EUROPEAN RADIATION RESEARCH 2012

15 Oct 2012, 08:00 → 19 Oct 2012, 18:00 Europe/Rome

Vietri sul Mare

Monday, 15 October

Registration

Opening address

Bacq & Alexander Lecture

1 Health risks of low doses of ionizing radiation: Looking for the undetectable?

Some parts from my winding path in the field of radiation biology and radiation protection research will be summarized. My initial focus was on high doses, where the target of interest, most often was DNA but in recent years it has moved to doses in the mGy range, where emphasis has been on changes in cellular metabolic pathways. Our special interest has been to search for biomarkers of exposure and of radiosensitivity which may help to understand the mechanisms of responses. A mechanistic understanding of the cellular responses to doses in the mGy range will be needed to bridge the lack of hard facts for risk estimates for cancer and non-cancer diseases obtained from epidemiological investigations to the mGy range and the requirement to come up with practical risk estimates. A challenge of similar magnitude is to grasp the importance of the dose rate effect. We are presently exploring in cellular model systems, the possibilities to reduce the dose rates from the commonly used Gy/min to a few µGy/min. The goals here are to look for dose rates where we can see transitions in the cellular responses at the level of proteomics and epigenetics and possibly relate these changes to a mechanistic view on the cellular responses. Keeping in mind that a dose rate of µGy/min is about 5 105 times higher than the normal background is indicative of the nature of the problem. Although the challenge for radiation biology research to provide more precise knowledge about health risks of low doses and dose rates is great, the new tools in molecular biology and the application of system biology approaches will have significant impact on our understanding of risks to humans and the environment.

Speaker: Dr Mats Harms-Ringdahl (Stockholm University, Sweden)

19:00 Welcome Reception

Tuesday, 16 October

Keynote Lecture 1

2 Models of Cancer Risks and Uncertainties for Radiation Exposures

Models of radiation carcinogenesis risks from terrestrial radiation or galactic cosmic rays (GCR) and solar particle events (SPE) in space are impacted by various uncertainties related to radiation quality, dose and dose-rate response models, and individual sensitivity. We describe NASA’s approaches to these problems. The use of age and gender specific models with uncertainty assessments based on human epidemiology data for low LET radiation combined with relative biological effectiveness factors (RBEs) and dose- and dose-rate reduction effectiveness factors (DDREF) to extrapolate these results to space radiation exposures are considered the current “state-of-the-art” [1]. The NASA Space Cancer Risk (NSCR) model (2010) [2-3] is based on recent radio-epidemiology data, however a key feature of the NASA model is the formulation of particle fluence and track structure based radiation quality factors for solid cancer and leukemia risk estimates, which are distinct from the ICRP quality factors, and shown to lead to smaller uncertainties in risk estimates. A new Bayesian analysis of uncertainties in the DDREF are summarized in our report. Many persons exposed to radiation on earth as well as astronauts are life-time never-smokers, which is estimated to significantly reduce radiation cancer risks. A key feature of the NASA radiation protection model is the classification of radiation workers by smoking history in setting dose limits. A major challenge to the current approach to cancer risk estimates are Non-Targeted Effects (NTE), which include bystander effects and genomic instability, which has been observed in cell and animal models of cancer risks. NTE’s could lead to significant changes in RBE and DDREF estimates for GCR particles, and distinct risk factors related to individual sensitivity and the potential effectiveness of radiation mitigator’s. We discuss methods to include NTE’s in uncertainty analysis and to design experiments to provide quantitative data for cancer risk assessment models. NSCR-2010 was reviewed by the National Research Council (2012) [1], and a revision to the NASA model, NSCR-2012 that addresses the recommendations from the NRC report. 1. NRC, The National Academy Press, Wash DC, 2012. 2. Cucinotta FA, Chappell LJ, and Kim MY. Space Radiation Cancer Risk Projections and Uncertainties-2010. NASA TP 2011-216155 3. Cucinotta FA et al. Radiat Res 176, 102-114, 2011; Health Phys (in press).

Speaker: Dr Francis Cucinotta (NASA)

Biological and Physical Dosimetry

3 MULTIBIODOSE: multi-disciplinary biodosimetric tools to manage high scale radiological casualties

In the event of a large scale radiological emergency biological dosimetry is an essential tool that can provide timely assessment of radiation exposure to the general population and enable the identification of those exposed people, who should receive immediate medical treatment. A number of biodosimetric tools are potentially available, but they must be adapted and tested for a large-scale emergency scenario. These methods differ in their specificity and sensitivity to radiation, the stability of signal and speed of performance. A large scale radiological emergency can take different forms. Based on the emergency scenario different biodosimetric tools should be applied so that the dosimetric information can be made available with optimal speed and precision. The aim of this multi-disciplinary collaborative project is to analyse a variety of biodosimetric tools and adapt them to different mass casualty scenarios. The following biodosimetric tools will be established, improved and/or validated: the dicentric assay, the micronucleus assay, the gamma-H2AX assay, the skin speckle assay and the blood serum protein expression assay. In addition EPR/OSL dosimetry in components of pocket electronic devises is investigated. The assays were chosen because they complement each other with respect to sensitivity, specificity to radiation and the exposure scenario as well as speed of performance. The project involves the key European players with extensive experience in biological dosimetry. Training is carried out and automation and commercialisation pursued. An operational guide will be developed and disseminated among emergency preparedness and radiation protection organisations. The final deliverable of this project will be establishment of a biodosimetric network that is fully functional and ready to respond in case of a mass casualty situation. Thus, the project will strengthen the European security capabilities by achieving tangible results. The project is funded by the FP7 Security program. URL: http://www.multibiodose.eu.

Speaker: Prof. Andrzej Wojcik (Stockholm University)

10:30 Coffee break

4 Synthetic diamond dosimetry

Diamond dosimetry has become attractive in radiotherapy, in particular since the introduction of high conformal radiotherapy modalities. With the modern techniques, patients are treated with non-uniform fields, with high dose gradients, variation in space and time of the dose rate and of the beam energy spectrum; the charged particle equilibrium condition is seldom fulfilled, and the perturbation introduced by the detector on the particle fluence strongly affects the measurement. One of the most relevant requirements for a dosimeter is to have small dimensions and high sensitivity. These characteristics should be added to the standard features of a good detector in radiotherapy: short- and long-term stability, linearity versus absorbed dose and dose rate, no energy or LET dependence, as well as no angular dependence of the response. What is more: for treatments characterized by time variation of the fluence, a fast response dynamics is a strong constraint to be met. An ideal dosimeter that fulfills all the above requirements does not exist; diamond, however, thanks to its material properties, seems quite close to satisfying them. Studies about natural diamonds for medical applications are found in the literature since ‘80s. All the existing papers highlight the favorable characteristics of diamond, but its use is limited because of the high cost of the detector. The use of diamond in dosimetry became more appealing when it was proven that synthetic diamonds could be grown in a reproducible way and at a low cost. However, for several years, the clinical use of polycrystalline (p) Chemical Vapour Deposited (CVD) diamond was hampered by its poor response stability. A way to reduce the effect of native defects on response stability is to use single-crystal diamond films. Today, single crystal (s) CVD samples of good quality are available and they can be successfully used also in IMRT applications. In addition to the quality of the material, other factors contribute to determining the performance of a dosimeter: encapsulation, electrical contacts, polarization voltage. Indeed, pCVD detectors used at zero bias, demonstrate an improvement of the dosimetric characteristics, also in IMRT and their performances can be close to those of sCVD diamonds. It is worth of notice that a pCVD diamond with suitable dosimetric characteristics opens up the possibility of developing a bi-dimensional device, useful for dose distribution verification in IMRT treatments.

Speaker: Prof. Marta Bucciolini (University of Florence Italy)

5 Radiation exposure in paediatric interventional cardiology in France

Children with congenital heart disease frequently undergo interventional cardiology procedures for diagnosis or intervention purposes. The complexity of these procedures may result in high radiation exposures. Given the high radiosensitivity of children, and their longer life span to develop radiation-health effects, an epidemiological cohort study is being launched in France in order to evaluate leukaemia and cancer risks among this specific population. In this context, precise retrospective evaluation of doses received by the children during paediatric interventional cardiology is needed. The study population includes all children who underwent at least one interventional cardiology procedure since 2000, who were under 16 years old at the time of the procedure. Up to now, 4500 children have been already included in the cohort but recruitment is still ongoing. On average, each child has undergone 1.3 interventional cardiology procedures, for a total of over 5,000 procedures. Nearly half of these were performed during the first year of life of the children involved. Dosimetric information have been collected and assessed over the period 2010-2011. Exposure parameters (dose x area product, fluoroscopy time and number of images) were retrieved from the dose-recording system and were tabulated and statistically analysed. The doses received at different organs were also evaluated using anthropomorphic phantoms (newborn, 1 year, 5 year, 10 year and 15 year old children) in which are inserted thermoluminescent dosimeters. Organ doses were calculated using software PCXMC 2.0, for different weight of the child at the time of the procedure. Finally, Dose Reference Levels will be established and effective doses will be calculated. Results will be presented according to different types of interventional cardiology procedures (diagnostic and therapeutic) and for different sub-categories of procedures, providing new information on typical levels of doses for paediatric interventional cardiology procedures in France.

Speaker: Dr Helene BAYSSON (IRSN)

6 Linking the Human Response to Unplanned Radiation and Treatment to the Nonhuman Primate Response to Controlled Radiation and Treatment: A New Approach to Compare Human and Animal Data

A new project funded by the U.S.National Institute of Allergy and Infectious Diseases, Radiation Nuclear Countermeasure program has been launched to link human data from radiation accidents to animal data resulting from controlled radiation studies in nonhuman primate (NHP) models. The aim of this project is to gain more insight into similarities and disparities concerning the pathophysiology of the acute radiation syndrome (ARS). A key objective is to determine whether the developmental sequence of the early signs and symptoms of ARS runs parallel in both systems. Other issues will focus on the similar consequences of acute and delayed radiation effects. Radiation-induced multi-organ failure as described in humans will be compared with that observed in the NHP models. We have initiated a comparison of radiation exposure dose versus signs, symptoms and consequent organ injury as a function of time in both humans and NHP. Materials& Methods: The human data derive from the Ulm SEARCH database (System for Evaluation and Archiving of Radiation Accidents based on Case Histories) which contains more than 800 case histories of radiation victims since 1945. The animal data are being provided by the NIAID-sponsored Preclinical Radiobiology lab at the University of Maryland. It is planned to build an”Interspecies Bridge”. Exposed individuals and NHP will be assigned to comparable categories for clinical severity and response categories (RCs) according to the METREPOL concept, where applicable. This will be done in an adapted mode for the animal model. A second step will focus on a comparative analysis of signs and symptoms within the RCs. Results: The method for”bridging“ human and NHP data proved to be successful. The initial results of this interspecies comparison and dose shift modeling are presented. Discussion: A validated interspecies comparison of relevant radiation dose effects and clinical data will be a sound approach to approve the use of radiation countermeasures in humans on the basis of relevant and controlled animal data.This interspecies approach should be expanded to include other animal species and models currently in use by the scientific radiation research community. This work is supported by NIAID contract HHSN272202000046C.

Speaker: Prof. Viktor Meineke (Bundeswehr Institute of Radiobiology affiliated to the University of Ulm)

7 The effect of KOH on Electron Paramagnetic Resonance signals measured in irradiated bone.

Electron Paramagnetic Resonance (EPR) dosimetry is based on ex vivo detection of radicals generated by ionizing radiation such tissues as tooth enamel and bone. This method was proved to be applicable in dosimetry of victims in radiation accidents. However, procedures regarding technical conditions of a reliable dosimetry in bone are still under study. The spectra of irradiated bones consist of an asymmetric component assigned to CO2- radicals generated in hydroxyapatite and a second one, symmetrical component, the origin of which is assigned to the bone’s organic content. The intensity of the asymmetric component was shown to be stable in time in both dry and water-soaked samples and is used for dosimetry. The aim of this study was to investigate the possibility of reduction of the “non-dosimetric” spectral components in irradiated human bone by chemical treatment and determination of consequences of this treatment on the dosimetric signal. The EPR study were performed on samples treated by 2M KOH and intensities of the two spectral components, numerically isolated from the measured EPR spectra, were calculated. The KOH hydrolysis of an irradiated bone resulted in a significant decrease of the “organic” spectral component and caused 25-30% reduction in the sample mass - apparently due to loss of organic bone constituents, which make about 30% of cortical bone. The signal measured in the deproteinized bone consisted only from the asymmetric signal of high stability. The intensity of this component per unit mass of the sample was about 6 % lower and its peak-to-peak amplitude was 7% higher than before the hydrolysis. The KOH treatment resulted in about 30-35 % loss in intensity of the signal attributed to CO2- radicals, however, the remaining dosimetric signal was not masked by the symmetric spectral component. It is concluded, that the KOH hydrolysis of irradiated bone considerably reduces the spectral components obscuring the dosimetric signal and can allow for increased accuracy of dosimetry in a low dose region.

Speaker: Mrs Karolina Krefft (Department of Physics and Biophysics, Medical University of Gdansk, Debinki 1, 80-211 Gdansk, Poland)

8 Gene expression and cYtokine Monitoring for Biodosimetry and RAdiation Sensitivity Screening (GYMBRASS)

One of the major hurdles for manned interplanetary space exploration is the increased cancer risk due to long-term exposure to cosmic radiation and solar particle events. However, besides cancer, also other adverse health effects may arise in astronauts as a result of exposure to cosmic radiation. In order to better understand and define the biological risks of cosmic radiation, it is important to identify biomarkers for exposure and for predicting individual sensitivity to radiation-induced biological damage. Based on preliminary experiments in which human peripheral blood mononuclear cells (PBMCs) from 10 different donors were X-irradiated (Pentak HF420 RX machine; 250 keV, 15 mA; dose rate= 0,25 Gy/min) with doses of 0 (control), 0.1 and 1 Gy, we have found evidence that gene expression signatures can be used to predict exposure to doses as low as 0.1 Gy, and possibly to even lower doses. Furthermore, our data show that the use of single exon expression signatures further enhances the sensitivity of the prediction analysis. Both at low and high doses, mainly genes involved in p53-related pathways (cell cycle arrest, DNA repair, apoptosis) were affected, although most of these genes showed a clear dose-dependent response. Finally, our data show that several genes expressed different transcript variants after irradiation. Some of these genes showed marked differences between different donors, indicating that the regulation of radiation-induced transcript variation may be important for the individual response to radiation. In our project we aim at identifying biomarkers (genes, exons, secreted proteins) for exposure of PBMCs to low (0.05 and 0.1 Gy) and moderate (1 Gy) doses of high-LET (linear energy transfer) heavy ions such as can be encountered in cosmic radiation. Concomitantly, the individual radiosensitivity of blood donors will be assessed using -H2AX staining. These data will then be integrated with the results from microarrays and protein assays (multiplex array system) to identify markers for individual radiosensitivity screening. All results will be compared to similar doses of low-LET X-ray irradiations in order to obtain more insight into the differences of the biological effects of different radiation qualities. Acknowledgements: Work supported by Belspo, PRODEX-9 project "MOSAIC-2 (42-000-90-380)".

Speaker: Prof. Sarah Baatout (SCK-CEN, Mol, Belgium)

Early Biophysical Processes

Convener: Alessandro Campa (ISS)

9 Geant4 calculations of microdosimetric variables for neutrons and light nuclei in tissue-like materials

Microdosimetry measurements with Tissue Equivalent Proportional Counters (TEPC) are routinely used to characterize the quality of radiation by obtaining distributions of lineal energy y. Radiation fields of therapeutic pencil-like nuclear beams in tissue-like phantoms are complex due to the admixture of secondary protons, neutrons and nuclei produced in fragmentation reactions [1]. We demonstrate the ability of our Monte Carlo model for Heavy-Ion Therapy (MCHIT) based on the Geant4 toolkit [2] to describe microdosimetry spectra measured in a water phantom irradiated by carbon nuclei [3]. Events of energy deposition to a TEPC sensitive volume are characterized by certain topology depending on the position of a TEPC in the phantom with respect to the primary beam. As demonstrated by calculations, microdosimetric spectra are defined by beam nuclei and nuclear fragments inside and close to the beam and by neutrons and protons far from it. Following the validation of MCHIT with microdosimetry spectra measured with quasimonoenergetic neutrons [4], we evaluate the contribution of secondary neutrons to the microdosimetry spectra in the water phantom irradiated by carbon nuclei. The equivalence of the microdosimetry spectrum measured with a macroscopic TEPC gas volume to one corresponding to a micrometer-size tissue volume is demonstrated by calculations. References: [1] I.A. Pshenichnov et al., Nucl. Inst. Meth. Phys. Research B 268 (2010) 604 [2] J. Allison et al., IEEE Transact. Nucl. Sci. 53 (2006) 270 [3] G. Martino et al., Phys. Med. Biol. 55 (2010) 3441 [4] Y. Nakane and Y. Sakamoto, Nucl. Instr. Meth. A 459 (2001) 552

Speaker: Dr Igor Pshenichnov (Institute for Nuclear Research, Russian Academy of Sciences and Frankfurt Institute for Advanced Studies, Goethe University)

10:30 Coffee break

10 Mechanistic modelling of radiation track structures, DNA damage induction and repair with PARTRAC

Mechanistic modelling of biological effects of ionizing radiation is an important tool complementary to experimental studies that allows testing hypotheses on radiation action and response mechanisms for improving our quantitative understanding of the underlying principles and systems. It helps analyse and interpret experimental observations and plays a crucial role in inter- and extrapolating data, frequently in a non-linear manner, beyond the limits of experimental protocols. PARTRAC is a state-of-the-art tool for Monte Carlo-based simulations of track structures, radiation-induced damage to DNA, and repair of the induced DSB via NHEJ [1]. The DNA repair module [2] describes stochastically the hierarchical action of repair enzymes processing the DNA ends, explicitly considering their spatial distribution and complexity in terms of nearby lesions. Experimental data on DSB and DNA fragmentation induced by photon and ion irradiations have been reproduced by PARTRAC calculations that account for the experimental DNA fragment length intervals and the adopted data analysis algorithms. For high-LET radiation the calculations predict significant yields of DSB associated with short DNA fragments below the threshold of detected fragment sizes. Recently, the non-linear DNA fragmentation measured at LBL after X-ray, N- and Fe-ion irradiation [3] has been compared to PARTRAC calculations in detail for 10-12 size intervals between 0.1 and 1120 kbp. Likewise the DNA repair module has been successfully adapted to measured LET-dependent repair kinetics after gamma-rays and different N-ion radiation qualities [4], relating differences in repair kinetics to variations in complexity of damage induced by radiations of diverse qualities. Recently, an alternative hypothesis, linking the DNA damage response with heterochromatic and euchromatic regions within the cell nucleus, has been suggested [5-6]. The methodology, assumptions and first modelling results obtained when including this structural level into the suite of PARTRAC tools will be presented in view of corresponding experimental data. Acknowledgement: Partially supported by the NoE “DoReMi” funded by EC. References [1] Friedland et al, Mutat Res 711, 28 (2011) [2] Friedland et al, Radiat Res 173, 77 (2010) [3] Löbrich et al, Int J Radiat Biol 70, 493 (1996) [4] Friedland et al, Int J Radiat Biol 88, 129 (2012) [5] Goodarzi et al, DNA Repair 9, 1273 (2010) [6] Jakob et al, Nucl Acids Res 39, 6489 (2011)

Speaker: Dr Werner Friedland (Helmholtz Zentrum München, Institute of Radiation Protection)

11 A dynamic radio-aerosol deposition and clearance model for the quantification of radiation burden and biological effects in the central airways

Elucidation of health effects of low dose radiation exposures is one of the most challenging issues of current radiation biology. Establishment of plausible dose-effect relationship for the range of low doses is hampered by numerous factors. Irradiation of humans for experimental purposes has serious ethical barriers. Animal experiments are also restricted. In these circumstances epidemiology, in vitro cell/tissue irradiation experiments and modelling are the suitable and complementary tools of radiation research. In this work, a numerical model has been developed in order to simulate the simultaneous action of particle deposition and clearance in the central airways. Particle deposition within a characteristic airway bifurcation (generations 4-5) was studied by computational fluid and particle dynamics methods. Clearance was modelled by determining the trajectories of particles trapped and propelled by mucus. Sources of radioisotopes were the deposition sites in the bifurcation and the radioactive particles cleared up from the deeper regions of the airways. These isotopes may decay while travelling through the studied bifurcation. Alpha decay of short lived radon progenies has been modelled by Monte Carlo techniques. Alpha tracks were generated. Their origins were the locations of the decays, while their direction was randomly selected. The 3D bronchial epithelium has been digitally reconstructed. The radiosensitive epithelial cells had ellipsoidal shapes. Cellular and cell nucleus doses were determined based on the intersections of alpha-tracks with the cells. Cell killing and transformation were determined based on the computed cell nucleus doses. The model has been applied for exposure conditions characteristic of homes. Based on our simulations the deposition of radioisotopes is highly inhomogeneous. Clearance is also nonuniform. Mucus velocities can be two orders of magnitude lower in the close vicinity of bifurcation peak resulting in delayed clearance of particles deposited nearby. The results demonstrate that the contribution of upclearing particles to the total activity in a central airways bifurcation is similar to that of the primarily deposited particles. Furthermore, decay patterns are less inhomogeneous than the deposition patterns. Cell inactivation and cell transformation curves seem to be linear, at least for the modeled very low doses. Present results may serve as inputs for complex radiation induced cancer models.

Speaker: Dr Árpád FARKAS (Centre for Energy Research, Hungarian Academy of Sciences)

12 Human mummification process and their impact on cell radiosensitivity: Computed Tomography model of Monte Carlo simulation

Johann Wanek 1,2, R. Speller 2, G. Royle 2, F. Rühli 1 1: University of Zürich, Centre for Evolutionary Medicine, Institute of Anatomy, Switzerland 2: University College London, Dept of Medical Physics and Bioengineering, United Kingdom Human tissue undergoes dramatic physicochemical changes as well as morphological alterations during the mummification process such as decreased cell volume and spacing. The exposure of ancient remains to radiation undergoing e.g. computed tomography imaging, is often accompanied by uncertainties regarding damage to ancient DNA in the ancient tissue. Consequently, anthropologists and forensic scientists are highly interested to know the probability of DNA destruction due to X-ray imaging. Up to the present, there have been no attempts to quantify the contribution of the direct effects of sparsely ionizing radiation on desiccated cells embedded in different phantom tissues. The aim of our studies is to investigate the impact of geometry and molecular weight variations on the radiation sensitivity of cells following mummification. A computed tomography model shall be presented for the irradiation of cell nuclei distributed by self-avoiding random walks in 3D (SAW). The results were calculated for phantom nuclei embedded in a cylinder composed of normal tissue phantom (NT) representing wet muscle tissue, dry muscle tissue (DT) representing dehydrated muscle tissue phantom or cortical bone (CB) phantom. Using Geant4 low energy extension, computed tomography (CT) scans were simulated in the energy range from 20 to 120 keV at a photon fluence of 1.1x1010 photons/cm2 and a sample rotation of 10 deg/step. A cluster of 81 ellipsoids with a dimension of 6x6x10 μm or 4x4x6 μm were placed at the isocenter of the CT model. The absorbed energy and the number of unaffected cells were calculated from protein phantoms. This study clearly showed that the number of unaffected cells increased from 37 to 69 % by changing the surrounding tissue phantom from NT to DT because of the reduced cell volume and molecular weight. These results are in close agreement with Hutchinson's observations that the radiation sensitivity of dry biological molecules can be estimated by their molecular weight. Reference: Hutchinson, F. Radiation inactivation of molecules in cells. The American Naturalist, Vol. 94, No. 874 (Jan.-Feb., 1960), pp. 59-70

Speaker: Mr Johann Wanek (University of Zurich, Centre of Evolutionary Medicine, Institute of Anatomy)

13 Geant4 implementation of an RBE calculation for ion therapy studies

Monte Carlo approach in hadrontherapy is widely used for the optimization of transport beam lines and/or dosimetric studies. In most of cases, in the clinical practice, depth dose distributions and lateral dose profiles are provided, without taking into account the biological effects of ionizing radiation. In case of ion beams, a mixed radiation field is produced, due to projectile fragmentation, and the biological effects related to the different secondary particles have to be correctly weighted with those related to the primary particles. It is so evident as, in such complex cases, Monte Carlo calculations for ion therapy may provide physical and RBE-weighted dose distributions in order to be routinely used in clinical practice. A general Monte Carlo tool for calculations in hadrontherapy has been developed. The application, whose name is “Hadrontherapy”, is based on the Geant4 toolkit, and it is included in the official Geant4 release inside the so called “advanced examples”. A new class has been created inside the Hadrontherapy application, with the scope to create an interface to the Local Effect Model (LEM), developed at GSI by M. Scholz and co-workers. Starting from some basic assumptions, LEM model is able to predict the biological action of different charged particle beams by considering their specific track structure and the different spatial energy distribution at the cell level. This class-interface has the aim of managing the output data of the LEM model, provided by the INFN group of Turin who deeply work in this concern since many years. These output data are integrated run-time with step-by-step information retrieved by the Monte Carlo codes. At the end of a simulation run, α and β parameters of the cell survival curve are obtained as a function of the penetration depth in water in a simple “sliced” geometry. These two parameters are averaged according to Zaider and Rossi formula, tacking into account in realistic way also the contribution due to the secondary particles produced by nuclear interactions of the primary beam. Moreover, RBE and biological dose are also stored in ascii files. Preliminary results will be shown, compared to experimental data of survival fraction for CHO cells, related to carbon ions at different entrance energies. Future developments are planned regarding the implementation of the recent versions of LEM model as well as other models with a different approach (MKM).

Speaker: Dr Francesco Romano (INFN Laboratori Nazionali del Sud, Catania, Italy - Museo Storico della Fisica e Centro Studi e Ricerche “E. Fermi”, Roma, Italy)

12:30 Lunch

DNA Damage and Repair

14 Investigating DNA repair centers in mammalian cells and their impact on dose-response linearity

We recently showed in non-malignant human breast cells (MCF10A) that at doses of X-ray above or equal to 1 Gy, radiation-induced foci (RIF) do not increase linearly with dose. RIFs were characterized by the local recruitment of DNA damage sensing proteins p53 binding protein (53BP1). Using a mathematical model of RIF kinetics we also showed that RIF induction rate increased with increasing radiation dose, whereas the rate at which RIFs disappear decreased. In addition, live cell imaging showed that RIF do not all appear synchronously after exposure, clearly demonstrating that counting RIF at a given time point only reflects a net number of damages. By integrating RIF kinetics over time one can infer the total number of RIF generated at a given dose. Mathematically correcting for the dose dependence of induction/resolution rates, we observed an absolute RIF yield that was surprisingly much smaller at higher doses: 15 RIF/Gy after 2 Gy exposure compared to ~64 RIF/Gy, after 0.1 Gy. Cumulative RIF counts from time lapse of 53BP1-GFP in human breast cells confirmed these results. We hypothesized that DNA damage was clustering into common regions of the nucleus more suited for DNA repair. Data collected so far suggest these ‘repair centers’ would be located into euchromatic regions of the nucleus with an average spacing of 1 to 2 μm. In this new work, we are now presenting RIF dose-kinetics from other mammalian cell lines (primary lines from humans and mice, immortal lines grown in 3D) to evaluate the universality of DNA damage clustering. We also introduce a new microfluidics approach integrated with an X-ray milli-beam on a fully automated fluorescent microscopy platform to accelerate RIF dose-kinetics quantification in an unbiased manner. Since mice cells have much better-defined heterochromatic domains, they are used primarily to test the impact of heterochromatin on DNA damage clustering. Cell-type response specificity is tested by having human breast cells grown on various extra-cellular matrix substrates, leading to specific cell differentiation. Our overarching goal is to characterize for low and high doses, RIF kinetics across species and within species and correlate RIF kinetics parameters and RIF frequencies to persistent DNA damage.

Speaker: Dr Sylvain V. Costes (Lawrence Berkeley National Laboratory, Berkeley, USA)

15 Low dose IR induces gene expression changes via DNA breaks and other pathways

The effects of low and protracted doses of ionizing radiation on humans are only partially understood. The shape of the dose response curve at low and protracted doses cannot be measured accurately using standard epidemiological methods. However, this information is needed for rationale policy decisions for setting exposure limits. Therefore, we set out to determine a gene expression signature that operates at low and intermediate doses using a panel of DNA double strand break repair deficient mouse models. We developed mouse models that lacked one or both of the major DNA double strand break repair pathways (nonhomologous end-joining and homologous recombination) and compared transcriptional responses in these DNA repair deficient mice with normal mice before and after irradiation with a relatively low dose of gamma radiation (200 mGy). Importantly, the changes in gene expression patterns were nearly identical in repair-deficient mice and in irradiated mice. We identified a gene expression signature that was very similar, but considerably more pronounced (up to 5-fold) in irradiated DNA repair deficient mice compared to normal mice, showing that the transcriptional responses are indeed caused by the DNA damage inflicted by radiation and that DNA repair deficient mice can be used as the proverbial ‘canary in the coalmine’ in the context of low dose radiation research. The identified gene expression signature includes Gadd45a, Gad45b, MDM2, BAG3, and CDKN1A (p21) and in addition allowed the identification of additional genes by correlation analysis. A distinct set of genes was consistently upregulated after low dose IR, but this did not change with DSB repair defects, suggesting that this part of the transcriptional responses is caused by other triggers than DSBs. We conclude that this transcriptional response can be used as a very sensitive read out for low levels of radiation exposure. This will be meaningful, as several genes from this signature have functions in known pathways that regulate cell proliferation and/or senescence and apoptosis, which are all relevant for carcinogenesis and (tissue) aging. This consistent gene expression signature defines a universal marker profile relevant for the general population, providing valuable tools for biomarkers, mechanistic understanding, prevention and therapy.

Speaker: Dr Dik C. van Gent (Erasmus MC)

16 In Vivo Gamma-Irradiation Low Dose Threshold for Suppression of DNA Double Strand Breaks Below the Spontaneous Level in Mouse Blood and Spleen Cells

There is a considerable controversy as to whether DNA damage induced by low doses and low dose rates of ionizing radiation is treated similarly by cellular defence machineries and what downstream delayed effects it causes compared to moderate to high doses. This constitutes the major challenge for the linear no-threshold model (LNT) currently used for radiological risk estimates. Among various DNA lesions induced by ionizing radiation DNA double strand breaks (DSBs) are of the most importance due to their potential to cause cell death, mutagenesis and carcinogenesis. The aim of this study was to examine accumulation of DNA DSBs in mouse blood leucocytes and splenocytes upon in vivo long-term chronic low dose gamma-irradiation. Animals were irradiated for 40, 80 or 120 days at a dose rate of 0.15 mGy/h, with total accumulated doses being 144, 288 and 432 mGy, respectively. DNA DSBs were measured in blood leucocytes and splenocytes using neutral comet assay. At the first time-point of 40 days we observed a slightly increased DSBs levels in exposed animals compared to control ones. Continuation of the exposure up to 80 days lead to a surprising finding that the DSB levels in the irradiated animals dropped below the control level. By day 120 of the irradiation, we still observed lower levels of DSBs in cells from exposed animals compared to unexposed controls, although the difference started to diminish. The effect at later times of irradiation could be explained based on the suggested inducibility of the DNA repair, once the threshold for signaling has been reached. We could further speculate that other mechanisms may be involved, such as the elimination of a hypersensitive and/or damaged sub-population of cells (e.g. by immune system). The question of whether low levels of DNA damage from low dose/dose rate exposures are dealt with by molecular repair systems in the same way as in the case of high dose exposures has a direct implication for radiological protection. This is due to a significant role played by cellular genome integrity machineries in carcinogenesis, with cancer being the most significant and formally accepted health consequence of ionizing radiation. Current LNT model used by authorities to calculate and control radiological risks has been largely criticized and debated. Our results provide further support for non-linearity in biological effects of low dose radiation, now at a molecular level of primary DNA lesions.

Speaker: Prof. Andreyan Osipov (Burnazyan Federal Medical Biophysical Center of the Federal Medical-Biological Agency)

17 DNA damage signal transmission by an ATM and PPM1G-HAUSP dependent pathway in response to ionizing radiation

The deubiquitylation enzyme USP7 (also known as HAUSP) plays a major role in regulating genome stability and cancer prevention by controlling the key proteins involved in the cellular DNA damage response, including Mdm2, p53, FOXO4 and PTEN. Deletion of USP7 leads to early embryonic lethality mainly due to increased p53 levels and cell proliferation arrest. Despite such an important role in controlling other DNA damage response proteins, USP7 itself has not been recognised as a target for regulation by DNA damage signalling pathways. We have recently found that USP7 regulation plays a central role in DNA damage signal transmission. We discovered that stabilisation of Mdm2, and correspondingly p53 down-regulation in unstressed cells, is accomplished by a specific isoform of USP7 (USP7S), which is phosphorylated at serine eighteen by the protein kinase CK2. Phosphorylation at serine eighteen stabilises USP7S and thus contributes to Mdm2 stabilisation and down-regulation of p53. Furthermore, following treatment of cells with ionizing radiation, dephosphorylation of USP7S by the ATM-dependent protein phosphatase PPM1G leads to USP7S down-regulation, followed by Mdm2 down-regulation and accumulation of p53. Interruption of this DNA damage signalling by PPM1G knockdown leads to improper coordination between DNA repair and replication, thus resulting in cell cycle arrest. Our findings uncover a novel DNA damage signal transduction pathway linking ATM, PPM1G and USP7S, and provide the mechanism as to how the DNA damage signal is quantitatively transmitted to coordinate a p53-dependent DNA damage response.

Speaker: Dr Dianov Grigory (University of Oxford)

18 Gamma-H2AX irradiation induced foci (IRIF) in cells exposed to a mixed beam of X-rays and alpha particles.

Increasing exposure of cancer patients to a mixed beam of high and low linear energy transfer (LET) ionizing radiation is an issue of growing concern. Little is known about the health effects of exposing organisms and cells to mixed beam irradiation. The effect of combined exposures has mainly been assessed with clonogenic survival or cytogenetic methods, and the results are contradictory. The gamma-H2AX assay has up to now not been applied in this context, and is thus a promising tool for investigating the early cellular response to mixed beam irradiation. In this study, VH10 human fibroblasts were irradiated with 241Am alpha particles, X-rays, or a combination of both at 37 ºC. Gamma-H2AX ionizing radiation-induced foci (IRIF) were scored for repair kinetics 0.5, 1, 3 and 24 h after irradiation (one dose per irradiation type), and for dose response at the 1 h time point. For dose response the effect of mixed beam was additive, and the relative biological effectiveness (RBE) for alpha particles (as compared to X-rays) was of 0.76 ± 0.52 for IRIF, and 2.54 ± 1.11 for large foci (LF). The repair kinetics for total number of IRIF in cells exposed to mixed beam irradiation was intermediate to that of cells exposed to alpha particles and X-rays. However, for mixed beam-irradiated cells the frequency and area of LF were initially lower than predicted and increased during the first 3 hours of repair (while the predicted number and area did not). Moreover, LF in mixed beam-irradiated cells were not phosphorylated to their full extent until 1 h after exposure. We hypothesize that the presence of low LET-induced damage engages the DNA repair machinery leading to a delayed repair of the more complex DNA damage induced by alpha particles.

Speaker: Mrs Elina Staaf (Centre for Radiation Protection Research, GMT department, Stockholm University, Sweden)

19 The interplay of DSB repair and cell cycle control

The DNA damage response pathways involve processes of double-strand break (DSB) repair and cell cycle checkpoint control. The coordinated interplay of these mechanisms is crucial for the maintenance of the genomic integrity as the progression through critical cell cycle phases with unrepaired DSBs can promote chromosome aberration formation. We recently demonstrated that the G1/S and G2/M checkpoints both have limitations. The G1/S checkpoint is very sensitive to DSBs and can exclude cells harboring a single DSB from the actively proliferating population. However, full G1/S checkpoint induction after ionizing radiation (IR) is a slow process requiring about ~4 h. Consequently, a time window is provided in which many cells enter S phase with high numbers of unrepaired DSBs, progress through S phase and exhibit elevated numbers of DNA damage foci and chromosome breaks in G2 and mitosis. Live cell imaging provided the opportunity to consolidate these findings by following single cells throughout S phase. Here, the possibility to track single GFP-53BP1 foci provided new insight into the dynamics of DSB repair and the formation of new DSBs in the context of replication. In contrast to the G1/S checkpoint, the G2/M checkpoint is initiated very rapidly but cells are released from G2 arrest prematurely before DSB repair is completed. Since DSB repair in G2 involves HR processes we speculated that cells enter mitosis with unresolved HR intermediates like Holliday Junctions. There is increasing evidence that in mitosis such structures can be resolved by Mus81- and Gen1-dependent mechanisms. However, using immuno-fluorescence microscopy, chromosomal approaches as well as live cell imaging technology we observed that some breaks are transferred into telophase and the subsequent G1 phase. We observed that cells which were irradiated in G2 and divided in the presence of unrepaired DSBs fail to efficiently repair the breaks in G1. This observation may suggest that structural alterations occur at the DSB ends during mitosis which render the breaks more difficult to repair in G1. We are currently elucidating the molecular processes which occur during Holliday Junction resolution in mitosis. In summary, both the G1/S and G2/M checkpoints have serious limitations, albeit different in nature, which likely contribute to the development of genomic instability.

Speaker: Dr Dorothee Deckbar (Darmstadt University of Technology, Radiation Biology and DNA Repair)

Radioecology

20 What can radiobiology and radioecology learn from each other?

Although traditionally separate disciplines, radiobiology and radioecology are closely interlinked, and a closer collaboration between them would be of great benefit to both fields. This talk will give a personal view on what the two disciplines can learn from each other, giving examples from current research. The most obvious area of overlap is radiation effects at the cellular and molecular level. Radioecology has adapted many molecular methods from radiobiology and other ‘white’ biology disciplines to examine radiation effects such as DNA damage and oxidative stress on non-human organisms. However, there is much more to be learnt, both in the use of novel methods but also in the understanding of the linkages between molecular or cellular level effects and what they mean for individuals, populations and even ecosystems. For radiobiologists, radioecology can offer a wide range of new model organisms (and knowledge of their biology) which can help answer questions about, for example, underlying mechanisms of radiosensitivity and adaptation to radiation. When it comes to the fate of radioactive substances in ecosystems, radioecology has traditionally focussed on transfer of radionuclides from the environment (e.g., water, soil) to organisms, and also between organisms in a foodchain (particularly to humans). It has also examined how properties of environmental chemistry affect bioavailability and uptake. Far less has been done on the cellular mechanisms of uptake, and those that may be radionuclide-specific. Here there may be much to learn from radiobiology and cell biology with their more in depth understanding of cellular mechanisms. Lastly, I will give a short introduction to STAR, an EU Network of Excellence in Radioecology, and highlight some of the planned research at the border between radioecology and radiobiology. Some of these cross-disciplinary issues are also included in a draft Strategic Research Agenda for Radioecology, on which feedback is welcomed.

Speaker: Dr Clare Bradshaw (Department of Systems Ecology, Stockholm University, Sweden)

21 Chronic lifetime ingestion of environmentally relevant doses of 226Ra leads to a transient growth perturbation in fathead minnow (Pimephales promelas, Rafinesque, 1820)

Purpose: Radium is a long lived actinide in the uranium decay chain. In living organisms it completes with calcium and thus can accumulate in organisms exposed to radium as a result of uranium mining and milling activities associated with the nuclear industry. Currently actinides are assigned an RBE (relative biological effectiveness) factor of between 20 and 40 but this value is highly uncertain as there are very few studies which measure biological effects following chronic exposure to low doses of radium. The aim of this study was to assess the impact of lifetime ingestion of environmentally relevant levels 226Ra on a common freshwater fish species. Methods: Fathead minnow (Pimephales promelas, Rafinesque) were obtained at the first feeding stage and established on a commercial fish food diet containing 226Ra in the activity range 10mBq/g-10Bq/g. They remained on this diet for 18 months and were sampled invasively at 1,6 and 18 months to assess biochemical indices and accumulated dose and non-invasively also at 12 and 15 months to assess growth. Results: Fish fed 10 and 100mBq/g diets showed a transitory dysregulation of growth at 6 and 12 months. Fish fed higher activities showed similar or less significant effects. Bioaccumulation at 1 month was below detection levels. At six months significant amounts of 226Ra were present but at 18 months they were gone and radium levels were at background in spite of the continued ingestion of the isotope. Assessment of bystander stress signaling throughout the time period showed a constant fish to fish signaling at all times and doses measured. Conclusions: Fathead minnow appear to bioaccumulate 226Ra initially and this is associated with growth dysregulation. However after 18 months an effective purging mechanism appears to be in place. The results may be important in the assessment of long-term environmental impacts of 226Ra.

Speaker: Dr Carmel Mothersill (Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Canada)

22 DATABASE ON RADIATION-INDUCED EFFECTS IN PLANTS FOR CRITICAL DOSES AND DOSE RATES ASSESSMENT

In recent years many efforts have been undertaken to develop a system of radiation protection for non-human biota. One of the key issues is an assessment of critical doses and doses rates for flora and fauna species at different exposure conditions. This work presents a database on radiation-induced effects in plants and an example of critical exposure values assessment on the base of data gathered for cultivated plants. Data on biological effects in plants (umbrella endpoints of reproductive potential, survival, morbidity, morphological, biochemical, and genetic effects) in dependence on dose and dose rates of ionizing radiation have been collected from scientific papers issued during last 50-60 years. Quantitative data are available for 80 species of cultivated and over 70 species of wild plants. Data are maintained as database in MS Access that contains nearly 19000 records (over 5000 datasets) at the moment; the work is ongoing. As critical exposure levels, there are considered doses producing 50% changes of biological effect at acute impact (ED50), or dose rates resulting in 10% changes at chronic exposure of plants (EDR10). Critical doses for different species are calculated from dose-effect dependences obtained with the corresponding data sets. Primary data are assesed for their quality according to several criteria. Three models (linear, logariphmic, and logistic) are tested for an applicability to fit a dose-effect dependence taking account of their goodness-of-fit and robustness of ED50 and EDR10 estimates. The critical doses and dose rates found from available information on reproduction and survival are presented. It is discussed to what extent critical radiotoxicity values can depend on a type of model chosen to fit dose dependence and quality of primary data. Findings obtained contribute to developing a unified concept of the environment and human protection from radiation and nonradiation contaminations.

