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Although the evolutionarily acquired role by cellular senescence as a powerful tumour suppressor is well acknowledged, ectopically senescing cells may exert deleterious effects on the tissue microenvironment, the most prominent of which is the so-called senescence-associated secretory phenotype (SASP), by which senescing cells may promote tumour progression. Chromosome aberrations, on the other hand, are the most validated biomarker for cancer risk and provide useful insights on mechanistical modes of ionising radiation action. The recently operational National Centre for Oncological Hadrontherapy (Centro Nazionale di Adroterapia Oncologica, CNAO) is the first Italian facility for cancer treatment with carbon ions. The main justification for the medical use of high –LET radiations rely on their greater effectiveness at tumour cell killing compared to low-LET radiations or lighter charged particles such as protons. Despite encouraging results on local tumour control from other centres worldwide that have adopted this therapeutic strategy, there still exists uncertainties surrounding late effects in the normal tissue. These can be brought about by sublethally damaged cells, such as those that enter senescence prematurely or those carrying transmissible genetic damage, which may lead to organ dysfunction/tissue disruption and secondary cancers. Therefore, we have studied the onset of premature cellular senescence by -galactosidase expression in post-irradiation serially cultivated cells as well as the induction of structural chromosome aberrations detected by FISH techniques in chemically-induced (calyculin A) metaphases 24 h after radiation exposure. Normal endothelial and epithelial cells were exposed to a 6-cm Spread Out Bragg Peak (SOBP) carbon ion beam (E = 246-312 MeV/n) at CNAO. Two doses (0.5 and 2 Gy) were delivered at various depths along the beam, with LET values ranging from 14 keV/m at the entrance to 100 keV/m at the distal SOBP position. Results will be illustrated and their implications for carbon-ion radiotherapy discussed.

The aim of this preliminary investigation was to assess whether human peripheral blood lymphocytes which have been pre-exposed to non-ionizing radiofrequency fields exhibit an adaptive response (AR) by resisting the induction of genetic damage from subsequent exposure to ionizing radiation. Peripheral blood lymphocytes from four healthy donors were stimulated with phytohemagglutinin for 24 h and then exposed for 20 h to 1950 MHz radiofrequency fields (RF, adaptive dose, AD) at an average specific absorption rate of 0.3 W/kg. At 48 h, the cells were subjected to a challenge dose (CD) of 1.0 or 1.5 Gy X-irradiation (XR, challenge dose, CD). After a 72 h total culture period, cells were collected to examine the incidence of micronuclei (MN). There was a significant decrease in the number of MN in lymphocytes exposed to RF + XR (AD + CD) as compared with those subjected to XR alone (CD). These observations thus suggested a RF-induced AR and induction of resistance to subsequent damage from XR. There was variability between the donors in RF-induced AR. The data reported in our earlier investigations also indicated a similar induction of AR in human blood lymphocytes that had been pre-exposed to RF (AD) and subsequently treated with a chemical mutagen, mitomycin C (CD). Since XR and mitomycin-C induce different kinds of lesions in cellular DNA, further studies are required to understand the mechanism(s) involved in the RF-induced adaptive response.

Radiation-induced chromosome aberrations (CAs) are amongst the very few radiobiological endpoints that can provide valuable mechanistic insights in the underlying radiation-matter interaction nanoscale processes. The mosaic of simple- and complex-type rearrangements radiation exposure can give rise to (inter- and/or intra-chromosome exchanges) directly links energy deposition patterns and these biological consequences, known to be of relevance for human health [1]. For this reason, CAs have been extensively studied following radiations of varying quality, i.e. LET. Traditionally, densely ionizing radiation yields CA spectra shifted towards a higher degree of complexity, which is thought to reflect the greater amount of clusterization of DNA damage associated with such radiation type [2]. However, relatively few data exist on the variation of CA induction and type along the Bragg curve for various ions [3]. This is of interest to assess the varying radiobiological effectiveness of the particle along its penetration depth (which may be of relevance for hadrontherapy) as well as to study differences among same-LET ions, i.e. the dependence upon the ion Z, hence track-structure effects. To this end, as part of the INFN-funded research project MIMO-BRAGG, we have collected data on CAs following irradiation at LNS-INFN cyclotron (Catania, Italy) and Centro Nazionale di Adroterapia Oncologica (CNAO, Pavia, Italy) for pristine 18O and 20Ne ion beams and a therapeutic SOBP 12C beam. Results will be shown and tentative conclusions including preliminary Monte-Carlo based simulations will be discussed. [1] Bonassi et al., Cytogenet Genome Res, 104 (2004) [2] Hada et al., Mutat Res., 711 (2011) [3] Manti et al, NIM B, 259 (2007)

