terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)-labelled DNA strand breaks were not observed, presumably due to rapid loss of DNA. Gamma irradiation of PFU prior to cytochalasin exposure caused a reduction in the frequency of highly polyploid cells (>16C), consistent with either a supra-induction of apoptosis or a reduction in the ability of PFU to reach high ploidies. We conclude that L5178Y-S PFU are deleted by apoptosis.

Manifestations and mechanisms of radiation-induced genomic instability in V-79 Chinese hamster cells.

PURPOSE: The relationship between different forms of persistent radiation damage in irradiated cells was investigated in order to identify a common underlying mechanism. MATERIAL AND METHODS: V-79 Chinese hamster cells were irradiated with different doses of X-rays, neutrons and alpha-particles. In the progeny of surviving cells, up to 4 weeks after irradiation, delayed reproductive death, delayed micronuclei, delayed appearance of dicentric chromosomes and delayed apoptosis were investigated in parallel. RESULTS: A similar dose-response relationship was found for all endpoints, with a steep rise at low doses to a plateau at doses > 3 Gy. The target for inducing genomic instability by alpha-particles is larger than the nucleus. All chromosomes are equally involved in delayed breakage reunion events. CONCLUSION: The results indicate that non-lethal radiation damage to an extranuclear target leads to a persistent increase in clastogenic activity in the surviving irradiated cells.

Genomic instability in Chinese hamster cells after exposure to X rays or alpha particles of different mean linear energy transfer.

Evidence has accumulated that radiation induces a transmissible persistent destabilization of the genome, which may result in effects arising in the progeny of irradiated but surviving cells. An enhanced death rate among the progeny of cells surviving irradiation persists for many generations in the form of a reduced plating efficiency. Such delayed reproductive death is correlated with an increased occurrence of micronuclei. Since it has been suggested that radiation-induced chromosomal instability might depend on the radiation quality, we investigated the effects of alpha particles of different LET by looking at the frequency of delayed micronuclei in Chinese hamster V79 cells after cytochalasin-induced block of cell division. A dose-dependent increase in the frequency of micronuclei was found in cells assayed 1 week postirradiation or later. Also, there was a persistent increase in the frequency of dicentrics in surviving irradiated cells. Moreover, we found an increased micronucleus frequency in all of the 30 clones isolated from individual cells which had been irradiated with doses equivalent to either one, two or three alpha-particle traversals per cell nucleus. We conclude that the target for genomic instability in Chinese hamster cells must be larger than the cell nucleus.