Speaker: Dr Alla Oudalova (Russian Institute of Agricultural Radiology & Agroecology)

23 Bioremediation of Radioactive Contaminants

The microbial reduction of metals has attracted recent interest as these transformations can play crucial roles in the cycling of both inorganic and organic species in a range of environments and, if harnessed, may offer the basis for a wide range of innovative biotechnological processes. Microorganisms can affect the stability and mobility of the actinides U, Pu, Cm, Am, Np, and the fission products Tc, I, Cs, Sr, released from nuclear fuel reprocessing plants. Under appropriate conditions, microorganisms can alter the chemical speciation, solubility and sorption properties and thus could increase or decrease the concentrations of radionuclides in solution and the bioavailability. Dissolution or immobilization of radionuclides is brought about by direct enzymatic action or indirect non-enzymatic action of microorganisms. Although the physical, chemical, and geochemical processes affecting dissolution, precipitation, and mobilization of radionuclides have been investigated, we have only limited information on the effects of microbial processes. The mechanisms of microbial transformations of the major and minor actinides and the fission products under aerobic and anaerobic conditions in the presence of electron donors and acceptors will be reviewed.

Speaker: Dr Changhyun Roh (Korea Atomic Energy Research Institute)

24 RPII Environmental Monitoring Response to the Fukushima Accident

This paper presents the results of additional environmental radioactivity monitoring carried out by the Radiological Protection Institute of Ireland, RPII, in 2011 following the accident at the Fukushima Dai-ichi nuclear power plant in Japan. It will also compare model predictions with measured air concentrations, demonstrating that the model predictions were broadly in agreement with the magnitude and timing of the observed radionuclide concentrations. The RPII carries out an environmental radioactivity monitoring programme on a continuous basis and has published the results in a series of reports, all of which are available on the RPII website, www.rpii.ie. On March 22nd 2011, in response to the deteriorating situation at the Fukushima Dai-ichi nuclear power plant in Japan RPII increased the frequency of sampling and analysis of air, rainwater and milk in Ireland in anticipation of the arrival of the radioactive plume in Europe. The aims of the monitoring were to assess the levels of radioactivity from the accident reaching Ireland and to provide data on which to base the RPII’s advice to the Government and public. Due to the decrease in levels of radioactivity detected from Fukushima to close to or below the limits of detection, the monitoring programme reverted to routine operation at the end of May 2011. Details of the re-assurance monitoring programme and results showing increased activities measured during this period will be presented.

Speaker: Ms Lorraine Currivan (Radiological Protection Institute of Ireland)

25 RADIOECOLOGICAL AND RADIOBIOLOGICAL STUDIES OF AQUATIC ECOSYSTEMS WITHIN THE CHERNOBYL ACCIDENT EXCLUSION ZONE

In spite of the 26 years, past after the Chernobyl NPP accident in 1986, self-purification of the main water bodies in the Chernobyl exclusion zone is extremely slow process. Therefore, ecosystems of the majority of lakes, dead channels and crawls possess high level of radionuclide contamination of all the components. The basic problems of radiation safety of the Chernobyl exclusion zone concern radionuclides wash-off with surface drainage water to river system, their export outside the exclusion zone and affection of the water quality in the Dnieper River. Undoubtedly, one of the most important and still insufficient studied problems of aquatic ecosystems within the exclusion zone is research of chronic impact of ionizing radiation on non-human biota. Our researches were carried out during 1997-2011 in Azbuchin Lake, Yanovsky (Pripyatsky) Crawl, cooling pond of the Chernobyl NPP, the lakes of the left-bank flood plain of the Pripyat River - Glubokoye Lake and Dalekoye Lake as well as the rivers Uzh and Pripyat within the Chernobyl accident exclusion zone. The main objects of radioecological monitoring were water, suspended matter, bottom sediments and hydrobionts of different taxonomy. The radionuclide content in biological tissues was measured for 28 higher aquatic plant species, 6 species of bivalve molluscs and gastropod snails as well as 18 species of fish. Our studies were conducted: (1) dynamics identification of radionuclide specific activity and distribution in the main abiotic and biotic components of aquatic ecosystems; (2) study of dynamic profiles of radioactive contamination levels in hydrobionts of different ecological groups and trophic levels; (3) assessment of the major factors, which determine distribution of radionuclides in the freshwater ecosystems; (4) study the seasonal dynamics of radionuclides content in macrophites and the role of main aquatic plant associations in processes of radionuclides distribution in aquatic ecosystems; (5) assessment of a possibility to use hydrobionts of different trophic levels as biological indicators of radioactive contamination of aquatic environment; (6) dose rate estimation due to external and internal sources of irradiation for different groups and species of hydrobionts; (7) evaluation of cytogenetical, hematological and parasitological effects dynamics as well as changes in producing capacity due to long-term radiation exposure of hydrobionts in conditions of water bodies within the Chernobyl ac

Speaker: Dr Dmitri Gudkov (Institute of Hydrobiology, Kiev)

15:30 Coffee break

Poster Session 1

26 Steady-state and pulse radiolysis study of selected alkaloids in aqueous DNA and human serum albumin (HSA) systems

Isoquinoline alkaloids widely coexist in many Chinese herbal medicines, e.g. Rhizoma Coptidis and Caulis Mahoniae. They possess antimicrobial, antidiarrheal and cardiovascular activities, and recently have been also suggested as potential drugs for treatment of the Alzheimer disease and as photo- or radiosensitizers in cancer therapy [1]. Small molecules bind to the DNA and HSA in different manners. Intercalation, groove binding and electrostatic intercalation are three major noncovalent binding modes of small molecules to DNA [2]. Human serum albumin (HSA) is known to have reversible binding to a large number of drugs, hormones and other small solutes in three homologous domains (named I, II and III). This binding is important in determining the transport and metabolism of such substances in the body [3]. Our spectroscopic and viscometric studies indicate that berberine is a partial intercalator, coralyne and sanguinarine are classical DNA intercalators, while binding to HSA is effective only for neutral (enolic) form of sanguinarine. In connection to the latter possibility we have undertaken steady-state and pulse radiolysis study of selected alkaloids: berberine, coralyne and sanguinarine in aqueous DNA and HSA (human serum albumin) systems. The main objective of our work was to determine reactivity of hydrated electrons and OH radicals towards the alkaloids embedded in DNA or HSA, and transient absorption spectra of one-electron reduction and oxidation products. Following reactivity of radiation-generated radicals towards isoquinoline alkaloids in aqueous systems of DNA and HSA, we inferred about the alkaloid binding to biomacrolecules. In the present work we report on rate constants of reactions of hydrated electrons with selected alkaloids embedded in DNA or HSA, and transient absorption spectra of one-electron reduction and oxidation products (formed due to reaction with OH radical and other species generated in the aqueous phase). The effect of steady-state e-beam irradiation on absorption spectra of the examined systems is also presented. The results are compared with those obtained for homogeneous alkaloid/water solutions. Acknowledgement : This work has been funded from the Polish budget for Science (research project N N204537339, 2010-2013). 1. Y. Li, W.Y. He, J. Tian, J. Tang, Z. Hu, X. Chen,. J. Mol. Struct. 2005, 79. 2. D. Suh, J.B. Chaires,. Bioorg. Med. Chem. 1995, 3. 3. T. Peters, JR. All about albumin : Academic Press, 1996

Speaker: Mrs Milena Marszalek (Technical University of Lodz)

27 C-centered paramagnetic species generated radiolytically in molecular sieves.

Radicals play an important role in many radiolytic and catalytic reactions. However, their structure and properties are difficult to study because of extremely high reactivity. Matrix isolation technique in noble gas matrices and trapping in zeolites are the methods of choice for studying electronic structure and geometry of radical ions in solids. We present the study of paramagnetic carbon centers generated radiolytically in molecular sieves with adsorbed carbon oxide. The combinations of Electron Paramagnetic Resonance technique and quantum chemical computation have been applied in order to identify the radical centers and define their geometry and reactivity. All samples after degassing and dehydration on vacuum line were exposed to 13CO and γ-irradiated in 60Co source at 77 K with dose of 5 kGy. The EPR spectra were measured in temperature range 100 – 370 K. In all samples slightly anisotropic doublets with different 13C hyperfine splittings were recorded just after irradiation. We assigned them to carbon centered radicals stabilized in different lattice sites. Based on the EPR measurements and DFT calculations we found that zeolite samples with high silica alumina ratio as: ZSM-5/13CO: Si/Al=38÷200, FAU Y/13CO: Si/Al=30÷80 and MOR/13CO: Si/Al=8 stabilize three type of carbon centres. In the first one (center A) •+CO radical cation interacts with oxygen located between Si and Al atoms -[≡Si-O-Al≡]•+CO. In the second one (center A`) it is bounded to oxygen located between two Si atoms [≡Si-O-Si≡]•+CO. For last carbon center we proposed the structure in which CO is attached to oxygen of terminal silanol group forming center B: [≡Si-O]•CO. In zeolites with Si/Al<5.1 as: LTA/13CO or FAU /13CO only centre A is stabilized. The values of 13C hyperfine splitting for centre A in various zeolites differ from Aiso=25.4 mT to Aiso=27.8 mT. In zeolites with stronger acid sites the higher A(13C) values were observed than in molecular sieves with weaker acid sites. We linked this change to the character of bonding between carbon and lattice oxygen. According to DFT calculations this bonding changes the character from covalent one in zeolites with weak acid sites to ionic in lattices with stronger acid centers. Our results show that the hyperfine splittings of +•13CO EPR doublet might be useful tool for probing the active sites in zeolite catalysts. This work was supported by the Polish National Science Centre grant No. 01/N/ST4/04025

Speaker: Mr Marcin Sterniczuk (Institiute of Nuclear Chemistry and Technology)

28 ABSOLUTE CROSS SECTIONS FOR THE FRAGMENTATION OF BIOLOGICALY RELEVANT MOLECULES AFTER IONISATION

The advent of heavy ions for cancer therapy has emphasised the need for novel dosimetric concepts that ideally should also account for the biological effectiveness of radiation. One approach to obtain such new dosimetric concepts is the field of nanodosimetry, where the particle track structure is taken into account by measurement [1] or by simulation [2] of ionisation cluster size distributions. The damage to DNA in the form of double strand breaks can be estimated from these distributions by means of phenomenological models that contain adjustable parameters, such as the conditional probability for an ionisation of the DNA molecule to result in a strand break [3]. In principle, the induction of a strand break due to an ionisation cluster in a DNA-segment could be modelled without free parameters if the cross sections for fragmentation of the molecular building blocks of the DNA backbone were known. In the present work, we show the first results from our crossed-beam experiment [4] for the absolute fragmentation cross sections after electron induced ionisation. Measurements have been made with electrons in the energy range of (50 – 500) eV irradiating supersonic jet targets of propane [5], methanol and tetrahydrofuran. References [1] S. Pszona, J. Kula, S. Marjanska, Nucl. Instrum. and Meth. Phys. Res. A 447, 601 (2000). [2] B. Grosswendt, Radiation and Environmental Biophysics 41, 103 (2002). [3] G. Garty R. Schulte, S. Shchemelinin, C. Leloup, G. Assaf, A. Breskin, R. Chechik, V. Bashkirov, J. Milligan, B. Grosswendt, Physics in Medicine and Biology 55, 761 (2010). [4] A. Arndt, W. Y. Baek G. Hilgers, R. Dörner, H. Schmidt-Böcking, H. Rabus, 1st Nano-IBCT Conference 2011, Book of Abstracts [5] P. Wang, C. R. Vidal, J. Chem. Phys 116 10 (2002).

Speaker: Mr Alexander Arndt (Physikalisch-Technische Bundesanstalt (PTB))

29 Radiolytic study of interactions of organic cations with heparin

Glycosaminoglycans are linear polysaccharides consisting of repeating disaccharide units. Because of the presence of negatively charged sulfate and carboxyl groups they are polyelectrolytes. They occur on animal cell surface and in the extracellular matrix. Glycosaminoglycans present on endothelium cell surface allow an interaction with the underlying subendothelium and blood components, but also with some xenobiotics, especially of cationic character. Vascular endothelium is an organ that plays an important role in proper functioning of cardiovascular system by its metabolic activity, and its dysfunction leads to various pathologies and diseases. The selected pyridinium salts, such as a metabolite of vitamin PP 1-methylnicotinamide (MNA), shows anti-inflammatory and anti-thrombotic activity [1]. These functions are connected with ability to modulate the secretory function of endothelium. For example anti-thrombotic activity of MNA is mediated by prostacyclin derived from vascular cyclooxygenase-2 [2]. The mechanism of action is not fully understood, but it is likely that the interactions of 1-methylnicotinamide with glycosaminoglycans present on endothelium, may play an important role. Here we present the results of the pulse radiolysis investigation on the binding of selected organic cations to heparin, one of glycosaminoglycans. These pulse radiolysis results are compared with steady-state spectrophotometry data obtained for methylene blue, a cationic dye whose interaction with heparin is well known. [1] J. Gebicki, A. Sysa-Jedrzejowska, J. Adamus, A. Wozniacka, M. Rybak, J. Zielonka, Pol. J. Pharmacol., 2003, 55, 109-112 [2] S. Chlopicki, J. Swies, A. Mogielnicki, W. Buczko, M. Bartus, M. Lomnicka, J. Adamus, J. Gebicki, Br. J. Pharmacol, 2007, 152(2), 230-239

Speaker: Malgorzata Jakubowska (Institute of Applied Radiation Chemistry, Lodz University of Technology, Lodz, Poland)

30 Radiolysis and photolysis of coralyne and sanguinarine water solutions.

Sanguinarine and coralyne are isoquinoline alkaloids isolated from plants. The use of these alkaloids as medicines by humans dates to several thousands of years ago and recently they are applied in cancer therapy. Naturally occurring alkaloids are effective in cancer treatment but its mechanism of action has not been fully elucidated. Sanguinarine is reduced upon entering the cell and then undergo redox cycling between the reduced and oxidized forms causing the cell death by apoptosis [1]. The one-electron reduction or oxidation reactions of alkaloids are important for understanding the mechanism of generation radicals taking part in apoptosis. The reactions of hydrated electron, carbon dioxide radical anion, hydrogen atom and Cl2•-, N3•, OH• radicals with coralyne and sanguinarine in aqueous solution have been studied by steady state and pulse radiolysis. The spectra of transient intermediates leading to the final products will be presented. The part of our presentation will be devoted to photoinduced electron transfer from excited triplet states to selected quenchers. References: [1] S.S. Matkar, L.A. Wrischnik, U. Hellmann-Blumberg, Archives of Biochemistry and Biophysics 2008, 477: 43-52 Acknowledgement: This work was supported by the grant N N204537339 from the Ministry of Science and Higher Education (Poland).

Speaker: Ms Anna Konarska (Institute of Applied Radiation Chemistry, Lodz University of Technology)

31 Reactivity of hydroethidine towards peroxyl radicals

Because of the fact that the oxidation product of hydroethidine (HE), 2-hydroxyethidium (2-OH-E+), is an unique, fluorescent marker for superoxide radical anion (O2•-), hydroethidine has become one of the most used fluorogenic probes for its intracellular detection. Although, HE can be used rather as a qualitative than quantitative probe for detection of superoxide, it is still worthwhile to examine the mechanism of its oxidation, and factors affecting formation of 2-OH-E+ [1]. Here we present results showing the reactivity of hydroethidine towards chloromethylperoxyl radicals like CH2ClO2•, CHCl2O2• and CCl3O2•, generated in oxygen saturated, water/propan-2-ol solution containing 4% (v/v) of proper halocarbon compound. We showed that chloromethylperoxyl radicals are able to oxidase HE with the second-order rate constant at pH=7.4 of k=2.74 x 108 M-1s-1, k=8.8 x 108 M-1s-1 and k=1.23 x 109 M-1s-1, for corresponding radicals, measured by pulse radiolysis. Spectral characteristic of the product formed upon one-electron oxidation of HE by RO2• radicals obtained under mentioned conditions was the same with that obtained from the irradiation of HE embedded in 1-butyl-3-methylimidazolium hexafluorophosphate (BMIM+PF6-) low-temperature glasses and characterized as hydroethidine radical cation (HE•+). The result were concluded by theoretical calculation using TD-DFT um052x/6-311+g(d,p) basis set. Results were depicted as an electrostatic potential and spin densities maps of hydroethidine radical cation (HE•+) and other species, which formation is highly possible under mentioned experimental conditions. [1]. Zielonka J., Vasquez-Vivar J., Kalyanaraman B. Nat. Protoc. (2008) 3, 8

Speaker: Mr Bartosz Michalowski (Institute od Applied Radiation Chemistry, Lodz University of Technology, Lodz, Poland)

32 CytoBayesJ: Software tools for Bayesian analysis of cytogenetic radiation biodosimetry data

The classical methods for dose estimation in radiation cytogenetics are now extremely well defined. However, classical statistical methodology requires that chromosome aberration yields are considered as something “fixed.” This 'frequentist' approach takes into account only the chromosomal damage which has been identified in a particular sample, and as such does not fully consider the intrinsically stochastic nature of aberrations. The alternative to the classical methodology is to use a Bayesian approach to data analysis. In the Bayesian framework, probability of an event is described in terms of previous beliefs and uncertainty. Previously existing, or prior, information is used in combination with experimental results to infer probabilities or the likelihood that a hypothesis is true. A number of authors have suggested that a Bayesian approach may be preferable for analysis of cytogenetic data and it has been shown that the Bayesian approach increases both the accuracy and quality assurance of radiation dose estimates. A software tool has been developed in order to bring Bayesian analysis to laboratory cytogeneticists. CytoBayesJ takes a number of Bayesian or ‘Bayesian like’ methods that have been proposed in the literature and presents them to the user in the form of simple user-friendly tools. These include: - Testing for the most appropriate model for distribution of chromosome aberrations amongst cells from the Poisson, Negative Binomial, Neyman type-A and Hermite models; - Calculations of posterior probability distributions for yields of chromosome aberrations; - Calculations of posterior probability distributions for radiation dose; - Bayesian methods of calculating detection limit and decision threshold. The software will be presented, together with details of testing with respect to cytogenetic data collected in a number of different radiation exposure scenarios by researchers who are active in the field of radiation cytogenetics. Overall, the applicability of the Bayesian approach to radiation cytogenetics will be further highlighted.

Speaker: Dr Elizabeth Ainsbury (Health Protection Agency)

33 RENEB – Realizing the European Network of Biological Dosimetry

Creating a sustainable network in biological and retrospective dosimetry that involves a large number of experienced laboratories throughout the European Union (EU) will significantly improve the accident and emergency response capabilities in case of a large-scale radiological emergency. A well organised cooperative action involving EU laboratories will offer the only chance for a fast and trustworthy dose assessment urgently needed in an emergency situation. In this regard the European Commission supports the establishment of a European network in biological dosimetry (RENEB) involving 23 organisations from 16 countries and mainly based on biological dosimetry laboratories. This will guarantee the highest efficiency in the processing and scoring of biological samples for fast, reliable results implemented in the EU emergency management. RENEB also implements recent developments in retrospective dosimetry. The network is based on 5 work packages: WP1 Operational basis: to create a “stand-by” basis based on coordination of the existing reliable and proven methods in biological and retrospective dosimetry. WP2 Basis for development: to expand and improve the network by implementing appropriate new methods and integrating new partners. WP3 Quality, Education and Training: to assure high quality standards by education and training activities. WP4 Sustainability: to establish a legal framework, harmonise the infrastructure and set up funding strategies. WP5 Dissemination: to strengthen the awareness of the network by connecting it to radiation emergency preparedness and response systems and by spreading of information. Since the project started in January 2012 the work-groups successfully implemented various tasks. A first training exercise to test for quality and efficiency within the RENEB laboratories was developed and realised. A plan of the first intercomparison was developed and realised. The RENEB web page is available online (www.reneb.eu), implying reporting sheets for new methodologies and potential new members. The first bulletin is edited and the project has been presented to interested communities and stakeholders in the field of emergency management (“Strengthening of Biological Dosimetry in IAEA Member States, IAEA, 2012), radiation protection (IRPA13) and radiation research (4th MELODI workshop 2012, European Radiation Research Conference 2012).

Speaker: Dr Ulrike Kulka (German Federal Office for Radiation Protection)

34 Intra- and inter-individual variability of the dicentrics frequency in human lymphocytes exposed to ionizing radiation at different temperatures

Dicentric assay is currently a gold standard for classic biodosimetry because it possess all features of an ideal assay: low background level, clear dose-effect relationship for different radiation qualities and dose rates, specificity to ionizing radiation, low invasiveness, good reproducibility and comparability of in vitro and in vivo results. This method is based on the assumption that the intra- and inter-individual variability of dicentric frequencies observed at a given dose point is similar. Since it is well known that the temperature during in vitro irradiation of human peripheral blood lymphocytes affects the level of dicentrics, it was interesting to verify whether the temperature influences the intra- and inter-individual variability in the donor’s sensitivity to irradiation. Peripheral blood was collected 3 times from 6 healthy donors. PLB were kept at 0°C , 20 °C and 37 °C for 30 minutes and exposed to 0; 0.5, 1, 1.5, 2; 2,5 and 3 Gy of gamma-rays. For the analysis of dicentric yield, chromosome preparations were obtained after 48 h of lymphocyte culture post irradiation. The results showed that the overall intra- and inter-individual variation of the dicentric yield (CV) at 0°C was higher than the intra- and inter-individual variation at 20ºC and 37ºC. The overall intra-individual CV of the dicentric yield at 0ºC was 1.3 times higher than at 20ºC and 37ºC. The overall inter-individual CV of the dicentric yield at 0ºC was about 1.2 times higher than at 20°C and 37 °C. The overall inter-individual CV for all doses and temperature points was 1.3 times higher than the overall intra-individual CV. This work was supported by grant number SP/J/143 339/11

Speaker: Prof. Anna Lankoff (Institute of Nuclear Chemistry and Technology, Centre for Radiobiology and Biological Dosimetry, Warsaw, Poland; Jan Kochanowski University, Dept. of Radiobiology and Immunology, Kielce, Poland)

35 Dose-response curves for Portuguese population: Chromosomal aberrations and Micronuclei assays

The main aim of a radiation study is to assess the risk of the population exposed to ionizing radiation. The work of the dose assessment may be very difficult because dose information about the population exposed is often indirect and incomplete. It is very important, therefore, to find a way to estimate reasonable and reliable doses of the population by a retrospective method such as biological dosimetry. Damage from occupational or accidental exposure to ionizing radiation is usually assessed by monitoring chromosome aberrations, in particular dicentrics chromosomes and micronuclei. The dicentric chromosome analysis is considered as the mainstay on the evaluation of biodosimetry. The dicentric assay can be reckoned as a sensitive method in comparison with other biodosimetric methods; the experimental procedures are rather practical and accurate even though microscopic observations are time-consuming. The cytokinesis blocked micronucleus assay is already validated to perform dose assessment and also to be used as a triage tool, since it’s easier and faster to evaluate samples. Both dicentric and micronuclei yield have linear-quadratic relationships with dose, enabling the correlation between dose exposure and biological effects to DNA. We performed dose-response calibration curves for both endpoints in order to estimate radiation dose in case of accidental radiological occurrences. It’s very important that the response to a large-scale radiological event can be given as quickly as possible. So the creation of a network like RENEB (Realizing the European Network in Biological Dosimetry) is of utmost importance. The existence of a biological dosimetry calibration curve in our country allowed us to be part of such a network.

Speaker: Dr Octávia Monteiro Gil (Instituto Tecnológico e Nuclear, Instituto Superior Técnico, Universidade Técnica de Lisboa)

36 Influence of image acquisition and analysis parameters on γ-H2AX dose-response curves in human lymphocytes exposed to ionizing radiation

The scoring of γ-H2AX foci as a measure for double-strand breaks (DSB) can be used to estimate exposure to the very low doses of radiation and shows that the yield of foci induced by ionizing radiation increases linearly with the radiation dose. These findings indicate that quantitation of γ-H2AX foci is a promising tool for biological dosimetry. Quantification of γ-H2AX foci can be achieved by various methods, but the fully automated assessment are a preferred approach for high-throughput biodosimetric analysis. Since the robust calibration dose-response curve is a prerequisite for accurate dose calculation, the factors influencing the measured number of γ-H2AX foci should be known. The aim of this study was to investigate the influence of various parameters of automated image acquisition and analysis on the slope of γ-H2AX foci dose-response curve. Peripheral whole blood was irradiated in vitro with 250 kV X-rays (0, 0.2; 0.4; 0.6; 0.8; 1; 1.5; 2; 3; 4 Gy, 1,2 Gy/min), incubated at 37°C for 30 min and then processed for γ-H2AX foci counting. Images of individual cells were captured and recorded as a training data files with an automated image acquisition and analysis system Metafer (Metasystems, Germany).These files were used to test various parameters dealing with automated cell selection (maximum concavity depth, maximum aspect ratio, minimum and maximum object area), thresholding (object threshold, upper threshold saturation area, minimum upper threshold in % of the gray level range) and spot counting (relative spot area, minimum spot distance, minimum and maximum intensity of a spot). The number of γ-H2AX foci obtained by applying various classifiers was used to construct dose-response curves, which were fitted to a linear model: y = c + αD and compared statistically to each other. The data obtained revealed that the image acquisition and analysis parameters significantly influence the slope of γ-H2AX foci dose-response curve. This work was supported by grant number WND-POIG.01.03.01-14-054/09

Speaker: Dr Maria Wojewódzka (Institute of Nuclear Chemistry and Technology, Centre for Radiobiology and Biological Dosimetry, Warsaw, Poland)

37 A novel analytical method of biological treatment plan optimization

Modern treatment planning allows the achievement of complex goals. Minimum and maximum doses cannot be considered as adequate figures of merit for the quality of a plan, and that is the reason why a complex set of parameters has to be evaluated when an irradiation plan is to be assessed. Particularly, radiobiological indices are useful tools for this evaluation, as they relate the plan to specific clinical goals. Therefore, when optimizing a treatment plan it is very useful to be able to characterize treatment plans, according to the closeness of their indices to a reference value (a goal or a previous result should be improved). A measure of closeness to the objective dose distribution is needed, and it has to be related to the values of the index. In this work, we propose a type of distance between dose volume histograms that can be used to biologically optimize a treatment plan. Given a DVH curve, the function F(z)=1-DVH is a distribution function for some random variable (absorbed dose for random points inside the tumour in this case). The Lévy distance between the distribution functions F and G, dL(F,G) can be defined and used as a metric between absorbed dose distributions. The problem to be considered is whether or not two DVHs inside a given Lévy distance correspond to tumour control probabilities within a specified interval of values. And the inverse problem is to establish the maximum Lévy distance between two DVHs in order to fulfil a constraint on TCP. TCP is represented as an operator on the set of probability distributions T. If T is an operator, and it is continuous, an upper and lower bound can be found on its values for distribution functions at a distance of R0 from F0. Therefore, given a tolerance on TCP, a set of DVH constraints can be given ensuring that tolerance is fulfilled. Any functional on DVHs can be treated according to this new theoretical framework, as long as it shows a minimum of good analytical properties of continuity and differentiability, as it is typically the case with radiobiological indices. This method is has a straightforward application, and shows great flexibility making easier a decision making process.

Speaker: Dr Francisco Cutanda Henríquez (Instituto de Salud Carlos III)

38 Establishment of an 192 Ir γ-ray calibration curve for lymphocyte dicentric assay in case of occupational or accidental exposure in industrial radiography

The analysis of dicentric chromosomes in human peripheral blood lymphocytes is the most reliable and accurate method for estimating a recent absorbed dose following external exposure to ionising radiation. The frequencies of dicentrics per grey vary depending on the variations in both the linear energy transfer (LET) and the relative biological effectiveness (RBE) of any particular radiation. Therefore, the conversion of an observed dicentric frequency into dose with reference to an in vitro calibration curve established for the type of radiation, which is the same to that of the radiation being studied, significantly improves accuracy of dose reconstruction by the dicentric assay. Since the most common radiation incidents are in industrial radiography using iridium sources, the aim of this study was to establish the calibration curve for 192Ir γ-rays as a reference in the Central Laboratory for Radiological Protection (Warsaw, Poland) for estimating the absorbed dose in case of undesired occupational or accidental radiation exposure. The conventional metaphase-spread chromosome technique was used to study the dose-response effects in irradiated in vitro lymphocytes of two healthy donors. The irradiation was performed with the Gammamat TSI-3 industry standard γ-ray projector containing the 192Ir source with an activity 2 644 GBq at that time. The dicentric data obtained for eight different doses of 192Ir γ-rays were fitted to a linear-quadratic model of chromosome aberration formation by low-LET radiation: Y=c+bD+aD2.To determine the best-fit coefficients the computer program CABAS and the maximum likelihood method were used. The coeficients obtained were: c=0,0010±0,0006; b=0,0322±0,0102 Gy-1; c=0,0541±0,0055 Gy-2. Established dose response curve was used for valuation of the dose for a worker accidentally overexposed to γ 192Ir industrial radiography source which, have become detached and must have been returned to the shielded container. The estimated mean dose was equal to 0,10 Gy, with the 95% confidence interval of 0,03-0,21 Gy, whereas the mean dose received on the basis of physical reconstruction of the accident was found to be 0,09 Gy. If the 60Co calibration curve was used, the obtained dose was 0,18 Gy with the 95% confidence interval of 0,06-0,32 Gy.

Speaker: Dr Katarzyna Golnik (Central Laboratory for Radiological Protection)

39 Sodium tartrate as ESR dosimetric material for low-dose measurement

Potential ESR dosimetric application of different compounds of sodium tartrate such as sodium tartrate dihydrate (NaTA), sodium bitartrate monohydrate (Na-bTA), and potassium sodium tartrate tetrahydrate (KNaTA) were investigated in the range of 0.5 – 50 Gy. Comparisons were also made with alanine (AL) [1], ammonium tartrate (AmTA) [2] and lithium formate (LiF) [3] samples. It is found that NaTA presents almost singlet ESR spectra with a shoulder at both sides of the singlet, while other compounds present more complex ESR spectra. From dosimetric point of view, NaTA has presented a better dose-response than other tartrate compounds (Na-bTA, KNaTA and AmTA) and AL. However NaTA has a good radiation dose response LiF has a slightly better response in the interested dose range. Nevertheless, the narrow linewidth of NaTA (~0.6 mT) makes it a good candidate to be used as a low-dose ESR dosimetric material [4]. [1] a) Regulla D.F., Deffner U., Appl. Radiat. Isot. 33, 1101–1114 (1982); b) Sharpe P.H.G., Rajendran K., Sephton J.P., Appl. Radiat. Isot. 47, 1171–1175 (1996). [2] a) Bartolotta A., D’Oca M.C., Brai M., Caputo V., De Caro V., Giannola L.I.,. Phys. Med. Biol. 46, 461–471 (2001); b) Marrale, M., Brai M., Triolo A., Bartolotta A., D’Oca M.C., Radiat. Res. 166, 802–809 (2006). [3] a) Lund E., Gustafsson H., Danilczuk M., Sastry M., Lund A., Vestad T.A., Malinen E., Hole, E.O., Sagstuen E., Appl. Radiat. Isot. 62, 317–324 (2005); b) Vestad T.A., Malinen E., Lund A., Hole E.O., Sagstuen E., Appl. Radiat. Isot. 59, 181–188 (2003). [4] a) Ikeya M., Hassan G.M., Sasaoka H., Kinoshita Y., Takaki S. and Yamanaka C., Appl. Radiat. Isot. 52, 1209–1215 (2000); b)Lund A., Olsson S., Bonora M., Lund E. and Gustafsson H., Spectrochim. Acta A. 58, 1301–1311 (2002). Acknowledgements: The authors want to thank for the financial support to the Turkish Research Council (TUBITAK), grant no: 110T825.

Speaker: Dr Hasan TUNER (Department of Physics, Faculty of Science and Literature, University of Balikesir, Balikesir, Turkey)

40 GAMMA DOSE RATE AND EFFECTIVE INDOOR GAMMA DOSE IN DWELLING OF CAMPANIA REGION

Ionizing radiation from natural sources such as cosmic rays and terrestrial radiation, is the major contribute to the exposure for public population. In particular gamma radiation is the largest contributor to the external dose of the humans. Today it is assessed that the majority of inhabitants in urban areas spends about 80% of their time indoor, so their exposure to ionizing radiation from building material could be relevant. For this reason it is necessary to monitor the external exposure in the confined environments in order to be able to provide an appropriate protection to humans. The aim of the present study is to evaluate the indoor gamma exposure starting from the measurements of dose in dwellings in Campania, a South Italy region characterized by a soil rich in radioactive isotopes, using Thermo Luminescencent Dosimeters TLD-100 (LiF:Mg,Ti). Additional purposes were the examination of the dependence of dose rate on materials, kind of building materials and measurement sites. Finally the corresponding effective mean dose rate for population was estimated. The annual dose rate, subtracting cosmic contribution, was found being 264 ± 111 nGy/h. Results of the survey show that the exposure in the coast zone homes is higher than that in the intarnal one. The analysis of effect of building materials has shown that houses made of cement and tuff have higher dose rate than houses constructed with a variety of other materials. The mean effective dose was estimated on the basis of the UNSCEAR model and was found to be 1.3 mSv/y, three times higher than Italian average.

Speaker: Dr Mariagabriella Pugliese (Dipartimento di Scienze Fisiche, Università degli Studi di Napoli Federico II, Napoli, Italy and INFN Sezione di Napoli)

41 Cytogenetic Assays For Assessment Of Ionizing Radiation-Induced DNA Damage

During humans lifetime,they are exposed to physical, chemical, and biological agents. Ionizing radiation as a ubiquitous environmental physical agent induces DNA damage directly (as a result of deposition of energy in cells) or indirectly (as a result of free radical formation and oxidative damage . And because of Radiation usage in the treatment of a wide range of malignancies. Therefore, simple and informative techniques to measure cytogenetic and molecular damage would be greatly valuable in studying genetic risk during radiotherapy. When physical dosimetry is unreliable, biological dosimetry based on the level of induced DNA strand breaks, chromosomal damage and gene mutations could be used. Cytogenetic assays are classical methods to detect chromosome aberrations (CAs), which have been used as end points of exposure to genotoxic agents. Various methods of monitoring biological effects have been suggested that should be considered as internal dosimeters in the detection of increased genotoxic and presumably also carcinogenic risks. Among them, there are different cytogenetic techniques that provide information about damage in individual cells: detection of chromosomal aberrations, sister chromatid exchanges, and micronuclei in peripheral blood lymphocytes. methods, such as dicentrics, micronuclei, and PCC, eventually in combination with chromosome painting, are currently among the most informative and widely used methods for acute exposure situations. One of the advantages of cytogenetic dosimetry is that this biological dosimeter can be assessed at any moment, whereas physical dosimeters are not always present on the subject.

Speaker: Ms najmeh assadi (babol university of medical sciences)

42 Preservation of chestnut fruits by gamma irradiation: inter-comparison of absorbed dose results using three types of dosimeters

Food irradiation is a well established process, approved by international organizations of food (FAO – Food and Agriculture Organization) and health (WHO – World Health Organization), and regulated in European Union by the Directive 1999/2/EC. Chestnuts are a seasonal fruit that must be post-harvest treated to meet the food safety regulations. Until recently, this was done with methyl bromide fumigation that was banned in March 2010 by the European legislation [EU, 2008] given no or few alternatives to the producers and agro-industry that process this product. Italy and Portugal are the two main producers of European varieties (Castanea sativa Miller) with 42700 kton and 22400 kton., respectively. In this context, irradiation could come as feasible alternative if the food product meets the needed quality parameters after processing. The main irradiation studies on chestnuts were done in Asian varieties and only recently in European varieties [Mangiacotti et al., 2009; Antonio et al., 2011]. Previously to the irradiation of chestnuts dosimetric studies were performed in a Co-60 experimental chamber with four sources, with a total activity of 267 TBq (6.35 kCi) in November 2011 (Precisa 22, Graviner Manufacturing Company Ltd, U.K.). The dose was estimated for a rectangular box with the dimensions of 15 x 15 x 7 cm, in the centre and corners, in two levels of the chamber using two types of dosimeters: a ionization chamber (model FC65-P, from IBA Group, Belgium); a Fricke solution, prepared in the lab following the standard ASTM E1026-92. Routine dosimeters, Amber Perspex (batch V, from Harwell Company, U.K.) were used to confirm the estimated doses. The comparison of results is showed and a dose mapping was built and is presented. The uniformity ratio, Dmax/Dmin, obtained was lower than 3, in accord with European legislation for food processing. EU, 2008. Commission Decision, 753/2008. O.J. L 258/68, 26th September. Mangiacotti, M. et al. 2009. Rad. Phys. Chem. 78, pp. 695-698. Antonio, A. L. et al. 2011. Food Chem. Tox. 49, pp. 1918–1923. Acknowledgements Agroaguiar Lda. for samples providing; ON.2/QREN/EU Project nº 13198/2010 for financial support; FCT grant to A. L. Antonio, SFRH/PROTEC/67398/2010.

Speaker: Dr Amilcar Lopes Antonio (1-School of Agriculture, Polytechnic Institute of Bragança, Portugal. 2-Department of Fundamental Physics, University of Salamanca, Spain. 3-IST/ITN, Nuclear and Technological Institute, Portugal)

43 Evolution of the Canadian Biological Dosimetry Network

In the event of a large scale radiological/nuclear emergency, biological dosimetry is essential for providing timely assessments of radiation exposure for the general population and to identify first responders who must be restricted from further exposure. The dicentric chromosome assay (DCA) is currently the accepted biodosimetry method for radiation dose assessment, however in a mass casualty scenario this assay is not well suited for providing timely dose estimates due to its time- and expertise-intensive nature. Over the past ten years, Canada has developed a program to increase biological dosimetry throughput using the DCA. As a first step, Canada developed a National Biological Dosimetry Response Plan (NBDRP) which is comprised of a network of four core reference laboratories and multiple clinical cytogenetic laboratories. The core laboratories participate in annual exercises to demonstrate their ability to provide accurate and timely triage quality dose estimates. In addition, the clinical cytogenetics students are being trained to expand our current DCA scoring capacity. Recently, two biological dosimetry laboratories from the United States have joined our network by taking part in our annual intercomparisons. To further increase the throughput of biological dose estimates, triage DCA scoring of 50 cells per sample had been adopted. This has now evolved into a “QuickScan” technique which has been evaluated and validated within the NBDRP, as an alternative rapid scoring approach. It has been shown that the DCA QuickScan can provide critical dose information at a much faster rate than the conventional DCA without sacrificing accuracy. The evolution of this network over the past 10 years will be reviewed with focus on how to maintain a functional network through annual exercises and intercomparisons and how Canada is prepared to assist on the international level through participation in networks such as the World Health Organization lead BioDoseNet. (Funded by the Chemical, Biological, Radiological and Nuclear Research and Technology Initiative)

Speaker: Dr Ruth Wilkins (Health Canada)

44 Dose assessment and consideration in the deep place of the skin at the time of liniment use

Purpose: For patients who experience radiation dermatitis during radiotherapy, we sometimes use pharmaceutical agents applied topically, such as creams and lotions. Irradiation of treated parts thus sometimes occurs. If a substance exists in the skin surface, the skin dose might be slightly affected by such exposure, and the agent could become a factor associated with radiation injury. The present experiment was conducted to evaluate and reproduce a state where topical agents were applied and surveyed. Methods: Measurements were made using dosimetry (parallel chamber). A MixDP (thickness, 50mm) was placed behind the dosimeter, and a MixDP (thickness, 2mm) was placed in front. Systemic contact dermatitis (SCD) was 100cm, the area of the radiation field was 10x10cm2. The radiation exposure comprised X-rays of 4MV and 6MV and electron beams of 9MeV and 12MeV. A wrap film placed on the 2mm-thick MixDP was considered as a control. Various medicines (Keratinamin Kowa ointment, RINDERON-VG lotion, RINDERON-VG ointment, Juvela ointment, two kinds of hand creams, and sunscreen (SPF30 PA++)) were applied to the wrap film and measured, and differences were analyzed. The agents applied had a concentration of about 0.5 g/10x10cm2. Results: When a topical cream was applied, 4MV X-ray values were +0.27% to +1.09% higher than control values. We found the largest difference using RINDERON-VG lotion, which was 1.75% higher than controls. With 6MV X-rays, a difference of +0.33% to +1.82% was seen with topical creams. Just like 4MV X-rays, with RINDERON-VG lotion, the difference was slightly larger, at +2.16%. The 9MeV electron beam was -0.70% to +0.20 different than the controls, while the 12MeV electron beam showed differences between -0.02% and +0.29%. Conclusion: With X-rays, significant differences arose between each energy level and each topical agent. RINDERON-VG lotion showed a larger difference than the other agents. With electron beams, no significant differences were seen. Dose differences were larger with lotions than with ointments. Given these findings, the factors affecting skin dose at a specific depth are considered to increase according to such factors as form or density. When an agent is used clinically, the dose increases several percent, and if the medicine does not require a specific method of use, major problems are unlikely to be encountered. However, if the medicine happens to become accumulated, observable influences appear likely to be produced.

Speaker: Mr Ryo Akima (Department of Diagnostic Radiology &amp; Radiation Oncology, Kochi Medical School)

45 ASSESSMENT OF THE FREQUENCY OF CHROMOSOMAL ABERRATIONS IN MAYAK WORKERS EXPOSED OCCUPATIONALLY

Objective: Assessment of structural genome damages in Mayak workers exposed occupationally to prolonged external gamma-radiation and/or internal alpha-radiation using mFISH. Material and Methods: Slides of 64 workers were analyzed in the study. Average age of the included individuals was 73.3±0.9 years. Workers included in the study were exposed occupationally to prolonged external gamma-rays and/or internal alpha-radiation from incorporated Pu-239. Cumulative dose of external gamma-exposure to the red bone marrow (RBM) was at the range from 0 to 2.7 Gy, absorbed dose of internal alpha-exposure to the RBM from incorporated Pu-239 was from 0 to 0.8 Gy. The analysis was performed based on the culture of peripheral blood lymphocytes. The analysis of chromosomal aberration frequency was carried out using the method of fluorescent in situ hybridization mFISH. Results and Discussion: Both stable and unstable chromosomal aberrations were observed in the studied group. In addition, complex chromosomal aberrations, i.e. aberrations including 3 or more breaks within 2 or more chromosomes were revealed. The frequency of stable aberrations was statistically significantly higher in the group of workers with cumulative absorbed dose of external gamma-radiation to the RBM over 0.5 Gy as compared with the group of workers with cumulative absorbed dose to the RBM less than 0.5 Gy. The frequency of stable aberrations in Mayak workers with cumulative absorbed dose of internal alpha-radiation to RBM over 0.02 Gy was statistically significantly higher as compared with the group of the workers who were exposed at lower doses. The frequency of complexes was statistically significantly higher among the individuals with cumulative absorbed dose of internal alpha-radiation from incorporated Pu-239 over 0.02 Gy (0.87±0.05 per 100 cells) as compared with the workers who were exposed at lower doses (0.13±0.05 per 100 cells). Relationship between the frequency of complexes with cumulative absorbed dose of external gamma-radiation to the RBM wasn’t revealed. Conclusion: The results of the present study has shown that the frequency of stable chromosomal aberrations in Mayak workers relates both to cumulative dose of external gamma-radiation to the RBM and cumulative absorbed dose of internal alpha-radiation to the RBM from Pu-239. The frequency of complexes depended only on cumulative absorbed dose of internal alpha-radiation to the RBM from incorporated Pu-239.