Linear Energy Transfer (LET) is the main physical parameter to compare charged particles to photons and predict the higher biological effectiveness of the former radiation. Cytogenetic damage complexity governs cellular radioresponse and reflects the peculiar manner with which energy is deposited by ions as described by the Bragg curve, and is therefore likely to change along ion penetration depth and with its track structure. Hence, ion beams of different Z but similar LETs may differ radiobiologically and construction of ???biological??? Bragg curves may improve modeling of this type of radiation. Moreover, there are still uncertainties on charged-particle non-cancer effects of relevance in cancer hadrontherapy. In fact, most experimental data are almost exclusively on tumour cell lethality and on the Spread-Out Bragg Peak (SOBP), overlooking sub-lethal damage induction on normal cells at various positions along the Bragg curve. Further, very little is known on non-targeted effects (NTE) by ion-irradiated normal cells expressing long-term sub-lethal damage. We studied the onset of Stress-Induced Premature Senescence (SIPS) by ???-galactosidase assay and the induction of chromosome aberrations (CA) by whole-chromosome FISH painting and mFISH along the Bragg curve for several ion beams on three normal cell lines (AG01522 fibroblasts, MCF-10A breast epithelial cells and endothelial HUVEC cells). Occurrence of NTE by medium-transfer design from irradiated prematurely senescing cells was studied at different time points after irradiation on unirradiated MCF10A and breast cancer epithelial MCF7 cells. End-of-track effects of up to 20 MeV 12C and 16O beams were studied at the 3-MV Tandem accelerator, Department of Physics, Naples; 60 MeV/u 16O and 20Ne beams were used at INFN-LNS cyclotron, Catania, to explore changing biological effectiveness along the Bragg curve. Data show SIPS being very effectively induced by ion irradiation, with a qualitative and quantitative dependence on ion type and Bragg curve position, persisting for up to 2 months post exposure. CA data also indicate a similar dependence: the elevated incidence of complex-type rearrangements as revealed by mFISH well agrees with these aberrations being a cytogenetic signature of high-LET radiation. However, the overall incidence of CA and SIPS point to a significantly greater efficiency of ion beams compared to x-rays even at very high LETs, contrary to the notion of a close-to-unity RBE above 200 keV/???m. The onset of SIPS at ion beam entrance may have important implications for hadrontherapy cancer patients. Finally, we observed a significant bystander effect by senescing cells manifesting itself with an increase in tumour cell proliferation, in agreement with in vivo reports of a Senescence-Associated Secretory Phenotype. Monte-Carlo modeling by Geant4 code is under way to correlate ion-track structure with such results.

In this study, we have pre-exposed human peripheral blood lymphocytes to non-ionizing radiofrequency fields (RF, 1950 MHz, UMTS signal, at 0.3 W/kg SAR) and then challenged them with ionizing radiation to examine the induction of adaptive response (AR). The results showed that 20 hours of pre-exposure of the cells to RF resulted in a significant reduction in the frequency of micronuclei induced by 1.0 and 1.5 Gy x-rays. Thus, the data indicated RF-induced AR.

L'efficacia radiobiologica nell'induzione della senescenza cellulare prematura, come effetto subletale di interesse per gli effetti tardivi da esposizione a particelle cariche, è stata studiata in linee cellulari umane normali per vari fasci di ioni accelerati. Esistono pochi dati sperimentali al riguardo; in aggiunta, il Linear Energy Transfer (LET), solitamente utilizzato per predire gli effetti generati dalle particelle cariche, non tiene conto delle possibili dipendenze dallo Z e varia significativamente lungo la profondità di penetrazione dello ione. E' pertanto possibile che l'efficacia nell'induzione di tale effetto vari lungo la curva di Bragg. I risultati preliminari sembrano confermare tale dipendenza.

Gli effetti radiobiologici della radiazione ionizzante di interesse in radioprotezione e adronterapia dipendono fortemente dalla qualità della radiazione. Ad alto LET, gli effetti della struttura di traccia di ioni accelerati giocano un ruolo che non è stato bene caratterizzato sia dal punto di vsita sperimentale che modellistico. Qui presentiamo dati sulla sopravvivenza clonogenica ottenuti esponendo linee cellulari tumorali intorno al picco di Bragg per fasci di ioni 12C, 16O e 19F accelerati al Tandem TTT-3 del Dipartimento di Scienze Fisiche, Università di Napoli Federico II. I risultati saranno discussi in relazione agli attuali modelli RBE-LET.

Several technologies developed by the Italian institute of nuclear physics (INFN) for fundamental physics have been applied to medical imaging and particle therapy techniques. The partnership with leading industries has always been crucial for successful applications. Within this framework is the implementation of a Treatment Planning System (TPS) for hadrontherapy with C-ion beams, but not exclusively, in partnership with the IBA Group, with the contribution of the TPS manufacturer Elekta. Several INFN research groups that have developed competencies in different scientific areas are cooperating to the task: experimental and phenomenological nuclear physics, Monte Carlo (MC) and techniques for numerical analysis, radiobiology and hardware development for monitoring purposes. A TPS prototype is currently being studied. In order to achieve a fast plan optimization, the dose distribution is computed using look-up tables obtained from MC simulations. These are performed using Fluka and an implementation of the Local Effect Model (developed by the GSI Biophysics group). Nuclear fragmentation experiments for C-ion are now performed at the INFN???s Laboratori Nazionali del Sud (LNS) (30-80 MeV/n). Further experiments are scheduled at SIS (GSI) in the framework of the FIRST experiment (200-400 MeV/n), in collaboration with other European Institutes (GSI, ESA, CEA). Radiobiological experiments are underway at LNS and Laboratori Nazionali di Legnaro, on rodent and human cells (C-ion @ 8-80 MeV/n). Lighter ions (A = 6-12) will also be used, to reproduce the effects of fragments (up to 20 MeV/u). These experiments will provide reliable data for the validation of the simulations and for further improvements of the physical and radiobiological models to be used in the TPS.

Stress-induced premature senescence (SIPS) is increasingly attracting interest as a cellular response to generalized sublethal stimuli, which include ionizing radiation (IR). Its timing can be modulated by the nature of the toxic agent while its underlying molecular mechanisms are not clear. Low doses of IR have been shown to induce SIPS in vitro in cell lines such as fibroblasts, this phenomenon being accompanied by premature differentiation. For the first time, here we demonstrate the occurrence of ectopic cellular senescence in human endothelial cells and its correlation with shortening of mean telomere length as assessed by Interphase Quantitative (IQ)-FISH. Carbon ions from the plateau region and from the Spread-Out Bragg Peak (SOBP), of interest for normal tissue and tumour vasculature hadrontherapy effects respectively, were accelerated at GSI (Darmstadt). Clonogenically iso-effective doses of low-LET radiation were used as reference. In our hands, the senescent phenotype was found to occur at early times post irradiation and was effectively induced by doses as low as 0.1-0.5 Gy of carbon ions. A persistent occurrence of senescing cells was found also in SOBP-irradiated cells, although at a lesser extent but despite the higher cell lethality known to be induced in SOBP. Mean length of telomeres, whose temporal stability is critical for maintenance of genomic stability and suppression of carcinogenesis, shows a reduction which is inversely related to LET, suggesting telomere length as a driving mechanism for SIPS after low LET irradiation but of no apparent relevance for carbon ion-induced senescence. These results will be discussed in relation to their implications for normal tissue adverse effects as well as desirable tumor vasculature impairment following hadrontherapy.