Speaker: Mrs Natalia Sotnik (Southern Urals Biophysics Institute)

46 Cytogenetic effects of chronic exposure of the Red Bone Marrow in humans

Population exposure to ionizing radiation in the Urals region (Russia) occurred as a result of failures in the technological processes at the Mayak plutonium facility in early 1950s. A major source of environmental contamination was releases of liquid radioactive wastes into the Techa River in 1949–1956 with peak amounts from September 1950 until November 1951. Over 30,000 persons living in villages located along the river downstream from the site of releases were exposed to significant levels of external and internal (mainly from 89, 90Sr) irradiation. The main pathways of exposure for the Techa riverside population were from drinking water and consumption of contaminated local foodstuff (mainly cow’s milk) and external gamma exposure due to proximity to bottom sediments and contaminated floodplain soils (Degteva et al. 2006). This paper includes the results of cytogenetic study of individuals exposed at the Techa River followed-up for over 40-year period (Akleyev et al. 1995). The main outcomes of the study were the following: • A significantly high level of unstable chromosomal aberrations (UCA) in T-cells of exposed individuals (RBM cumulative dose range from 0.1 Gy to 3.0 Gy) was observed during the whole period of observation. • The frequency of UCA decreased by a factor 2-3 over the last 30 years. • The level of spontaneous UCA in rural residents remained stable over the whole period and was 0.6-0.8 dicentrics and rings per 1000 cells. • The frequency of translocations in exposed individuals differed significantly from non-exposed group of the same age (Bauchinger et al. 1998, Degteva et al. 2005). • A significant linear dependence (dose range from 0.5 Gy to 3.6 Gy) between the radiation induced translocations and individual RBM dose from incorporated 89, 90Sr was found (Vozilova et al. 2012). The research was partially funded from the European Community's Sixth Framework Programme under grant agreement № FP6-516478 (“Southern Urals Radiation Risk Research”) (SOUL) and the European Community's Seventh Framework Programme under grant agreement № FP7-249675 (“Epidemiological Studies of Exposed Southern Urals Population”) (SOLO).

Speaker: Ms Alexandra Vozilova (Russian Federation)

47 THRESHOLD LIMITS IN CYTOGENETIC INDICATION AND BIODOSIMETRY IN TERMS OF CHRONIC EXPOSURE

In spite of the great variety of bioindication and biodosimetry methods, World Health Organisation (WHO) and International Atomic Energy Agency (IAEA) accept dose reconstructions by cytogenetic methods based on the analysis of specific chromosomal aberrations (DIC, FISH techniques) on metaphases from peripheral blood cells. Capabilities and characteristics of cytogenetic indication and biodosimetry are well-studied after acute exposure (IAEA, 2011), while the number of studies on bioindication and biodosimetry after chronic exposures is quite limited. The objective of the current study was to assess threshold limits for cytogenetic indication after chronic external gamma-ray exposure. mFISH was applied to assess the yield of chromosomal aberrations in peripheral blood lymphocytes among Mayak workers chronically exposed to external gamma-rays. To ensure reliable bioindication based on the analysis of the chromosomal aberrations yield in peripheral blood lymphocytes after occupational chronic exposure, the yield must be greater than “background” level. The yield of stable chromosomal aberrations per 100 cells measured in the control group (comprising similar aged individuals with no occupational exposure) and equal to 1.760.33 was considered as “background” level. Based on our method of dose threshold assessment using dose distribution functions (Osovets S.V., Azizova T.V., Day R.D. et.al. 2011, 2012), a dose threshold for bioindication after chronic external gamma-ray exposure was estimated equal to about 1.0 Gy based on the yield of stable chromosomal aberrations. Bioindication threshold assessed based on the yield of stable chromosomal aberrations >1.760.33 (61 individuals) indicates that individuals were chronically exposed to external gamma-rays in total absorbed dose to red bone marrow > 1.0 Gy. Due to the small study groups the estimates obtained are to be considered as preliminary. However, limits obtained should allow for correct planning future research on development the biodosimetry system for assessment of total doses of chronic exposure, based on the yield of stable chromosomal aberrations in peripheral blood lymphocytes. The current study was funded by the Federal Ministry of Defense of the Federal Republic of Germany withtin the framework of the contract M / SAB X / 9A001 «Biological indication and Dosimetry of chronic exposure».

Speaker: Dr Sergey Osovets (Southern Urals Biophysics Institute)

48 NK cells and their response to ionizing irradiation

Natural killer cells (NK cells) belong to white blood cells which care for anti-tumour and anti-viral control in organism. They can be distinguished from other lymphocyte subpopulations by specific surface markers detection. Our study is aimed at a sensitivity of NK cells to ionizing gamma irradiation. Generally, lymphocytes induce cell apoptosis in a consequence of gamma irradiation. Nevertheless individual lymphocyte subpopulations and subsets differ a lot in their sensitivity to irradiation. It has been published data describing peripheral blood NK cells as a radiosensitive subpopulation with can be used as a good ex vivo biodosimetric marker within human lymphocytes (Vokurkova et al., 2006). Contrary to ex vivo design, the in vivo results indicate human NK cells less sensitive than other lymphocytes I peripheral blood (Chambers et al., 1998; Louagie et al., 1999). To understand this discrepancy we studied changes in peripheral blood NK cells after ionizing irradiation with the aim to compare in vivo (whole-body irradiated experimental animals) and ex vivo (irradiation of isolated peripheral blood) experimental designs of a large mammalian swine model and small rodent model of Wistar rats. NK cells were identified by specific monoclonal antibodies and their relative representation in blood was analysed by multicolour flow cytometry. Our results indicate that porcine as so as rat NK cells respond to irradiation as the most relatively radioresistant population. They changed in vivo similarly to ex vivo nevertheless processes in vivo manifested in significantly shorter time-frame. Obtained results will be useful for a retrospective biodosimetry and back estimate of absorbed dose of ionizing irradiation. This work was supported by Ministry of Defence of the Czech Republic (The institutional support for a long-term organization development plan 1011, project No. OVUOFVZ200806 and project No. OVUOFVZ200809).

Speaker: Ms Lenka Zárybnická (Faculty of Military health Sciences, University of Defence)

49 Gene Expression Analysis in Human Peripheral Blood Lymphocytes for Biodosimetric Applications after Low and High Dose Gamma-Irradiation

In case of a large-scale radiation accident with involvement of individuals without physical dosimeters it is important to identify individuals who have received a moderate to high radiation dose to ensure proper medical care. As current methods are time-consuming, a fast and reliable method based on gene expression alterations is developed. Human blood of 3 male and 3 female healthy donors, belonging to 3 different age classes, was irradiated ex vivo with 0, 0.02, 0.1, 0.5, 1, 2 and 4 Gy (γ-rays, Cs-137). Peripheral blood lymphocytes (PBL) were isolated and cultured for 6, 24 and 48 h in the medium- and high dose range (0.5 – 4 Gy) and for 24 and 48 h after low dose irradiation (0.02 and 0.1 Gy). Subsequently RNA and proteins were isolated and RNA was applied for processing whole human genome microarrays (Agilent) to analyze expression profiles. In the medium- and high dose range the most robust altered genes were selected for further qRT-PCR and protein expression analysis. To examine the radiation-specificity of the candidate genes, PBL were exposed to the DNA-damaging agents Paracetamol (25 and 200 µg/ml) and Mitomycin C (0.1 and 0.4 µg/ml) for 6, 24 and 48 h and gene expression was accordingly analyzed. By a p-value and fold-change driven gene selection 9 genes were identified in the low dose range and 16 genes in the medium- and high dose range allowing a radiation dose prediction accuracy of 96% independently on the time-point post irradiation up to 48 h. For 6 predictive genes in the medium- and high dose range and for two genes in the low dose range the observed radiation-induced gene expression profiles were confirmed and validated by qRT-PCR measurements in pooled and non-pooled samples. Additionally, qRT-PCR analysis revealed that the radiation dose predictive genes are highly radiation-specific when compared to exposure with Paracetamol or Mitomycin C. Protein expression analysis showed only for two genes a weak correlation between gene and protein expression after irradiation. In vitro gene expression analysis in human PBL based on whole human DNA-microarray data allowed identifying a rather small set of radiation dose predictive and radiation-specific genes with a high potential for biodosimetric applications in vivo after low-, medium and high dose exposure. Funded by Bundesministerium für Bildung und Forschung (BMBF), Grant numbers 02NUK005A and 02NUK005D. Supported by Kompetenzverbund Strahlenforschung (KVSF).

Speaker: Dr Ralf Kriehuber (Radiation Biology Group, Department of Safety and Radiation Protection, Forschungszentrum Jülich, D-52425 Jülich, Germany)

50 A metaphase finder using single manufacturer's product

Metaphase finder is a tool for biodosimetry that finds metaphase chromosomes among blood cells on slide glasses. It consists of microscope, auto-focus system, automated X-Y stage, camera and computer. It does the image diagnosis of the microscopic images of the slide glasses, and displays the positions of metaphase cells. A metaphase finder is used for the personnel worked at Fukushima nuclear plant to know how much dose they irradiated. The author and colleagues have already reported a low-cost metaphase finder system (1), using commercially-available products. But the system was a combination of multiple manufacturers and then it requires much knowledge and skill to the resellers and users. Now, the author reports here a new metaphase finder system. The main difference is that the new system is made by the catalogue products of single manufacturer. And also, its software is written in macro language for ordinal microscope controller, which is also shown on the catalogue. Except macro program, no custom made parts is used. The system becomes more compact and less price. It will be another choice for resellers and users. REFERENCE (1) Akira Furukawa, Masako Minamihisamatsu, and Isamu Hayata: Low-cost metaphase finder system, Health Physics, Volume 98, Number 2, 2010.

Speaker: Dr Akira Furukawa (National Institute of Radiological Sciences (Japan))

51 Radiation dose estimative using computational murine model from Gd-159 nanostructured radiopharmaceutical

Different therapeutic radiopharmaceuticals containing the P-32, Re-188, Sr-89, and Y-90 radioisotopes have been used effectively in alleviating bone pain resulting from metastases, synovectomy, and tumors. These radioisotopes all present the ability to emit high doses of beta radiation, leading to the death of tumor cells. Similarly, the Gd-159 isotope emits negative beta (1001 keV) and gamma radiation (main energy: 363.54 keV) suitable for therapeutic applications in nuclear medicine. Previous in vitro studies showed that Gd-159-DTPA-BMA complex presents a high in vitro cytotoxicity against tumor cells. However, when administered internally, this radioisotope does not accumulate in target tissues, being rapidly eliminated through renal excretion. Thus, seeking to solve these problems, different pharmaceutical formulations consisting of nanostructured carriers, such as liposomes, have been proposed to reduce the toxicity in non-target organs, especially in patients with chronic kidney diseases, while increasing the effective concentration and contact time in target tissues. In the present work, a computational tool was developed to simulate this effect, which occurs in experiments in vitro, helping to predict the possible results, saving time and resources, presenting a possible direction for studies of interest. The computer model was formed by a murine voxel phantom coupled to the Monte Carlo code MCNP5. The murine voxel phantom was constructed from 157 axial tomographic images using a PICKER SeleCT/SP CT scanner. The voxels dimensions were 0.71 x 0.71 x 1.5 mm3, equivalent to 118 columns, 76 lines and 157 slices. All murine phantom organs have homogeneous composition and homogeneous density. The elemental composition and mass density for the phantom organs and tissues were taken from the International Commission on Radiation Units and Measurements (ICRU) Report 46. The results obtained by the computational simulation, using biodistribution data in mice, showed to be in good agreement with the experimental data obtained in vitro and presented errors of less than 5%. This indicates that the computational tool can be used in future studies for more accurate analysis of biokinetic effects caused by radiopharmaceuticals in target organs and its vicinity.

Speaker: Dr ADRIANO M. SANTOS (Nuclear Technology Development Center - National Commission on Nuclear Energy)

52 Development of nuclear safety and radiological protection methods for the nuclear power engineering’s current and future needs.

The strategic research project entitled “Technologies Supporting Development of Safe Nuclear Power Engineering” is a response to the demand for the Poland’s energetic safety optimization in the light of nuclear power engineering introduction. The strategic project will link research performed by Polish research teams with that which is carried out abroad and provide Polish scientists and experts specialized in nuclear industry with training opportunities. Its implementation shall contribute to the solution of problems posed by spent nuclear fuel and radioactive waste. In addition, the project shall lead to the adoption of legal and factual regulations on radiological protection, which, in its turn, will result in the increased social appreciation of the nuclear power engineering expansion in Poland. Task nr 6 of the project entitled “Development of nuclear safety and radiological protection methods for the nuclear power engineering’s current and future needs” is implemented by research network comprises Institute of Nuclear Chemistry and Technology, National Centre for Nuclear Research, Institute of Nuclear Physics Polish Academy of Sciences and Central Laboratory for Radiological Protection as the research network leader. One of the objectives of the project is to develop bioassays based on molecular biomarkers that can identify radiation-exposed individuals and that can provide individual radiation dose assessments to enable triage and optimal medical management. To this end we analyzed the expression of DNA damage responsive genes in ex vivo irradiated whole blood from three healthy donors. Analyzed genes were as follows: GADD45A, CDKN1A, MDM2, BBC3, SESN2, BAX, DDB2, ATF3, PLK3, GDF15 and BCl2. Blood was X-irradiated with a doses of either 0 Gy; 0,6 Gy; or 2 Gy and gene expression was measured by real-time PCR at 6, 12, 24, and 48 hours after irradiation. The preliminary data suggest that the analysis of expression profiles of the selected genes in whole blood may be very useful for fast identification of irradiated samples and therefore is a promising molecular biomarker for radiation biodosimetry. This work was supported by grant number SP/J/143 339/11

Speaker: Dr Kamil Brzoska (Institute of Nuclear Chemistry and Technology, Warsaw, Poland)

53 Retrospective dose assessment by EPR and OSL in mobile phones

In the retrospective dose assessment of individuals potentially exposed to ionizing radiation after an accident, dosimetry with inert materials can complement or be used as an alternative to biodosimetry assays. In the framework of the EU funded project Multibiodose (www.MULTIBIODOSE.eu), it is proposed to implement a combined system of dose assessment based on the use of EPR and OSL measurement in mineral glass of the window displays and in electronic components from a same mobile phone. This work is being carried out with the objective to develop and validate a method for dose assessment in a mass-casualties scenario. The first part of this work was dedicated to evaluate the properties of the EPR and OSL signals of types of glass and electronic components in mobile phones and to classify them according to three criteria: a) availability in portable electronic devices, b) presence of a radiation specific signal immediately after irradiation and c) presence of a remnant radiation induced signal 10 days after irradiation. Twenty-five mobile phones of different brands and models were analyzed at each partner laboratory, leading to a total of 75 devices covering 61 different models. All mobile phones contained electronic components presenting a radiation induced signal detectable by OSL and about 85% of mobile phones presented a detectable radiation induced EPR signal in glass. The detection limit of the OSL signal was lower than 1 Gy. The radiation sensitivity was found to be lower for the EPR/glass than for the OSL/components. For both glass and electronic components the signals were still clearly detectable 10 days after irradiation. In all electronic components a signal loss of about 50% after 10 days was observed, whereas in most glass samples the signal loss was not detectable or within the measurement uncertainty. The complementarities of EPR and OSL properties support our hypothesis about the advantages offered by using two independent measurement methods on the same device. A European intercomparison will be organized through the Eurados network (www.Eurados.org) in 2012 in laboratories owning OSL and EPR equipments, with the aim of spreading the expertise on these techniques in a laboratory network to develop large-scale capacity of measurement. ACKNOWLEDGEMENTS The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 241536.

Speaker: Dr Paola Fattibene (Department of Technology and Health, Istituto Superiore di Sanità, Rome, Italy)

54 Estimation of entrance surface dose and compliance with diagnostic reference levels for selected plane film X-ray radiographic procedures in Poland

Introduction: In 2011 National Centre for Radiation Protection in Health Care (NCRPHC) together with sanitary inspections accomplished the pilot project for estimation of the entrance surface dose (ESD) for the most frequent plane film X-ray examinations. As a result the compliance with diagnostic reference levels (DRLs) in routine practice of the supervised departments were analysed. Materials and Methods: During the survey the measurements of air kerma and DAP were performed using the measuring equipment of sanitary inspections (ionisation chambers and semiconductor dosimeters). The following types of X-ray examinations were studied: scull (AP/PA and LAT), chest (PA and LAT), lumbar spine (AP, LAT and LSJ) and pelvis (AP). A number of data were collected i.e.: brand, type, year of the manufacturing, total filtration, exposure settings (kV, mAs, table-source distance, radiation field area) and average number of the particular examination per year. Accordingly to the methodology given by NCRPHC all measurements were performed for exposure settings routinely used for a reference patient (170 cm hight and 70 kg weight). Results: Sixty-seven radiology departments in 8 of 16 districts have been inspected. The measurements were performed for the total number of 407 radiographic examinations. The reviewed radiology departments used 24 brands of X-ray equipment. The dominant manufacturers were GE, Philips and Siemens in comparable proportions. The oldest X-ray equipment was manufactured in 1979 and the newest one in 2010. The minimum, maximum and mean values of ESD were compared to the national DRLs. For all the examinations the values of effective dose were calculated using the PCXMC 2.0 software. Conclusions: The comparison of the ranges and mean values of ESD and DAP has shown a reasonable agreement with previously published data. The DRLs has been exceeded in a considerable number of radiology deprtments. In most of the cases the recorded values of the high voltage were lower than the values recommended in the publication of EUR 16 260 EN.

Speaker: Dr Katarzyna Majchrzak (National Centre for Radiation Protection in Health Care, Lodz, Poland)

55 Measures of DNA damage sensitivity correlate bladder cancer cell treatment sensitivity in vitro and outcome in vivo.

Bladder cancer patients suffer significant rates of treatment failure, including high rates of recurrence and poor outcomes for advanced disease. If mechanisms to improve tumour cell treatment sensitivity could be identified and/or if patient tumour response could be predicted, it should be possible to improve both control and survival rates, i.e. by selecting the most appropriate treatment for those patients with correspondingly sensitive/responsive tumours. Previously, we have shown that induction of radiation-induced DNA damage as measured by alkaline Comet assay (ACA) correlates cell radiosensitivity for a panel of human bladder cancer cell lines in vitro. In the present study we have shown that ACA measures of Cis-platin and mitomycin-C-induced damage also correlate cell chemosensitivity in vitro, with there being predominantly the same rank order for chemosensitivity as for radiosensitivity. Furthermore, ACA studies of radiation-induced damage in different bladder cancer cell DNA substrates (nuclei & nucleoids vs. intact parent cells) suggest that it is a feature retained in the prepared nucleoid bodies that is responsible for the relative damage sensitivity of bladder cancer cells, suggestive of differences in the organisation of the nuclear DNA within resistant/sensitive cell lines. Finally, analysis of tumour samples reveals that lower ACA measures of DNA damage sensitivity correlate with poorer outcomes following treatment; notably this includes the observation of lower measures of induced damage being significantly associated with local recurrence of non-invasive disease and this being a better predictor than the presence of high-risk histology (G3 pT1). In conclusion, this study demonstrates that mechanisms which govern treatment-induced DNA damage are central to bladder cancer cell treatment sensitivity and supports an association between DNA damage resistance and aggressive tumour phenotype in this cancer model.

Speaker: G Jones (University of Leicester)

56 Estimation of transgenerational effects of radiation in male germ cells of mice using high-density microarray CGH platform

We used two methods to estimate genetic effects of radiation. The first method was the comparative genomic hybridization (CGH) with high-density microarray containing 2.1 million probes (HD-2 microarray), which permits high-resolution examination of copy number changes (CNV: deletions and amplifications) throughout the whole genomes between two DNA samples. Refinement of the experimental and analytical protocols allowed accurate detection of small (2-3 kb, harboring only two adjacent probes) to large deletions which were previously characterized. The HD-2 microarray CGH approach was used for detection of deletion/amplification mutations among the genomes of 80 F1 mice derived from 4-Gy gamma irradiated spermatogonia and 80 control mice. A total of 22 mutations, 10 in the exposed group (6 deletions in 6 mice, 4 amplifications in 4 mice) and 12 mutations in the control (7 deletions in 7 mice, 5 amplifications in 3 mice, i.e., 1 mouse had 3 amplifications) were detected. The second was Restriction Landmark Genome Scanning (RLGS), a method which visualizes several thousand DNA fragments as spots and permits the detection of an autosomal deletion as a half-normal intensity spot. We applied the RLGS method for a genome-wide assessment of the induction rate of deletion mutations. Examinations were made on 1,007 progeny (502 control and 505 derived from spermatogonia exposed to 4 Gy of X rays). The results showed 1 deletion mutation in the un-irradiated paternal genomes of 502 offspring (0.2%) and 5 deletions in the irradiated paternal genomes of 505 offspring (1%). The deletion sizes ranged from 2 to 13 Mb. If the frequencies are taken at face values, the net increase was 4 deletions after an exposure of 4 Gy, or 1 deletion per Gy per individual if a linear dose response is assumed. Since the present RLGS screened about 0.2% of the total genome, the probability of any deletion mutation induced in the whole genome is estimated as 500 times (i.e. 1/0.002) of 0.2%, or about 1 per Gy. On the other hand, the CGH method using HD-2 microarray, containing 2.1 million probes distributed at approximately 1-kb intervals, searched deletion mutations at about 70% of the whole genome. The number of deletion mutation in the 80 exposed offspring is much smaller than that expected from the RLGS results (about 220; 80 × 1 deletion/Gy ×４Gy × 70%). The results imply that the number of large deletions induced by radiation exposure maybe considerably smaller than currently estimated.

Speaker: Dr Jun-ichi Asakawa (Dept. of Genetics, Radiation Effects Research Foundation)

57 Analyses of CT induced DNA damage to determine radiation sensitivity of different age groups, especially young children – a pilot study in the frame of the EU EPI – CT project

The increasing use of paediatric computed tomography (CT) worldwide has raised the question of possible late effects from exposure to ionising radiation. The European collaborative EPI-CT project aims at studying the cancer risks and the underlying biological effects in an international cohort study. The project is coordinated by the Section of Environment and Radiation at the International Agency for Research on Cancer (IARC). Eighteen centres from Belgium, Denmark, Germany, Finland, France, Luxemburg, the Netherlands, Norway, Spain, Sweden and the United Kingdom are cooperating in this project to enrol approximately one million patients. The overall objective is to inform about dose reduction and optimisation in paediatric CT. The aim of the biological part of the study is to compare different biomarkers for radiation exposure and to test their sensitivity in clarifying the biological mechanisms behind low dose hypersensitivity observed in CT examined paediatric patients. The work is divided into a number of distinct and complementary tasks which will allow to study the effects of CT exposure using a variety of approaches, including assessment of DNA damage, mainly through monitoring chromosomal aberrations and y H2AX foci. In this context, an in vitro feasibility study to investigate age-dependent radiosensitivity is conducted at the BfS in Germany. Blood samples from three different age groups ranging from newborns (umbilical cord blood), young children (2 – 5 years) to adolescents (>18 years) are being collected and investigated for different radiation-induced DNA damage parameters. Blood samples are being irradiated in a CT scanner. Chromosomal aberrations as well as the induction and repair of DNA double strand breaks have been investigated using y H2AX foci analyses for two different age groups (adults and umbilical cord blood). Preliminary results will be presented at the ERR meeting. Final results of this pilot study will be used for evaluating the feasibility of conducting a larger study allowing sufficient statistical power for estimation of age- and sex-dependent radiosensitivity. Funding comes from EC - (FP 7 under Grant Agreement n ◦ 269912)

Speaker: Mrs Ursula Oestreicher (Bundesamt für Strahlenschutz)

58 TOPORS modulates H2AX discriminating genotoxic stresses

H2AX plays an important role in chromatin reorganization implicated in DNA repair and apoptosis under various DNA damaging conditions. In this study, the interaction between TOPORS and H2AX was verified in vitro and in vivo using mammalian cell extracts exposed to diverse DNA damaging stresses such as ionizing radiation, doxorubicin, camptothecin, and hydrogen peroxide. In vitro assays for ubiquitination revealed that TOPORS functions as a novel E3 ligase for H2AX ubiquitination. TOPORS was found to be dissociated from H2AX proteins when cells were exposed to oxidative stress, but not replication-inducing DNA damaging stress. The protein stability of H2AX was decreased when TOPORS was ectopically expressed in cells, and oxidative stresses such as hydrogen peroxide and ionizing radiation induced recovery of the H2AX protein level. Therefore, these biochemical data suggest that TOPORS plays a key role in the turnover of H2AX protein, discriminating the type of DNA damaging stress. [ This work was supported by Grant No. 2010T100100303 from the MKE, Republic of Korea.] Key words : H2AX, camptothecin, doxorubicin, ionizing radiation, hydrogen peroxide.

Speaker: Dr CHA SOON KIM (Radiation Health Research Institute, KHNP)

59 Differences in correct and incorrect rejoining of DNA double-strand breaks in human fibroblasts having different radiosensitivity

DNA double-strand breaks (DSBs) in irradiated cells are primarily responsible for the biological effects of ionizing radiation. The restitution of the broken ends depends on repair potential of the cells and determines their fate: survival, transformation or death. It has been suggested that differences in DSBs repair capacity between individuals and cultured cell strains may explain the differences in radiosensitivity observed in vivo and in vitro. We tested this hypothesis using two human primary fibroblast cultures the 1st having normal radiation-sensitivity (NRS) and the 2nd having hyper radiation-sensitivity (HRS) that was derived from a phenotypically normal pediatric patient treated for medulloblastoma with reduced doses consequent to a family history of hypersensitivity to radiation reminiscent of chromosomal fragility syndrome (Alsbeih et al. Radiother/Oncol 66(3), 2003). Cell survival was determined by the clonogenic assay, chromosomal aberrations by FISH painting of chromosome 4, DSBs rejoining and fidelity by PFGE. Using Southern blotting, genomic DSBs rejoining was determined through the use of a probe for Alu repetitive sequence, while DSBs repair fidelity was measured in a 3.2 Mbp Not I restriction fragment on chromosome 21, with the probe D21S1 specific for that DNA fragment. NRS displayed normal radiosensitivity with a survival fraction at 2 Gy (SF2) of 0.35 while HRS was significantly more sensitive (SF2=0.15). HRS also incurred a higher level of chromosomal aberrations after 2 Gy than NRS. Residual genomic DSBs 24 h after 80 Gy were slightly higher in HRS compared to NRS. In both cells, however, this residual damage was small accounting for ≤ 10% of the initial damage in the whole genome. Measuring rejoining of correct and incorrect ends in the Not I restriction fragment revealed significantly higher misrejoining frequency in HRS when compared to NRS. The residual damage at 24 h was 10% and 40% of the initial damage (80 Gy) for NRS and HRS, respectively. Conclusions: differences in radiosensitivity are associated with differences in DSBs repair at low (chromosomal aberrations) and high radiation doses (PFGE). Measuring DSBs repair fidelity in specific regions of the genome could provide better resolution and a more accurate estimate of radiation-induced DNA damage. (KFSHRC work supported by KACST under LT-CNPSTI 9-MED749-20, RAC#2010 005; MDA work supported by NCI under CA06294).

Speaker: Dr Ghazi Alsbeih (King Faisal Specialist Hospital & Research Centre)

60 Super resolution microscopy of ion induced repair foci in human HeLa cells

High LET irradiation of living cells using heavy ions generates a high amount of DNA double strand breaks (DSB) in close vicinity to each other along the ion track. Various repair proteins cluster to the damage sites, e.g. γH2AX and 53BP1, and form repair foci of a gross size of about 1 µm. Due to the fact that one focus covers more than one DSB, a fine structure within the focus can be expected. First indications for such a fine structure were found in wide field images of cells taken one hour after irradiation with 55 MeV carbon ions by a 5 × 5 µm² matrix performed at the ion microprobe SNAKE. While a typical focus with the diameter of about 1 µm can be easily resolved using a conventional fluorescence microscope, its substructures cannot be resolved due to the diffraction limit of about 250 nm in conventional fluorescence microscopy. Therefore, for analyzing foci fine structures systematically, we utilize super-resolution microscopy techniques like structured illumination microscopy (SIM), STED, or localization microscopy (SPDM) which provide a lateral resolution of about 130 nm (SIM) to 50 nm (SPDM) fwhm. Since also with these techniques the lateral resolution is better than the z-resolution we used an irradiation configuration, where the cells are irradiated at a small-angle to the image plane. Thus, the whole ion track appears as a line within one layer of a 3D microscope image. Due to these improvements the super resolution images indicate clearly a fine structure when e.g. 53BP1 is stained with two colors. For quantification the Pearson correlation coefficient is calculated for a pixel wise shift of one color channel with respect to the other (Van Steensel approach [1]). With this, it is clear that there is a fine structure of a scale of about 200 nm. This becomes obvious by an extra correlation peak of about 200 nm FWHM. Using the same Van Steensel approach with images, where one color marks 53BP1 and the other γH2AX, we show that there is no total correlation of the fine structure between 53BP1 and γH2AX on the 200 nm scale. Using the product of the difference of the mean (PDM) 2D profiles similar 200 nm structures become visible. In addition, this approach probably show the location of multiple DSB within a single focus with high resolution when the two damage markers colocalize in small regions inside the focus but anticorrelate in larger sourrounding regions. [1] B. van Steensel et al., Journal of Cell Science 109, 787-792 (1996)

Speaker: Judith Seel (Universität der Bundeswehr München)

61 The study of DNA damage in lymphocytes and granulocytes after whole body irradiation in mice

Whole body ionizing gamma irradiation represents genotoxic stress, which has a decisive influence on all blood cell lines. Lymphocytes belong to the most radiosensitive cell lineages. After exposure of the organism to ionizing irradiation they die by apoptosis, the extent of which depends on the reeived dose. Bone marrow contains a reserve pool of functionally mature granulocytes, which might be readily mobilized into peripheral blood. While peripheral blood lymphocytes represent one of the most sensitive cellular tools for biodosimetric studies, granulocytes have not been in the focus of biodosimetric studies so far. We have compared a biodosimetric potential of peripheral blood and bone marrow lymphocytes and granulocytes by quantifying γ-H2AX expression. Material and Methods: Cells were isolated from the heparinized peripheral blood and bone marrow from sham-treated control and irradiated female mice (doses of 1-3-5-7-9 Gy) one hour after treatment. The level of histon H2AX phosporylation was detected by FITC-conjugated anti-phospho histone H2AX (Ser139) monoclonal antibody (Millipore, USA) in lymphocytes (lymphogate in the FSC/SSC dotplot and CD45+Gr1- surface phenotype) and granulocytes (CD45+GR1+ cells with higher SSC parameter) by surface immunophenotyping followed by intranuclear staining and flow cytometry. CyAn flow cytometric analyzer (Beckman Coulter) and Summit 3.4 software were used for data acquisition and analysis. Results: At lower doses (1-7 Gy), the intensity of H2AX phosphorylation appears to be higher in the granulocyte population than in lymphocytes both in peripheral blood and bone marrow. At higher does (9 Gy), H2AX is phosporylated to the same degree in both of major leukocyte populations studied. Conclusions: Fast biomarkers associated with DNA damage like γH2AX offer an opportunity to evaluate the received dose immediately after irradiation. Blood lymphocytes have commonly been used for visualization of double strand breaks (DBS) early after radiation exposure. We have shown that granulocytes are as good biomarker as their lymphoid counterparts, the granulocyte population appears to be even more sensitive at lower doses. In addition, the use of granulocytes for the mean of cellular fluorescence of γ-H2AX could be profitable at higher doses of irradiation, when the number of lymphocytes is severely reduced.

Speaker: Zuzana Sinkorova (University of Defence)

62 Application of FISH technique to study chromosome 1, 2 and 4 in colorectal cancer patients

Colorectal cancer is one of the most commonly occurring malignancies in both men and women. However, application of radiotherapy might be beneficial depending on individual susceptibility or even may cause risk of secondary cancers and others complications. Therefore, studies on influence of endogenic or egzogenic factors on the body’s response in modeling a therapeutic procedure are still required. Many reports indicate the importance of FISH technique in revealing sensibility to structural rearrangements such as stable aberrations as translocations and many other abnormalities in individual’s chromosomes. This technique has significant impact for public health as a biomarker of applied in human monitoring for the health effects of exposure, in particular the cancer risk. The aim of this study was to compare vulnerability to the induction the aberrations in chromosome pairs: 1, 2 and 4 by X-ray’s challenging high dose (2 Gy) in lymphocytes from colorectal cancer patients (age range with 39 – 73) with results from control group-healthy subjects (age range 25 – 60), of various occupations, including nuclear medicine workers. We wanted to find out if their cellular radiosensitivity can be associated with predisposition to cancer induction as well as any endogenic or egzogenic factors i.e.: occupational exposures, smoking habit or diets. Cells were examined in metaphase after application the three-color FISH technique. Preliminary results showed extensive variability in susceptibility to radiation, expressed in significantly elevated or lowered frequency of aberrations for chromosome 1, 2 and 4 for various patients with colorectal cancer, when compared to average of control group. Results are in an agreement with our previous findings from classic cytogenetics, that have shown higher than in a control group level of chromosome damage and strong variation between individuals. Additionally our results suggest, that application of challenging dose and FISH technique may identify patients more sensitive or resistant to radiation as well as a role of a life style related factors modifying their vulnerability to the therapeutic treatment. Secondly, our results from control group, also show to what extent diagnostic or occupational exposures might alter susceptibility to other genotoxic and health risk. Acknowledgments Work partly supported by NCBiR Research Task No. SP/J/6/143339/11, KBN 501/G/538, MNiSzW N N404034039, CIOP-PIB National Programme.

Speaker: Dr Justyna Miszczyk (Institute of Nuclear Physics PAN, Kraków, Poland)

63 DNA end resection is required for the repair of complex lesions in human G1 cells

We study DNA double strand break (DSB) repair with heavy ion radiation. As densely ionizing radiation it generates strictly localized DSBs within the nucleus and thus, represents an excellent tool for investigating the recruitment of repair factors to DSBs by immunofluorescence. Applying this technique we were able to show that with increasing lesion complexity DSBs induced in the G1 cell cycle phase require resection since RPA is located at DSBs in G1. This was surprising as DSB resection is mostly known as a prerequisite of homologous recombination in the S and G2 cell cycle phase. We further revealed that the nucleases CTIP, MRE11, and EXO1, which are already known for resection in G2 are also crucial for the observed resection in G1. Their concomitant down regulation by RNA interference completely prevents resection in G1. Further, our data reveal that resection of ionizing radiation induced DSBs is important for their repair and thus cell survival. In our presentation we will further discuss the regulation of DSB resection in G1 upon heavy ion irradiation.

Speaker: Nicole Averbeck (GSI Helmholtzzentrum für Schwerionenforschung)

64 Single and double strand breaks of DNA in peripheral blood lymphocytes of chronically exposed individuals

Our purpose was investigation DNA single-strand breaks (SSB) and double-strand breaks (DSB) levels in peripheral blood lymphocytes exposed individuals 60 years after exposure. Two groups of humans was formed: exposed (51 subjects) and non-exposed (24 subjects). In the group of exposed individuals were identified individuals with chronicradiation syndrome (CRS) – 8 individuals, and with leucopenia – 14 individuals. Groups are similar by age, sexual and ethnic structure. We used comet assay alkaline and neutral variants. The parameters of assessing the level of DNA breaks were used the percentage of DNA in tail (PDNA) and tail moment (TM). In peripheral blood lymphocytes of exposed individuals in comparison with non-exposed we detect increase values of parameters of SSB: PDNA 4,55±0,32 % vs 3,11±0,52 % (р=0,025), TM 0,66±0,08 %*mcm vs 0,23±0,06 %*mcm (р=0,001). During comparison non-exposed with subgroups of exposed was received following values of parameters PDNA and TM: in group of exposed with CRS 5,08±0,81% and 0,85±0,21 %*mcm (р=0,06 and р=0,022 resp.), in group of exposed with leucopenia 4,96±0,71% and 0,76±0,16 %*mcm (р=0,046 and р=0,007 resp.), in group of exposed without CRS and leucopenia 4,18±0,38% and 0,55±0,11 %*mcm (р=0,022 for TM). In peripheral blood lymphocytes of exposed individuals in comparison with control we detect increase values of parameters of DSB: PDNA 13,67±0,56 vs 10,30±0,70 % (р=0,001), TM 2,94±0,28 %*mcm vs 1,91±0,26 %*mcm (р=0,001). During comparison non-exposed with subgroups of exposed was received following values of parameters PDNA and TM: in group of exposed with CRS 15,67±1,01 and 3,81±0,75 %*mcm (р=0,001 and р=0,041 resp.), in group of exposed with leucopenia 13,36±0,93 % and 3,06±0,38 %*mcm (р=0,014 and р=0,021 resp.), in group of exposed without CRS and leucopenia 13,27±0,83 % и 2,64±0,40 %*mcm (р=0,01 for PDNA). Thus for peripheral blood lymphocytes of chronically exposed individuals increase DNA SSB and DSB level is character. Most differences were detected for exposed individuals with CRS and leucopenia.

Speaker: Ms Alyona Pogodina (Ural Research Centre for Radiation Medicine)

65 Ionizing radiation induced genomic instability in human fibroblasts and modulating effects of telomerase activities in different processes of DNA DSB repair

There have been suggestions that the telomerase system is involved in radiation induced genomic instability. The aim of this study was to investigate the involvement of telomerase in the development of chromosomal damage and its repair at different stages of the cell cycle following an exposure of human fibroblasts to ionizing radiation. We examine the relationship between several response criteria, including cell survival, chromosomal damage (using micronucleus), G2 induced chromatid aberrations (using conventional G2-assay as well as chemically induced premature chromosome condensation assay), and double strand breaks of the DNA (using γ-H2AX and Rad 51) in two cell lines, BJ human foreskin fibroblasts and BJ1-hTERT- a telomerase-immortalised BJ cell line. To distinguish cells in G1, S and G2 phase, cells were co-immunostained for CENP-F, a protein whose expression is tightly cell cycle-regulated. Cells were also immunostained for the protein Cyclin A and the DSB marker 53BP1. Following X-ray-irradiation (doses in the range of 0.1-6 Gy), for cell survival and micronuclei, where the overall effect is dominated by the cells in G1 and S phase no difference was found between the two cell types; in contrast, when radiation sensitivity at G2-stage of cell cycle was analysed, a significantly higher sensitivity was observed for the BJ cells in comparison to the BJ1-hTERT cells. Therefore, it can be concluded that telomerase appears to be involved in DNA double strand break repair processes namely in G2 phase. The data, taken overall, reinforce the idea that hTERT or other elements of the telomere/telomerase system may defend the chromosomes integrity by influencing the repair at G2 phase of cell cycle in human fibroblasts.

Speaker: Dr Otilia Nuta (Radiation and Environmental Science Centre, DIT, Republic of Ireland; Department of Toxicogenetics, Leiden University Medical Center, the Netherlands; Health Protection Agency, Centre for Radiation, Chemical and Environmental Hazards, Oxfordshire, U.K.)

66 PROTON RADIATION DOSE INFLUENCE ON NUMBER OF DSB'S IN CANCER CELLS

In this investigation pancreatic cancer cells MIA PaCa-2 were irradiated with various doses of proton radiation: from 1 Gy to 30 Gy. Experiments were done using Tandetron 4110 accelerator in Center for Physical Sciences and Technology(which is in Vilnius, Lithuania). Irradiation was executed when cells formed monolayer. Distance from exit window to sample was 22 mm. Diameter of proton exit window was 0.175 mm, window was cover with 5 um Mylar film. Properties of proton beam was evaluated with GEANT 4 (energy losses in Mylar and air, range in matter, processes of interaction, spectra of electrons created by protons etc.) and MCNPX (beam radius dependence on distance from proton exit (to the atmosphere) to the sample. Initial energy of protons were 1.6 MeV, beam radius (FWHM) at sample plane was 0.85 mm. It is important to know beam radius to assure even dose distribution. It was calculated number of protons and created electrons per cell and per DNA volume. From modeling results (processes of interaction) possible number of lesions in DNA was evaluated. From these numbers presumptive number of DSB's per cell could be calculated. Actual number of DSB's was checked using flow cytometer. Survival using colony method was also measured for dose control.

Speaker: Mindaugas Malcius (Center for Physical Sciences and Technology, Vilnius, Lithuania)

67 The influence of end resection by CtIP on the Artemis dependent DNA double-strand break repair pathway in G1 and G2 phase after high- and low-LET irradiation

The major repair pathways for DNA double-strand breaks (DSBs) are non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ is active in all cell cycle phases and repairs DSBs with fast kinetics. It represents the major repair pathway in G1 and G2. In contrast, HR is the main repair pathway for replication-associated DSBs in S phase and also repairs a subfraction of DSBs in G2. In G1 and G2 a small fraction of DSBs, representing those located in heterochromatic regions, is repaired with slow kinetics. This slow repair component represents HR in G2 phase but an NHEJ process in G1. Deficiency of the endonuclease Artemis leads to a significant repair defect after X-irradiation in the slow repair component in G1 and G2. In G2, this Artemis dependent defect is associated with HR-mediated DSB repair, but the precise role of Artemis remains unclear. One model is that Artemis endonuclease activity processes secondary or cruciform structures which preferentially arise during the resection of DSBs in heterochromatin. To test this model we depleted CtIP, a factor necessary for DSB end resection and HR, in an Artemis deficient background and irradiated the cells with X-rays. Our results indicate that CtIP depletion completely rescues the Artemis repair defect in G1 and G2, suggesting that in the absence of CtIP DSBs are repaired independently of Artemis by NHEJ. In contrast to X-rays, irradiation with high-LET particles leads to a pronounced DSB-repair defect in Artemis-downregulated G1 and G2 cells. The repair defect was more severe compared to Artemis-depleted cells irradiated with X-rays; almost all DSBs remained unrepaired up to 72h post IR and the lack of repair was independent of the chromatin status of the DSBs. Interestingly, depletion of CtIP alone also induced a pronounced repair defect after high-LET irradiation in G1 and G2, which is in stark contrast to the irradiation with X-rays. This leads to the assumption that almost all DSBs induced by high-LET radiation undergo resection by CtIP irrespective of the cell cycle phase and their chromatin-status. Furthermore, co-depletion of CtIP and Artemis revealed that CtIP-depletion cannot rescue the Artemis-repair defect after high-LET irradiation. In summary, almost all DSBs induced by high-LET irradiation undergo resection by CtiP and further processing by Artemis, irrespective of their chromatin status; in G2-cells these resected and processed DSB are repai

Speaker: Dr Sandro Conrad (Technical University of Darmstadt)

68 Accumulation of non-DSB oxidative clustered DNA lesions in irradiated BRCA1 deficient cells affects mitigation of radiotoxicity and enhances chromosomal instability

Radiation can increase significantly the level of oxidative stress that cell have to sustain and mitigate. Persistent of such stress can lead to unrepaired or misrepaired DNA lesions the accumulation of which is considered for many the initiation point for chromosomal instability, neoplastic transformation and human pathogenesis. Indeed, the presence of high oxidative stress in human patients or mice carrying BRCA1 mutations, in a homozygous or heterozygous form, is associated with a varying level of different lesion repair deficiencies, chromosomal instability, premature aging, high frequency of lymphoma, and ovarian or mammary tumour formation. In order to evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild type MCF-7 cells. Cell lines with DNA-PK repair deficiencies and peripheral blood lymphocytes from breast cancer patients with BRCA1/2 germ line mutations were also used. Repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) and single strand breaks (SSBs) was measured in cells exposed to γ-ray doses (3-5 Gy). Given that radiosensitivity and chromosomal instability is related to accumulation of chromatid breaks, parallel experiments were performed in the induction of chromatid breaks after G2-phase irradiation by means of a standardized G2-assay that we have recently proposed. Independent monitoring of the γ-H2AX foci was also performed and metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells were classified as highly radiosensitive since they showed a significant accumulation of OCDLs and chromatid breaks compared to MCF-7. BRCA1 partial expression contributed significantly in the overall repair of OCDLs. HCC1937 cells irradiated in plateau phase, surprisingly they demonstrated at metaphase a much higher level of unrejoined single chromatid lesions and a decreased yield of dicentrics chromosomes in comparison to the yields obtained in MCF-7 cells. Therefore, accumulation of OCDLs can lead to surviving cells with a high level of chromosomal aberrations and instability prone to transformation, suggesting a genuine role of BRCA1 in the mitigation of radiotoxicity, chromosomal instability and the development of breast cancer.