Introduction: Endothelial cell and capillaries are particularly sensitive to radiation. The importance of alterations in endothelial cell and capillaries caused by ionizing radiation has been evident since the first observations by researchers. Radiosensitivity of endothelial cell and biological markers of effects such as, particularly, premature cellular senescence assessed by b-galactosidase assay and the behaviour of the capillary loops in subjects radio-exposed recorded with “in vivo capillaroscopy” are the topic of this research. Over the last 20 years there has been a considerable development of the procedures involving risk of exposure to ionising radiation in departments undertaking interventions under radiological monitoring. Materials and Methods: The induction of SIPS (stress-induced premature senescence) and its relationship with telomere length, which drives physiological senescence, were investigated in vitro by exposing a model system (HUVEC or human vein endothelial cells) to the GSI carbon ion beam used for therapeutic purposes, at both the LET values incurred by normal (plateau region) and tumour (SOBP or Spread-Out Bragg Peak) cells. X-ray irradiation was used as reference. The examination in vivo of capillaries was carried out in 16 workers (radiologists and medical staff) aged between 42 and 67 years and length of service between 9 and 22 years exposed to radiation during the procedures of interventional radiology, using a ‘Videocap’ (DS-Medica, Milan) video-capillaroscope with optical probes of 200x. The multiparametric capillaroscopic examination has been integrated with the classification of different profiles with reference to morphology and hemorheology. Results: As early as 2 weeks from irradiation, many more cells stain positive for b-galactosidase in the progeny of irradiated cells than in that from controls. At such early times, doses as low as 0.1-0.5 Gy from the plateau region of the Bragg curve were more effective than 2 Gy SOBP at inducing SIPS. In general, plateau-irradiated HUVECs showed more senescent cells than those from x ray irradiation. A part of senescing cells significantly higher than control was also observed following SOBP irradiation despite the incidence of lethal damage being associated with its higher LET. The protocol adopted in radio-exposed subjects during the procedures of interventional radiology, has shown an augmentation of abnormalities of the capillaroscopic examinations suggesting infraclinic effects of ionising radiation connectible with length of service. Conclusions: Ionising radiation efficiently cause SIPS in the HUVEC model system, exhibiting a dependence upon LET;The senescent phenotype occurs at early times post irradiation and is induced by doses as low as 0.1-0.5 Gy of 12C ions from the plateau region of the Bragg curve;Telomere length attrition may be a driving molecular mechanism for SIPS after low LET-irradiation but is of no apparent relevance for 12C ion-induced senescence. In the medical staff radioexposed, the use of in vivo capillaroscopy seems an effective way of controlling the effects of chronic professional radiation doses accumulated over many years of service and it seems of great interest as a biological indicator of effects for radiological prevention.

La radioprotezione degli astronauti è cruciale per il successo di missioni spaziali di lunga durata come quelle programmate su Marte. La scelta dei materiali per la schermatura della radiazione cosmica va ottimizzata al fine di ridurre il rischio di danno biologico all???equipaggio da essa prodotto. La frammentazione nucleare di ioni di alta energia e alto Z incidenti su tali schermi può infatti modificare la qualità della radiazione. Saranno presentati spettri di frammentazione ottenuti analizzando rivelatori CR-39 esposti a ioni accelerati e schermati con un sistema automatico, sviluppato presso il nostro laboratorio. Particolare cura è stata posta nella misura di coppie di tracce sovrapposte.

This work aimed at measuring cell-killing effectiveness of monoenergetic and Spread-Out Bragg Peak (SOBP) carbon-ion beams in normal and tumour cells with different radiation sensitivity. Clonogenic survival was assayed in normal and tumour human cell lines exhibiting different radiosensitivity to X- or γ-rays following exposure to monoenergetic carbon-ion beams (incident LET 13–303 keV/μm) and at various positions along the ionization curve of a therapeutic carbon-ion beam, corresponding to three dose-averaged LET (LETd) values (40, 50 and 75 keV/μm). Chinese hamster V79 cells were also used. Carbon-ioneffectiveness for cell inactivation generally increased with LET for monoenergetic beams, with the largest gain in cell-killing obtained in the cells most radioresistant to X- or γ-rays. Such an increased effectiveness in cells less responsive to low LET radiation was found also for SOBP irradiation, but the latter was less effective compared with monoenergetic ion beams of the same LET. Our data show the superior effectiveness for cell-killing exhibited by carbon-ion beams compared to lower LET radiation, particularly in tumour cells radioresistant to X- or γ-rays, hence the advantage of using such beams in radiotherapy. The observed lower effectiveness of SOBP irradiation compared to monoenergetic carbon beam irradiation argues against the radiobiological equivalence between dose-averaged LET in a point in the SOBP and the corresponding monoenergetic beams.

Among the most important obstacle to long-term space exploration by human beings there is the health risk due to the exposure to high charge and energy ions present in the galactic cosmic rays. The shielding is the simplest solution to protect the crews but the nuclear fragmentation events modify the radiation field. In recent years alternative materials are under investigation in order to enhance the shielding properties: therefore is crucial to measure the interaction cross section because biological effects are related to the radiation quality. In this work we report the measurement of the total charge changing cross section of 1 A GeV iron ions on different targets of interest in space radiation protection. An automatic system, realized in our laboratory, was used to analyze the CR-39 detectors. The system comprises an inverted microscope with motorized stage, a CCD camera, a motion control device and the computer code TRANA written in Labview language. The code uses the Danielsson algorithm for track recognition and determines the geometrical parameters and the central brightness of the tracks. The analysis of an image (about 200 X 300 micron^2) takes 1-3 seconds, depending on the number of observed tracks. Special care is taken for couples of overlapping tracks.

Complicazioni nel tessuto sano sono inevitabili in radioterapia. Nonostante la loro probabilità si riduca usando ioni carbonio invece dei fotoni, non è noto se la maggiore efficacia biologica dei primi in termini di inattivazione clonogenica si mantenga anche per il danno alla progenie cellulare. Mentre la senescenza cellulare prematura insorge in risposta a vari stress citotossici, non se ne conoscono meccanismi e dipendenza dalla qualità della radiazione. Riportiamo dati sulla senescenza indotta in un sistema modello (HUVEC) da un fascio di ioni carbonio usato a fini terapeutici (GSI, Germania) e da raggi X, rapportandoli a misure della lunghezza telomerica media.

Al fine di garantire un'efficace Radioprotezione degli astronauti, è necessario studiare come la radiazione cosmica interagisce con nuovi materiali che stanno trovando ampie applicazioni nella costruzione di moduli gonfiabili per i veicoli o le stazioni spaziali, in quanto non vengono danneggiati a seguito di eventuali impatti con meteoriti. Prove sull'efficacia di questi materiali, dal punto di vista radioprotezionistico sono in corso sulla Stazione Spaziale Internazionale e sono state effettuate anche presso il Brookhaven National Laboratory per poter valutare la dose dovuta a particelle HZE, poiché nello spazio il campo è misto e le sue componenti sono quindi difficilmente separabili.