Speaker: Dr Georgia Terzoudi (Radiobiology Laboratory, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, National Centre for Scientific Research “Demokritos” Athens, Greece)

69 Involvement of chromatin organization at different cell-cycle stages in the conversion of DNA lesions into chromatid breaks and exchanges

The formation of diverse chromosomal aberrations following irradiation at different cell-cycle stages remain a long standing controversy, probably because most of the studies have focused on elucidating the enzymatic repair mechanisms involved using simple DNA substrates. Yet, recognition, processing and repair of DNA damage occur within the nucleoprotein complex of chromatin which is dynamic in nature, capable of rapid unfolding, disassembling, assembling and refolding. Using conventional cytogenetics and premature chromosome condensation to visualize interphase chromatin, we discuss in the present work the current status of knowledge and evidence to support the hypothesis that chromatin organization at different cell-cycle stages is an important determinant in the conversion of sub-microscopic DNA lesions into chromatid breaks and the formation of exchanges. Specifically, using G2-checkpoint abrogation by caffeine, we have investigated the formation and repair kinetics of chromatid breaks during G2-M transition and the correlation of G2-checkpoint efficiency to prevent chromatid breakage with G2 chromosomal radiosensitivity. Our results demonstrate that radiation-induced chromatid breaks during G2-M transition are irreparable. This observation is important since it justifies the use of an increased yield of chromatid breaks at metaphase following G2-irradiation, as a reliable predictive biomarker for individual radiosensitivity. Furthermore, we present data to demonstrate the vital importance of chromatin organization and the proximity of breaks in determining the kinds and frequencies of exchanges. When cells are irradiated at metaphase and chromosomes are analyzed in daughter cells in the subsequent G1 or G2 phase, the ratio of inter-change to intra-change it shifts to almost entirely to the intra-change category and even more to the intra-arm intra-change. Consequently, the type and yield of radiation-induced chromosomal aberrations at a given cell-cycle-stage depends on the combined effect of DNA repair processes and chromatin organization, which is cell-cycle-regulated and subject to up- or down-regulation following mutagen exposure or genetic alterations. This hypothesis is used to revisit unresolved issues and in particular to explain the variability in radiosensitivity observed at various cell-cycle-stages, among mutant cells and cells of different origin, or among different individuals.

Speaker: Dr Gabriel Pantelias (Radiobiology Laboratory, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, National Centre for Scientific Research “Demokritos” Athens, Greece)

Non-Ionizing Radiation

70 Interactions between the electric fields and the cell membranes: from the basic mechanisms to the clinical applications

When electric pulses are applied to cells in vitro and even in vivo, the main interaction occurs at the level of the cell membranes. Indeed, cell membranes are not conductive elements while both the external and the internal media, resulting in the generation of transmembrane potential differences. Due to the thinness of the membrane (5 nm), the resulting local electric field at the membrane can be extremely high resulting in the destructuration of the membrane (electroporation). Then the membranes remain permeable for several minutes, even after the delivery of an ultra-short pulse of a few nanoseconds of high field magnitude. This electropermeabilization allows molecules that usually cannot cross the plasma membrane (nonpermeant molecules) to enter the cells. One of the in vitro popular applications of the electroporation is the delivery of nucleic acids (DNA or RNA, short or long), which can also be performed in vivo and which has already moved to clinical trials. However, the most achieved application is the antitumoral electrochemotherapy that was conceived and developed by our team. After successful preclinical and clinical trials, electrochemotherapy is now routinely used to treat cutaneous and subcutaneous tumors in about 90 cancer centers in the EU. Clinical trials are dealing now with the treatment on deep seated tumors like bone metastasis, unresectable liver metastasis, etc. The fundamentals of the interactions of the electric pulses with the membranes were actually unknown. Very recent results of our team will be presented and discussed as they bring new insights that may explain the mechanisms of the membrane electropermeabilisation. Interestingly, they raise the question of the limits between the ionizing and the non-ionizing radiations.

Speaker: Dr Lluis M. Mir (UMR 8203 Vectorology and Anticancer Therapies, CNRS, University Paris-Sud, Institut Gustave Roussy, 94805 Villejuif, France)

71 How consistent and coherent are the available estimates of glioma risk in relation to mobile phone use?

Over 60 studies on mobile phone use and intracranial tumours in adults have been carried out to date, while one study only has examined this relation in children. This brief overview focuses on malignant brain tumors in adults (labeled “glioma” from the most frequent morphotype). The 24 studies on glioma risk in adult mobile phone users published by the end of 2011 include 2 cohort studies of mobile phone subscribers, 16 country-specific case-control studies, 3 pooled analyses of published primary studies, and 3 different analyses of the Interphone international study, carried out on the whole data-set from the 13 participating countries, or on data-subsets. Available studies are far from consistent. Moderate degrees of variation in effect size attributable to heterogeneity are observed in the meta-analysis of risk estimates for ever vs never regular use, as well as for short-term mobile phone use, with combined relative risks around the null value in both cases. The combined relative risk is slightly increased among long-term uses (≥10 years), but the original measures of effect are highly heterogeneous and the finding is entirely attributable to the Örebro study series. Possible distortions from recall and selection or participation bias complicate the interpretation of findings from case-control studies, while non-differential exposure misclassification is an issue for the subscribers’ cohort studies. Side-validation studies allowed to attribute at least in part the deficits in risk observed in the Interphone studies to participation bias, and the increased risk among heavy users to recall bias and/or systematic exposure error. The exposure index used in the Danish cohort study (subscriber status) was shown to have low sensitivity but very high specificity, which means that the downward bias expected from random exposure measurement error is of minor importance in the analyses of risk in low-prevalence strata such as long-term users. The increased glioma risks observed in some epidemiologic studies are not compatible with the incidence rates of glioma recorded in the Nordic countries and the US in middle-aged adults (40–59 years) during the past 20 years, assuming induction periods up to 10 years. A modest risk increase among heavy users, on the order of that seen in the Interphone study (40%), cannot yet be excluded, and there are still few data on cancer risk beyond 15 years since start of mobile phone use.

Speaker: Dr Susanna Lagorio (National Centre for Epidemiology, Surveillance and Health Promotion - National Institute of Health, Rome (Italy))

72 The effects of Electromagnetic pulse on migration of T lymphocytes

Junye Liu, Huawei Zhang, Xia Miao, Guirong Ding, Yurong Li, Guozhen Guo Department of Radiation Medicine, Fourth Military Medical University School of Preventive Medicine, Xi’an 710032, Shaanxi, China * This work is supported by National Natural Science Foundation of China, No. 30972430 and 81072272. Background: It has been found in our previous study that electromagnetic pulse (EMP) could increase the lymphocyte number in murine spleen and inhibit proliferation of splenocytes, which suggests EMP induced accumulation of lymphocytes in spleen. This study was aimed to evaluate the effects of EMP on T lymphocyte migration. Methods: Balb/c mice were exposed to EMP radiation. The in vitro migration directed by chemokines SDF-1 and CCL5 of T lymphocytes from spleen, thymus was evaluated with transwell assay. Results: The mice received 200 times of irradiation of 200 kV/m EMP and sacrificed at designated time points. In vitro migration assays revealed that EMP increased migration of splenocytes and thymocytes to SDF-1 and CCL5. The enhanced effects of EMP emerged 1d after radiation, reached to its peak at 7d after EMP radiation. In another experiment, the mice were exposed to 200 kV/m of EMP radiation with different exposure times (100-400 times) and migration assays were performed 7d after radiation. It was shown EMP could improve T lymphocyte migration in a dose-dependent manner. Conclusion: EMP radiation could increase chemotaxis of T lymphocytes in time-dependent and dose-dependent manners, suggesting that EMP could improve the recruitment of T lymphocytes to inflammation sites and thus influence the adaptive immunity. It will be interesting to further explore the underlying mechanisms. Keywords: electromagnetic pulse (EMP); chemotaxis; lymphocyte; SDF-1; CCL-5

Speaker: Prof. Junye Liu (Fourth Military Medical University)

73 The nucleotide pool sanitization enzyme hMTH1 protects cells from mutation induced by UVA

The exact mechanism by which UVA radiation induces mutations is not yet fully understood. It is known that UVA can induce different types of DNA damage via production of free radicals. Free radicals that react with nucleic acids may give rise to mutations, sister chromatid exchanges and chromosomal aberrations. UVA can also oxidize dNTPs in the nucleotide pool. Oxidized dNTPs such as 8-oxodGTP and 8-oxodATP can be incorporated into the DNA during replication and cause mutations. This process can be inhibited by the nucleotide pool sanitization mechanism, mediated by the hMTH1 protein, in which oxidized dNTP is dephosphorylated to dNMP (e.g., 8-oxo-dGTP to 8-oxo-dGMP). 8-oxo-dGMP dephosphorylates to 8-oxo-dG which is then excreted to the extracellular matrix. The aim of this study was to investigate the protective role of hMTH1 toward UVA induced cytotoxicity and mutagenicity. For this purpose human B lymphoblastoid cell line (TK6) was transfected with shRNA directed against hMTH. The expression level of hMTH1 in the transfected cells was investigated by Western blot technique. A mutation frequency assay was done to study mutation frequency induced by UVA in the transfected and non-transfected TK6 cells. In transfected TK6 cells, a significantly increased level of mutations was observed after UVA irradiation. (Fotouhi A. et al., 2011). To investigate what types of mutations that are induced by UVA in the transfected and non-transfected cells, mutations spectra in the Thymidine kinase (Tk) gene was determined. The most prominent type of mutation found was base pair substitutions, more precisely GC>AT transitions in non-transfected exposed TK6 cells whereas AT>GC transitions dominated in transfected TK6 cells. The results imply that UVA induces mutations primarily by oxidizing 8-oxodATP in the nucleotide pool.

Speaker: Ms Asal Fotouhi (Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholms university)

74 Single-strand DNA breaks in human hair root cells exposed to mobile phone radiation

Purpose: To analyze the short term effects of radiofrequency radiation (RFR) exposure on genomic deoxyribonucleic acid (DNA) of human hair root cells. Subjects and methods: Hair samples were collected from 8 healthy human subjects immediately before and after using a 900-MHz GSM (Global System for Mobile Communications) mobile phone for 15 and 30 minutes. Single-strand DNA breaks of hair root cells from the samples were determined using the ‘comet assay’. Results: The data showed that talking on a mobile phone for 15 or 30 minutes significantly increased (p< .05) single-strand DNA breaks in cells of hair roots close to the phone. Comparing the 15-min and 30-min data using the paired t-test also showed that significantly more damages resulted after 30 minutes than after 15 minutes of phone use. Conclusions: A short-term exposure (15 and 30 minutes) to RFR (900-MHz) from a mobile phone caused a significant increase in DNA single-strand breaks in human hair root cells located around the ear which is used for the phone calls.

Speaker: Dr SEMRA TEPE ÇAM (Turkish Atomic Energy Authority)

75 The synergistic effects of ultrasound and laser on the enhancement of transdermal insulin delivery in diabetic rats

The cardinal goal of this study was to examine the effect of 3-MHz ultrasound and/or laser on transdermal insulin delivery and glucose level in diabetic rats. Diabetes was induced in 45 male rats using Streptozotocin. Animals were randomly divided into 9 groups of 5 animals each. A leak-proof chamber containing either normal saline or insulin was placed on the shaved abdominal skin of the rats. These groups include; rats with no intervention (G1); those treated only with normal-saline (G2); those treated only with insulin (G3); those exposed to laser with normal-saline in the chamber (G4); those exposed to laser with insulin in the chamber (G5); those exposed to ultrasound with normal-saline in the chamber (G6); those exposed to ultrasound with insulin in the chamber (G7); those exposed to both laser and ultrasound with the chamber filled with normal saline (G8) and finally those exposed to both laser and ultrasound with the chamber filled with insulin (G9). The level of blood glucose was measured at minutes 0, 15, 30, 45, 60, 75, 90, 105, and 120 after starting the experiments. The minute 0 refers to the exact time that the anesthetic was administered (before starting irradiation with either laser or ultrasound). Non-parametric tests including Wilcoxon, Kruskal –Wallis, and Mann-Whitney were used for data analysis. These results clearly indicate that the glucose level in rats exposed to both irradiations (laser plus ultrasound) was significantly less than those of exposed to any of these two irradiations alone. To the best of our knowledge, this study is the first investigation that assesses the combination effect of these two physical interventions in physical agents- mediated drug delivery. These findings may open new horizons in non-invasive drug delivery.

Speaker: Dr Ali-Reza Mehdizadeh (Shiraz University of Medical Sciences)

Non-Targeted Effects of Radiation

76 Implications of Non-targeted effects for advanced radiotherapy

Our understanding of the mechanisms of radiation response in biological systems has been changing from studying the role of radiation damage at the DNA and cellular level to integrated models of tissue and whole organism responses. Key to this has been a growing appreciation of bystander responses where cells respond to their neighbours being irradiated and intercellular signalling mechanisms play a role. Bystander responses have been observed locally between cells but also over longer ranges including between tissues in the intact organism. Microbeam approaches, where localised radiation beams can be delivered, have been key experimental tools which are allowing the delineation of mechanisms underpinning bystander signalling in a range of models. For advanced radiotherapies, delivery of dose into tumours is highly modulated in both space and time. A major consequence is the production of dose-gradients across tumours and normal tissues as dose is “painted” into the treatment volume. Little is known about the underlying biological rationale optimising these approaches and the role, if any bystander signalling will play. More recently studies with modulated photon beams have begun to address these issues to develop biologically driven models for radiotherapies which can take into account any impact of bystander signalling. This is leading to a redefinition of bystander signalling, away from the hit versus non-hit signalling hypothesis, to one where the response of cells and tissues to radiation exposures is via an integrated response to oxidative damage.

Speaker: Prof. Kevin Prise (Queen's University Belfast)

77 Implications of non-targeted effects of radiation exposure for radiation protection

“Non-targeted effects” is an all embracing term to include biological effects observed in cells that were themselves not irradiated, but were the progeny of an irradiated cell, or received signals from an irradiated cell. These non-targeted effects include a variety of endpoints usually associated with exposure to ionizing radiation, e.g., chromosomal rearrangements, mutation induction and cell killing. In terms of radiation protection, we have traditionally assumed that the irradiated volume is associated with potential risk for developing the delayed effects, including cancer, but there were negligible risks to the non-exposed tissues. Non-targeted effects however, suggest that the volume at risk for delayed effects may be greater than the volume actually irradiated. Nevertheless, there is only a risk if these non-targeted effects are detrimental, if they are beneficial, the implications for radiation protection may be very different. This presentation will discuss the implications of non-targeted effects for radiation protection given these different scenarios.

Speaker: Dr William F. Morgan (Pacific Northwest National Laboratory, Richland, WA 99352, USA)

78 TNFR2/p75 Signaling Induces Delayed Radiobiological Bystander Responses in BM-derived EPCs: Implications for Development of Mitigating Factors

Tumor necrosis factor(TNF) binds two receptors TNFR1/p55 and TNFR2/p75 and activates several signaling cascades. Ionizing radiation (IR) increases tissue levels of TNF. TNF signaling regulates numerous cytokines/chemokines that may mediate IR-induced non-targeted effects (NTE), a phenomenon where cells that are not directly “hit” by IR exhibit IR effects via signals received from distant IR cells. Little is known about the role of p55 or p75 in regulating NTE in bone marrow (BM)-derived endothelial progenitor cells (EPCs). Medium transfer experiments (MTE) were performed ex-vivo with BM-derived EPCs from WT, p55 knockout(KO) and p75KO mice. EPCs were irradiated with 1Gy of γ-IR, then IR-conditioned medium (CM) was collected at 1, 5, 24 hrs, and 3, 5 days post-IR. Filtered (0.22µm) IR-CM was transferred to naïve non-IR EPCs of the same genotype. After 24 incubation, CM from IR EPCs was processed for ELISA profiling (16 genes) and IR EPCs from p55KO mice were processed for microarray profiling. CM-treated EPCs were processed for p-H2AX/p53BP1 staining for presence and decay of double strand breaks (DSB). In WT EPCs the peak of mean p-H2AX foci/cell was at 24h, whereas in p55KOs the number of p-H2AX foci/cell were decreased twice on day 5 (9±0.8 vs 4.8±0.6, p<0.01, WT vs p55KOs). These finding indicate that altered TNF signaling inhibits early NTEs (hours) in BM-derived EPCs. Compared to WT, delayed (5 days) NTEs were increased in IR-CM-treated p55KO EPCs (3.8±0.4 vs 8.5±1, p<0.02, WT vs p55KO), suggesting significant role of TNF-TNFR/p75 signaling (the remaining active receptor in p55KO EPCs) in mediating delayed NTEs. ELISA profiling of 16 proteins in IR-CM over 5 days post-IR showed 200-1600% increases (p<0.002, p55KO vs WT) in cumulative levels of TNF, IFNr, IL1α, IL1β, IL6, EGF, MIP-1α, MCP-1, GM-CSF. Microarray profiling of γ-IR p55KO EPCs revealed 3999 significantly expressed genes (one-way ANOVA, p<0.05) with 1179 genes with false discovery rate (FDR) <0.05 (day 5: 371 genes were 2-fold up/down), and 194 at FDR<0.01 (day 5: 93 genes were 2-fold up/down), a significant increase in gene transcription 5 days post-IR. We conclude that TNF ligand-receptor axis regulates NTEs in naïve EPCs and suggest that restoring TNF signaling balance could represent a preventative/mitigating measure for inhibition of delayed NTEs in tissues adjacent or distant from primary IR target.

Speaker: Mr Sharath Sasi (Tufts University)

79 125I-monoclonal antibodies triggercell membrane-mediated bystander effects.

Objectives The observation of radiation-induced bystander response has important implication for understanding the efficiency of radiotherapy particularly after low-dose exposure. We investigated the role of bystander phenomena in cells exposed to low-dose radioimmunotherapy (RIT) using 125I-monoclonal antibodies (mAb). Methods: Contribution of bystander effects was assessed using standard medium transfer experiments between donor and receiver cells. To identify the role of the internalization of mAbs in the bystander response, cells were incubated in the presence of sodium azide, a drug blocking the internalization of antigens. The role of cell membrane rafts in 125I-mAbs-induced bystander effects was investigated by incubating donor cells undergoing RIT with either Methyl-β-cyclodextrine or Filipin, two lipid raft disruptors. Finally, the potential role of oxidative processes in bystander effects was tested using N-Acetyl-L-Cystein (NAC) or DMSO, two radical scavengers. Results: Following treatment with anti-CEA or anti-HER1 125I-mAbs, we observed a decrease in survival of both donor(to 60% of control) and receiver cells (to 25% of control), confirming the presence of bystander effects. No cell killing in receiver cells was observed after donor cells were treated with the non-targeting mAb125I-PX, indicating the specificity of the latter response. Importantly, when anti-CEA or anti-HER1 125I-mAbs were trapped at the surface of donor cells, no significant difference in survival was observed, compared with the above results, obtained in response to internalized 125I-mAbs, both for donor or receiver cells. These results suggest that internalization of 125I within the cell is not required for the generation of a cytotoxic bystander response. In addition, the disruption of lipid rafts significantly increased clonogenic survival in donor (P<0.001) and receiver cells (P<0.05), indicating the important role of the cell membrane and more specifically of lipids raftsin the production of bystander effects. Furthermore, the observation that NAC increased the survival of donor and receiver cells indicated the involvement of oxidative stress in bystander effects associated withtreatment using125I-mAbs. Conclusions: These results provide evidence that the cell membrane plays an essential role in the mediation of bystander responses induced by125I-mAbs.

Speaker: Dr Salomé PAILLAS (IRCM, INSERM)

80 Radiation-induced adaptive response in fruit flies with mutations in DNA repair genes

Low doses of ionizing radiation induce a wide range of biological effects including hormesis, adaptive response and hypersensitivity that modulate lifespan. However, the molecular mechanisms underlying the lifespan effects of low-dose irradiation are poorly understood. DNA repair is one of the key mechanisms of cell response to different types of stress including ionizing radiation. The purpose of this work is to investigate the role of genes of base and nucleotide excision repair (PCNA, XPC, XPF, D-Gadd45 homologues) and genes of DNA double-strand breaks repair (BLM, Rad50, Rad51, Rad54 homologues) in the radiation-induced adaptive response in Drosophila melanogaster. The lifespan of flies with Canton-S wild-type and w1118 genotypes and flies with mutations in DNA repair genes were estimated under different irradiation conditions: 1) without irradiation; 2) chronic exposure at 40 cGy dose rate induced by Ra226 source during preimaginal developmental stages (10 days); 3) acute exposure at 30 Gy dose rate induced by Co60 source after imago eclosion (30 minutes); 4) successive irradiation by both doses. It was found that the chronic low-dose irradiation induced the radioadaptive response to the acute exposure in Canton-S wild-type flies. The negative effect of the acute irradiation on the lifespan declined 2 times after pre-irradiation with low-doses. Radioadaptive response was persisted but was less expressed in flies with heterozygous mutations in PCNA, XPC, Rad51, Rad54 homologues, and was absented in flies with homozygous and heterozygous mutations in XPF, D-Gadd45, BLM homologues. Obtained results demonstrate that investigated DNA repair genes play a significant role in radiation-induced adaptive response at an organism level in vivo.

Speaker: Prof. Alexey Moskalev (Institute of biology of Komi Science Center of RAS)

81 Evaluation of the NF-kB pathway dynamics after exposure to gamma radiation

Background: NF-κB transcription factors are critical regulators of immunity, stress responses, apoptosis and differentiation. A variety of stimuli coalesce on NF-κB activation. NF-κB-dependent transcription is not only tightly controlled by positive and negative regulatory mechanisms but also closely coordinated with other signaling pathways [1]. Indeed, the NF-kB pathway is activated in response to a wide range of physical and chemical stress stimuli (e.g. ionizing radiation, signaling proteins) and lead to an up- or down-regulation of several molecules, amongst which its own inhibitor. Purpose: Investigate the temporal dynamics of activation of NF-kB and its pathway-related molecules and look at correlations, if any, with external stimuli such as different doses of gamma rays exposure and/or medium change. Experimental setup and Methods: Human skin fibroblasts AG01522 were grown until confluence. Then the culture medium was replaced with new one and after 45 minutes the flasks were exposed to different doses of gamma rays (up to 5 Gy) from a 60-Co source. ELISA Assays were performed to evaluate the amount of NF-kB in the nuclear extracts while other related proteins (e.g. Akt, Erk 1/2) were investigated in the cytoplasmic samples, through Western Blot and ELISA Assay. Interleukins (e.g. IL-6, IL-8) released into the medium were investigated as well, through the previously described techniques. Results: Temporal dynamic studies performed for NF-kB investigation show a fast activation of this transcription factor after culture medium change. This environmental stimulus greatly perturbs this pathway that reaches again the homeostatic equilibrium several hours later. The comparison between sham-irradiated and irradiated cells did not show any statistically relevant additional perturbation to the “in vitro” system, already perturbed by the medium change. Investigations of the other molecules related to this pathway show a perturbation of their expressions with timings comparable to the one of NF-kB. Conclusion: The effects of ionizing radiation exposure on the activation of NF-kB, subsequent to the change of medium in confluent cell cultures, appear to be negligible, or even absent, up to 5 Gy of gamma rays. [1] Oeckinghaus A. et al., Nature Immunol, 12(8):695-708, (2011) This work was partially supported by the European Commission (EC Contract FP7 EURATOM project “EPIRADBIO” and “DOREMI”).

Speaker: Mr GABRIELE BABINI (University of Pavia, Physics Department - INFN, Section of Pavia)

Wednesday, 17 October

Keynote Lecture 2

82 Biomarkers - the challenge to personalize radiation risk

All radiation-late effects after sub-lethal exposure are mediated by additional confounding factors, which can be inherited, or exogenous or purely stochastic in nature. In their combination, they form a network of a few known, but to a large degree yet unknown parameters that make radiation-associated late effects appear as non-deterministic events. During the pathogenesis of radiation-associated late events, pre-existing genetic determinants can have a profound influence onto individual risk, such a sever germline-mutations in DNA repair pathways (Brca1/2, Fanc,Nbs, XP), stress-response genes (ATM, Rb1, Cdkn2a) or genes that regulate other cellular functions (P53, Ptch, Blm). In their most sever forms, mutations in these genes cause clinical syndromes that will contra-indicate any unnecessary radiation exposure. The big challenge in the new genetic era, however, is the discovery of low-penetrance variations in these genes or the associated molecular pathways, which can increase radiation-risk without a pre-existing syndromic condition. In addition to hypomorphic mutations, even bigger challenges are genetic variants in regulatory elements of such genes or in non-coding mRNAs that can influence the protein level. In a more direct relation of the disease pathogenesis after radiation-exposure are biomarkers which can signal early or intermediate steps of the disease progression. More a challenge for the future than current medical practise are the identification and validation of markers to diagnose cancer and non-cancer disease at an early pre-clinical stage, when therapeutic interventions might still block a further disease progression. Abnormal levels of certain cytokines in the peripheral blood, but also tumor markers from disseminated tumor cells or the detection of rare, disease-specific miRNA species in serum might have the potential to identify persons, who are not only under an increased risk following a radiation-exposure and/or a congenital susceptibility, but who warrant an immediate medical care. This network of early genetic and later disease-based biomarkers can therefore help not only to prevent high-risk persons from any unnecessary radiation exposure, but also to reduce the health consequences of an accidental or unavoidable irradiation.

Speaker: Dr Michael Rosemann (Institute of Radiation Biology, Helmholtz-Center Munich, Germany)

Normal Tissue Damage

83 Cardio-pulmonary consequences of thoracic irradiation in rats

The risk of early radiation pneumonitis limits the radiation dose and efficacy of radiotherapy of thoracic tumours. Besides lung dose, in rats (1) and patients (2) dose to the heart was shown to be a risk factor of radiation pneumonitis. Here we investigated whether the enhanced damage caused by combined heart and lung irradiation can be understood from their individual effects on cardio-pulmonary physiology. First we investigated whether the enhanced increase in respiratory rate observed after combined heart and lung irradiation, as compared to irradiation of the lung alone is reflected in the classical signs of radiation pneumonitis. Indeed, we showed that heart irradiation enhanced inflammation and fibrosis. Next we investigated whether early changes in heart function could be observed during the peak of radiation pneumonitis. Although the heart is regarded as a late responding tissue, we did observe reduced FDG uptake and perivascular fibrosis 8 weeks after irradiation. Moreover, we observed increased left ventricle (LV) end-diastolic pressure and relaxation time, both correlating excellently with the perivascular fibrosis of the LV. Altogether this indicates that heart irradiation induces early LV diastolic dysfunction. Lung irradiation alone resulted in irradiated-volume dependent pulmonary vascular remodelling leading to increased pulmonary artery pressure and pulmonary perivascular oedema (3). Moreover, we observed an increase in relaxation time of the LV, suggesting also lung irradiation impairs LV diastolic function. Taken together these results indicate that heart and lung irradiation both induce loss of left ventricle diastolic function albeit through different mechanisms. In agreement with the above we found that heart irradiation leads to enhanced levels of interstitial oedema, whereas both heart and lung irradiation induce perivascular oedema as a known consequence of diastolic dysfunction. Our results explain the enhanced inflammation and fibrosis observed after combined lung and heart irradiation. In summary, heart and lung irradiation independently impair cardiac diastolic performance. In our rat model heart irradiation induces early subclinical patho-physiological changes that, if combined with lung irradiation may manifest as an enhanced risk and severity of radiation pneumonitis. 1. van Luijk P et al., Cancer Res. 2005. 2. Huang EX et al., Acta Oncol. 2011. 3. Ghobadi G et al., Thorax 2012 .

Speaker: Dr Peter van Luijk (Dept. of Radiation Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands)

10:30 Coffee break

84 Normal tissue damage – models of second tumor induction after radiotherapy

In developed countries, more than half of all cancer patients receive radiotherapy at some stage in the management of their disease. A radiation-induced secondary malignancy can be the price of success if the primary cancer is cured. Therefore, there is increasing concern regarding radiation-related second cancer risks in long-term radiotherapy survivors and a corresponding need to be able to predict cancer risks at high radiation doses. Of particular interest are second cancer risk estimates for new radiation treatment modalities such as intensity modulated radiotherapy, intensity modulated arc-therapy, proton and heavy ion radiotherapy. The long term risks from such modern radiotherapy treatment techniques have not yet been determined and are unlikely to become apparent for many years, due to the long latency time for solid tumor induction. Most information on the dose-response of radiation-induced cancer is derived from data on the A-bomb survivors who were exposed to γ-rays and neutrons. Since, for radiation protection purposes, the dose span of main interest is between zero and one Gy, the analysis of the A-bomb survivors is usually focused on this range. With increasing cure rates, estimates of cancer risk for doses larger than one Gy are becoming more important for radiotherapy patients. Therefore in this review, emphasis was placed on doses relevant for radiotherapy with respect to radiation induced solid cancer. Simple radiation protection models should be used only with extreme care for risk estimates in radiotherapy, since they are developed exclusively for low dose. When applied to scatter radiation, such models can predict only a fraction of observed second malignancies. Better semi-empirical models include the effect of dose fractionation and represent the dose-response relationships more accurately. The involved uncertainties are still huge for most of the organs and tissues. A major reason for this is that the underlying processes of the induction of carcinoma and sarcoma are not well known. Most uncertainties are related to the time patterns of cancer induction, the population specific dependencies and to the organ specific cancer induction rates. For radiotherapy treatment plan optimization these factors are irrelevant, as a treatment plan comparison is performed for a patient of specific age, sex, etc. If a treatment plan is compared relative to another one only the shape of the dose-response curve is of of importance.

Speaker: Prof. Uwe Schneider (University of Zurich and Radiotherapy Hirslanden)

85 Ionizing radiation induces acute and chronic modifications of the transcriptional profile of endothelial cells: A Molecular-functional system biology approach

Normal tissue damage after radiation therapy is characterised by a chronic altered phenotype of endothelium. Molecular mechanisms involved in the initiation and the acquisition of a chronic activated phenotype of endothelial cells after radiation exposure remain unclear. The aim of this work is to characterize in vitro molecular actors involved in both the acute and late activated phenotype of endothelial cells. Methods : Human Umbilical Vein Endothelial Cells (HUVEC) were exposed to a single dose of 2 Gy, 20 Gy or a fractionated dose of 10 x 2 Gy. Expression profiling was performed using a Taqman Low density array (TLDA) approach. Gene signature (~500 genes) associated with immune response, apoptosis, angiogenesis, inflammation and protein kinase related genes was performed 0.5, 1, 2, 3, 4, 7, 14 and 21 days after irradiation. Moreover functional assays (Migration and interactions with blood cells) were performed 21 days after irradiation. Results: Irradiation modifies very rapidly the phenotype of endothelial cells after 2 and 20 Gy. Interestingly 21 days after irradiation, gene expression profile analyses reveal a strong persistent altered molecular profile of HUVECs associated with chronic modifications of functions. Moreover, results showed a specific acute molecular signature at 2 and 20 Gy but also numerous mRNA expression levels modified from 12 hours and remainder modified 3 weeks after 20 Gy. Finally, comparison of the single and the fractionated dose of 20 Gy reveal both specific molecular signatures associated with the total dose and with the regimen of the exposition. Conclusion : The molecular profile of endothelial cells exposed to ionising radiation is very rapidly modified and kinetic analyses showed that HUVECs acquire a chronic pathological phenotype until 3 weeks after radiation exposure. Our results confirm numerous previous data published in the literature (overexpression of IL-6, IL-8, ICAM1, E-selectin… ) and reveal modifications, both in acute and chronic response, of the expression levels of numerous molecular actors not described until now as radiation-responsive genes. Our study suggests that our in vitro model is a useful approach to characterise molecular mechanisms involved in the initiation and the acquisition of a radiation–induced chronic pathological phenotype. This model will allow us the molecular modelling of radiation-specific endothelial cell response.

Speaker: Dr Fabien MILLIAT (Institute for Radiological Protection and Nuclear Safety)

86 FISH analysis of chromosome damage in whole blood as indicators of late radiation toxicity after radical radiotherapy in prostate cancer

Whole chromosome fluorescence in situ hybridization (FISH) allows for the detection and identification of chromosome translocations in metaphase spreads. Previous research has shown that radiation induced translocations correlate with both acute and late effects after radiotherapy [1]. This study has examined the incidence of translocations, after exposure to in vitro radiation, in both normally responding patients (pts) and those exhibiting late effects after radiotherapy treatment to evaluate FISH as a method for predicting radiosensitivity. Patients were selected from a randomized trial evaluating the optimal timing of Dose Escalated Radiation (76 Gy) and short course Androgen Deprivation Therapy for intermediate-risk prostate cancer. In the first 350 pts entered on trial with mature follow-up (mean 78 months), 3% developed grade 3 late proctitis. Blood samples were taken from this radiosensitive cohort (10 pts) along with matched control pts (20 pts) with no late proctitis. Whole blood samples were exposed to 0 or 4 Gy and cultured according to the IAEA recommended methods. Staining was carried out according to the standard protocol provided by the manufacturer of the probes (Cytocell 1,2,4 Direct Probe). At least a 1000 metaphase spreads, or up to 100 translocations per sample were scored according to the PAINT system. While both groups were statistically similar at 0 Gy, preliminary results indicate that after an in vitro dose of 4 Gy, the radiosensitive group had significantly higher rates of translocations (average=2.51, SD=0.36) compared to the control group (average=1.89, SD=0.23) (p<0.002). Further statistical analysis will investigate different types of damage including differentiating between stable and unstable damage. These results confirm previous studies and indicate that the analysis of translocations using FISH after in vitro irradiation correlates with clinical response to radiation. This cytogenetic assay should be considered as a predictor of radiosensitivity. Reference [1] S. Neubauer, J. Dunst, and E. Gebhart, "The impact of complex chromosomal rearrangements on the detection of radiosensitivity in cancer patients," Radiother. Oncol., vol. 43, no. 2, pp. 189-195, May1997.

Speaker: Ms Lindsay Beaton (Health Canada)

87 TP53INP1 gene is implicated in radiation-induced senescence

BACKGROUND AND PURPOSE: Tumor protein 53-induced nuclear protein-1 (TP53INP1) encodes two nuclear protein isoforms (TP53INP1 and TP53INP1) and transcription is activated by p53. Overexpression of TP53INP1 promotes apoptosis and cell cycle arrest in different tumor cell lines. Therefore, TP53INP1 appears as a key element in p53-mediated cell death and cell cycle arrest, induced by cellular stress. Moreover, it was shown that TP53INP1 interacted with p53 and these interactions modified the transcriptional activity of p53 on several target genes such as CDKN1A, PIG-3 and MDM2. The objective of this study was to assess whether TP53INP1 plays a functional role in regulating cellular responses to IR. METHODS: F11hTERT (telomerase immortalized) human fibroblast cells were used in the study. To investigate the role of TP53INP1 in radiation response the gene was silenced by the stable transfection of shRNAs using lentiviral vectors. Cells were irradiated direct and bystander effects were assessed by following survival using colony-forming assay and by investigating mitochondrial DNA deletions by quantitative real time PCR Alterations in cell cycle distribution were analyzed by flow cytometry, while radiation-induced senescence was studied with SA--Gal staining. Autophagy changes were measured by Acridine Orange staining and the expression of TP53INP1, GDF-15, GADD45A and CDKN1A was measured by real-time PCR. RESULTS: We demonstrate here that IR results in dose dependent expressional changes of TP53INP1 in both irradiated and bystander fibroblast cells. We constructed stable fibroblast cell lines in which the expression of TP53INP1 was constitutively knocked-down by RNA interference. TP53INP1 was required for IR induced maximal elevation of CDKN1A and GDF-15 expressions. Likewise, autophagy and senescence was deregulated following irradiation in the absence of TP53INP1. TP53INP1 deficient cells showed resistance of the G2-delay, and the proliferation rate was higher compared with wild type F11hTERTcells. Finaly, we showed that TP53INP1 proficiency is important for clonogenic survival after radiation. CONCLUSIONS: These data reveal novel functional roles for TP53INP1 in cell cycle, survival and responses to IR. Taken together, we concluded that autophagy impairment induces premature senescence through a TP53INP1-dependent manner in primary human fibroblasts.

Speaker: Dr Hargita Hegyesi (NRIRR "Frédéric Joliot-Curie" National Research Institute for Radiobiology and Radiohygiene)

Oxidative Stress

88 Clustered DNA lesions: From Chemistry, Formation, DNA repair and biological importance

In cells and tissues there is a constant radiolytic attack that has exogenous or endogenous (intracellular) oxidative origin. During these attacks, although not in all cases, the primary damage is being induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Examples of such species are the hydroxyl radical (OH·), O2·-, singlet oxygen (1O2) and peroxynitrite to others. Intracellularly, ROS are primarily created in mitochondria as a natural byproduct of natural metabolism of oxygen but also from cells of the immune system such as macrophages. Human tissues have developed through millions of years of evolution several sophisticated mechanisms such as radical scavengers, antioxidant enzymes catalase and superoxide dismutase and elaborate DNA repair mechanisms. The oxidatively-induced DNA damage consists of a variety of lesions of small to high importance and dangerous for the cell i.e., isolated base lesions or single strand breaks (SSBs) to complex lesions like double strand breaks (DSBs) and other non-DSB oxidatively-induced clustered DNA lesions (OCDLs). In this presentation, I will discuss the current status of knowledge and evidence on the chemistry and formation mechanisms, involvement of intracellular oxidative stress and DNA damage in human pathology and possible use of these parameters as stress biomarkers. At the same time, I will discuss controversies related to potential artifacts inherent to specific methodologies used for the measurement of oxidatively-induced DNA lesions in human cells and tissues.

Speaker: Prof. Alexandros Georgakilas (East Carolina University)

10:30 Coffee break

89 Protein hydroperoxides: key mediators of radiation damage to proteins and complex biological systems

Proteins are major targets for oxidative damage in biological systems due to their high abundance and rapid rates of reaction with radicals and other reactive species (e.g. UV-induced 1O2). Exposure of proteins to radicals generated by radiation, metal ion/hydroperoxide systems, peroxyl radicals, peroxynitrite, and activated white cells, results in the formation of protein-derived radicals. Subsequent reaction of these species in the presence of O2 yields new reactive groups on proteins including hydroperoxides and 3,4-dihydroxyphenylalanine (TIBS, 1993, 18, 437-441; Biochim Biophys Acta 2005, 1703, 93-109). These protein-derived species are long-lived, can diffuse from their site of generation due to slow enzymatic detoxification, and can be detected in intact cells. Subsequent reactions of these intermediates can result in secondary damage, with this occurring via at least two mechanisms: metal-ion mediated reduction of the hydroperoxide to alkoxyl radicals (Biochem J, 1995, 305, 643-649; Arch Biochem Biophys, 1996, 336, 163-172), and direct 2-electron reaction with readily oxidised materials (thiols and thioethers). Reaction of peptide and protein hydroperoxides with thiol (Cys) groups appears to be of particular biological importance, as this can result in inactivation of critical thiol-dependent enzymes. Thus enzymes involved in protein turnover (cathepsins and the proteasome; Free Radic Biol Med, 2006, 40, 1539-1548; 2011, 50, 389-399), metabolism (GAPDH), signalling (protein tyrosine phosphatases; Free Radic Biol Med, 42, 1543-1551), ion homeostasis (Ca2+-ATPases), redox maintenance Free Radic Biol Med, 2010, 48, 1071-1078; 49, 1505-151) and apoptosis are all readily inactivated by peptide- and protein hydroperoxides. Inactivation occurs in a concentration-, time- and structure-dependent manner with hydroperoxide consumption and thiol oxidation occurring in parallel; in some cases sulfenic acid intermediates are detected. Some protein hydroperoxides are more effective than H2O2, probably as a result of the longer biological lifetime. Overall, these data support the hypothesis that hydroperoxides formed on oxidized proteins may contribute to cellular dysfunction and altered redox signalling in systems subject to oxidative stress by inducing strand breaks and mutagenic lesions in DNA (Biochem J, 1998, 330, 1059-1067; 1999, 344, 125-134, inhibiting key cellular enzymes, altering cellular redox status and signalling, and depleting antioxidants.

Speaker: Prof. Michael J. Davies (The Heart Research Institute)

90 RADIATION PROTECTIION WITH NEWLY SYTHESIZED SCHIFF BASE COPPER COMPLEXES DERIVED FROM L-HISTIDINE AND PYRIDINECARBOXALDEHYDES

Efficient bioactive antioxidant properties are exhibited by copper organic complexes along with their potential for prevention and/or treatment of ionizing radiation injury is mainly related to SOD- and Catalase-mimetic activities, as well as their respective facilitation of specific metalloelement-dependent enzyme de novo syntheses enabling them to prevent the accumulation of toxic metabolic products, promote biochemical, cellular, and tissue repair processes required for recovery from radiation injury including survival of lethally irradiated mice and rats. The new copper complexes of Schiff bases derived from L-histidine amino acid and isomeric 2-, 3-, 4- pyridinecarboxaldehydes were synthesized and studied for radioprotective activities in white rats. The compounds were administered to rats in deionized water at doses of 10 mg/kg or 40 mg/kg using subcutaneous or oral modes of administration. In 1 hour or 24 hours after treatment animals were exposed to single X-ray irradiation at 6.5 Gy (LD50/30). Animals irradiated without preliminary treatment served as Control. Radioprotective effects of the compounds were assessed through determination of the indices of rats’ survival and their average life span in 30 days post exposure, as well as evaluation of cytogenetic state of bone marrow cells in survived animals. Additional sets of experiments were undertaken involving animal exposure to X-rays at 5 Gy in 1 hour post the preliminary subcutaneous injection of 10 mg/kg compounds to rats in order to elucidate the influence of compounds studied on SOD and Catalase activities. On 3, 7, 14 and 28 days after irradiation SOD and Catalase activities were analyzed in blood of animals. The research demonstrated that Schiff bases derived from L-histidine amino acid and isomeric 2-, 3-, and 4- pyridinecarboxaldehydes possess no radioprotective activities or they are weakly expressed , while their copper(II) complexes are effective radiation protective agents against radiation-induced lethality in rats at subcutaneous injection 1 hour before exposure, as well as at oral administration 24 hours prior to irradiation. SOD- and Catalase-mimetic activity of the compounds studied allowing to protect organism from radiation-induced oxidative stress, as well as cytogenetic activity of copper complexes to a certain extent predetermine radioprotective ability of these metallocomplexes. Acknowledgements: This work was supported by ISTC A-1764 Project.

Speaker: Dr Margarita Malakyan (Scientific Centre of Radiation Medicine and Burns)

91 Free radicals and sulfur-containing proteins: Assessment of structural modifications and damages in lipid domains

Effects of radical stress in the biological environment is a very active field of research connecting varies discipline in life science. Exposure of proteins to free radicals may cause structural and functional changes. In particular, a single radical event that leads to an initial damage involving sulfur-containing amino acid residues could produce a reactive species able to damage another cell compartment such as lipid domains. In this contest, damages to some sulfur-containing proteins (i.e. ribonuclease A, metallothioneines, human serum albumin, substance P) were considered to elucidate the effects of radical stress exposure on the overall protein structure. Experiments were carried out in both aqueous solutions and vesicle suspensions. Free radical generation, mimicking an endogenous radical stress, was obtained by gamma-irradiation of aqueous solutions. By changing the appropriate conditions of irradiation, a selection of the reacting radical species was carried out. The protein degradation due to radical exposure was evaluated by Raman spectroscopy [1-3]. In fact, Raman spectrum can provide valuable information on both amino acid side chains (i.e. S-S, Tyr, Trp, Cys-Metal) and conformational changes in the protein secondary structure. Protein structure and amino acid content resulted to play an important role in blocking the ready access of free radicals both to the sulfur-containing residues and the active site, so strongly affect both the radio-sensitivity of proteins. By using a biomimetic model based on unsaturated lipid vesicle suspensions the occurrence of tandem protein/lipid damage was shown [1-2]. In fact, protein degradation is accompanied by structural alteration of unsaturated lipids forming liposome vesicles, which changed the naturally occurring cis geometry to the trans configuration. In fact, the reactions of reductive reactive species with methionine residues and/or sulfur-containing ligands afford diffusible sulfur-centered radicals, which migrate from the aqueous phase to the lipid bilayer and act as isomerising species of the double bonds. [1] A. Torreggiani, J Domènech, R.Orihuela, C.Ferreri, S. Atrian, M. Capdevila, C. Chatgilialoglu Chem. Eur. J. 15 (2009) 6015. [2] A.M. Salzano, G. Renzone, A. Scaloni, A. Torreggiani, C. Ferreri, C. Chatgilialoglu Mol Biosyst. 7 889 (2010). [3] Z. Jurasekova, A. Tinti, A. Torreggiani Anal. Bioanal. Chem. 400 (2011) 2921.

Speaker: Dr Armida Torreggiani (ISOF - CNR)

92 (5'R)- and (5'S)-5',8-cyclo-2'-deoxyguanosine lesions

Diffusible hydroxyl radicals (HO•) are known to react with DNA either by hydrogen abstraction from the 2-deoxyribose units or by addition to the base moieties. The majority of HO• attacks occur at the base moieties. However, there is growing evidence that the oxidation of 2-deoxyribose in DNA plays a critical role in the genetic toxicology of oxidative stress and inflammation. About the half of HO• attacks toward the sugar unit of DNA occurs at H5' with the formation of C5' radical, that gives an intramolecular attack to the C8 position of the purine base, generating a unique cyclic base-sugar adduct (purine 5',8-cyclo-2'-deoxynucleosides). These tandem-type lesions are observed among the DNA modifications [1,2]. Inspired by the mode of formation of these lesions, the diastereomeric forms (5'R and 5'S) of 5',8-cyclo-2'-deoxyguanosine have been synthesized and fully characterized, as well as their corresponding phosphoramidates. This approach facilitates the quantitative determination of such lesions in biological samples as biomarker of free radical damage and is propedeutic for preparation of modified oligonucleotides for biochemical/biophysical studies related to specific diseases and impairment of enzymatic repair. Different oligonucleotides models which can occur in double strand and G-quadruplex structures are used to test the reactivity of hydroxyl radicals towards the sugar backbone of DNA. In this context, radiation-induced transformations have been considered in depth for the characterization of reaction intermediates by pulse radiolysis and the products formation by gamma-radiolysis. [1] Chatgilialoglu C, Ferreri C, Terzidis MA, Chem. Soc. Rev. 2011, 40, 1368–1382. [2] Belmadoui N, Boussicault F, Guerra M, Ravanat JL, Chatgilialoglu C, Cadet J, Org. Biomol. Chem. 2010, 8, 3211–3219.

Speaker: Dr Chryssostomos Chatgilialoglu (ISOF-CNR)

93 Oxidation of methionine in model peptides. A mass spectrometry and IRMPD characterization of the final products

It is known that methionine residues are easily oxidized non enzymatically to their sulfoxide form, and it has been proposed that this process in conjunction with its repair by methionine sulfoxide reductases would be a protective mechanisms towards oxidative stress. However oxidation of methionine does not always lead to the sulfoxide and in addition the susceptibility to oxidation of Met residues in a protein does vary. We have reinvestigated the oxidation of several peptides containing Methionine either in C- or N-terminal by OH radicals produced by radiolysis and by photolysis. In all cases we have identified the final products by MSn. in addition we have characterized the sulfoxide forms by Infra Red Multiple Photon Dissociation (IRMPD) coupled to MS. We show that Methionine sulfoxide is always formed albeit in various quantities. In addition other products were characterized. In oxidation photo-sensitized by benzophenone derivatives, adducts are formed. Thus the biological consequences of the oxidation of Methionine in oxidative stress must be revisited.

Speaker: Dr Chantal Houée-Levin (Laboratoire de Chimie Physique, Université Paris Sud, 91405 Orsay, France)

12:30 Lunch

Chromatin Remodelling and Radiation Epigenetics

94 Chromatin remodeling in response to DNA damage

Our cells are constantly under attack from both endogenous and exogenous genotoxic agents. These genomic insults result in potentially harmful DNA lesions, the most dangerous of which is the chromosomal DNA double strand break (DSB). In response to a DSB cells activate a multitude of responses, including chromatin structural changes, cell cycle checkpoints and DNA repair. How cells signal and repair DSBs within chromatin is however poorly understood. Through genetic screens in yeast and worms, we identified several factors, including chromatin remodelers, as novel regulators of the DSB response in humans. We found that these factors assemble directly at DSB sites to orchestrate the signaling and repair of these lesions and promote cell survival in response to genotoxic insult. The results of this ongoing work will be presented at the meeting.

Speaker: Dr Haico Van Attikum (Leiden University Medical Center)

95 Radiation Epigenetics: Does ionizing radiation affect DNA methylation?

Several manifestations of the radiation response such as bystander effects, adaptive processes, or transgenerational effects suggest involvement not only of genetic but also of epigenetic mechanisms. These might include transcriptional changes mediated by small RNAs, histone modifications or DNA methylation. Altered methylation of CpG sites can modify gene expression, induce genomic instability and, finally, result in carcinogenesis. How this cascade is driven by radiation is still widely unknown, although there are several reports available on altered methylation patterns following radiation exposure in plants, rodents and human cells. In our analyses we focused on repeated exposure of cell lines to ionizing radiation which can lead to adaptive response. While the importance of epigenetic mechanisms in adaptation to repeated stress exposure is known for a variety of agents (e.g. cisplatin, tobacco smoke), their role in fractioned ionizing radiation needs to be elucidated. We treated MCF7 breast cancer cells with repeated fractions of 2 Gy, resulting in total doses of 10 and 20 Gy. Methylation patterns were determined by methyl-CpG immunoprecipitation (MCIp) followed by global methylation profiling on CpG island microarrays. Cells receiving a total dose of 10 Gy showed a delay in cell division but started regrowing after 14 d, and exhibited similar radioresistance as mock treated cells. MCIp profiling revealed methylation changes in several CpG islands 48 h after fractionated irradiation with 10 and 20 Gy. These methylation differences were quantitatively validated on bisulfite-converted DNA by MassArray technology, a method involving PCR, base-specific cleavage, and MALDI-TOF mass spectrometry. Significant hypomethylation was mainly confirmed in CpG units associated with the transcription factor FOXC1 (P<0.001) and TRAPPC9 (p<0.001) which is involved in NFkB signalling. In summary, we showed differences in epigenetic DNA methylation in cells recovering after fractionated irradiation. Together with published evidence, our data support a role of DNA methylation in the radiation response. Further studies need to clarify the functional impact of these changes

Speaker: Dr Odilia Popanda (German Cancer Research Center (DKFZ))

96 The influence of hypothermia on the ionizing radiation-induced γ-H2AX foci formation, micronuclei and clonogenic survival in A549 cells in relation to chromatin organization

It is well known that hypothermia during in vitro irradiation of human cells affects the level of clonogenic survival, frequency of chromosome aberrations and DNA supercoil rewinding. The mechanisms of the hypothermia effect are not known but it was suggested that it may be related to chromatin condensation. Elucidation of the mechanisms of the hypothermia effect is interesting and important both from the perspective of understanding the cellular response to radiation and of the impact that the effect may have on the interpretation of experimental results. With this in mind, the aim of our study was to examine the influence of hypothermia on the ionizing radiation-induced dose-response relationship for γ-H2AX foci formation, micronuclei and clonogenic survival (SF) in relation to chromatin organization. In this study, two sets of experiments were performed. In the first experiment, A549 cells were kept at 0 °C and 37 °C for 30 minutes and then exposed to 0; 0.5, 1, 1.5, 2; 2,5 and 3 Gy of γ-rays In the second experiment, A549 cells were exposed to histone deacetylase inhibitor trichostatin A (TSA) for 18 hr prior to irradiation at 0°C and 37 °C. After irradiation samples were processed for analysis of clonogenic survival, micronuclei and gamma-H2AX foci. Our results revealed that the frequency of micronuclei in cells exposed at 0°C to γ-rays was lower than after exposure at 37°C. The SF of cells exposed at 0°C γ-rays to gamma-rays was higher than after exposure at 37°C. No effect of temperature was observed on the number of γ-H2AX foci. The pretreatment of TSA radiosensitized A549 cells. The results dealing with the influence of hypothermia on the ionizing radiation-induced effects in relation to chromatin organization will be presented during the ERRS conference.

Speaker: Dr Halina Lisowska (Jan Kochanowski University, Department of Radiobiology and Immunology, Kielce, Poland)

97 What can we learn from localization microscopy? New insights into radiation induced changes of the chromatin nanostructure

Spatial Position Determination Microscopy (SPDM) has become one of the established localisation microscopic techniques that enable effective light optical resolution in the nanometre range even in 3D conserved cell nuclei. It is based on the application of labelling fluorophores that can be switched between two different spectral states (e.g. off/on) to achieve a temporal isolation and thus a spatial separation of the signal molecules. A subsequent computational calculation of these “blinking” events allows the determination of precise positions of the individual labelling molecules as well as the measurement of their spatial distances even if they are considerably below the conventional optical resolution limit. Using SPDM, the nuclear nanostructure and arrangements of euchromatin (EC) and heterochromatin (HC) in irradiated and non-irradiated HeLa cells was investigated after nucleosome labelling via fluorescent proteins and specific antibodies against EC or HC, respectively. In non-irradiated cell nuclei, theoretical approaches of chromatin modelling and statistical analyses revealed a non-random organization of nucleosomes below the 100 nm scale. The sensitivity concerning identification of nanosized structures was proven by changing the labelling site in dimensions of single nucleosomes. Changes of the arrangements of EC and HC regions were measured in terms of distances and molecule densities during repair processes. Besides the nucleosome pattern of H2A and H2B, conformations of EC and HC regions were found to be subjected to different changes due to radiation dose and during repair processes. Image information obtained by SPDM measurements and calculations may offer new insights into the understanding of repair mechanism and support new types of dose-efficiency correlations. Funding by the Federal Ministry for Environment, Energy and Reactor Safety (BMU) is gratefully acknowledged.

Speaker: Prof. Michael Hausmann (Kirchhoff-Institute for Physics, University of Heidelberg)

98 Epigenetic changes in human fibroblasts after low dose rate γ radiation

Recently, the epigenetic mechanisms involved in cancer development have come in focus. An epigenetic event refers to changes in gene expression controlled by histone modifications (acethylation) and/or DNA methylation (gene silencing). Accumulated evidence indicates that epigenetic modifications including persistent proteomic changes are among the delayed effects of ionizing radiation (IR). It has been suggested that radiation induced genome instability and transgeneration effects may be epigenetically mediated. Understanding the epigenetic mechanisms related to low dose and low dose rate radiation may also provide valuable information with significant implications in radiation protection and cancer prevention. To investigate the protein expression profile and identify the modified proteins, we have performed 2D-PAGE analysis in primary human fibroblast cells (VH10) after exposure to IR at low dose rate. In the present study, human fibroblast cells were exposed to 1 Gy of gamma radiation delivered at 4.1 mGy/h. To investigate the late effect of IR, cells were sub-cultured and repeatedly collected from 3 hours up to 28 days post-irradiation. 2D-PAGE analysis was performed on cells collected at 3h, 7 and 28 days post-irradiation to study the proteomic changes. Our preliminary results based on analysis of 2D-PAGE gels by PD-Quest software indicate that changes in protein expression can be observed at least 28 days post-irradiation. Notably, most of the modified proteins at 28 days post-irradiation were down regulated. Further, the results on protein profiles at different post exposure times and proteins sequenced by mass spectrometry will be presented.

Speaker: Ms Sara Shakeri Manesh (Stockholm University)

99 Circulating MicroRNAs as Biomarkers in Radiation Therapy of Head and Neck Cancer Patients

MicroRNAs (miRNAs) are present in stable extracellular forms in body fluids such as liquor or blood plasma. Changes in the levels of circulating miRNAs of cancer patients are associated with malignant progression and have proven useful for the prediction of therapy responses. This study is intended to investigate if therapeutic irradiation induces changes in the plasma miRNA profiles of head and neck cancer patients. The aim is to establish plasma miRNAs as non-invasive biomarkers to predict tumor response in radiation therapy. MiRNA expression profiles of blood plasma samples from 7 head and neck cancer patients were analyzed before and after local irradiation in 2 fractions of 2Gy each. Plasma samples of an age and sex-matched collective of 6 healthy donors were investigated in parallel. Real-time PCR with TaqMan™ low-density miRNA-cards allowed quantification of a pool of 377 miRNAs in the plasma. CT-values were normalized to the mean value of all detected (Ct ≤ 35.5) miRNAs in each case. Changes in miRNA-quantity after irradiation were calculated via ∆∆-CT-method and student’s t-test was applied to determine differentially expressed miRNAs. Single Primer Assays were performed for selected miRNAs in order to validate the changes. Out of 377 investigated miRNAs approximately 100 were detected in patient plasma. After exposure on average 15 miRNAs showed increased expression, while 24 miRNAs showed lower expression levels. These include “switch on” and “switch off” miRNAs which are exclusively detected with or without irradiation. Remarkably, 26 miRNAs were identified as responding to irradiation in at least 4 out of 7 patients. Additionally, tumor specific miRNA signatures were identified by comparative cluster analysis of the miRNA profiles of nonirradiated patient plasma and miRNA profiles of plasma from healthy donors. In this pilot study we identified changes of the miRNA-levels in the plasma of head and neck cancer patients after therapeutic irradiation. Furthermore we detected miRNA signatures that are specific for head and neck cancer patients. A correlation of the obtained results with clinical outcome data and a validation in further patients is in progress. The quantification of radiation-responsive miRNAs in blood plasma may provide a minimally invasive way to predict the radiation therapy response of tumor patients, thus potentially leading to a more individualized treatment in the future.

Speaker: Ms Isolde Summerer (Helmholtz Zentrum Muenchen)

New Cancer Treatment Modalities

100 Radiosensitization by Au nanoparticles

Over the last couple of decades, nanoparticles have attracted considerable interest for their possible use and exploitation in a wide range of applications. For radiotherapy, doping cancerous cells with high Z materials represents an ideal way to boost the dose to the tumour volume without compromising the dose to the healthy normal surrounding tissues. Gold nanoparticles (GNPs) in particular can be easily functionalized to be preferentially uptaken by specific tumour cells offering very selecting targeting strategies. Enhancement of radiosensitivity in GNP doped cells has been reported with some contradiction in literature especially for high energy X-rays where no physical dose enhancement is expected. Further investigations on the nanoparticle-cell interactions are therefore required to improve our understanding at the nano- and molecular-scale and optimise the use of GNPs in radiotherapy. A mathematical model based on the Local Effect Model (LEM) used for heavy ions indicates that radiosensitization is not just due to a dose enhancement factor but complex lesions may be created in proximity of the nanoparticles by showers of secondary Auger electrons. Through clonogenic cell survival assays, we have confirmed a radiosensitising effect for a panel of cancer cell lines (MDA-231s, DU145s and T98s) in the presence of GNPs but no for normal human fibroblasts (AGO-1522). Interestingly, these cell lines differ greatly on the type of cell death induced when exposed to GNPs and radiation as seen by Annexin/PI flow cytometry analysis. The different levels of radiosensitization are likely to be due to differential GNP uptake as well as different mechanisms triggered by the nanoparticles uptake. The GNPs alone have been shown to cause DNA damage in MDA-231s and DU145s but not in T98Gs while pilot data from microbeam experiments show that cytoplasmic irradiation alone in the presence of GNPs results in significant increase of DNA damage, suggesting cytoplasmic organelles are sensitive to GNPs and irradiation. Further data indicates that the observed increased DNA damage and radiosensitisation is due to the depolarisation of mitochondria directly caused by significant increases in reactive oxygen species upon exposure to gold nanoparticles. Radiosensitization caused by GNPs seems therefore be the result of combination of physical (dose and lesion complexity enhancement) and biological effects (mitochondria inactivation).

Speaker: Dr Giuseppe Schettino (Queen's University Belfast)

101 Laser-accelerated particle beams for radiotherapy: where do we stand now?

Radiotherapy using charged particles has attracted increasing interest. The superior dose distribution of protons and heavy ions is hoped to translate into excellent dose conformation to the target and sparing of normal tissues. Whether the potential improvements in outcome justify the costs of particle therapy is, however, heavily discussed. The high costs are mainly due to the higher expenses for ion generation, acceleration and beam lines. With the advent of ultrafast high energy lasers, the idea of laser-driven acceleration of particles suitable for therapeutic applications has arisen, combined with the hope for a reduction of costs and required space. The energies achieved at present are still far from those required for radiation therapy. In addition, many technical questions regarding for example energy selection, repetition rate and beam preparation and transport remain unsolved. Apart from these technological developments, it is important to address potential differences in the radiobiological effects between laser-accelerated particles and those accelerated conventionally. Particle beams delivered from laser acceleration will be pulsed. In radiotherapy settings, ultra high dose rates of >109 Gy s-1 are expected at the tumor voxels. Within the Munich-Centre for Advanced Photonics (MAP) we performed a series of systematic comparisons of biological endpoints after irradiation with pulsed and continuous proton beams. We established a pulsed proton beam at the ion microbeam SNAKE at the Munich tandem accelerator to apply macroscopic doses of a few Gy within <1 ns. The irradiations are performed with monoenergetic protons which enables us to perform irradiations with conventional dose rates for direct comparison with the same beam quality. At the dose rates investigated, the RBE of the pulsed and the continuous irradiation mode did not differ significantly for a variety of endpoints such as colony forming ability, induction of chromosomal damage, alteration of cell cycle distribution, and others. Very recently, by combining advanced acceleration and beam transport strategies we were able to generate nanosecond quasi-monoenergetic proton pulses with a table-top laser system, delivering high doses of several Gy to human tumor cells in a single shot. Preliminary measurements of gamma-H2AX foci were in agreement with other proton RBE values in conventional beams at comparable proton energies.

Speaker: Dr Anna Friedl (University of Munich, Department of Radiation Oncology)

102 Indications for Intensity Modulated Radiation Therapy (IMRT) for the Treatment of Large Left Breast Volumes

PURPOSE: To determine indications for the use of an IMRT technique for large volume size left breast cancers. MATERIALS/METHODS: Retrospective review of Stage 0-III left breast cancers with large volumes treated with conservation therapy from April 2011 to January 2012. Computer tomography simulation was used to design fields. Patients were treated supine and received 2 Gy fractions to 50 Gy to the whole breast followed by an electron or 3D boost of 16 Gy using 6, 15 or mixed 6/15 MV photons. A variety of techniques including electronic compensation (E-comp), field in field (FinF), and 3D with wedges (3DW) were compared. Dosimetric evaluations were made of the PTV, lung, heart and contralateral breast for each technique. RTOG skin toxicity grades, treatment data, and breast volumes were obtained by chart and treatment plan review. RESULTS: A total of 30 patients were treated, and all patients received chemotherapy. Volumes ranged from 718-3296 cc (ave.1483.33 cc). Separations ranged from 20-35 cm (ave. 23.87 cm). During treatment there were 63% Grade 1, 37% Grade 2, 0% grade 3/ 4 RTOG skin toxicity; no treatment breaks recorded. E-comp plans resulted in better coverage of the V95 with improved dose homogeneity of the PTV. This was seen especially in breast volumes > 2400 cc. Also, there was a reduction in V110 and V115 in the FinF and Ecomp plans compared to conventional 3DW technique. In terms of dose to the contralateral breast Ecomp had a slight advantage (1%). No significant difference seen in the LT lung V20 and Heart V25 and Contralateral Breast V5 between Ecomp or FinF which was better than 3DW. CONCLUSIONS: For women with large breast size Ecomp planning significantly improved dose homogeneity decreased acute skin toxicity and less hot spot value. These factors grow more important in women with large breasts, who may experience more Grade 3/ 4 skin toxicity and increased pain resulting in a lower quality of life with standard tangential fields. Therefore, we suggest using an Ecomp technique for left-sided breast cancer in the following situations: 1) breast volume >1500cc 2) separation of >25cm or 4) combination of large volume/cup size with separation > 22 cm or vice versa, 5) the use of mixed beams. For patients with large breast volumes supine IMRT treatments can provide good dose homogeneity, spare dose to critical structures, and may be preferable to the prone breast irradiation.

Speaker: Ms sally sheim (NCCCR)

103 12C ion beam dose distribution in presence of medium inhomogeneities: comparison between different measurements and simulations with the treatment planning system for particles TRiP98

Heavy-ions beams offer several advantages compared to other radiation such as low lateral scattering and high biological effectiveness (RBE) in the Bragg peak region, making them particularly attractive for the treatment of radio-resistant tumors localized close to organs at risk [1]. To cope with these unique properties, a dedicated treatment planning system (TPS), TRiP98, was developed and clinically used in the carbon ion pilot project [2,3]. It is now used as a research prototype. The theoretical models and experimental databases included in TRiP98 are presently mainly based on measurements in water. This approximation can be applied successfully to reproduce many biological tissues with the exception of bones, where the presence of heavy elements, like calcium, might change significantly the composition of the resulting mixed radiation field. Presently, the TriP98 physical beam model takes into account for the bone only its density variation, but it neglects difference between bones and water in attenuation of the primary and production of secondary particles through nuclear fragmentation. However, a detailed knowledge of the particle field at each point of the treatment area is crucial for an accurate estimate of the actual dose. A previous study [4] investigated the influence of different types of bone on the carbon particles range. In the present work we aim at understanding the influence of medium inhomogeneities on the 12C beam dose distribution. For the experiment, a bone target was placed inside a water phantom. The irradiation of the target volume positioned partially behind the bone target and partially directly in water was simulated with TRiP98 and the dose at the interface measured at several depths along the primary beam direction. Absolute dose measurements were achieved with a system of pin-point ionization chambers, while the relative dose distribution was investigated with two different solid states detectors: thermoluminescence detectors of type TLD-700 and alanine ESR pellets. The position of the target volume, the thickness and the type of bone were changed to investigate the influence of the inhomogeneity in different quasi-clinical scenarios. The experimental results were compared with the values predicted by TRiP98. 1. M. Durante et al., Nature Rev. Clin. Oncol. 7, 37 (2010) 2. M. Krämer et al., Phys Med Biol. 45, 3319 (2000) 3. M. Krämer et al., Eur. Phys. J. D 2010 60, 195 (2010) 4. O. Jaekel et al., Med. Phys. 28 (2001)

Speakers: Dr Antonio Carlino (Physics Department, University of Palermo, Italy; Biophysics, GSI Darmstadt, Germany;INFN Section of Catania, Italy), Prof. Marco Durante (Biophysics, GSI Darmstadt, Germany; Physics Department, Technische Universitat Darmstadt Germany)

104 A systematic review of the use of proton therapy for the treatment of hepatocellular carcinoma: geographical distribution, technique modality delivery and effectiveness.

Purpose: To investigate the “state of the art” of proton therapy in the management of hepatocellular carcinoma (HCC) in terms of 1) the worldwide adoption of this technique , 2) the proton delivery modality and 3) the treatment toxicity and the reported outcome. Materials and Methods: A systematic review of published reports for the period 1985-2012 was performed according to the PRISMA guidelines with the following inclusion criteria: at least 5 patients, English language, at least 1 year follow up. Results: Inclusion criteria were met by 7 studies (3 prospective, 4 retrospective) from the initial 657 citations retrieved. As of May 2012, six proton centers (2 in USA, 3 in Japan, 1 in China) published their results for the treatment of HCC for a total number of 878 patients. All the facilities adopted delivery techniques based on passively-scattered proton beams. Organ motion was managed by respiratory gating with the use of implanted fiducials in most cases; 4D CT was used in the latest series from USA. Gross tumor volume to clinical target volume (CTV) margins ranged from 0.5 to 1 cm; CTV to planning target volume margins were up to 2.1 cm even with the use of gated techniques. Various schedules of treatment were adopted, depending on tumor stage and location: the total dose delivered ranged from 52 to 84 Gy RBE in 4-38 fractions. Nine cases of radiation induced liver disease were reported; 29 non hematologic G3 toxicities occurred. Eight skin-subcutaneous tissue and 4 gastrointestinal G3 late toxicities were registered along with 3 rib fractures; 3 cases of bile duct stenosis were experienced in the earliest series from Japan. Local control was superior to 80% in all series, with a peak of 90.2% at 5 years in one series from Japan. Survival rates at 5 years were available for the Japanese series, ranging from 23.5% to 44.6%; a 55.9% survival rate at 5 years was registered in the report from Tsukuba for child-pugh A patients. Conclusion: The encouraging results of the pioneeristic experiences from Japan enabled the slow but steady spread of the use of protons in the treatment of HCC in Western countries. Prospective data are needed; the increasing number of particle therapy centers with new treatment delivery techniques (i.e. active scanning) represents an opportunity to compare protons with the other, non surgical local therapies currently available for HCC treatment, such as x-ray radiotherapy, chemoembolisation and radiofrequency ablation.

Speaker: Dr Francesco Dionisi (ATREP Agenzia provinciale per la Protonterapia, APSS Trento)

15:30 Coffee break

Poster Session 2

105 ACCUMULATION OF RADIONUCLIDES IN VEGETABLE SOME CROPS IN ZONES OF THE ARMENIAN NPP

Artificial radionuclides (RN, 90Sr-T1/2=28.6 years, 137Cs-T1/2=30.1 years) dangerous for health are released to ecosystems because of human influence in the field of nuclear energetics RN can penetrate into human organism through water-soil-plant chain. Armenian NPP (ANPP, opened in 1976, operated till 1989, reoperated in 1995) is in a densly populated area of the Ararat Valley with intensive agriculture. ANPP uses the water of the River Metsamor. Unbalance waters of the ANPP fall into the Metsamor River which is used for irrigation of the soils. Since 1996 we have carried out radiomonitoring researches in water- soil-plant ecosystems in zones of the ANPP with a radius of 2-15 km. The aim of the studies is to develop practical radioprotective activities for getting ecologically pure food. The results of investigations have shown that the content of 90Sr fluctuated between 2.2-8.8 Bq/kg and 137Cs between 4.0-13.1 Bq/kg in vegetable crops (basil, celery, dill, parsley, lettuce, pepper, tomato, eggplant, marrow, cucumber, cabbage, potato, cauliflower) which were grown in gray soils and irrigated with the water of the Metsamor River. It is caused by joint influence of many factors, especially biological peculiarities of crops (vegetation duration, mineral nutrition, form of expansion of roots in the soil, watering regime, anatomical structure of leaves, their size etc.). We can conclude that RN penetrated into crops through irrigation water, soil as well as atmosphere. It also turned out that in soil-plant system the observed ratio (OR=90Sr/137Cs in plant¸90Sr/137Cs in soil) <1 for crops, that is, 137Cs accumulated in the crops more than 90Sr. The OR>1 and the absorption of 90Sr exceeded 137Cs for vegetable marrow and cucumber. Accumulation coefficient (AC=content of RN in the plants¸content of RN in soil) AC 90Sr>AC 137Cs 1,3 times for cucumber and AC 137Cs >AC 90Sr 1.2 - 3.0 times for other crops. There is a positive strong comparative connection between OR and AC90Sr for vegetable crops (correlation coefficient is r=0.81±0.14) and there is negative weak comparative connection between OR and AC137Cs (r=-0.30±0.23). RN content in crops hasn’t increased during 2002-2011and doesn’t exceed the MACL. We can conclude that, ANPP doesn’t have any radioecological danger for people who live in the Ararat Valley, as well as residents of neighboring regions. The researches have been implemented since 2011 in the context of project 11-1f262 of Ministry of Science of RA.

Speaker: Mrs Laura Ghalachyan (G.S. Davtyan Institute of Hydroponics Problems NAS RA)

106 WATER STREAM HYDROPONICS AS A RADIOECOLOGICALY PROFITABLE BIOTECHNOLOGICAL METHOD FOR PRODUCITON OF PLANTING MATERIAL

Artificial radionuclides (ARN, 90Sr-T1/2=28.6 years, 137Cs-T1/2=30.1years) dangerous for health are released to ecosystems because of human influence in the field of nuclear energetics. ARN can penetrate into human organism and cause different diseases. At the institute (30km radius zone of the Armenian NPP) a modern system-water stream hydroponics (cylindrical, gully, continuous) with polymeric film usage was worked out for plants soilless production which is cheaper for 5-6 times and is radioeclolgicaly more profitable as compared with the existing classical hydroponicums with reinforced concrete plots. The results of experiments have shown that cylindrical hydroponics in comparison with others modules promoted productivity increase of Sweet Basil (Ocimum basilicum L.) 1.3-2.9 times, at the same time increasing the output of essential oils (1.2-3.6 times), extractive substances (1.5-2.6 times), flavonoids (1.4-2.7 times) and tannins (1.3-1.9 times) in the planting material. Activation of most important physiological processes of plant (total content of water (2-10%), content of free water (7-35%) and chlorophyll a+b (11-33%) in leaves) has been clearly noticed in cylindrical system, which along with other factors provides high productivity of Sweet Basil. The results of radiochemical researches have shown that the planting material of Sweet Basil obtained by water stream hydroponics is smaller than the planting material obtained by classical hydroponics with β-sum activity (1.1-1.2 times) and content of ARN (90Sr 1.2-1.3 times and 137Cs 1.3-1.4 times). Probably it happened due to providing the best air-water-heat nutrient regime for cultivation of plants and reducing radioecological tension (reduction of nutrient solution expense, stream method of push, covering of substrates with films, prevention of ARN penetration in substrates and overground parts of plants from the atmosphere).The use of nutrient solution irretrievable push principle and closed ecological system, allows to exclude enviromental pollution and minimizes the danger of diseases, spread of pests and weeds. It is important to note that ARN content doesn't exceed the MACL in planting material obtained by classical and water stream hydroponics method. We think these researches will create the prerequisites for including practically unused areas in production sphere and obtain ecologically clear and qualitative raw material which will somewhat solve the environmental and biodiversity problem

Speaker: Mr Khachatur Mairapetyan (Hydroponics ploblems institute NAS RA)

107 IRRADIATION OF LUMINOUS MARINE BACTERIA BY TRITIUM

Increase of radioactive contamination in the environment makes the effects of radiation on living organisms very important. Detailed investigations of irradiation of living organisms were conducted using simple assay systems. All biological assay systems are nonspecific and integral. These principal properties account for the field of application of bioassays. The use of microorganisms, of marine luminous bacteria in particular, is currently favoured. The tested parameter is luminescent intensity of bacteria, which can be easily measured instrumentally. Chronic effect of Tritium, beta-emitting nuclide, on luminous bacteria Photobacterium phosphoreum was studied. Tritium is a widespread radionuclide, its content increases in environment now; evaluation of Tritium effect on living organism is of great importance. Purpose of this work was to study chronic effect of Tritium (0.01–100 MBq/L) on physiological characteristics of bacteria. It was shown that the presence of Tritium up to 0.1 MBq/L of activities in nutrient media increases bacterial growth and the presence of Tritium of activities exceeded 0.1 MBq/L suppresses it. Sample of exponential stage of growth (6 hours) revealed the absence of distinct effect of Tritium on BL. Sample of stationary stage of growth (22 hours) demonstrated bioluminescence (BL) activation (Irel > 1) up to 8 times. After 95 hours, inhibition of BL intensity (Irel < 1) was observed. Luminol chemiluminescence method used to evaluate amount of peroxides in Tritium solutions. It was shown, that the presence of Tritium of different activities doesn’t change peroxide content in solutions. Activation can be caused by processes of media ionization, but not peroxide effects. Activation of bacterial growth and BL intensity can be a result of intensification of electron transfer followed by the increase of rates of biochemical processes.

Speaker: Mrs Maria Selivanova (Siberian federal university)

108 Microarray analysis of the transcriptional response to different doses of ionizing radiation in Salmonella enterica serovar Typhimurium

Microarrays can be used to measure the expression of thousands of genes to identify the changes in expression between different biological states. To define the repertoire of Salmonella typhimurium genes responding to different doses of ionizing radiation (IR), 1, 10, and 100 Gy, transcriptome dynamics were examined using DNA microarrays. At least 74 genes were induced and 38 genes were repressed 2-fold or more by a dose of 100 Gy, while 26 genes were induced and 29 genes were repressed by a dose of 10 Gy. In particular, only 2 and 4 genes were up- and down-regulated significantly after 1 Gy IR exposure, respectively. We found that four SOS genes (umuD, dinP, dinG, and uvrB) are strongly and moderately induced by 100 and 10 Gy IR, respectively, but not by 1 Gy IR. However, interestingly, most of the genes showing the dose-dependent increase like the four SOS genes were found to be Salmonella prophage genes such as Fels-1 and Gifsy-1 and -2. In addition, heat shock genes (dnaK and groELS) also showed the dose-dependent expression. These results suggest that a dose of 1 Gy is not sufficient to provoke an IR response in bacteria. Considering the high expressional level of prophage genes compared to SOS genes, it is likely that an additional transcriptional regulatory mechanism may play a role in the induction of prophage genes.

Speaker: Dr Dongho Kim (Korea Atomic Energy Research Institute)

109 INVESTIGATION OF RADIOACTIVITY IN THE HYDROPONIC AND WILD MEDICINAL RAW MATERIAL OF THYME

In the consequence of anthropogenic influence the artificial and natural radionuclids (RN) penetrate into the environment then through ecological chain enter in biogeochemical circulation which leads to their undesirable accumulation in the living organisms. The study of artificial (90Sr, 137Cs) and natural (U) RN accumulation in hydroponic and wild plants of Thymus (T) marshallianus Willd. and Thymus serpyllum L. has particular interest, as Armenia is located in the technogenic influence zone of Armenian Nuclear Power Station (ANPS). The use of Thyme in medicine is conditioned by the existence of valuable essential oil content and biologically active substances which have range of pharmacological activities such as antimicrobial, antiviral, antioxidant, etc. The species of Thyme are included in the composition of tea, herb collections and drugs. Radiochemical analyses were carried out in hydroponic (Yerevan 30km zone) and natural (Dilijan approximately 90km zone) areas of technogenic influence of ANPS. Results of the study illustrated, that the accumulation of artificial and natural RN by T. marshallianus and T. serpyllum was different in the same rate of radioecological stress. T. marshallianus grown in wild and hydroponic conditions exceeded T. serpyllum by β-activity -1.2 and 1.1; 90Sr -1.4 and 1.4; 137Cs -1.1 and 1.5; U - 1.4 and 1.1 times respectively. The content of the artificial RN in hydroponic and wild T. marshallianus was nearly the same, while the U content was different. Accumulation of U in T. marshallianus grown in wild conditions exceeded hydroponic one 1.2 times which is probably due to its high content in the soil compared with hydroponics. The content of U and 90Sr in T. serpyllum from both areas was the same, whereas wild plants accumulated 137Cs 1.4 times more than hydroponic plants. It was found that the content of β- radiate RN in the wild plants of both species of Thyme was 1.1 times lower as in hydroponics. However, in the total β-activity part of 90Sr (1.1 times) and 137Cs (1.4 times) in the hydroponic plants was lower compared with wild plants. It has been confirmed that the content of controlled RN in the hydroponic and wild vegetative raw material of both species of Thyme does not exceed the M.A.C.. Therefore, the elaborated hydroponic method can contribute to the creation of cultivated areas of Thyme and receive ecologically pure medicinal raw material. The researches were implemented in the context of project 11-1F-264.

Speaker: Dr Elmira Sargsyan (Institute of Hydroponics Problems NAS RA)

110 Risk assessment of lake fish exposure due to 137Cs radiation

Over the last few years increasing attention has been paid to the elements required for the estimation of risk of non-human biota related to ionizing radiation. Freshwater biota of a small humic lake, called Red, which became meromictic some thirty-five years ago due to the inflow of a large amount of humic water, was investigated. The bottom areas of this lake are related to the favorite sites of the tench (Tinca tinca) winter torpor. Long-term studies data of 137Cs concentrations in Red lake water and sediments were used as inputs for ERICA software. Dose rates for all default freshwater biota were assessed. Additionally, these values were evaluated for the tench with real parameters. It was found that the total dose rate of tench due to 137Cs radiation is found to be 8.45∙10-2 µGy h-1, which is much less than ERICA screening value of 10 μGy h-1. However, this result shows that the fish according to its spending winter torpor period in the sediments with the elevated radiocesium content receives the extra external dose, comparing with that due to natural radionuclides, that can cause genetic changes.

Speaker: Dr Marina Konstantinova (CENTER FOR PHYSICAL SCIENCES AND TECHNOLOGY)

111 Two-phase migration model of the radionuclide transfer from water into the lake bottom sediments

In scientific literature studying migration of radionuclides from water into soil and bottom sediments, the process is described by one-phase models. The main drawback of such models is related to the fact that the radionuclide diffusion coefficient in sediments decreases with time. However, analyses of radionuclide vertical profiles in carbonate bottom sediments as well as results of laboratory experiments show that the process proceeds in two temporal phases. The first- fast migration stage is governed by the light ion transfer. The resulting vertical profile of the radionuclide activity concentration in sediments is exponentially decreasing with depth. The second migration phase is slow. It involves the main significant processes: bioturbation, sedimentation as well as the first order kinetic reactions and radioactive decay. In the case of accidental appearance of radionuclides in water systems the one-phase model cannot be properly employed to predict the long-term consequences of radionuclide migration into media where they are mostly accumulated- bottom sediments and soil solid phase. Application of the correct model of radionuclide behaviour in the radioactive waste storage environment and in water saturated soils is of great concern as well. The two-phase mathematical model of the radionuclide vertical migration in the water saturated solids (bottom sediments and soil) and its solution are presented. The model sensitivity analysis revealed the main important parameters affecting the radionuclide migration rates. This research was funded by a grant (No. MIP-041/2012) from the Research Council of Lithuania.

Speaker: Dr Evaldas Maceika (State scientific research institute Center for Physical Sciences and Technology)

112 RADIOECOLOGICAL EVALUATION OF HYDROPONIC MEDICINAL RAW MATERIAL OF HYPERICUM PERFORATUM L.

Contamination of environment by various wastes from industries, transports, agriculture and nuclear power stations is an actual subject for comprehensive study. Constantly, numerous radionuclides (RN) penetrate in the nature and accumulate extensively by plants and through ecological chain soil-plant-animal-human inpour into biogeochemical circulation resulting in their undesireable accumulation in the organism. The investigations were conducted in Ararat Valley which is in the zone of technogenic tension with radius of 30 km from Armenian Nuclear Power Station. During the research was studied the accumulation of artificial (90Sr, 137Cs) and natural (U) RN in aerial flowering parts of Hypericum (H.) perforatum L. cultured in open air hydroponics. In hydroponic conditions plants were cultivated on volcanic slag and gravel substrates. As control was used plants grown in soil. Pharmacological value of H. perforatum is owing to the rich composition of biologically active compounds (flavonoids, hypericin, pseudohypericin, hyperforin, tannins, etc.) which have antiviral, anticancer, antioxidant, antidepressant, and other properties. The researches indicated that, at the same radioecological stress, growth conditions have significant influence on accumulation of RN by plants. Medicinal raw material of H. perforatum observed in soil conditions by the content of U, 90Sr and 137Cs exceeded 1.1; 1.1; 1.8 times hydroponic plants on gravel, and 1.1; 1.3; 1 7 times on volcanic slag respectively. The part of 90Sr and 137Cs in total β-activity in hydroponic plants was less compared with soil control: on volcanic slag 1.9 times and on gravel 1.8 times. In hydroponic and soil plants the accumulation of 137Cs exceeded 90Sr, while in control its content was 3.4 times higher. The medicinal raw material obtained from both hydroponic substrates did not show any significant differences by 137Cs content, whereas the content of 90Sr in plants received from gravel 1.2 times exceeded that of volcanic slag. Consequently, the results of the study indicated that hydroponic plants from ecological aspects are much more pure than soil ones. Nevertheless, all studied variants of medicinal raw materials by the content of controlled artificial RN overall did not exceed the Maximum Allowable Concentration and corresponds to the requirements of international standards. Therefore, for receiving ecologically clean medicinal raw material of H. perforatum the hydroponic culture is more preferable.