Gli effetti biologici tardivi della radiazione ionizzante sono di interesse per i danni al tessuto sano derivanti dal trattamento radioterapico. Ioni come il carbonio risultano in una dose a queste cellule molto ridotta rispetto ai fotoni, ma la maggiore efficacia di queste particelle nel causare morte clonogenica, essenziale per eradicare tumori radioresistenti, potrebbe tradursi in un???efficacia altrettanto elevata anche per altre forme di danno radioindotto. In vitro la senescenza è inducibile da vari agenti: in questi casi essa non appare come stadio fisiologico che prelude all???esaurimento del potenziale proliferativo e alla differenziazione terminale della cellula, ma si configura come vero e proprio meccanismo alternativo a morte e trasformazione neoplastica. Tuttavia, l???accumularsi di tale fenotipo in vivo può compromettere la funzionalità del tessuto interessato e, in ultima analisi, favorire l???insorgenza di tumori secondari attraverso la destabilizzazione del genoma. Vanno quindi compresi dipendenza di tale risposta dalla qualità della radiazione (ioni vs. fotoni) e meccanismi (ruolo degli oncogeni vs. telomeri). Abbiamo studiato la senescenza nel sistema modello endoteliale HUVEC per un fascio di carbonio usato per radioterapia (GSI, Germania). In parallelo, è stata misurata la lunghezza media telomerica dalle intensità di fluorescenza di un marcatore pantelomerico e uno centromerico. Raggi X hanno funto da radiazione di riferimento. I dati fin qui raccolti indicano che la progenie delle cellule esposte entra in senescenza prima di quanto facciano colture non irraggiate, con una complessa dipendenza sia dal LET che dalla riduzione di lunghezza telomerica.

La radioterapia intraoperatoria (IORT) è una recente modalità di trattamento che prevede la somministrazione di una singola dose di elettroni in sede di intervento chirurgico. Le condizioni di irraggiamento prevedono ratei di dose e di dose per impulso molto più alti rispetto alle radioterapia convenzionale con LINAC (200 e 20 volte maggiori, rispettivamente), con nuovi problemi dosimetrici e radiobiologici da affrontare. Cellule di carcinoma mammario sono state irraggiate con elettroni prodotti dall???acceleratore per IORT dell???azienda Ospedaliero-Universitaria di Pisa e con raggi X presso l???Università di Napoli Federico II per la misura della sopravvivenza clonogenica e la valutazione dell???efficacia biologica relativa.

Shielding is the only countermeasure currently available for exposure to cosmic radiation during space travel. We compared aluminum (Al) and polymethylmethacrylate (PMMA, or lucite) shields of 20 g/cm^2 thickness using 1 GeV protons accelerated at the NASA Space Radiation Laboratory. The dose rate increased after the shield, and the increase was more pronounced after the Al than the PMMA shield. No significant differences in the induction of chromosomal aberrations were observed in human lymphocytes exposed to the same dose with no shield or behind the Al and PMMA blocks. However, the biological effectiveness per incident proton was increased by the shields. Simulations using the General-Purpose Particle and Heavy-Ion Transport Code System (PHITS) show that the increase in dose is caused by target fragments, and aluminum produces more secondary protons than PMMA. Nevertheless, the spectrum of particles behind the shield is confined within the low-LET region, and the biological effectiveness is consequently similar.

Computer code calculations based on biophysical models are commonly used to evaluate the effectiveness of shielding in reducing the biological damage caused by cosmic radiation in space flights. Biological measurements are urgently needed to benchmark the codes. We have measured the induction of chromosomal aberrations in human peripheral blood lymphocytes exposed in vitro to 56Fe-ion beams accelerated at the HIMAC synchrotron in Chiba. Isolated lymphocytes were exposed to the 500 MeV/n iron beam (dose range 0.1-1 Gy) after traversal of 0 to 8 g/cm2 of either PMMA (lucite, a common plastic material) or aluminum. Three PMMA shield thickness and one Al shield thickness were used. For comparison, cells were exposed to 200 MeV/n iron ions and to X-rays. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid cell-cycle selection produced by the exposure to high-LET heavy ion beams. Aberrations were scored in chromosomes 1, 2, and 4 following fluorescence in situ hybridization. The yield of chromosomal aberrations per unit dose at the sample position was poorly dependent on the shield thickness and material. However, the yield of aberrations per unit ion incident on the shield was increased by the shielding. This increase is associated to the increased dose-rate measured behind the shield as compared to the direct beam. These preliminary results prove that shielding can increase the effectiveness of heavy ions, and the damage is dependent upon shield thickness and material, and heavy ion energy and charge.