Speaker: Ms Anush Vardanyan (Institute of Hydroponics Problems, National Academy of Sciences, Republic of Armenia)

Slides Vardanyan_poster_(2).pdf

113 ENVIRONMENTAL ANALYSIS OF RADIOLOGICAL AGENT IDENTIFICATION LABORATORY AT BRAZILIAN ARMY TECHNOLOGICAL CENTER

Radiological analysis at Brazilian Army Technological Center (CTEx) has been performed since 1980s, in order to be applied environmental monitoring. Nowadays, analytical techniques such as gamma spectroscopy and total alpha/beta counting are performed by Chemical Biological and Nuclear Defense Division (DDQBN). Its major role is to support CTEx in development of searching about homeland security against Chemical, Biological, Radiological and Nuclear (CBRN) threats. As the Radiological Agents Identification Laboratory (LIAR) is being set up at DDQBN, advanced detection devices, including two Hiperpure Germanium (HPGe) spectroscopy systems, became available. The LIAR follows safety requirements enforced by Brazilian Nuclear Energy Commission (CNEN), being responsible for performing environmental radiological monitoring of the CTEx area. So far, no presence of radiological contamination has been found. Moreover, if any is identified, decontamination procedures will be immediately deployed. In addition, since CTEx is located next to the Guaratiba Archeological and Biological Park, the need for keeping accurate records of the radiological history of the site is even greater. Thus, this work is aimed at describing the environmental radiological measurements performed by LIAR at the analysis about sediment of Guaratiba mangrove swamps by determining U238, Th232, K40 and Cs137 concentrations. The procedures for collection, preparation, packaging and analysis of samples are also described. Finally, the work considers some future perspectives of using LIAR for performing environmental radiological analysis services, setting up database as well as acquiring further specific training experience so as to engage in more extensive homeland security activities.

Speaker: Mr Jorge Alberto Valle da Silva (Brazilian Army Technological Center - CTEx)

114 HEALTH STATUS OF POPULATIONS AFFECTED BY THE CHERNOBYL ACCIDENT

For a long time it was considered that genetic effects of irradiation are related only to direct DNA damage. But a lot of evidence suggests the existence of a phenomenon of information transfer from irradiated cells to non-irradiated (radiation induced bystander effect, RIBE). The specific nature of these factors and mechanism of RIBE are still unknown. Effects of bystander factors induced in vivo in blood of people affected by the Chernobyl accident are of particular interest. For the first time such studies were performed by Dr. I. Emerit in 1990, when it was shown that blood sera from liquidators of the Chernobyl accident or residents of areas contaminated by radionuclides contained an increased level of bystander factors. In Emerit studies own lymphocytes of serum donors were used as a test-system. In the present study a new method was developed which allows evaluation of the effects of bystander factors from sera of different groups of populations on immortalized culture of human keratinocytes. This method has serious advantages as compared to that which was used earlier: used cells are not previously irradiated and could be cultivated for a longer period of time, they are more sensitive as compared to human peripheral blood lymphocytes, experiments could be easily repeated, allowing simple comparison and interpretation of the obtained results. Using this method, we evaluated the level of damaging bystander factors in blood of areas irradiated by the Chernobyl accident. Affected population groups included liquidators of the Chernobyl accident and people living and working in areas of Gomel region contaminated by radionuclides. The analysis has shown that bystander factors persist in Chernobyl liquidator blood samples for more than 20 years since irradiation. The data suggest that blood sera contain bystander factors, which are able to induce micronuclei and decrease metabolic activity of HPV-G cells. These factors could be neutralized by antioxidant substances. The discovery of the nature of such factors will facilitate better understanding of radiation damage mechanisms. Knowledge of these mechanisms is increasingly important for cancer radiation therapy –for using special correcting coefficients taking into consideration effects of bystander factors. The ability to neutralize and modify these factors may help to decrease significantly the effects of whole body radiation exposure.

Speaker: Dr Pavel Marozik (Institute of Genetics & Cytology NAS Belarus, Minsk, Belarus)

115 Reduction of Spontaneous Mutagenesis by Bystander Cell Death in V79 Cells

Bystander responses have generated considerable interest in the field of modern radiobiology because of their non-liner relationship with low-dose radiation. Moreover, potential carcinogenic risks are considered to be increased by bystander responses because harmful consequences such as cell death and DNA damage typically induced in bystander cells. Using the synchrotron X-ray microbeam irradiation system developed at the Photon Factory, KEK (Japan), we demonstrated that nitric oxide (NO)-mediated bystander cell death was biphasically enhanced in a dose-dependent manner. Here, we irradiated 5 nuclei of V79 cells　(1.0×10^5 cells / dish) using 10 μm × 10 μm 5.35 keV X-ray beams. We measured the mutation frequency at the HPRT locus in bystander cells. The mutation frequency with the null radiation dose was 2.6 × 10^−5 (background level) that decreased to 5.3 × 10^−6 with a dose of approximately 1 Gy (dose absorbed by the nuclei of irradiated cells). At higher doses, the mutation frequency was the same as the background level. A similar biphasic dose response was observed for bystander cell death. Bystander cell death and HPRT mutation frequency were significantly correlated (p < 0.05). The correlation between these responses indicated that bystander cell death and mutagenesis in bystander cells were responses to the same or related stimuli. Oxidative damage of nucleotides by reactive oxygen species (ROS) is thought to play an important role in spontaneous mutagenesis. Intracellular ROS levels are known to be persistently high in genetically unstable cells because these cells have low antioxidative activity. Further, a recent study showed that exposure to NO causes mitochondrial degeneration and subsequent cell death in cells with low antioxidative activity. Thus, we hypothesize that genetically unstable cells might be selectively killed by bystander responses because of their low antioxidative activity, given that NO is an important mediator of bystander cell death. As a result, the antioxidant ability of the surviving cell population may have increased, and hence, mutagenesis may have been suppressed in the bystander cells. Our results suggested that radiation-induced bystander responses can enhance the selective killing of genetically unstable cells in the bystander cell population and that this selective cell death might act as a protective mechanism against increases in non-lethal, potentially carcinogenic damage, e.g., mutations.

Speaker: Dr Munetoshi Maeda (Proton Medical Research Group, Research and Development Department, The Wakasa Wan Energy Research Center, WERC)

116 Low dose/dose rate gamma irradiation facility for in vitro biological systems

The response of in vitro biological systems to low dose and low dose rate irradiations is a topic that is important both for the determination of still unclear basic mechanisms and for its implications in radiation protection and therapy (late response of healthy tissue). The study of low dose/dose rate effects requires experiments performed under strictly controlled conditions where the dose and the dose-rate are evaluated with great accuracy and kept as uniform as possible on the sample. The realization of a dedicated irradiator appears to be the most convenient approach, since its design can be optimized for low dose-rate exposures of cultured cells under physiological conditions, protracted for several weeks. Here we present a gamma irradiation facility for the exposure of cultured mammalian cells to dose rates ranging from few μGy/h to some tens of mGy/h. The irradiator has been designed at the Istituto Superiore di Sanità (ISS), and, as of June 2012, it is in the phase of construction. Once fully operational, the facility will make use, alternatively and depending on the dose rate required, of three Cs-137 sources with different activities: 37 MBq, 740 MBq and 18.5 GBq. Each source will be housed in an appropriate holder placed at the center of an irradiation chamber. These three chambers are made of lead hollow cylinders with internal and external radius of about 14 mm and 84 mm respectively, and about 130 mm high. The top is closed by a suitable electrically removable lead cap. For a cell irradiation experiment, one of the irradiation chambers will be put into a shielded CO2 cell culture incubator. Also the design of the incubator shielding has been carried out at the ISS. The facility will be particularly suitable for non targeted effects studies. The most relevant characteristics of this especially designed irradiator are: 1) a dose rate uniformity on the sample at least of 95% and 2) a percentage of 662 keV (energy of the photon released in the Cs-137 decay) photons impinging on the sample greater than 80%. The dose will be evaluated with Geant4 simulations and measured with dosimeters such as TLD, OSL, alanine and/or ionization chamber. A monitoring system of the critical parameters (temperature, CO2, humidity) will also be installed. The realization of the facility has been made possible by a special EU funding, through a dedicated task, “LIBIS”, of the EURATOM Network of Excellence “DoReMi” (7th FP).

Speaker: Dr Giuseppe Esposito (Istituto Superiore di Sanità and INFN Gr. Coll. Sanità, Roma, Italy)

117 Bleomycin - induced bystander effects and stress response

Purpose: In the present study, we propose to evaluate the ability of bleomycin to induce bystander effects and the stress response mechanisms that might be involved in this process. Materials and Methods: L929 fibroblasts were treated with different concentrations (less than 60 µg/ml) of a DNA damaging agent, bleomycin. These cells were assessed for DNA damage, cell death and induction of stress response pathways (heat shock cytoplasmic stress, ISR and ER stress, mitochondrial stress). The media preconditioned was used to analyse induction of bystander damage. The assays to estimate the bystander effect are: DNA damage (micronuclei), clonogenic survival, induction of stress response by qPCR for specific markers of organellar stress. Results: We observed an increased DNA damage in L929 fibroblast treated with bleomycin. This increase is proportional with the concentration used. The level of micronuclei is also increased in bystander cells but does not depend on bleomycin concentration. Preliminary data show a consistent transcriptional down-regulation of all the stress response factors tested (i.e. Hsp70 Cyt, Hsc70cyt, BiP, CHOP, Hsp60mit) in the bystander cells and their down-regulation in the direct treatment with the exception of Hsp70cyt. This indicates that bleomycin might induce an arrest of transcription both in direct treatment and in bystander cells. Conclusion and prospective: Bleomycin induced bystander response reflected in increased DNA damage. Unexpectedly, stress response pathways markers are inhibited at transcriptional level. The stress response mechanisms involved in this process will be further investigated together with the role of reactive oxygen species and nitric oxide.

Speaker: Dr Iulia Diana Savu (Horia Hulubei National institute of Physics and Nuclear Engineering – IFIN-HH)

118 The influence of the number of irradiated cells in the spatial distribution of bystander effects

The need to better understand the risk associated with the exposure to low doses of ionizing radiation is the driving force of several studies currently being performed. In view of adaptive responses, bystander effects and cell death, the validity of the linear non-threshold relationship between dose and subsequent effects has been challenged. The way cell populations react to low radiation doses cannot be fully explained with a model obtained extrapolating to the low dose region the behavior of cells´ response to high radiation doses. Bystander effects are non-targeted effects induced in cells that were not irradiated but were in contact with irradiated ones or received signals from the culture medium. Cell death therefore appears to be a main safeguard mechanism, in particular necrosis or apoptosis. In the low dose region either bystander effects or cell death are pivotal to understand the underlying mechanisms of cellular response. The aim of the present study was to evaluate the influence of the number of irradiated cells in the non-irradiated areas. To analyze the spatial effects of the bystander signal, according with the number of irradiated cells, we exposed only a part of the cells plated on the cell dish to α-particles. The cell dish has 3.5 cm of diameter and depending on the study, approximately, 3/4 or 1/2 of this area was shielded by aluminum foils, being the irradiated areas differ from each other by a factor of 2. In order to pursue this objective a human lung cancer cell line, A549, was exposed to several doses of α-particles emitted by 210Po, namely 5, 10, 50 and 100 mGy. The non-irradiated areas were divided in two concentric areas with the same number of cells. Making use of the radiation induced extranuclear/extracellular technique γ-H2AX, we quantified, in situ the cellular damage, by means of DSBs and using the apo-trace organic molecule we quantified the cell death via apoptosis. Our results indicate that the cellular induced damage decrease with the distance to the irradiated area. In addition, we observed an increase of cellular damage in the non-irradiated areas when a higher number of irradiated cells are considered. We can also conclude that the number of irradiated cells influence the cellular damage induced in non-irradiated areas that were either in contact with or received signals from irradiated cells.

Speaker: Mrs Ana Belchior (Instituto Superior Técnico, Instituto Tecnológico e Nuclear)

119 In vitro and in vivo gene expression studies highlight differential expression profiles of low doses of ionizing radiation when compared to high doses

DNA microarrays are widely utilized to study cellular responses of complex nature, and when studying ionizing radiation, especially when the interest is low doses, we are faced with the dilemma of technique sensitivity, except for γ-H2AX foci. Previous studies have shown that microarrays are effective in the determination of pathways and genes that can be induced by ionizing radiation. In our study, we examined the global gene expression profiles of whole blood samples before and after exposure to a typically low and high X-irradiation dose (0.05, and 1 Gy), at a rate of 30 mGy/min (250kV, 1.6 mA, 1 mmCu).We investigated the gene expression profiles after 8 hours. Gene Set Enrichment Analysis (GSEA) revealed two distant dose-dependent profiles. In contrast to high doses, we found that a low dose of 0.05 Gy showed higher statistical ranking of immune-related pathways that are mainly involved in response and/or secretion of growth factors, chemokines and cytokines. This response is typically related to bystander effects and tissue response. On the other hand, a domination of classical radiation response was ranked higher at 1 Gy; these include tumor suppressor protein 53, apoptosis, DNA damage and repair. In order to validate our in vitro conclusions we moved to an in vivo study. Blood samples were collected from prostate cancer patients undergoing arc therapy before and 24 hours after the first fraction of radiotherapy. Dosimetric calculations showed that the circulating blood of these patients received a dose in the range 0.03-0.1 Gy. RNA was isolated and DNA microarrays were performed on a total of 7 patients. We found 890 genes that were significantly modulated between controls and irradiated samples (FDR <5%). Pathway enrichment analysis showed the involvement of the significant genes in immune modulation, cytokine secretion, DNA damage, and cell cycle. Some of these genes (XRRC4, BMX, and TNFAIP6) were confirmed using qRT-PCR. GSEA analysis revealed similar pathways as observed in the individual gene list analysis, with more emphasis on the involvement of certain immune related pathways such as toll-like receptors and and MAPK. Both in vitro and in vivo results show that low doses of ionizing radiation can elicit an immune response involving intercellular communication and probably increased radiation-induced damage. This work is supported by the Federal Agency of Nuclear Control (FANC-AFCN, Belgium) and by the Euratom FP7 EPI-CT project.

Speaker: Mr Houssein El Saghire (Faculty of Life Sciences and Medicine, Ghent University AND Radiobiology Unit, Belgian Nuclear Research Centre, Belgium)

120 The LNGS underground facility for biological experiments in reduced radiation environment

All humans are continuously exposed to ionizing radiations (IR) both from natural sources, such as cosmic rays and radioactive decay products, and from man-made sources, as in diagnostic radiology, nuclear medicine or radiotherapy. Although much is known about the effects of exposure to medium/high doses of IR, biological and health effects at low doses are still broadly debated. Among the effects observed at low doses both in vivo and in vitro, the adaptive response is defined as the capability of cells pre-exposed to low doses of IR or chemical mutagenic agents to acquire resistance to moderate or high doses of the same or a different agent. We hypothesized that environmental radiation, representing a source of chronic very low dose/dose-rate exposure, may condition the response of living systems to acute exposure to genotoxic agents, also including IR itself. The Gran Sasso National Laboratory (LNGS) of the Italian Institute for Nuclear Physics (INFN), located under the Gran Sasso mountain, offered us a unique opportunity to test such hypothesis. With respect to the external reference laboratory, the radiation spectrum is different and the overall dose rate is about 80x lower. To minimize accumulation of 222Rn, the underground laboratory is equipped with a ventilation system that captures air from outside the highway tunnel and pumps it inside the laboratories. After the pioneristic experiments conducted in S. Cerevisiae, that indicated a progressive sensitization to mutagenic drugs in yeasts kept under reduced environmental IR conditions, we set up a cell culture facility to study the response of more complex biological systems, such as rodent and mammalian cells. Data obtained with V79 Chinese hamster fibroblasts and human TK6 lymphoblasts indicated that cells cultured in a reduced radiation environment are more susceptible to DNA damage induced by physical agents, show a greater susceptibility to apoptosis, have a reduced ROS scavenging capacity and present alterations of the cellular trascriptome with respect to cells grown in a “reference” radiation laboratory. Other underground laboratories in the world are starting biological experiments and strong attention has been manifested by the international community to the results already obtained at the LNGS. Low dose research activity is still in progress using in vitro biological systems and the future plans foresee the set up of an animal housing facility for in vivo studies.

Speaker: Dr Maria Antonella Tabocchini (Istituto Superiore di Sanità & INFN RM1. Gr coll Sanità, Rome, Italy)

121 Dose and Spatial Effects in Distant Bystander Radiation Signaling In vivo: Implications for Tumorigenesis

The aim of this work was to investigate the dose and spatial dependence of abscopal radiation effects occurring in vivo in the mouse, along with their tumorigenic potential in radiosensitive Patched1 heterozygous (Ptc1+/-) central nervous system (CNS). Ptc1+/- mice, carrying a germ-line heterozygous inactivating mutation in the Ptc1 gene, and uniquely susceptible to radiation damage in neonatal cerebellum, were exposed directly to ionizing radiation (1, 2 or 3 Gy of X rays) or treated in a variety of bystander protocols. In partial-body irradiation experiments, the animals’ head was fully protected by suitable lead cylinders while the remainder of the body was exposed to X-rays. Apoptotic cell death was measured in directly irradiated and shielded cerebellum at different time points after irradiation, while tumor development was monitored in lifetime groups. The same end-points were measured in mice irradiated with 3 or 10 Gy of X rays using different shielding geometries. We show a clear dose dependence of in vivo transmission of radiation signals to non-target mouse CNS. While dose-dependent cell-death was observed in off-target cerebellum for all doses and shielding conditions tested, a conspicuous lack of bystander response for CNS tumorigenesis occurred at the lowest dose of 1 Gy in body-exposed/head-shielded animals. The shielding geometry could also significantly modulate tumorigenesis as a function of dose. Our study shows a clear interplay of radiation dose and exposed tissue volume in abscopal effects occurring in mouse CNS under conditions relevant to humans. The results may help understanding the mechanisms of long-range radiation signaling in harmful effects, including carcinogenesis, occurring in off-target tissues.

Speaker: Dr Simona Leonardi (ENEA CR Casaccia, Rome, Italy)

122 TGF-β and the Radiation Bystander Effect in Human Peripheral Blood mononuclear cells – preliminary results and potential applications

The radiation bystander effect, whereby cells not directly exposed to ionizing radiation exhibit characteristic signs of radiation damage was first observed twenty years ago and remains an active area of research within radiobiology. This effect has since been demonstrated in a number of experimental systems; the literature confirms the relevance of the bystander effect to human peripheral blood lymphocytes, which are routinely harvested for the monitoring of radiation workers. Recently attempts have been made to connect the bystander effect to another potential factor for the assessment of low-dose risk, the radioadaptive response. One of the signal molecules, widely mentioned in the context of the bystander effect is TGF-β, a secretable protein factor implicated in apoptosis via the SMAD pathway, and cell cycle regulation via p15 and p21. When secreted, TGF-β has been proven to act as an autocrine signaling factor and to interact with the immune system, preventing the activation of lymphocytes, and colocalizing with inflammation sites. Seeing the potential to produce a bystander effect in the systems we routinely use in our laboratory, we have conducted two preliminary experiments with peripheral blood mononuclear cells (PBMCs) pooled from 8 and 21 donors, respectively, in which we irradiated PBMCs in the presence or absence of serum with 3.3 Gy of 60Co γ-rays and incubated non-irradiated cells with medium taken from irradiated ones. The preliminary results seem to confirm the presence of a bystander effect in our system, with cellular viability dropping off some 7-8% 24h after irradiation and more significantly (~25%) in the cells which were irradiated in the presence of serum; medium transfer had similar observable effects to irradiation in the absence of serum. TGF-β levels also remained relatively constant when PBMCs were irradiated in the absence of serum, but increased sharply with irradiation plus serum. Subsequent experiments will aim to confirm the presence of a bystander effect through analyzing the following parameters: cellular viability, apoptosis, DNA damage (comet test-neutral and alkaline), micronuclei, SCEs, intracellular ROS, yH2AX foci, and quantification of TGF-β and SMAD-3. In case a bystander effect is solidly established, we will continue with our experiments, potentially using an occupationally exposed cohort in order to screen for an adaptive response, or observing the effects of proteasome inhibitors on the system.

Speaker: Mr Peter Ostoich (National Centre for Radiobiology and Radiation Protection, Sofia, Bulgaria)

123 Effects of EMP Exposure on Rat Brain Microglia

Objective: To investigate the effect of electromagnetic pulse (EMP) exposure on rat brain microglia and its possible mechanism. Methods: Adult male SD rats and primary cultured rat brain microglia cells were exposed or sham exposed to EMP at 200 kV/m for 200 pulses, the repetition rate was 1 Hz. The activation of microglia was determined by CD11b immunocytochemistry, the protein and phosphorylated protein levels of Mitogen-Activated Protein Kinase (MAPK ) pathway signal molecules were determined by Western Blot, the levels of cytokines and nitric oxide (NO) in brain cortex and primary cultured microglia cells were determined by the enzyme-linked immunosorbent assay (ELISA) and commercial kit at 1, 6, 12 and 24 h after EMP exposure. Results: It was found that the the number of CD11b immunopositive cells increased at 1h , reached the peak at 6h, and recovered to sham level at 24h after EMP exposure, moreover, the levels of cytokines (such as TNF-α and IL-10) and NO also transiently changed within 24hs after EMP exposure. In addition, The protein level of p38 and phosphorylated p38 but not ERK and JNK transiently increased after EMP exposure both in vivo and in vitro studies. p38 inhibitor (SB203580) pretreatment partly prevented EMP-induced the alterarion in secretion function of microglia. Conclusions: These results suggested that EMP exposure could activate rat brain microglia and affected its secretion function, and p38MAPK pathway was involved in this process. This work was supported by the Research Fund of National Natural Science Foundation of China [31170798，81072272 and 81102091]

Speaker: Prof. Gui-Rong Ding (Department of Radiation Medicine, Fourth Military Medical University)

124 Health Effects of Occupational Exposure to Pulsed Microwave Radiation in Military Radar Personnel

Radar workers are exposed to pulsed high frequency electromagnetic fields. In this study, health effects of these radiations in personnel who routinely work with radar systems are investigated. The 28-item General Health Questionnaire was used as a self-administered tool for assessment of general mental health and mental distress. One hundred workers occupationally exposed to radar radiations (2-18 GHz) participated in the study. Visual reaction time was recorded with a simple blind computer-assisted-visual reaction time test. To assess the short term memory, Wechsler Memory Scale-III test was performed. Twenty to thirty nine percent of the radar workers reported different problems such as needing a good tonic, feeling run down and out of sorts, headache, tightness or pressure in the head, insomnia, getting edgy and bad-tempered. Furthermore, 47% of the radar workers reported feeling under strain. In response to this question that if they have been able to enjoy their normal day to day activities, 31% responded “less than usual”. It was also shown that work experience had significant relationships with reaction time and short term memory indices i.e. forward digit span, reverse digit span, word recognition and paired words. To the best of our knowledge, this is the first study to show that occupational exposure to radar microwave radiation leads to changes in somatic symptoms, anxiety and insomnia, social dysfunction, and severe depression. Altogether these results indicate that occupational exposure to radar microwave radiations may be linked to some adverse health effects.

Speaker: Mr Shahram Taeb (Shiraz University of Medical Sciences)

125 Effect of surgical stress and immobility of the hormone Ghrelin and body weight and food consumption in adult male rats

Introduction: Ghrelin is a hormone that was discovered in 1999 by kojima where stomach cells that secrete this hormone increases growth hormone secretion and the amount of food consumed has a significant role stress can increase blood levels of Ghrelin is the purpose of this study surgical stress and immobility in periods 7 and 14 days on the secretion of Ghrelin and body weight and food is consumed. Methods: In this experimental study of 60 adult male rats were used in animals were randomly divided into 6 groups. The first group and second group served as controls were studied groups 3 and 4 were operated and then after 7 and 14 days of weighing to the blood sampling was performed, groups 5 and 6 also stress immobility, respectively. were for 7 and 14 days and then blood samples were taken and Ghrelin levels were measured in serum samples. Results: Ghrelin levels in serum had no significant effect on surgical stress. Stress immobility Serum Ghrelin levels were significantly increased. Effects stress Ghrelin serum levels greater than 14 groups on 7-day stress is immobility. Stress immobility for 14 days significantly decreased body weight than the control group. (Mean ± sem) food intake for both the significant increase in stress. Conclusion: Ghrelin increased serum level of immobility stress, weight loss and food intake is increased. Key words: Surgical stress, immobility stress, Ghrelin, male rat

Speaker: Mr mojtaba keshavarz (Msc Of Developmental Biology , Department Of Biology,Islamic azad university of jahrom , jahrom,, Fars, Iran.)

126 Retrospective (1992-2009) analysis of data on oxidative homeostasis state in clean-up workers of Chornobyl accident aftermath

Increase of chronic diseases number among Chornobyl Nuclear Power Plant (ChNPP) accident clean-up workers require more detailed study of mechanisms forming pathological background for such clinical implications. The prolonged disturbances of oxidative homeostasis (OH) on the top of radiation induced molecular-genetic disorders can be a ponderable factor for morphofunctional changes which afterward might realize into chronic clinical manifestations. Thus, investigation of OH status in irradiated persons allows understanding the character of biochemical changes in remote period depending on absorbed doses. ОH status was studied in 355 males clean-up workers aged (48.62±0.55) who was irradiated in 1986-1988 in dose range (1.22±0.06) Gy during elimination of ChNPP accident aftermath. The investigation term (1992-2009) was divided on three periods: I -1992-1995, II - 1996-1999, III - 2006-2009. The controls were formed by 177 practically healthy men without radiation contact in accordance to each period. The levels of lipoperoxidation products (LPP) - phospholipids with isolate, double and triple bonds, tiobarbiture acid reactive species (TBARS), сarbonyl proteins (CP), reduced glutathione (GSH), erythrocyte superoxidedismutase (Zn/CuSOD), catalase (E-CAT), glutathione peroxidase (GPx) and activities are determined in peripheral blood samples. The integral factor of antioxidant status (FAOS) for estimation of ОH derangement was calculated as well. The disturbance of OH in all examinees was observed during the whole investigation term. It was revealed that in persons irradiated in dose >0.25 Gy the integral FAOS level dropped by 1.52 times (p=0.001) since I to III period against the background of LPP accumulation with double, triple bonds, TBARS, CP, GSH levels and decreased activity of E-CAT, switching over of antioxidant protection to GPx and while the decline of Zn/CuSOD activity was marked only in persons with absorbed doses >2.00 Gy. The LPP and CP content, Zn/CuSOD activity and FAOS level dynamic of changes correlated with absorbed doses and did not connect with age factor in examined persons during investigation term. Thus, the enhancement of OH disturbances in clean-up worker of ChNPP accident as postaccidental terms increased, peculiarities of which correlated with absorbed dose, possibly is a result of individualized sensitivity to irradiation that forms the pathobiochemical background for development and maintenance of steady clinical implications

Speaker: Ms Alla Kubashko (Research Center for Radiation Medicine)

127 The serum levels of 8-oxo-dG as a biomarker of individual radiosensitivity after in vitro exposure of whole blood.

About 20% of patients receiving radiotherapy will develop adverse reactions to the therapy and 5% of these patients will develop severe reactions. Theoretically, all cancers could be controlled if a sufficiently high radiation dose could be delivered to the target. However, doses used in radiotherapy are adapted to the tolerance of the most sensitive patients. Thus, radiotherapy would greatly benefit from a diagnostic tool providing information on individual radiosensitivity. The long term goal of this project is to provide diagnostic tools to help to individualize the therapy to improve tumor control, thus providing patients with better quality of life and reducing the health care costs. Low linear energy transfer ionizing radiation produces a wide spectrum of DNA damages. The majority of the primary radiation effects are mediated by reactive oxygen species (ROS). The dominant forms of radiation-induced base modifications include 8-oxo-dG. If 8-oxo-dG is formed in DNA it can lead to GC→TA transversions. However, the nucleotide pool is also a target of ROS, where 8-oxo-dGTP can be formed. 8-Oxo-dGTP can be incorporated into DNA opposite an A, which can lead to AT→CG mutations1. We have previously shown that urinary 8-oxo-dG could be used as a marker for individual radiosensitivity in breast cancer patients2. We think that the observed differences in urinary levels of 8-oxo-dG in breast cancer patients undergoing radiotherapy may reflect individual capacity to handle oxidative stress induced by radiation in the exposed healthy tissue. Based on our previously published data, we hypothesized that leucocytes in whole blood from sensitive patients can be used to monitor their repair capacity of 8-oxo-dGTP formed in response to radiation. To test this hypothesis we have developed a highly sensitive ELISA assay for determination of serum level of 8-oxo-dG as a biomarker of oxidative stress. Whole blood were collected from radiosensitive and non-sensitive breast cancer patients and exposed to gamma radiation. The serum level of 8-oxo-dG was monitored for each individual in exposed and non-exposed samples. The result indicates that this assay may have the potential to be used to identify the most sensitive patients. 1) Evans M D, et al., Mutat Res 2004; 567, 1-61. 2) Haghdoost S, et al. Int J Radiat Oncol Biol Phys 2001; 50, 405-410.

Speaker: Mrs Sara Skiöld (Stockholm University)

128 LIPID PEROXIDATION IN TISSUES OF MICE UNDER IRRADIATION AT LOW DOSES AND DIFFERENT DOSE RATE

Up to now the question is discussed dose or dose rate is the more significant for the development of biological consequences under the irradiation of animals especially at low doses. Earlier in laboratory experiments and investigations on rodents caught at areas with different levels of the radioactive contamination the high sensitivity of the lipid peroxidation (LPO) regulatory system parameters in tissues of mice to the ionizing radiation action at the low doses was shown. Parameters of the LPO regulatory system in murine tissues with different antioxidant (AO) status were studied before and after gamma-irradiation at the dose of 15 cGy (the dose rate were 0.01; 0.25 and 9 cGy/min); the X-ray exposure at the dose of 16 cGy (the dose rate was 44 cGy/min) and at the doses less 1.5 mGy under changing dose rate within action. The spleen relative mass in groups of irradiated mice is mainly due to the total X-ray dose under changing dose rate, and the analogical index in the liver irradiated mice depends on the initial value of parameter for control. Experimental data demonstrated the different sensitivity of LPO parameters to the low intensity irradiation at the low doses depending on the dose rate and changing of these parameters being kept for a long time after action. The initial value of parameters is found to exert the more substantial influence on the spleen and liver relative mass, the phosphatidylcholine (PC) and lysoforms shares in the tissue phospholipids (PL) of mice. The content of TBA-reactive substances in the blood plasma is the most sensitivity parameter to the action of X-ray at low doses. The reliable changes and substantial influence of the dose rate, AO status of tissue and the duration after irradiation are revealed for the molar ratio of [sterols]/[PL], the PC/phosphatidylethanolamine ratio and the ratio of sums of more easily oxidizable to more poorly oxidizable fractions of PL. The X-ray changing dose rate primary effect on the formation of biological consequences is revealed at doses less than 0.5 mGy in the blood plasma and more than 0.5 mGy in brain. The experimental data testify to the complicated nonlinear character of the biological effects under the action of ionizing radiation at low doses on mice. Besides, the biological effectiveness of irradiation under the different dose rate is substantial depended on the AO status of tissues and the duration after irradiation.

Speaker: Prof. Lyudmila Shishkina (no)

129 Reactivity of Resorufin-based Fluorogenic Boronate Probe PC1 Towards ROS/RNS and Its Use in The Studies of Peroxynitrite Reactivity

It is known that aryl boronates can react with hydrogen peroxide, and hypochlorite with the formation of corresponding phenols. Previously, we have shown that arylboronic acids react rapidly, directly and stoichiometrically with peroxynitrite with the formation of corresponding phenols, as major products (85-90%). High reactivity toward peroxynitrite makes them promising probes for the detection of peroxynitrite in vitro and in vivo, and a useful tool for the studies of peroxynitrite reactivity. Here we present the results of the study on the reactivity of boronate fluorogenic probe PC1 toward selected ROS/RNS (e.g. peroxynitrite and peroxynitrate). The boronate probe PC1 was also used in the kinetic studies on the reactivity of peroxynitrite towards selected salen-manganese complexes EUK. Here we show that there is a strong correlation between reported catalase activity of salen-manganese complexes EUK and the second-order rate constants of their reaction with peroxynitrite. For better understanding of the factors affecting the oxidation of boronates we also carried out some experiments with model para-substituted aryl boronic acids.

Speaker: Dr Adam Sikora (Institute of Applied Radiation Chemistry, Lodz University of Technology, Lodz, Poland)

130 Lack of Oxidative Stress after Short-Term Exposure to Naturally Elevated Levels of Gamma Radiation in Ramsar

Ramsar, a city in northern Iran, has areas with some of the highest recorded levels of natural radiation among inhabited areas measured on the earth. In some areas of Ramsar, residents receive an annual radiation dose from background radiation that is about 13 times higher than the 20 mSv/y that is considered as a dose limit for radiation workers. These residents have lived for many generations in these areas of high background radiation. In 2002 we reported on the induction of adaptive response in residents living in high background radiation areas of Ramsar. It is believed that the adaptive response involves the transcription of many genes and the activation of a wide variety of signaling pathways that trigger specific cell defense mechanisms. In this study, 53 Wistar rats were randomly divided into five groups of 10-12 animals. Animals in the 1st group were kept for 7 days in an outdoor area with normal background radiation while the 2nd, 3rd, 4th and 5th groups were kept in 4 different outdoor areas with naturally elevated levels of gamma radiation in Ramsar. A calibrated RDS-110 survey meter, mounted on a tripod approximately 1 meter above the ground, was used to measure exposure rate at each location. On days 7 and 9 blood sampling was performed to assess the serum levels of catalase (CAT) and malondialdehyde (MDA). On day 8, all animals were exposed to a lethal dose of 8 Gy gamma radiations emitted by a Theratron Phoenix (Theratronics, Canada) Cobalt-60 (55 cGy/min) at Radiotherapy Department of Razi Hospital in Rasht, Iran. Results obtained in this study indicate that high levels of natural radiation cannot induce oxidative stress. CAT and MDA levels in almost all groups were not significantly different (P=0.69 and P=0.05, respectively). After exposure to the lethal dose, CAT and MDA levels in all groups were not significantly different (P=0.054 and P=0.163, respectively). These findings indicate that short-term exposure to extremely high levels of natural radiation (up to 196 times higher than the normal background) does not induce oxidative stress.

Speaker: Prof. SMJ Mortazavi (Shiraz University of Medical Sciences)

131 Reduction of Peroxyl Radicals of Amino Acid Derivatives by Urate

Proteins are important targets of partly reduced oxygen species (PROS) that are responsible for biological damage under oxidative stress. Protein oxidation results in the formation of amino acid radicals (AA•) randomly on the protein surface; in the presence of oxygen, further reaction may yield protein peroxyl radicals and protein hydroperoxides. All these species are likely to propagate damage. Urate, a low-molecular antioxidant, is able to repair tyrosine and tryptophan radicals in various proteins in vitro.1-2 We have studied the reaction of urate with the peroxyl radicals (AAOO•) of N-Ac-Gly-amide, N-Ac-Ala-amide and N-Ac-Pro-amide. All amino acid peroxyl radicals studied here were reduced by oxidizing urate to the urate radical. The reaction was followed in the range 320-400 nm, where urate radical has a strong absorption. Interestingly, the rate of urate radical formation is independent of the urate concentration with rate constants of ca. 103 s–1, which suggests initial formation of an adduct between AAOO• and urate that then further decays to form urate radicals. It is to be stressed that reaction of AAOO• with urate gives rise to hydroperoxides (AAOOH) that are also reactive molecules. Our results suggest that urate could “repair” protein radicals (AA•) and prevent biological damage in vivo, given its high concentration in living organisms. For ascorbate, the principal endogenous antioxidant, we reported already rate constants for the repair of tyrosine and tryptophan radicals in various proteins in vitro3 and we recently measured rate constants close to 107 M–1 s–1 for its reaction with AAOO• (unpublished data). The urate radical produced upon repair of protein radicals is, in turn, reduced by ascorbate. The net result is loss of ascorbate with conservation of urate. [1] B.M. Hoey and J. Butler Biochim. Biophys. Acta 1984, 791, 212-218. [2] A.S. Domazou, H. Zhu, and W.H. Koppenol, Free Radic. Biol. Med. 2012, 52, 1929-1936. [3] A.S. Domazou, W.H. Koppenol and J.M. Gebicki, Free Radic. Biol. Med. 2009, 46, 1049-1057.

Speaker: Anastasia S. Domazou (Swiss Federal Institute of Technology Zurich (ETH Zürich))

132 The effects of gamma-radiation-induced damage on environmental isolates of the baker’s yeast Saccharomyces cerevisiae

Recent work has shown that proteins are critical targets in irradiated bacteria governing their survival. Our laboratory and others have shown that DNA and protein damage are uncoupled in radiation-resistant prokaryotes exposed to gamma-rays. Radiation-resistant bacteria such as Deinococcus radiodurans are extremely resistant to protein oxidation, but they are as susceptible to radiation-induced DNA damage as naturally sensitive cells. The model ‘Death by Protein Damage’ in irradiated cells asserts that the efficiency of DNA repair, and thus survival, depends on the antioxidant status of a cell1. The question of whether or not proteins are critical targets in irradiated simple eukaryotes remains unanswered. If the above model extends to irradiated simple eukaryotes, this work may provide new opportunities to study and control oxidative stress in higher eukaryotes, including mammalian cells and their cancer cell counterparts.   Protein protection in D. radiodurans is based on the accumulation of manganous (Mn2+) metabolite complexes which catalytically scavenge superoxide and related reactive oxygen species (ROS) generated by water radiolysis. When reconstituted in vitro, the Mn2+ complexes of D. radiodurans were highly radioprotective of proteins, but not DNA. Studies in Saccharomyces cerevisiae has shown that yeast accumulate Mn2+together with secondary metabolites (e.g., trehalose and glutathione) as part of the stress response, indicating that these organisms may form similar protective complexes.   We have found that many yeast qualify as extremophiles based on their ability to grow under high-level chronic gamma-irradiation, and survive high-dose acute irradiation.  The sequenced lab strain FY1679 displays 10% survival (D10) at 900 Gy, which corresponds to ~60 DSBs per haploid genome. Some environmental strains of S. cerevisiae are even more resistant, displaying D10 values around 3 kGy, which corresponds to ~150 DSBs/haploid genome. By comparison, D. radiodurans can survive ~160 DSBs/haploidgenome following gamma-ray exposure. We are currently investigating radiation-induced DNA and protein damage to determine if proteins are the critical targets in irradiated yeast. We aim to further our work in this eukaryotic model system by examining the small molecules accumulated in these strains to determine their contribution to radiation resistance.   1. Daly M.J. (2011) DNA Repair 11:12-21. (http://www.sciencedirect.com/science/article/pii/S1568786411003193

Speaker: Ms Maggie Wear (Uniformed Services University of the Health Sciences)

133 The Protective Effects of Vitamin C and E, Against Oxidative Stress Induced by Sulfasalazine in theTestis of Male Adult Rats

Background & Objective: Sulfasalazine (SASP) is a drug used for the treatment of inflammatory bowel diseases. SASP causes testicular damage. The aim of this study was to verify whether or not an additional supply of vitamin E or C or together prevent the toxic effect of SASP on the rat spermatogenesis. Materials & methods: Adult male wistar rats (150-200 gr) were divided into five groups (n=8) control and four experimental groups. The experimental groups were treated with SASP (600 mg/kg/day by gavage feeding ) for 14 successive days. Additionally the experimental group 2 were treated with the vitamin C (20 mg/kg/body weight/daily), the experimental group 3 received vitamins E (200 mg/kg body weight/daily) and the experimental group 4 received daily vitamin C and vitamin E 20 and 200 mg/kg body weight, respectively through the same root. At the end of the experimental period the mean body weight growth and the ratio between body and testis weight were calculated and compared with the control groups, Spermatogenesis Parameters such as, daily Sperm count, percent of motility, viability, Daily sperm production (DSP), testicular histopathology, serum testosterone levels and the level of Malondialehyde (MDA) were evaluated. The data were analyzed using Danken and one way variance test and the P≤ 0.05 were considered significant. Results: Sperm count, percent of motility, viability and DSP were decreased in SASP treated animals. A significant decrease in the number of sertoli cells, Leydig cells, spermatogonia and spermatid were seen in the testis of SASP treated animals. The level of MDA was increased in the SASP treated group. However, Co-administration of vitamin E and C with SASP reduced the level of MDA and deleterious effects of SASP on the sperm parameters and testicular histology. Finally, the Co-administration of vitamin E and C had the most protective effects when compared with the control groups. Conclusion: The results suggested that additional supply of vitamin E and C protect against SASP–induced oxidative stress in the rat testis. Keywords: Sulfasalazine,Vitamin C and E, Spermatogenesis

Speaker: Mr ahmad mozafar (Msc Of Developmental Biology , Department Of Biology,Islamic azad university of jahrom , jahrom, Fars, Iran.)

134 Late oral mucosa alterations after radiotherapy for head and neck cancer: cytological study of radiation induced epithelial cells injury

Introduction and aims: Radiotherapy for head and neck (H&N) district malignancies may induce permanent tissue damage leading to multiple life-long side effects that place patients at continual risk for oral sequelae. In our study late toxicity after radiotherapy for H&N cancer has been assessed by exfoliative oral cytology. Matherials and methods: Eligibility criteria were: age between 30 and 80, a minimum of two-years follow-up after completion of 3D conformal radiotherapy for H&N solid tumors, with oral mucosa in the fields of exposure of therapy. From december 2011 to may 2012, 14 consecutive patients were enrolled at their programmed follow-up visit. They were 11 males and 3 females, mean age was 66.5 years (range 52-76), mean follow-up after radiotherapy was 47.3 months (range 24 –98), mean dose was 65.6 Gy (range 50-70.2) with conventional fractionation, mean overall treatment time was 58.8 days (range 43-88). Tumor prevalent localization was oropharynx and oral cavity (11 out of 14 patients). Seven underwent postoperative radiotherapy and six received concomitant chemotherapy. Smears were collected by scraping left and right cheeck mucosa with wooden tongue spatulas, then transferred to 4 dry glass slides (2 for each cheeck), fixed immediately with Biofix® and stained with modified May Grunwald Giemsa staining. Smears were examined under 400X power using a light microscope. Results: All the smears collected showed cytological alterations: typical “folding” appearance of epithelial cells, a particular cellular phenotype marker of radiation injury, with enlarged and hypertrophic nucleus. In order to graduate the presence/absence of this specific cellular phenotype, we counted the folding cells as a percentage of all cells per dry glass slide for each cheek, made the average and finally scored them. On our preliminary data, we observed that in 9 out of 14 patients there was a linear relation between dose delivered and oral mucosa injury but in 5 out of 14 patients there was no correspondence between dose delivered and score of folding cells; 4 patients showed the same score in left and right cheek but dose was not equally distributed between the two sites and 1 patient conversely showed symmetric distribution of dose but not of score. We can hypotize that also immune system plays a key role in the variability of individual response of oral tissue to radiation damage; our data need to be supported by a larger sample.

Speaker: Dr Patrizia Massaro (Fondazione I.R.C.C.S Policlinico San Matteo, Pavia)

135 hUCB-MSC ameliorates irradiation-induced intestinal injury in murine model

Purpose: Gastrointestinal syndrome after high-dose acute radiation exposure (GI-ARS) is life-threatening problem. In this study, we examined the radiation mitigation effects of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) on intestinal damage of gamma-irradiated rats. Methods: to investigate the effect of hUCB-MSC on mitigation of intestinal injury following gamma-irradiation, animals were divided into three group; non-irradiated control group, non-treated group and hUCB-MSC transplanted group after irradiation. The animals were exposed 14 Gy in abdomen and sacrificed at 4 days and 8 days after irradiation to examine the various pathological changes related to intestinal injury. hUCB-MSC (1x107cells/animal) was intravenously injected at 4 hours after irradiation. Result: All animals showed severe diarrhea and piloerection after irradiation. We found histopathological mechanisms that radiation-induced intestinal injury was characterized by remarkable shortened villi, decreased crypt, edema of neural plexus and cellular degradation of ganglions. hUCB-MSC transplanted group was significantly increased the proliferation activity of ileac crypt, compared with irradiation controls. The hUCB-MSC transplantation reduced irradiated intestinal morphological changes such as crypt size and villus height. In addition, the administration of hUCB-MSC increased the expression of platelet derived growth factor receptor-α (PDGFR-α), involved in villus morphogenesis and ubiquitin c-terminal hydrolase-l1 (UCH-L1), a marker of neuronal cells. These results suggest that hUCB-MSC has the protective effects not only on the intestinal cells but also on the neuronal cells in the small intestine. Conclusion: Although further study will be needed about time point, route of injection or combined treatment with other drugs, our results suggest that hUCB-MSC administration is useful for regeneration of intestinal cell and reconstruction of organic structure against radiation-induced GI syndrome.

Speaker: Mr Sehwan Shim (Korea institute of radiological &amp; medical sciences)

136 Prediction of normal tissue radiosensitivity in gynecological and prostate cancer patients from polymorphisms in XRCC1, XRCC3 and OGG1 genes and radiation-induced apoptosis in peripheral blood lymphocytes

Several observations indicate that normal tissue hypersensitivity may be related to certain genetic factors. In view of the importance of DNA repair in cell and tissue response to radiation, single nucleotide polymorphisms (SNPs) in the genes responsible for DNA damage signaling and repair pathways seem to be suitable candidates in the search for the genetic basis of clinical normal tissue radiosensitivity. The aim of our study was to investigate whether SNPs selected in XRCC1, XRCC3 and OGG1 genes are associated with an increased risk of adverse reactions to radiotherapy in patients treated for gynecologic and prostate cancers. Furthermore, the reliability of radiation-induced apoptosis in the prediction of acute reactions within the studied population was investigated. The study population consisted of 30 women with cervical and uterine cancer and 30 man with prostate cancer. All woman received a tumor dose of 50,4 Gy in 28 fractions and all man received a tumor dose of 74 Gy in 37 fractions. Acute normal tissue reactions were scored according to RTOG criteria. Peripheral blood from cancer patients was collected before radiotherapy and irradiated in vitro with a dose of 2 Gy. The frequency of apoptotic cells was determined by flow cytometer after FITC-Annexin-V staining. Polymorphic sites in XRCC1 (194Arg/Trp, 399Arg/Gln), XRCC3 (IVS5-14), and OGG1 (326Ser/Cys) were examined by (PCR)-restriction length fragment polymorphism. The results of the present study showed that the only XRCC1 codon 194 variant showed a significant protective effect in prostate cancer patients (p = 0.006). Gynecological cancer patients with low frequency of radiation-induced apoptosis showed a greater grade of complications than patients with high frequency of radiation-induced apoptosis (p = 0,031). This work was supported by stipendium to Aneta Węgierek-Ciuk"Wiedza i Gospodarka - rozwój kompetencji naukowych i biznesowych dla wzrostu konkurencyjności gospodarki regionalnej"współfinansowanego ze środków Europejskiego Funduszu Społecznego.

Speaker: Ms Węgierek-Ciuk Aneta (Dept. of Radiobiology and Immunology, Jan Kochanowski University, Kielce, Poland)

137 Cytokine secretion profiles and signaling pathways analysis of endothelial cells exposed to high doses of ionizing radiation

Vascular injury is one of the most common effects of ionizing radiation on normal tissues due to the high radiation sensitivity of endothelial cells. The knowledge of molecular mechanisms involved in endothelium dysfunction following radiation exposure is needed to identify therapeutic targets and to develop strategies to prevent and/or reduce effects of irradiation. Methods : To clarify some molecular mechanisms involved in this response, we examined irradiation-responsive proteins in cultured primary human umbilical vein endothelial cells (HUVEC) using multiplex suspension bead arrays (Bio-Plex suspension array system). In an attempt to study the long term response of irradiated endothelial cells, protein secretion and signaling pathways were studied after exposure of HUVEC to doses of 0, 2 and 20 Gy. The secretion of 27 cytokines (interleukines, chemokines, growth factors) and the levels of 5 intracellular phosphoproteins (Akt, ERK1/2, JNK, P38 MAPK, P53), with their respective total forms, were assayed 1, 2 and 3 weeks after ionizing radiation exposure. Results : Out of the 27 cytokines, 4 proteins (IP-6, Eotaxin, IL-6 and RANTES) were clearly overexpressed in the culture medium after 21 days. On the other hand, the levels of the intracellular phosphoproteins were all overexpressed 21 days post-irradiation, suggesting that signaling pathways were continuously activated in long-term cultured irradiated HUVECs. Conclusion : These results clearly indicate that the survival fraction of irradiated HUVEC always display a modified phenotype 3 weeks after ionizing radiation exposure. This phenotype could reflect the continuous expression of stress and inflammatory signals in the survival fraction of irradiated HUVECs.

Speaker: Mr Georges TARLET (Institute for Radiological and Nuclear Safety)

138 Is late radiation toxicity dependant on genotype? A study of patients with osteoradionecrosis

Approximately 20% of cancer patients undergoing radiotherapy (RT) experience some kind of adverse reaction and 5% experience severe reaction in healthy tissues. Recent data have highlighted the importance of individual radiosensitivity dependent on genotype, but there is a paucity of studies on late adverse effects, since these endpoints are difficult to determine. In this study, we investigated patients with mandibular osteoradionecrosis (ORN), a late adverse effect following RT in head & neck cancer. ORN is characterized by loss of blood supply leading to hypoxia, hypocellularity and tissue necrosis with severe pain and possible pathological fractures. Treatment for ORN is resection of necrotic tissue, often followed by free tissue transfer of vascularized bone. This procedure is demanding and the risk of recurrence is evident. Since ORN seems to be dependent on individual susceptibility, it is important to study the association with genotype, since ORN not only limits the patient´s quality of life, but also is tremendously costly for the healthcare system. As of today, there is no predictive assay available that could identify highly radiosensitive patients among the normally radiosensitive, which makes RT treatment plans based on individual radiosensitivity impossible. Several theories exist regarding mechanisms behind severe late adverse effects that occur after RT. Chronic inflammation/oxidative stress, dysregulated wound healing and reduced DNA repair capacity have all been proposed as key factors in the development of adverse late effects. However, the influence of genetic variations has never been studied for ORN, which is a well-defined adverse late effect of RT and probably dependent on individual susceptibility. In a study of 74 patients, blood was collected from 37 patients with ORN (RTOG score 4) along with 37 age-, dose- and TNM-matched controls (RTOG score 0) from the Department of Oral and Maxillofacial Surgery at the Karolinska Hospital in Stockholm. Blood was irradiated with 0, 0.005 and 2 Gy γ-rays and the levels of 8-oxo-dG 1 hour post irradiation were measured to assess the capacity to handle radiation-induced oxidative stress. Also, single nucleotide polymorphisms (SNPs) in candidate genes involved in oxidative stress (SOD2, eNOS), inflammation (HIF1A), wound healing (TGFβ1) and DNA repair (ATM, XRCC1, XRCC3) were investigated. The results (not yet available at this time) will be presented.

Speaker: Mr Karl Brehwens (Centre for Radiation Protection Research, GMT Department, Stockholm University, Sweden)

139 Early impairment of spatial cognition after local brain irradiation with carbon ions

Cerebral dysfunction is one of the major concerns associated with radiotherapy of brain tumors and acute lymphoblastic leukemia. Side effects such as learning impairment and cognitive dysfunction are reported on the central nervous system (CNS) tissue after exposure to radiation during cancer therapy. This study investigated early effects on the cognitive function and microvessel density (MVD) after local brain irradiation with carbon ions using mice. Irradiation was carried out a 290 MeV/u 5 mm SOBP (Spread out Bragg Peak) carbon beam with 7.6 cm diameter produced by the Heavy Ions Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Sciences, Japan. The irradiation dose was set at level known to produce vascular change followed by necrosis, which appeared the late period after irradiation with 30 Gy. The whole of brain was irradiated, excluding eyes and brain stem. The mice irradiated with single dose of 30 Gy showed deficit in short-term working memory assessed at 36 hr after irradiation, whereas mice receiving carbon irradiation showed no deficit in long-term reference memory. At 16 weeks after irradiation, the irradiated mice showed marked learning impairment compared with age-matched controls and the irradiated mice showed substantial impairment of working memory. Histopathological observation revealed no abnormal finding in the irradiated brain at 1 week after irradiation, although irradiated mice showed marked neuronal degeneration at the hippocampus within CA1 to CA3 layers at 16 weeks after irradiation. In the irradiated group, neuronal cells in the hippocampal CA1-3 areas were reduced by 30-49%. However, MVD of the hippocampus decreased rapidly from 1 week after irradiation. The cerebral MVD also declined from 1 to 12 weeks and radiation vasculopathy generally was dose and time dependent. It is possibility that radiation-induced degeneration of neuronal cells caused by same mechanism as the ischemia follow vasculature breakdown. These results furthermore suggested that high LET beam with carbon-ions have severely induce neuronal degeneration by vasculature breakdown.

Speaker: Dr Nobuhiko Takai (Pharmaceutical Sciences, Nagasaki International University)

140 Association of DNA Damage and Repair with Radiotherapy Induced Acute Skin Reactions in Breast Cancer Subjects

With improving overall survival rates, there is emerging focus on the quality of life after adjuvant cancer treatment. During radiotherapy, unacceptable normal tissue toxicity is the major limiting factor for delivering a tumoricidal dose. Therefore, it is of great importance to determine whether variations in inherent cellular radiosensitivity along with extrinsic factors have greater influence on individual differences in the normal tissue adverse reactions. Assays and parameters to predict, what severity of the skin reactions will develop after the treatment remains absolutely necessary for clinicians to make therapeutic choices for personalized therapy. Aim of the present investigation is to assess the usefulness of γH2AX assay in predicting the radiotherapy induced normal tissue response among breast cancer patients. Breast cancer patients (n=42) selected for the study were treated with radiation therapy using Linac X-ray linear accelerator. The individual radiosensitivity was determined by the microscopic scoring of γH2AX foci in peripheral lymphocytes collected prior to radiotherapy, and irradiated in vitro with 2 Gy X-rays. The DSB damage and repair capacity (as residual damage) at different repair time points (0.25, 3 and 6h) was analyzed. Acute skin adverse reactions were scored by Radiation Therapy Oncology Group (RTOG) criteria. There exists a clear interindividual variation in radiosensitivity among the subjects (CV=15.54%) with reference to their residual damage. Based on the toxicity grading criteria patients were grouped in to non-over-reactors (G0 & G1) and over-reactors (G2 & G3). These groups showed a significant difference (P=0.0282) in their residual damage levels. Linear regression analysis of initial damage, residual damage (3h & 6h) with RTOG grades revealed a significant association (R2=0.1175, P=0.0263) at 6h of post repair time point with radiotherapy induced acute skin reactions. Although, this study has limited sample size we could able to associate the residual damage with the development of acute skin reactions. With additional sample size this study may have more valid findings with clinical implications for tailoring radiotherapy. The facilities of Manipal University and the financial support from Department of Biotechnology, Government of India (BT/01/COE/06/02/07) are gratefully acknowledged.

Speaker: Prof. Satish B S Rao (Division of Radiobiology & Toxicology, Manipal Life Sciences Centre, Manipal University)

141 Variation in tumour and normal tissue radiosensitivity in patients with breast cancer as detected by the alkaline comet assay

INTRODUCTION: For high survival cancers such as breast cancer, the ability to identify radiosensitive patients in advance would allow personalized treatment to lower adverse side effects. The alkaline comet assay appears to be a highly sensitive and reproducible test to measure DNA breaks induced by ionizing radiation. To date, most studies using the comet assay to assess radiosensitivity in the normal tissue have relied on peripheral blood lymphocytes (PBL). However, it has previously been demonstrated that the comet assay is also capable of detecting instrinsic differences in radiosensitivity between breast cancer cell lines and between cells extracted from tumours of different patients with advanced breast tumours undergoing palliative treatment. METHODS: The aim of this study was to evaluate the ability of the alkaline comet assay to detect differences in DNA damage following ex vivo irradiation of normal and tumour cells from different patients with breast cancer. Patients with operable breast cancer were prospectively recruited from the local breast unit. Prior to histopathological fixation, representative samples of tumour and normal breast tissue were extracted from the surgical specimen and cells were submitted to the alkaline comet assay after gamma irradiation at 0, 2, 4, 6, and 8 Gy using standardised protocols with a Raji standardised cell line as reference. RESULTS: DNA damage in both normal and tumour cells increased with radiation dose as measured by percentage of tail DNA. The relative increases were greatest at doses of 2 and 4 Gy. Tumour cells had higher baseline levels of DNA damage than normal tissue. There was noticeable inter-patient variability in radiation response. Correlation with acute tissue toxicity after radiotherapy to the breast will be presented. CONCLUSIONS: To our knowledge, this is the first study directly assessing breast tumour and normal tissue sensitivity in a standard patient population by means of the alkaline comet assay. The results confirm the potential of the alkaline comet assay to predict tissue radiosensitivity at clinically relevant doses. If validated, it should enable clinicians to determine individual patient and tumour radiosensitivity and thus allow more personalised breast cancer treatment.

Speaker: Mr Tim Rattay (University of Leicester)

142 The role of apoptosis in the oral mucositis – experimental studies

Oral mucositis (OM) is a frequent dose-limiting side effect of radiotherapy of head-and-neck tumours. The pathophysiological basis of the response of the mucosal lining is the radiation-induced impairment of proliferation in the germinal layer of the mucosal epithelium. The contribution of apoptotic processes in both the epithelium and the associated endothelium are currently discussed controversially. Ceramides are essentially involved in apoptotic processes. Therefore, inhibition of ceramide-associated signal transduction was performed to determine the role of apoptosis in an established mouse model of OM. Irradiation was administered as single dose or fractionated treatment with 5x3 Gy/week over 1 week (days 0-4) or 2 weeks (d 0-4 and 7-11), each followed by graded test doses (d 7 or 12, respectively). Single dose or test irradiations were performed with graded doses (5 dose levels, 10 animals) in order to generate complete dose effect curves. Mucosal ulceration, corresponding to confluent mucositis grade 3 of the RTOG/EORTC classification, was analysed as the clinically relevant endpoint. Two ceramide inhibitors were tested: Desipramin, an acidic sphingomyelinase inhibitor, and Fumonisin B1, a ceramide synthase inhibitor. In combination with single dose irradiation, the drugs were administered from day -3 until first diagnosis of ulcer (D) or healing (H) of all ulcers. During 1 week of fractionation, mice were treated from day -3 until day 7, D, or H. During 2 weeks of fractionation, the drugs were applied from day -3 to day 7, 14, D or H, or only during the second week of fractionated irradiation (from d 7 to d 14). In combination with single dose and one week fractionated irradiation, neither Desipramin nor Fumonisin B1 had any significant effect on OM. In combination with fractionation over 2 weeks, treatment with Desipramin showed a significant increase of ED50 value only when administered over the longest time-period (i.e. from d -3 to H). Fumonisin B1 reduced the incidence of mucosal ulcerations in all application protocol during 2 weeks of fractionation with marginal significance. Our results indicate that inhibition of ceramide synthesis via two different pathways influenced the radiation response of oral mucosa only at certain administration protocols, which were associated with “repopulation” processes in the tissue. The findings indicate that apoptosis does not play a predominant role in the pathophysiology of radiation-induced OM.

Speaker: Dr Eva Bozsaky (Medical University Vienna, Christian Doppler Lab for Medical Radiation Oncology)

143 Follistatin is a potential mitigator of radiation induced fibrosis and a predictor for radiosensitivity

Late normal tissue side effects from cancer radiotherapy can manifest as fibrosis and severely affect quality of life. Decreasing the treatment dose to spare the normal tissues and avoid fibrosis interferes with the efficiency of tumour control. Discovery of novel treatments to avoid adverse responses to radiation would benefit radiotherapy outcomes immensely. Exon expression analysis performed in primary fibroblasts derived from cancer patients who developed fibrosis or not, has revealed that activin and its inhibitor, follistatin, are induced by IR. Activin A is a member of the TGFβ cytokine superfamily and is a potent regulator of the inflammatory response and tissue fibrosis. Follistatin binds activin A with high affinity and results in its inactivation. We have also measured intracellular and secreted follistatin in IR-treated fibroblasts over time and found that protein levels correlated well with the detected IR-induced follistatin and activin A mRNA levels. Furthermore, comparison between fibrosis patients and control samples revealed lower levels of follistatin transcripts in patients who developed severe fibrosis. Hence, the data indicate a potential role for follistatin in the radiation response and ultimately fibrosis mitigation. Further characterization of the cellular signalling and transcriptional targets of follistatin and associated factors will provide novel biological insights into the molecular pathology of fibrosis and establish potential therapeutical targets for fibrosis prevention and treatment.

Speaker: Alesia Ivashkevich (Centre for Innate Immunity and Infectious Disease, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria, 3168, Australia)

144 Ionizing radiation induces acute and chronic modifications of the transcriptional profile of endothelial cells

Normal tissue damage after radiation therapy is characterised by a chronic altered phenotype of endothelium. Molecular mechanisms involved in the initiation and the acquisition of a chronic activated phenotype of endothelial cells after radiation exposure remain unclear. The aim of this work is to characterize in vitro molecular actors involved in both the acute and late activated phenotype of endothelial cells. Human Umbilical Vein Endothelial Cells (HUVEC) were exposed to a single dose of 2 Gy, 20 Gy or a fractionated dose of 10 x 2 Gy. Expression profiling was performed using a Taqman Low density array (TLDA) approach. Gene signature (~500 genes) associated with immune response, apoptosis, angiogenesis, inflammation and protein kinase related genes was performed 0.5, 1, 2, 3, 4, 7, 14 and 21 days after irradiation. Irradiation modifies very rapidly the phenotype of endothelial cells after 2 and 20 Gy and gene expression profile analyses reveal a strong persistent altered molecular profile of HUVECs. The molecular profile of endothelial cells exposed to ionising radiation is very rapidly modified and kinetic analyses showed that HUVECs acquire a chronic pathological phenotype until 3 weeks after radiation exposure. Results showed a specific acute molecular signature at 2 and 20 Gy but also numerous mRNA expression levels modified from 12 hours and remainder modified 3 weeks after 20 Gy. The comparison of the single and the fractionated dose of 20 Gy reveal both specific molecular signatures associated with the total dose and with the regimen of the exposition. Our results confirm numerous previous data published in the literature and reveal modifications, both in acute and chronic response of the expression levels of numerous molecular actors not described until now as radiation-responsive genes. Our study suggests that our in vitro model is a useful approach to characterise molecular mechanisms involved in the initiation and the acquisition of a radiation–induced chronic pathological phenotype.

Speaker: Valérie Buard (Institute for Radiological Protection and Nuclear Safety)

145 Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays

Recent reports suggest that extranuclear targets in cytoplasm may have a role in mediating radiation effects in mammalian cells exposed to ionizing radiation (Tartier et al., 2007, Maeda et al., 2010). Mitochondria, a kind of major organelles, are distributed throughout cytoplasm. They contain their own genome, and mediate essential cell functions, such as generation of ATP and regulation of cell death. Mitochondria generate reactive oxygen species (ROS) as by-product of respiration for ATP production. If cells fail to reduce ROS, it might give the cells oxidative stress. In addition, dysfunctions of mitochondria have been known to be involved in a wide variety of diseases. Radiation effect on mitochondrial functions, however, remains to be fully elucidated. As the first step to understand the cytoplasmic effects of radiation, we have examined mitochondrial morphology in mammalian cells exposed to X-rays. Mitochondria are continuously fusing or dividing during mitosis, or in response to environmental condition changes. It is also known that mitochondrial morphology dynamically change with cell cycle progression. In this study, after irradiation of X-rays (150kVp) to human or mouse cells, we labeled mitochondria by Mitotracker Red and analyzed kinetics of mitochondrial morphology by a live-cell imaging technique using a fluorescence microscope (KEYENCE, BZ-9000) equipped with a time lapse imaging system. Cell cycle stages were identified by simultaneously staining cell nuclei with Hoechst33342. Mitochondrial images were captured every 15 or 30 minutes for 6 days after irradiation. We report the relation between the morphological change of mitochondria and radiation induced cell cycle arrest.

Speaker: Dr Miho Noguchi (Advanced Science Ressearch Center, Japan Atomic Energy Agency)

146 The effect of increasing X-ray doses on normal and malignant cell migration

A novel, synchrotron-based approach, known as microbeam radiotherapy (MRT) has the potential to revolutionise the way radiotherapy is performed. MRT uses a lattice of kilovoltage X-ray microbeamlets, with each beamlet being 10-50 microns wide with centre-to-centre separation of 100-300 microns. It is not known why normal tissues can tolerate such high doses of radiation, or why tumor response to MRT is so effective when only a fraction of their volume is irradiated by the lattice of microbeams. Our group previously showed that tumor cells showed extensive migration within 24 hours post-MRT. By contrast, in normal skin, peak dose-irradiated cells showed minimal evidence of migration up to 3.5 days post irradiation. The rapid intermixing of lethally irradiated cells with undamaged cells within the tumor may accentuate cell-mediated communication, thus providing a plausible explanation for the effectiveness of MRT despite the fact that less than half the cells in the tumor are lethally irradiated by the lattice of X-rays. The aim of the project is to investigate how cell migration is influenced by MRT and conventional radiation. We use time-lapse microscopy (live cell imaging) coupled with sophisticated image processing techniques to track individual cells within the well chamber slides. Briefly, we irradiate well chamber slides containing cultured normal and malignant cells with a range of doses using a conventional Co-60 radiation source and on the Imaging & Medical Beamline at the Australian Synchrotron in Melbourne. We cultured cells in high and low density conditions to investigate migration across wounds (confluent cells) and for quasi-isolated cells. Preliminary statistical analysis of the data suggests that while the averaged displacement of cells may not vary significantly when cells are irradiated with doses of 2, 5, or 10 Gy of conventional radiation; the higher doses caused an increase in the variance of cell displacements. Preliminary synchrotron MRT experiments have shown increasing doses causing increasing migration speed. However the synchrotron MRT experiments suffered from unknown dosimetry and more precise experiments are required. The quantitative cell migration data we will generate will provide new and novel insights into basic tumour biology and will deepen our understanding of cell migration in the presence of ionising radiation.

Speaker: Dr Jeffrey Crosbie (University of Melbourne)

147 A NEW THYROID SPECTROMETER BASED ON A LaBr3(Ce) DETECTOR FOR OCCUPATIONAL INTERNAL RADIATION EXPOSURE MONITORING

The Whole Body Monitoring Laboratory from IFIN-HH, Magurele, Romania, performs, periodically, according to its Quality Assurance Programme, in vivo monitoring of the thyroid in order to be determined the I-131 retention by gamma spectrometry. The thyroid measurements are required, strictly, from radiation protection reasons, for people involved in nuclear medicine activities from hospitals and from radiopharmaceuticals production centres where there is a potential risk of occupational internal contamination with I-131 through inhalation. The goal of the thyroid monitoring is to determine the amount of I-131 activity incorporated and to estimate the equivalent and effective doses. Reliable results suppose accurate radiation measurements which depend on the type of the in vivo thyroid monitoring system and on its correct calibration for the detection of the I-131 gamma energy lines. The retained activity in thyroid is quantified by processing the peak spectra of the main I-131 line of 364.5 keV. The current thyroid spectrometer from IFIN-HH is based on a 2”x 2” NaI(Tl) scintillation detector that is traditionally used in gamma spectrometry for the detection of photons with energies higher than 50 keV. Despite its poor resolution, the NaI(Tl) detector was considered suitable for the detection of I-131 in thyroid because of its high detection efficiency and its room temperature operation mode. Taking into account the developments on nuclear detectors based on inorganic scintillation materials, namely the Cerium-doped Lanthanum Bromide detector, manufactured by Saint-Gobain Crystals, it became challenging to test and evaluate this detector for in vivo thyroid monitoring, because of its attractive features as very good energy resolution, high gamma detection efficiency, very fast light output decay, operation at room temperature and detector small sizes. A 1.5“ x 1.5” LaBr3(Ce) detector was available to replace the NaI(Tl) detector of the thyroid gamma spectrometer, in the same measurement geometry. Background and radionuclide spectra were acquired in order to be evaluated its main characteristics for a qualitative and quantitative analysis, namely, the resolution, the efficiency and the Minimum Detectable Activity at the I-131 energy line of 364.5 keV. There were performed the energy calibration using standard gamma etalon point sources and the efficiency calibration using a thyroid phantom and Monte Carlo simulation for calibration method validation.

Speaker: Dr MIRELA ANGELA SAIZU ("Horia Hulubei" National Institute for Physics and Nuclear Engineering)

148 Effect of Internal Contamination with HTO on the Innate Anti-Tumour and Inflammatory Reactions in Mice

One of the significant sources of internal radiation exposure of workers and members of the public is tritium, a β--emitting isotope of hydrogen that binds with hydroxyl radicals to form the easily internalized tritiated water (HTO). As with other exposures to low-LET ionizing radiation deposition of HTO in the body leads to absorption of energy in the sensitive targets which, in the long run, can apparently instigate or promote tumour development. Thus, we aim in the present project granted by the Polish National Science Centre is to estimate whether internal contamination of mice with HTO modifies the development of pulmonary tumour metastases and whether this effect can be associated with alterations in the anti- or pro-neoplastic functions of macrophages and NK lymphocytes. The studies will be carried out on two strains of mice (BALB/c and C57BL/6) that differ in their sensitivity to ionizing radiation and whose pro-inflammatory and macrophage-type responses are differently expressed. Mice will be intraperitoneally injected with HTO at such activities that the total absorbed doses of radiation delivered to a mouse will be 0.01, 0.1 or 1.0 Gy; control mice will be injected with PBS. Starting on day 7 post-injection the following parameters will be assessed: cytotoxic activity of NK cells, nitric oxide production by macrophages (as a marker of the cytolytic function of these cells against susceptible tumour targets), spleen and bone marrow cellularity, leukocyte and thrombocyte counts in peripheral blood, serum levels of selected pro- and anti-inflammatory cytokines, and development of the induced tumour nodules in the lungs. Thus far, we have conducted only preliminary studies using BALB/c and C57BL/6 mice i.p. injected with HTO at total absorbed doses of 0.01 Gy (a dose encountered in some occupational settings) and 0.1 (upper limit of the low-dose region) only to find that peritoneal macrophages obtained from the two strains produced elevated amounts of NO, but in the BALB/c macrophages this effect was pronounced after internal contamination with HTO at 0.1 Gy, whereas in the C57BL/6 macrophages after contamination at 0.01 Gy. The obtained results will broaden our understanding of bio-medical outcomes of internal contamination with tritium by furnishing evidence of its anticipated pro- or anti-neoplastic and/or pro- or anti-inflammatory effects.

Speaker: Dr Ewa M. Nowosielska (Military Institute of Hygiene and Epidemiology)

149 CHANGES IN RADIOSENSITIVITY TO GAMMA-RAYS OF LYMPHOCYTES FROM HYPERTHYROID PATIENTS TREATED WITH I-131

The therapeutic application of I-131 is a widespread clinical practice for the treatment of thyroid cancer and hyperthyroidism. Although it has almost no side effects in adults, it is desirable to optimize the applied dose to each patient to maximize the clinical result with acceptable damage to healthy tissues. A previous study showed the occurrence of an adaptive response (AR) in peripheral blood lymphocytes (PBL) in a considerable fraction of patients who received this treatment, although a large inter-individual difference was observed. The aim of the present study was to assess the existence of an AR, in terms of DNA and chromosome damage, in PBL of patients undergoing I-131 therapy in view of developing personalized treatments. The study considered 18 patients (together with 10 healthy donors matched for age and gender) treated with I-131 for hyperthyroidism at the Dept. of Nuclear Medicine of Policlinico Gemelli (Rome). A combination of in vivo and ex-vivo approaches was used as follows: a) extraction of lymphocytes from patients immediately before and 1 week after I-131 administration; b) in vitro irradiation of these cells with acute doses of gamma rays (challenging doses) c) measurement of radiation-induced damage in terms of H2AX histone phosphorylation and of micronuclei (MN) induction (indicative of DNA and chromosome damage, respectively), d) evaluation, for each patient, of the occurrence of AR or sensitization by comparing the effect of the challenging dose on the samples taken before and 1 week after I-131 administration. The results obtained have shown: - a large inter-individual variability of PBL response to the challenging dose both in terms of -H2AX assay and MN induction, in contrast to more uniform responses in healthy donors; - no significant dependence of this response on age or gender; - an AR after treatment in 36% of patients, and a sensitization in 29% as evaluated by -H2AX assay, and in 39% and 22%, respectively, in terms of MN induction. - a scarce correlation between the responses in terms of DNA and chromosome damage, likely related to a large individual variability of repair capability in these patients. These findings indicate that besides the proposed “protective effect” of the radio-metabolic therapy there could be a sensitization effect, and support the need of properly assessing the individual patient’s response in developing any individually tailored treatment.

Speaker: Dr Valentina Dini (Departement of Technology and Health, Istituto Superiore di Sanita', Rome, Italy; INFN, Sez. Roma1- Gruppo Collegato Sanità, Rome, Italy)

ERRS General Assembly

Thursday, 18 October

Keynote Lecture 3

150 The pathology and the pathogenesis of late radiation-induced heart diseases after low and high radiation doses

Contrary to historical concepts of the radioresistance of organs with slow cell turnover, the heart is one of the most radiosensitive organs in the human body. The risk of death from radiation-induced heart diseases is significantly increased after high radiation doses (>40 Gy in Hodgkin’s disease), after intermediate doses (<10 Gy mean heart dose in breast cancer) and after low doses (<1 Gy in Japanese A-bomb survivors). Despite a plethora of in vivo studies performed in experimental animals (dogs, rabbits, rats and mice) during the last 40 years, neither the precise pathology, nor the pathophysiology or the pathogenesis of fatal radiation-induced heart diseases have been clarified. The wide range of radiation doses to the heart which have been proven to cause fatal radiation-induced heart diseases suggests that different pathogenic mechanisms are involved. After high radiation doses to large parts of the heart, pericarditis has been the most frequent radiation-induced heart disease in the past, yet this complication is very rare with modern treatment plans. High local doses to coronary arteries have been suggested to increase the risk of atherosclerosis and myocardial infarction, however, this could not be reproduced in rodent experiments. The most common pathology after local irradiation of the heart of rodents is progressive general and focal rarefication of the microvasculature. The different pathogenic mechanisms depend strongly on anatomical dose distributions in the heart. As long as these are not well understood, any plan based on NTCP models is unsafe. Future research in experimental animals should concentrate on in vivo and ex vivo correlations of molecular and cellular alterations with histopathological damage in the irradiated heart in relation to local dose. Experiments on cell lines in vitro are of limited value unless integrated into a comprehensive in vivo/ex vivo research programme. The most promising approach would be prospective studies in radiotherapy patients correlating anatomical dose distribution in the heart to the distribution of alterations quantified with advanced functional imaging procedures.

Speaker: Klaus-Rüdiger Trott (University College London Cancer Institute and Università degli Studi di Pavia)

Charged Particles in Medicine and Space Research

151 CNAO advances in proton and carbon ion patient treatment

CNAO, Centro Nazionale di Adroterapia Oncologica, is the first hadrontherapy facility in Italy with both proton and carbon ion (maximum energy 400 MeV/u) therapeutic beams. It is equipped with three treatment rooms with fixed horizontal and vertical beam lines with active spot scanning dose delivery system. Patient treatment with protontherapy (PT) has started in September 2011 after physical and biological beam qualification. Treatments with carbon ion radiotherapy (CIRT) will start in September 2012 after the conclusion of the physical and biological commissioning of the CNAO carbon ion beam and the definition of the beam RBE (Radiobiological Effectiveness). In vitro and in vivo experiments are currently running for the CNAO carbon ion beam qualification within the frame of national (INFN – Istituto Nazionale di Fisica Nucleare) and international (NIRS- National Institute of Radiological Sciences, Chiba, Japan) research collaborations. At present, all PT patient treatments are performed within clinical protocols specifically authorized by the Italian Ministry of Health and aiming at the Certification for the routine clinical practice. Eight protocols have been approved so far for patient PT: skull base chordoma and chondrosarcoma, spinal chordoma and chondrosarcoma, reirradiation of recurrence of spinal chordoma and chondrosarcoma, meningioma, brain carcinoma, glioblastoma, re-irradiation of head and neck cancer, and boost after photons RT in locally advanced head and neck cancer. Three new protocols have been approved for CIRT as part of the CNAO Certification process: salivary gland adenoid cystic cancer, recurrent salivary gland pleomorphic adenoma and reirradiation of recurrent rectal cancer. A wider cancer ethiology are expected to be treated at CNAO in the near future as part of the routine clinical practice for cancer patients.

Speaker: Dr Roberto Orecchia (CNAO Centro Nazionale Adroterapia Oncologica, Pavia, Italy, and European Institute of Oncology, Milano, Italy)

10:30 Coffee break

152 Charged radiation in the International Space Station: the ALTEA measurements

Radiation is a fundamental issue in preparing the near future human space exploration missions. The knowledge of the cosmic radiation field characteristics in the environments where astronauts are going to live during their interplanetary voyages is a mandatory step towards the minimization of the radiation risks below acceptable thresholds. Most recent results from radiation biology ask for a detailed knowledge of this radiation flux, discriminating charge (Z), input energy, rates (often referred to as the parameters describing the radiation quality). Measurements should therefore be able to determine the radiation environment with this detail, also to provide elements for a throughout validation of radiation models. ALTEA is a facility developed with ASI grants which includes a system of advanced active silicon detectors operating in the International Space Station (ISS) since 2006. ALTEA is the only detector in operation in the ISS, able to provide information on the radiation quality. In this talk we will show recent results achieved with ALTEA, during a 3D survey of the USLab, and the most recent tests on possible shielding materials performed in Columbus (ALTEA-shield, ESA sponsored experiment). A quite significant asymmetry of the high LET (Linear Energy Transfer) radiation due to the shielding provided by the different racks and experiments in the ISS has been demonstrated in these measurements, as well as a negligible variation of the High-Z elements during a solar particle event (December 2006 SPE). Also a significant overestimation of iron in CREME96 based models is shown. Iron is a very important ion in the radiation risk assessment, accounting for a significant percentage of the space radiation induced cancer risk, and this discrepancy must therefore be properly addressed. Previous results obtained with ALTEA and aimed at the understanding of the anomalous Light Flashes phenomenon (perception of light without light) reported by astronauts since Apollo 11, will be also briefly showed. In these experiments the particles impinging in the brain of the astronauts were measured together with their brain electrophysiological dynamics, to study the modification of the electrophysiological activity due to particle passages.

Speaker: Dr Livio Narici (Tor Vergata University, Roma, Italy)

153 Carbon ion irradiation suppresses metastasis related genes in human prostate carcinoma cells.

Hadrontherapy is a form of external radiation therapy, which uses beams of charged particles such as carbon ions. Compared to conventional X-ray therapy, the main advantage of hadrontherapy is the precise dose localization along with an increased biological effectiveness. This high ballistic accuracy allows depositing the maximal dose to the tumor, while damage to the surrounding healthy tissue is limited. First results obtained from prostate cancer patients treated with carbon ion therapy, show good local tumor control and survival rates. However, the impact of hadrontherapy on cancer metastasis is not well characterized yet. Previous studies show that hadrontherapy may inhibit metastasis by suppressing cell motility and migration. In contrast, clinical studies show evidence that X-rays might promote the metastatic potential of cancer cells. In the present study we investigated the effect of carbon and X-irradiation on changes in metastasis related genes in a human prostate adenomacarcinoma cell line, PC3. PC3 cells were irradiated with various doses (0, 0.5, 1 and 2 Gy) of accelerated 13C-ions (75 MeV/u; LET = 33.4 keV/µm) at the GANIL facility (France). A similar experiment with X-rays (Pantak HF420 RX machine; 250 keV, 15 mA; dose rate= 0,25Gy/min) was performed at SCK•CEN. RNA was extracted 2h, 8h and 24h after irradiation. So far, samples irradiated with 0, 0.5 and 2 Gy (carbon ions and X-rays) were selected for further whole genome transcriptomic analysis using micro-arrays (8h time-point). After labeling samples were hybridized to Human Gene 1.0 ST Array chips (Affymetrix). Gene expression profiles are currently being analyzed using Partek software. Our initial results demonstrate that carbon irradiation induced different effects at the level of gene expression compared to X-rays. For instance, within a set of genes related to cell motility and migration we found seven genes (APC, NEXN, MYH10, CCDC88A, ROCK1, FN1 and MYH9) with a significant fold change of < -3 after 2 Gy of carbon ion irradiation which were not affected by X-rays. Although these findings need further validation, they seem to support the abovementioned data concerning the inhibitory effects of hadrontherapy on cancer metastasis. In conclusion, this study helps gaining more insight into genes and pathways differentially regulated following exposure of cancer cells to different radiation qualities thereby leading to a better biological evaluation of various types of radiotherapy.

Speaker: Dr Marjan Moreels (Radiobiology Unit, Belgian Nuclear Research Centre, SCK•CEN, Boeretang 200, 2400 Mol, Belgium)

154 Radiobiological properties of accelerated ion beams along the Bragg curve

Radiation quality is the determinant of the radiobiological effectiveness of ionising radiation since the molecular damage at the DNA level mainly responsible for observed cytogenetic effects mirrors the pattern of energy deposition by radiation tracks. It is known that high-LET radiations, whose energy loss in the traversed matter is described by the Bragg curve, are more effective than x- or gamma rays at causing acute effects. However, despite the growing use of accelerated ion beams for cancer treatment (e.g. hadrontherapy), substantial uncertainties surround the radiobiological consequences of exposure to charged particles as far as long-term and non-cancer effects are concerned, particularly for the consequences on normal tissue at the beam entrance or in the distal part of the spread-out Bragg peak (SOBP) in therapeutic set up. Moreover, although the relative biological effectiveness (RBE) of particle radiation has been traditionally related to the radiation linear energy transfer (LET), the latter parameter has consistently proven inadequate to interpret particle radiation effectiveness. Hence, a radiobiological characterization of the Bragg curve is needed by which “biological” Bragg curves can be derived. To this purpose, we have measured the induction of lethal and sub-lethal damage in human normal and cancer cells at various positions along pristine and modulated ion beams accelerated at the superconducting cyclotron of the LNS-INFN laboratory (Catania, Italy) and at the 3-MV TTT-3 Tandem accelerator of the Department of Physical Sciences, University of Naples Federico II. In particular, 62 MeV 1H and 62 MeV/n 12 C ions beams, both monoenergetic and with a SOBP have been used at LNS, while effects near ion track end have been studied at Naples facility for 12C, 16O and 19F ion beams of up to 20.3 MeV. The endpoints studied include cell killing assayed by clonogenic survival, chromosomal aberrations as detected by FISH painting and cellular premature senescence by beta-galactosidase activity. Our preliminary results support the idea that DNA damage and cellular response vary significantly along the ion path and that track structure ought to be taken into account to better model charged particle biological effectiveness.

Speaker: Dr Lorenzo Manti (University of Naples Federico II and INFN, Section of Naples, Italy)

155 INVERSE DOSE RATE EFFECT FOR INDUCTION OF CHROMOSOMAL ABERRATIONS IN HUMAN G0 AND G1 LYMPHOCYTES AFTER PROTRACTED ОR VERY HIGH DOSE RATE NUCLEAR REACTOR RADIATION EXPOSURE

Cytogenetic studies of neutron dose rate effect were carried out at the BARS-6 pulse reactor (IPPE, Obninsk, Russia) using human lymphocytes in G0 and G1 (15 hours after PGA stimulation) stages. Blood samples in plastic tubes were irradiated in water/ice-filled flasks with unfiltered radiation (Dn/Dg=1.5-2) from two unshielded metallic active zones. Equal doses in the range 0.3-1.8 Gy were given at the exposure time of 65 microsecond (burst) or during 1 hour (protracted irradiation mode). Metaphase preparations of the 1st mitosis were conventionally stained. Aberrations recorded were dicentrics, centric and acentric rings, and excess acentric fragments. In both stages the same dependences were observed: the efficiency of the protracted irradiation was higher for induction of all types of chromosomal aberrations (CA), total CA yield, and cells with CA as compared with the burst irradiation. The temperature of irradiation (20oC or 0oC) did not affect the results. The cytogenetic efficiency difference was highest for the induction of dicentrics (1.81/G1,1.78/G0), followed by total CA yields (1.64/G1, 1.44/G0), acentric rings, and was the least for the induction of centric rings and excess acentrics (ca. 1.1).Thus, an inverse dose rate effect for the induction of CA in human lymphocytes exposed to reactor radiation at dose rates of 0.8-4.5∙cGy/min or 0.5-2.7 MGy/min was observed. The dose curves were fitted by linear equations in G0 for all CA types and by linear-quadratic equations in G1 for dicentrics and total CA. The yield of CA in lymphocytes was higher at G0 stage than at G1 for both irradiation modes which was in line with known data for neutrons. For both irradiation modes an overdispersion of chromosomal aberration distributions in cells was observed. We may speculate that accompanying gamma-radiation (35-40% of the dose) induces multiple SSBs resulting in relaxation of supercoiled DNA/chromatin making it more susceptible to neutron lesion formation and fixation. This process is, perhaps, of little importance during the short burst because chromatin has not enough time to relax. Other studies at this reactor showed that pulse irradiation was more effective - CA in mice bone marrow, concentration of malonic dialdehyde in lyposomes, nearly isoeffective - yeast and bacteria survival, albumin peroxides, thymus cell death, less effective - G(Fe) in Fricke solution, suggesting that the neutron DRE depends on the complex interplay of different mechanisms.

Speaker: Dr Vladimir Potetnya (Medical Radiological Research Center)

156 Biological Consequences of Unrepaired Clustered DNA Lesions in response to High-atomic Number and Energy Particles in Monolayer and Organotypic Cell Cultures

Clustered DNA damages induced by ionizing radiation are refractory to repair and may trigger carcinogenic events for reasons that are not understood. Here, we employ an in situ method to directly monitor induction and repair of clustered DNA lesions at the single-cell level. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages, but not the physical location of these damages within the sub-nuclear domains, determined the cellular ability to repair these damages. Importantly, examination of metaphase cells derived from HZE particles irradiated cells revealed that the extent of chromosome aberrations directly correlated with the levels of unrepaired clustered DNA lesions. In addition, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We found that, unlike simple DSBs, complex DNA lesions induced by iron particles were irreparable in organotypic 3D culture. Levels of expression of multiple DNA damage repair pathway genes were significantly reduced in the organotypic 3D culture compared with those in 2D culture providing molecular evidence for the defective DNA damage repair in organotypic culture. Further, when differentiated cells with unrepaired DNA lesions re-entered the cell cycle, they manifested a spectrum of gross-chromosomal aberrations in mitosis. Our data suggest that down-regulation of multiple DNA repair pathway genes in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis. As the organotypic 3D model mimics human lung, it opens up new experimental approaches to explore the effect of radiation in vivo and will have important implications for evaluating radiation risk on human lung carcinogenesis and cancer therapy.

Speaker: Dr Aroumougame Asaithamby (Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Texas, USA)

Radiation Carcinogenesis

157 Radiation-induced cancer in the mouse: when dose matters

Exposure to ionizing radiation produces deleterious consequences in humans, including cancer. Although quantitative radiation effects are well established at high doses, considerable uncertainties persist about radiation-induced cancer in the low-dose region. Given our current state of knowledge, the model applied by regulatory bodies is based on the evidence that DNA damage, causing mutation and cancer, increases linearly with dose, and therefore relies on a linear extrapolation of cancer risk from high to intermediate and low doses. A more complete understanding of the mechanisms of action of radiation at low doses might help define the shape of the dose-response curve, decreasing the uncertainties associated with cancer risk estimates in the low-dose region. The use of tumor data from animal studies is necessary as a complement of epidemiological studies of human populations to develop estimates of relative risk at low doses. In addition, animal experiments provide valuable insights into the mechanisms of radiation interaction with living cells and organisms, allowing clarification of the pathways of tumorigenesis and of the factors modifying radiation risks. We have employed a mouse model of radiogenic CNS cancer to assess the tumorigenic potential of exposure to low-intermediate doses of radiation. Our in vivo findings have identified a linear dose–response in the low-dose region for several biological endpoints, such as tumor-free survival, apoptosis, and tumor induction in the range of 100-500mGy. This presentation will provide examples of recent tumor data from mouse studies demonstrating important implications of radiation dose on the final tumor outcome. These data highlight the complexity underlying low-dose radiation carcinogenesis, and might contribute to more complete understanding of low-dose cancer risk.

Speaker: Dr Simona Pazzaglia (Laboratory of Radiation Biology and Biomedicine, ENEA , Rome, Italy)

10:30 Coffee break

158 Radiation biomarkers in thyroid cancer

Childhood thyroid carcinoma incidence is significantly increased after exposure to ionizing radiation. Such an increased thyroid cancer risk in children and adolescents has been observed after the Chernobyl accident in contaminated areas even at moderate or low doses of 150 mGy and below. So far, the molecular mechanisms underlying radiation-induced carcinogenesis in the thyroid gland have not been identified, yet. In order to gain understanding in this field radiation biomarkers that point to deregulated genes and pathways need to be discovered. Several studies on post-Chernobyl thyroid cancers aimed to identify radiation-specific gene expression signatures by using gene expression arrays. We have used an approach in which we integrated a genomic radiation marker that we previously identified by array CGH with mRNA expression data of the genes located in this genomic region. This allowed identifying genes that were altered in both gene and mRNA copy number. Using this approach we found an exclusive association of gain of the chromosome band 7q11.22-11.23 in papillary thyroid carcinomas (PTCs) from patients that were exposed to the Chernobyl radioiodine fallout at very young age. CLIP2, a candidate gene from this chromosomal band was specifically and exclusively overexpressed in the exposed cases at the mRNA and protein level (IHC) in tumours from exposed patients. Systems biology characterisation based on time-series mRNA expression data from cell culture models after specific perturbation of CLIP2 expression are currently carried out in order to identify the CLIP2 interactome and to gain insights into the functional impact of the gene in radiation-associated carcinogenesis of PTCs. The novel radiation markers provide first important insights into the mechanisms of radiation-related carcinogenesis of young onset PTC and underpin the concept of radiation-specific carcinogenesis. We will deepen this knowledge using new systems approaches and will integrate it with epidemiology in order to improve risk estimation for radiation-induced thyroid cancer.

Speaker: Prof. Horst Zitzelsberger (Helmholtz Zentrum München, Research Unit of Radiation Cytogenetics)

159 Kidney cancer mortality and ionizing radiations among uranium miners

Introduction: A significant excess of kidney cancer mortality risk was observed among French uranium miners, chronically exposed at ionizing radiations (IR). However this excess did not appear associated with radon exposure. This relationship is studied in German cohort, which is ten times larger than the French cohort. Method: This cohort includes 58987 uranium miners, follow-up between 1946 and 2003. 154 kidney cancer deaths occurred in this period. Kidney cancer mortality risk attributable to IR exposure is assessed with proportional hazard model with the age in time scale. Results: The mean time of follow-up was 33.8 year, with a total of 1994420 person-years. The mean age was 44.3 years and cumulative radon, gamma radiations and uranium dust exposures was respectively 241.2 WLM (Working Level Month), 40.8 mSv and 3.5 kBq.h.m-3. Kidney cancer mortality risk did not increase significantly with each cumulated exposure even after adjustment for exposure rate. Hazard Ratio obtained was for radon, 1.012/100WLM (CI95%=[0.916; 1.043]), gamma radiations, 1.000/mSv (CI95%=[ 0.998 ; 1.002]) and uranium dust, 1.003/kBq.h.m-3 (CI95%=[0.9785; 1.022]). Discussion: First analyses did not reveal significant link between kidney cancer mortality and IR exposure. Further analysis will be performed on several sub-population and other models will be tested.

Speaker: Mr Damien Drubay (IRSN/PRP-HOM/SRBE/LEPID)

160 Genetic background and 227Thorium as risk factors in biologically based models for induction of bone cancer in mice

We find evidence that the different susceptibility of BALB/C and CBA/Ca mice to bone cancer after 227Thorium injection is mostly due to different promotional responses to radiation. In BALB/C x CBA/Ca back-crossed mice we analyzed the specific contribution of two individual loci in the carcinogenic process. This analysis suggests that the two high or low risk alleles are either acting on promotion, or on the background parameters. Taken together with the comparison of CBA/Ca and BALB/C mice, this suggests that the two loci are candidates for modifying radiation-induced promotion.

Speaker: Dr Wolfgang Heidenreich (Helmholtz Zentrum München, Neuherberg, Germany)

161 Lineage tracking of the p21 promoter-activated cells to identify the cell of origin for radiation carcinogenesis

Ionizing radiation is a well-known carcinogen, but the cell of origin (target cells) is still unidentified for radiation carcinogenesis. Whatever the nature of target cells, the irradiated tissue may accumulate radiation damage causing cancer, if the damaged target cells are not excluded from the tissue through its cell death or tissue turnover. The identification of target cells hence requires a model system that can chase the fate of cells responding to radiation and their descendants within the tissue. For this aim, we have started to set up such system. Among various radiation-responsive genes, p21 (Cdkn1a) was chosen to mark the promoter-activated cells in response to radiation, considering its high signal-to-noise ratio in many tissues. The targeting construct was designed to express CreERT2 (bacteriophage P1-derived Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) in the cytoplasm upon activation of the p21 promoter, which in turn translocates into the nucleus upon administration of 4-hydroxytamoxifen (OHT). The Cre-activable floxed Rosa26 reporter mouse crossed with the p21-CreERT2 mouse expresses the reporter gene (e.g., LacZ or tdTomato) in CreERT2-expressing cells and their descendants upon p21 promoter activation and OHT administration, thereby allowing lineage tracking of the p21 promoter-activated cells and clarification of tissue turnover after irradiation. In the p21-Cre-ERT2-Rosa26 reporter mouse further crossed with the cancer-prone mouse (e.g., ApcMin/+), it can be revealed whether the resulting tumors are derived from the p21 promoter-activated cells and their descendants upon irradiation and OHT administration. This poster shall describe the current progress in this project.

Speaker: Dr Nobuyuki Hamada (Radiation Safety Research Center, Nuclear Technology Research Laboratory, Central Research Institute of Electric Power Industry (CRIEPI), Tokyo 201-8511, Japan)

12:30 Lunch

Awardees 1

162 Molecular genetic markers for assessment of individual radiosensitivity of cancer patients

Ionizing radiation (IR) is used for radiotherapy of different cancer diseases including lung cancer. This pathology extends to considerable part of working men decreasing quality of life. Modern cancer treatments include the exposure at significant dose of IR. It's focused on local body area. Dose size depends on disease severity and health status of the patient. But medical regimen is very strict, therefor individual differences between patients are obliterated. The important task is to find balance between irradiation load for organism and irradiation sufficiency for tumor destruction. Monitoring analyses give information about tumor size, degradation rate of tumor and hematopoiesis suppression. The obtaining of information is faster with usage of molecular markers. It makes it possible to decide about a continuation of the treatment and its total amount correctly. As the markers we used the ratio of nuclear and mitochondrial DNA amounts in plasma of cancer patients. Resent study have demonstrated the existent of a considerable amounts of DNA in the plasma after different type of influences on human organism including IR. The presence of one or the other DNA type may show the predominant process that causes this DNA in the plasma (nuclear or mitochondrial). We suggest the irradiation of IR during radiotherapy is prevalent. The process is the main reason of free DNA presence in plasma. Method. During the study we analyzed plasma samples from 9 patients with lung cancer. Plasma was derived from whole blood with centrifugation. DNA isolation was performed from 500 mkl. Ratio of nuclear and mitochondrial DNA copies was assessed with Real-Time PCR. Plasma DNA was used as a template. The copies number of mitochondrial gene ND1 coding first subunit of NADH-dehydrogenase was compared with the copies number of nuclear fragment of beta-actin gene. Beta-actin is highly conserved protein and its gene is usually used as a loading control. Results. The results of Real-Time PCR were obtained for moments “before” and “after 1 course” of treatment for each patient. Comparison of data revealed several reactions on radiotherapy. It was shown the increasing both mitochondrial and nuclear DNA. The degradation rate of tumor may be estimated with these data. Conclusions. There is a probability of normal tissue irradiation so we suppose the free DNA level in blood is the complex answer on genotoxic factor. That may reflect the individual radiosensitivity of the patient.

Speaker: Irina Soboleva (ITEB RAS)

163 Hsp90 Inhibition with NVP-AUY922 Protracts Tumor Growth and Prolongs Median Survival of Xenograft Tumor Bearing Mice after Radiation

In its function as an ATP-dependent molecular chaperone the heat shock protein 90 (Hsp90) facilitates the correct folding and maintenance of other proteins, commonly known as Hsp90 clients. These clients include many oncogenic proteins, as transcription factors, growth factor receptors, steroid hormone receptors and various signaling proteins. Protein stability is hampered in solid tumors, due to diffusion limited supply with nutrients and oxygen, resulting in cellular stress and a high demand for molecular chaperones. Therefore, targeting the Hsp90 chaperone function is a promising approach for radiosensitisation. This study explores the antitumor efficacy of the novel Hsp90-inhibitor NVP-AUY922 combined with ionizing radiation (IR) in in vitro and in vivo experiments. For the in vitro experiments, the human HT 1080 fibrosarcoma cell line was treated with 200 nM NVP AUY prior to IR, under normoxic or hypoxic conditions. Inhibition of Hsp90 increased radiosensitivity, regardless of the oxygen supply of tumor cells, as determined by reduction of the colony forming ability. Treatment with NVP-AUY922 caused a depletion of the relative radioresistant S-phase and an accumulation of cells with G2/M DNA content. This increase of cells in the G2/M-phase of the cell cycle prior to IR enhanced sensitivity to radiation, as determined by flow cytometrical measurement of cell cycle and DNA double-strand breaks. To generate human tumor xenografts, we inoculated HT 1080 cells in the flanks of female immunosuppressive SCID mice. When tumors reached a volume of about 100 mm³, mice were treated i.p. with 30 mg/kg NVP-AUY922 24 h prior to IR with 2 Gy. Mice weight and tumor volume was monitored 3 times weekly. In addition, control, drug-treated and/or irradiated tumors were analyzed for the expression of certain marker proteins. Treatment of tumor bearing mice with NVP-AUY922 before IR resulted in a protraction of tumor growth rates, as well as in a prolonged median survival time. Analyzing the protein expression levels by western blot and immunhistochemistry, we observed that various proteins, including p-AKT and Hsp90, are affected by Hsp90-inhibition, indicating an in vivo activity of NVP-AUY922. This preclinical study clearly demonstrates the radiosensitizing abilities of NVP-AUY922 in hypoxic and normoxic in vitro experiments as well as in human in vivo xenografts, and might have import implications for the treatment of solid tumors.

Speaker: Mr Sebastian Kuger (Department of Radiation Oncology, University Würzburg)

164 Direct and inverse dose and dose rate effects of low dose pulsed X-rays on DNA double-strand breaks repair in human cells

Effects of ionizing radiation registered in cells after low dose irradiation are still poorly understood. In several studies both direct and reverse effects of dose and dose rates observed in this dose range. Pulsed mode of irradiation is a useful way to understand not only the effects of dose and dose rate but also to distinguish between effects of dose rate within pulse and average dose rate during the time of irradiation. Thus, the aim of this study was to analyse effects of dose and frequency of pulsed X-rays on level of radiation-induced DNA double-strand breaks and their repair kinetics in human peripheral blood lymphocytes and MOLT-4 cells in vitro. Analysis of radiation-induced γH2AX and 53BP1 repair foci was used for assessment of DNA double-strand breaks (DSB) in these cells. Cells were irradiated by pulsed X-rays using custom high-power electric pulse generator Sinus-150 (current intensity 4 kA, peak energy 90-100 keV, pulse length 4 ns) with various total doses formed by change of rate within pulse and fixed number of pulses - 4000. Dose-response curve of radiation-induced foci of both proteins had shown deviations from linearity to higher effect in 12-32 mGy dose range and lower effect at 72 mGy. Number of residual radiation-induced γH2AX and 53BP1 foci at 18 h after irradiation depended on frequency of X-ray pulses: highest effect was registered at 13 pulses per second for peripheral blood lymphocytes and at 8 pulses per second for MOLT-4 cells. It suggests that frequency effect is influenced by individual cell features. For better understanding the role of average dose rate and dose rate within pulse in observed effects we exposed human peripheral blood lymphocytes to 32 and 72 mGy with various average dose rates (1.9-43.2 mGy/min) and dose rates within pulse (0.03-2.25 mGy/ns). It was shown that both effectiveness of DNA DSB repair and fraction of cells with γH2AX pan-staining used as a marker of apoptosis at 18 h after irradiation were depended on dose rate within pulse. Inverse effect of dose rate within pulse was observed in the range 0.5-1.0 mGy/ns with the total dose of 32 mGy. At lower dose rate within pulse direct effect was shown for both effectiveness of DNA DSB repair and fraction of cells with γH2AX pan-staining. This study was supported by RFBR grant 12-04-00893, Ministry of Education and Science of Russian Federation (projects P1080, 16.512.11.2063).

Speaker: Dr Stanislav Vasilyev (Institute of Medical Genetics, Tomsk, Russia)

165 NVP-AUY922 and NVP-BEP800 selectively radiosensitize tumor cell lines but not normal skin fibroblasts

In previous studies we have described the radiosensitizing potential of novel heat shock protein 90 (Hsp90) inhibitors NVP-AUY922 and NVP-BEP800 in normoxic and hypoxic conditions (Stingl et al. 2010, Djuzenova et al. 2012). However, the therapeutic potential of Hsp90 inhibitor as a radiation sensitizer will strongly depend on a selective increase in the radiosensitivity of tumor cells over normal cells. In present study we examined the impact of NVP-AUY922 and NVP-BEP800 on radiation response of two normal fibroblast cell lines HFib1 and HFib2 compared with the two established tumor cell lines (A549 and SNB19). The cell lines tested were treated with Hsp90 inhibitor (200 nM) 1 hour before IR, irradiated in the drug-containing medium and kept thereafter up to 48 h after irradiation. Thirty minutes, 24 h and 48 h after drug-IR treatment cells were plated in Petri dishes for the colony survival assay. Furthermore we determined the expression of Hsp90 and its clients, several survival (Akt, Raf-1, survivin etc.) and cell cycle associated (Cdk1, Cdk4 and pRb) proteins. Interestingly, we found that Hsp90 inhibitors NVP-AUY922 and NVP-BEP800 did not radiosensitize the both tested fibroblast lines (HFib1 and HFib2) if inhibitors are kept in culture medium for 30 min and 24 h post-IR. An extended exposure (48 h) of irradiated fibroblasts to NVP-AUY922 sensitizes them to radiation, however, to a much lesser extent than that was observed in case of tumor cells. Moreover, the prolonged incubation of normal fibroblasts with the Hsp90 inhibitors did not affect their plating efficiency indicating that Hsp90 inhibitors were not toxic to normal tissue. This conclusion can be supported by the comparison of D10 values 48h post-IR, which decreased after treatment with NVP-AUY922 from 8 to 3.9 Gy for SNB19 cells, whereas in HFib1 and HFib2 from 6.5 to ~5.5 Gy. Further finding of the study is the fact that Hsp90 inhibition and IR up-regulated the expression of Hsp90 and Hsp70, but to much lesser level than that in tumor cell lines A549 and SNB19. The expression of several other marker proteins in normal fibroblasts after combined drug-IR was mostly similar to that in tumor cell lines. To summarize, our data show that the Hsp90 inhibitors NVP-AUY922 and NVP-BEP800 can preferentially radiosensitize tumor cells, while affecting non-malignant cells to a much lesser extent.

Speaker: Ms Natalia Niewidok (Department of Radiation Oncology, University Hospital Wuerzburg)

166 Telomere length does not correlate with radioresistance in human TK6 limphoblasts

Many and differents are the proposed mechanisms leading to resistance to ionizing radiation treatment. Among them, an inverse relationship between telomere length and radioresistance has been recently advanced. Investigating such a relationship in TK6 lymphoblasts, we found that clones originating from cells survived to 4 Gy of X-rays showed a significantly higher telomere length if compared with clones grown from untreated cells. The lengthening observed was not attributable to a radiation-induced increase in telomerase activity as demonstrated by TRAP assay performed in the dose range of 1-10 Gy. Given the evidence that TK6 whole population was characterized by heterogeneity in cellular mean telomere length, we tested the hypothesis that a process of selection may favourite cells with longer telomeres (more radioresistant cells) following exposure to irradiation. In order to do it 15 independent TK6 clones were selected and characterized for telomere length on the basis of q-FISH and flow-FISH analysis. Among the screened clones four characterized by long telomeres and four characterized by short telomeres were tested for their radiosensitivity by means of clonogenic assay and D0s obtained were compared with D0 of the whole population. The results obtained showed that, in our experimental conditions (cell model, radiation doses and telomere lengths) no significant relation was observed between D0 and telomere lengths. Overall, these results led us to reject the initial hypothesis and to exclude a relevant role played by telomere length in the phenomenon of radioresistance.

Speaker: Dr Francesco Berardinelli (University of rome "roma Tre")

167 Age-related alterations in stress-response gene expression induced by chronic low-dose gamma-irradiation in Drosophila melanogaster

The purpose of this research is to investigate the role of genes of DNA breaks recognition (ATM, ATR homologues), base and nucleotide excision repair (PCNA, XPC, XPF, Gadd45 homologues) and double-strand breaks repair (BLM, Rad50, Rad51, Rad54 homologues), and apoptosis (p53, Gadd45) in formation of specific long-term effects of chronic low-dose gamma-radiation in Drosophila melanogaster. The relative expression levels were estimated in imago of Canton-S wild-type flies divided into two groups: 1) without irradiation; 2) chronically exposed by gamma-radiation at 40 cGy dose rate induced by Ra226 source during preimaginal developmental stages (10 days). The levels of expression of ATM, ATR, PCNA, XPC, XPF, BLM, Rad50, Rad51, Rad54 homologues (but not p53 and Gadd45) were 1.5-2.6-fold increased after chronic low-dose irradiation. The increase of stress-response gene expression is a possible source of cellular and organism resistance mechanisms activation via more efficient functional recognition and utilization DNA damages, one of the factors of biological effects formation under low-dose gamma-radiation exposure such as hormesis and radiation-induced response. The effect was persisted in ATM, PCNA, XPF, BLM genes during Drosophila lifetime. It is possible that higher expression levels of these genes provide the long-term effects of chronic irradiation.

Speaker: Ekaterina Plyusnina (Institute of Biology, Komi SC, UrB of RAS)

168 Effect of low doses of high-LET radiation on mice and their offspring in vivo

In the present work we investigated the influence of low doses of high-LET radiation on the cytogenetic damage in bone marrow and thymus cells and solid tumor growth in mice and their offspring, using the radiosensitivity and “adaptive response” (AR) tests. Two-month-old SHK male mice were used. All groups of animals were exposed to doses of 0.17 – 30.8 cGy (1 cGy/day) in the radiation field behind the concrete shield of the Serpukhov accelerator with 70 GeV proton energy, that simulates the spectral and component composition of radiation fields formed in the conditions of high-altitude flights. After that mice were additionally irradiated with X-radiation according to the scheme of AR: 0.1 Gy + 1.5 Gy. Adjuvants such as dibazole and calcium chloride were used as adaptogens. After 28 h, the animals of all groups were killed by the cervical dislocation. Bone marrow specimens for calculating micronuclei (MN) in polychromatic erythrocytes (PCE) were prepared by a conventional method. The weight of thymus was determined from the ratio of average weight of organ to average weight of animal in the group. The growth of solid tumor was estimated by measuring the size of the tumor at different times after inoculation of ascitic cells s.c. into the femur. It was found that: 1) high-LET irradiation of mice with all doses leads to an increase in the level of cytogenetic damage compared with the level of spontaneous lesions; the levels of PCE with MN were similar in mice irradiated with all doses; radiosensitivity to the dose of 1.5 Gy of X-radiation does not differ from those of unirradiated animals. 2) low-doses of high-LET radiation do not induce cytogenetic AR in bone marrow and thymus cells as opposite to low doses of chronic X-radiation. The combined irradiation of mice with the dose of 0.2 and 0.3 Gy and adjuvants does not induce the AR as well. 3) the mean size of the tumor in mice depends on the dose value: the dose of 0.25 cGy decreases the tumor size but the dose of 30 cGy has no influence. However irradiation of mice with this dose in the presence of adjuvants increases the tumor growth. 4) in F1 generation from males irradiated in the presence or absence of adjuvants the level of cytogenetic damage, radiosensitivity to the dose of 1.5 Gy of X-rays and ability to induction of AR in bone marrow and thymus cells do not differ from that of unirradiated mice offspring; the mean size of the tumor depends on dose value and pretreatment of mice by adjuvants.

Speaker: Ms Svetlana Sorokina (ITEB RAS)

169 Application of the FISH technique to estimate individual susceptiblity to radiation

Radiotherapy plays an important role in the treatment of many cancers. Therefore, there are many approaches and strong needs to establish the most appropriate method that would maximize efficiency of therapy and minimize the side effects and late health risk. It should also allow rapid and reliable screening of individuals and stratifications due to lower or upper radiosensitivity for modeling and optimizing a therapeutic procedure and eliminating the highest risk. The aim of this study was to compare variability in the individual susceptibility to the induction by challenging dose of the aberrations in chromosome 1 in prostate cancer patients (PC) in comparison with benign prostatic hyperplasia (BPH) persons. Whole blood samples were irradiated with a challenging dose of X-rays, cultured for 3 days, harvested in a metaphase and referred to the FISH procedure using probes for chromosome 1. Results of our studies have shown differencies in susceptibility to radiation, expressed in significantly elevated or lowered frequency of aberrations for chromosome 1 for cancer patients when compared to BPH group. After irradiation significantly higher levels of all the studied chromosome 1 aberrations, except for deletions, in the case of PC patients were revealed (% AB.C(1) 5.09 ± 1.44 vs 2.70 ± 1.21; p<0.001). Additionally, almost five times higher frequencies of acentric fragments were observed in lymphocytes of cancer patients in comparison to BPH group (on average 2.29 ± 0.96 vs 0.48 ± 0.27, p<0.001). Lymphocytes of the same patients before irradiation (in vivo level) showed significantly higher levels of all studied chromosome 1 aberrations, except for translocations. To estimate individual radiosensitivities after subtracting in vivo levels, the same trends were observed as for the cells after irradiation. In the case of PC patients significantly higher levels of all the studied chromosome 1 aberrations were observed, except for deletions with the most prominent differences for frequencies of acentric fragments (2.27 ± 1.16 vs 0.46 ± 0.24; p<0.001) in comparison to BPH group. Our results demonstrate that the frequency of acentric fragments detected as the response to a challenging treatment can be proproposed as a very efficient predictor of susceptibility to planned treatment. Limitation of analysis of acentric fragments only after challenging irradiation may greatly speed up the diagnosis.

Speaker: Dr Justyna Miszczyk (Institute of Nuclear Physics PAN, Kraków, Poland)

170 A canonical non-homologous end-joining pathway involving CtIP-dependent end-resection promotes chromosome translocation formation

Misrepair of DNA double-strand breaks (DSBs) can lead to chromosomal translocations, a driving force in carcinogenesis. DSBs induced by exogenous agents such as ionizing radiation (IR) are repaired with fast and slow components representing repair events in euchromatin and heterochromatin, respectively. The fast component involves canonical non-homologous end-joining (c-NHEJ) in G1- and G2-phase. The slow component represents homologous recombination in G2, but its nature in G1 was unclear. Here, we show that DSBs repaired with slow kinetics in G1 undergo CtIP-dependent end-resection, although the extent of resection is much more limited than in G2. This CtIP-dependent component involves the c-NHEJ factors Ku70/80, DNA-PKcs and LigIV. Thus, wild-type (wt) G1-cells repair DSBs by two c-NHEJ processes, a fast CtIP-independent and a slow CtIP-dependent process. To investigate how these two processes contribute to the formation chromosomal translocations in G1, we used premature chromosome condensation (PCC) combined with three-color fluorescence-in-situ-hybridisation (FISH). In wt cells, some translocations are formed with fast kinetics but most translocations arise with slow kinetics. CtIP depletion does not affect translocation formation of the fast process but diminishes translocation formation of the slow process. Hence, CtIP-dependent c-NHEJ contributes substantially to the formation of radiation-induced translocations which, however, can also arise by CtIP-independent c-NHEJ. Interestingly, although most DSBs and chromosome breaks (>80%) are repaired with fast kinetics, the majority of translocations form at later times with slow kinetics. This demonstrates that slow CtIP-dependent c-NHEJ is more error prone than CtIP-independent c-NHEJ.

Speaker: Ms Julia Künzel (Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany)

Awardees 2

171 DISTRIBUTION OF RADIONUCLIDES IN WATER-SOIL-PLANT SYSTEM IN DIFFERENT ZONES OF THE ARMENIAN NPP

Since 1996 we have carried out radiomonitoring researches in water-soil-plant ecosystems in zones of the Armenian NPP (ANPP) with a radius of 2-15 km and 30km (surroundings of IHP, Yerevan). ANPP is in a densely populated area of the Ararat Valley with intensive agriculture (there are 64 villages and 3 towns in 2-15km radius zone of the ANPP). The Ararat Valley is in the south-west of Armenia, 850-900m above sea level and is distinguished by hot dry climate conditions and precipitation is 300-350mm with intensive agriculture. The aim of the studies is to reveal the peculiarities of migration and accumulation of controlled artificial radionuclides (ARN, 90Sr, 137Cs) in water-soil-plant system of agrocenosis and to develop activities based on them for getting radio-ecologically safe agricultural products. Since 2011, the radiomonitoring investigations have been implemented in the context of project 11-1f262. The results of investigations have shown that natural waters, grey carbon soils and different plants in 2-15km radius zone of the ANPP exceeded the ones in 30 km radius zone with content of ARN. Here is the list of natural waters in the vicinity of the ANPP according to content of ARN: Akna Lake>Metsamor River>drinking water. The drinking water of the ANPP exceeds that of IHP with content of 90Sr 1.8 times and the water of the Metsamor River exceeds 1.4 times the water of the Hrazdan River which flows through the territory of IHP. According to data of 1996-2011 the content of 90Sr exceeded the content of 137Cs in natural waters of the three studied zones. So, according to data of 2011 90Sr exceeded 137Cs 9.4 times in artesian water of the ANPP; 9.0 times in the River Metsamor, 12.2 times in Lake Akna; 5.0 times in artesian water of IHP; 7.1 times in the River Hrazdan. The soils (0-30cm) in the vicinity of the ANPP exceed the soils of IHP with content of 90Sr and 137Cs 1.8 and 1.4 times. The distribution of ARN in both zones depends on the depth of soil layer. The maximum content of ARN is in the upper 0-10cm of the soil layer. It turned out that the plants (the grass 1.5;1.2 times, the leaves of thuja-1.7; 1.2 times, the leaves of oak- 1.5; 1.3 times) and fruits (mulberry-1.2; 1.4 times, apricot-1.4-1.6 times) grown in the vicinity of the ANPP exceeded the indices of IHP with content of 90Sr and 137Cs. In the vicinity of ANPP mulberry, apricot, the leaves of thuja and oak exceeded the same indices of the IHP with β sum activity 1.3;1.1; 1.2 times.

Speaker: Ms Ani Asatryan (Institute of Hydroponics Problems, NAS RA)

172 Whole-body irradiated Balb/c mice can be protected against experimental Francisella tularensis infection by passive transfer of immunity

Francisella tularensis (F. tularensis) is a highly virulent, intracellular pathogen. An efficient immune response is dependent on T cell-mediated immune responses and IFN-gamma production during first days after F. tularensis LVS (LVS) infection. Nevertheless, there is also an evidence that B cells, as well as antibodies, are necessary for mice to develop a resistance against primary and secondary infection by LVS The understanding of the poorly described role of humoral immunity is more than important for the effort to develop effective prophylactic procedure against the infection with Francisella virulent strains. We utilized gamma-irradiated mice model for studies of the protective role of anti-F. tularensis antibodies in order to partially eliminate cellular responses and also address the responses in immunocompromised host. The gamma-irradiation by doses greater than 3 Gy completely impairs the resistance to infection and causes a disbalance of cytokine production in mice. Mice sublethally irradiated using a 60Co irradiation in the total dose of 4 Gy did not survive an intradermal infection with 10e2 CFU of F. tularensis LVS in comparison to their unirradiated counterparts. However, the passive transfer of immune sera from LVS immunized mice protected sublethally irradiated mice against the challenge with otherwise lethal LVS infection, led to the decrease of the IL-beta, IL-4, IL-6 or TNF-alpha serum levels, increased the level of IFN-gamma and conversely, had a minimal effect on the levels of these cytokines in organ homogenates when compared to the nonimmunized counterparts. The necessity of cell-mediated immunity and cytokines for the protective effect of antibodies is still a controversial issue. Our results clearly demonstrate a significant effect of passive transfer of immunity performed by the transfer of specific anti-LVS antisera. Furthermore, our data conclude that in spite of changes in cytokine production after sublethal irradiation, specific antibodies are still able to protect the mice against lethal LVS infection. Moreover, the first evidence of combination of successful passive transfer of specific antisera and subsequent active immunization of immunocompromised animals is demonstrated. In summary, we demonstrate that B cell-mediated effector responses together with the induction of T cell-mediated immunity both play an important role and this should be taken into the account in the design of new vaccines.

Speaker: Mrs Klara KUBELKOVA (University of Defence, Faculty of Military Health Sciences, Center of Advanced Studies)

173 Infrared light induced the transgenerational genomic instability on mice

In the last decade, the phenomenon of adaptive response (AR) has attracted considerable attention of investigators. It is considered as a form of cell defense from mutagenic action of different factors. Therefore the search for adaptogens of physical and chemical nature which are able to transform the organisms to a new adapted state similarly as low doses is an actual problem. Various devices, based on the action of electromagnetic waves of the infrared subspectrum are currently used in clinical practice. Previously, in our experiments it was shown that exposure of mice to infrared light (IRL) as well as low doses of X- and γ-rays, induced AR in bone marrow cells, remained unchanged thymus weight after irradiation with a challenging dose (1.5 Gy) and decreased tumor growth rate. In the present work, we investigated the induction of AR in hemopoietic organs (bone marrow cells and thymus) and Ehrlich carcinoma growth rate on the mice offspring (F1, F2, F3) born from IRL (850 nm, 101 Hz, 22 mW/cm2) irradiated males. To induce the AR the standard scheme of radiation (0.1 Gy + 1.5 Gy) was used. The level of cytogenetic damage was assessed in bone marrow cells using a micronucleus test. The weight of the thymus was determined from the ratio of the average weight of the organ to the average weight of animals in the group. The influence of the adaptive exposures on the tumor growth was estimated by measuring the size of the tumor at different times after the inoculation of ascitic cells into the femur. Our investigation of three generations from males irradiated by IRL demonstrated that: the level of cytogenetic damages did not differ from that of unirradiated mice, the radiosensitivity decreased, the animals did not induce cytogenetic AR, the thymus weight decreased and the tumor growth rate did not differ from that of the offspring from unirradiated males. The obtained experimental data demonstrated the induction of genomic instability on three generations of mice irradiated by infrared light.

Speaker: Mrs Alsu Dyukina (Institute of Theoretical and Experimental Biophysics of RAS)

174 Neutron dosimetry by means of Electron Spin Resonance (ESR) technique

Along with the Neutron Capture Therapy (NCT) development and with the use of thermal neutrons for radiotherapeutic purposes, many efforts have been devoted to the beam characterization in order to optimize the therapy procedures. Reliable dosimetric measurements should be able to determine the various components (neutronic and photonic) of the mixed beam usually employed for therapy. We have studied the effect of the additive such as gadolinium and 10B-boric acid on the neutron sensitivity of alanine ESR dosimeters exposed to a gamma and mixed (n, gamma) field mainly composed by thermal neutrons. We have chosen both this additive nuclei because of their very high capture cross section to thermal neutrons. Furthermore, in the nuclear reaction with thermal neutrons particle, which in turn release their energy in the neighbourhood of the reaction site, are ejected. We completed our study through a Monte Carlo simulation aimed at obtaining information about the reliability of this powerful tool in predicting the response enhancement achievable with the addition of gadolinium and 10B-boric acid in alanine dosimeters. These computational values obtained through simulation are compared with the experimental results.

Speaker: Dr Maurizio Marrale (Department of Physics University of Palermo)

175 Reduction of porphyrins in aqueous solution in the presence of DNA. Pulse radiolysis studies.

Porphyrins and related tetrapyrrolic compounds have attracted interest of scientists from many areas due to their immense biological importance and their fascinating physical, chemical, and spectroscopic properties. Synthetic tetrapyrrole pigments, which selectively localize in tumor cells, can be applied in photodynamic therapy (PDT). Under certain conditions, porphyrins can also act as radiosensitizers. Thus, a reasonable combination of radiotherapy and PDT might be really promising anticancer treatment.[1] The subject of this study is reactivity of two cationic porphyrins: meso-tetrakis (4-N-methylpyridyl) porphine (TMPyP) and meso-tetrakis [4 - (trimethylammoniumyl) phenyl] porphine (TAPP) in the buffer solution of DNA. From pulse radiolysis studies we concluded that the free base porphyrins (P) are reduced to their radicals (P*-), which subsequently disproportionate to yield the phlorin and chlorin. These two main reduction products can be distinguished by their reactivity to oxygen[2] (especially in the presence of DNA). It was observed that charges on the side chains of porphyrin molecules affect site of porphyrin localization within the DNA helix. Moreover, DNA shows a strong protective effect as it partially isolates porphyrin molecules from molecular oxygen and from the hydrated electron. This work has been founded from the Polish budget for science (research project NN 204 537339, 2010 - 2013). References: 1. Luksiene, Z., Kalvelyte , A., Supino, R., 1999. On the combination of photodynamic therapy with ionizing radiation. J. Photochem. Photobiol. B: Biol. 52, 35–42. 2. Baral, S., Neta, P., Hambright, P., 1984. Spectrophotometric and kinetic studies of the radiolytic reduction of several pyridylporphyrins and their metal complexes. Radiat. Phys. Chem. 24, 245-255.

Speaker: Mr Tadeusz Strózik (Institute of Applied Radiation Chemistry, Lodz University of Technology – The Faculty of Chemistry)

176 Radiation-induced transgenerational carcinogenicity: a study with a tumour susceptible mouse model

The inheritance of an increased susceptibility to spontaneous or induced carcinogenesis after paternal irradiation has been suggested, but it remains a controversial issue, also because its mechanisms are still unknown. To further test this hypothesis, we set up experiments employing Ptch1+/- mice that are prone to developing spontaneous and radiation-induced cancer and, as such, could enhance an inherited tumour susceptibility phenotype. Wild-type male CD-1 mice were irradiated with 1 Gy X-rays and mated 42 days later with untreated Ptch1+/- females to test transgenerational effects transmitted by spermatogonial irradiation. After mating, sperm of irradiated fathers were collected for the analysis of genetic and epigenetic alterations. Half of the progeny was enrolled in a lifetime carcinogenicity study to test the transmission of increased spontaneous cancer incidence; the other half was irradiated on postnatal day 2 (P2) with 1 Gy X-rays, according to previous results showing the sensitivity of cerebellum cells at this stage for the development of radiation-induced medulloblastoma. Tumour incidence of both groups was compared with historical laboratory data obtained on the progeny of non-irradiated fathers. In parallel, the level of spontaneous and radiation-induced DNA damage and repair in P2 cerebellum cells was analyzed by comet assay in the progeny of irradiated and unirradiated fathers. In addition, the possible transmission of genomic instability was investigated in bone marrow and spleen cells by comet and micronucleus assays. Data collected so far show a nearly significant increase of the incidence of radiation-induced medulloblastoma in the progeny of irradiated fathers, whereas no difference is observed in the spontaneous tumour incidence between the progeny of irradiated and unirradiated fathers. The dose-effect relationships for DNA damage induction in P2 cerebellum cells obtained in the progeny of irradiated and unirradiated fathers did not differ. The study of DNA repair in these cells and of genomic instability in bone marrow and spleen cells is in progress. Overall, although preliminary, these data suggest that Ptch1+/- mice could represent a promising tool to investigate transgenerational genomic instability and carcinogenesis. To elucidate the molecular mechanisms of possible transgenerational carcinogenesis, tumours will be characterized for Ptch1 Loss of Heterozygosity/epigenetic silencing and gene expression profiling.

Speaker: Dr Lorena Paris (Unit of Radiation Biology and Human Health, ENEA, Rome, Italy; Department of Ecology and Biology, University of Tuscia, Viterbo, Italy)

177 Does Resveratrol affect radiation-induced chromosome damage? A multiparametric study on human lymphocytes exposed in vitro

Resveratrol (3,5,4'-trihydroxy-trans-stilbene RESV) is a phytoalexin produced by plants as a defensive response against fungal attacks. During last decades a huge amount of experimental data demonstrated that RESV exerts various beneficial effects on human health. Controversial data are present in literature about the ability of RESV to affect DNA molecule integrity both in cancer and normal cells. However, poor literature data are dealing with the protective effect exerted by RESV treatment towards DNA damage induced by exogenous and endogenous exposure of normal cells both in vivo and in vitro. We focused our attention on a putative radioprotective activity of RESV. Since controlled exposure to ionising radiation (IR) is one of the most effective treatments of cancer patients an important area of research concerns the identification of radiation modifiers/protectors that when present prior to or shortly after the irradiation exposure could reduce/minimize the normal tissue damage. In this regard RESV might be considered an interesting candidate. We investigated the ability of RESV to modulate the chromosome damage induced by IR using the Cytokinesis-Block Micronucleus (CBMN) assay and the Chromosomal Aberration (CA) test on human peripheral lymphocytes. In order to simulate the in vivo condition, blood samples obtained from healthy donors were treated with RESV and subsequently exposed to IR before mitogenic stimulation (G0 phase). While RESV by itself does not induce any DNA damage, we surprisingly found a controversial effect in combined treatment (RESV+IR): a reduction of IR-induced MN and an increase of IR-induced CA (in particular charged to dicentric chromosomes). These results suggested a deeper analysis of the influence of RESV both on DNA repair efficiency and on cell viability. The modulation by RESV of DNA repair has been analysed through Comet assay and γ-H2AX foci induction. The influence of RESV on cell viability has been assessed through apoptosis and cell growth analysis.

Speaker: Dr Emiliano Basso (Department of Biology - Roma Tre University)

178 CELLULAR AND MOLECULAR EFFECTS INDUCED BY 45 MeV C-IONS AND GAMMA RAYS IN NORMAL HUMAN PRIMARY FIBROBLASTS.

Investigation of the mechanisms underlying the biological effects induced by densely ionizing radiation has relevant implications in both radiation protection and therapy. In particular, the possible advantages of hadrontherapy with respect to conventional radiotherapy in terms of high conformal tumor treatment and sparing of healthy tissues are well known. Further improvements are limited by lack of radiobiological knowledge, particularly about the specific cellular response to the damage induced by particles of potential interest for tumor treatment. This study investigates early and late effects induced in AG01522 normal human primary fibroblasts by C-ions or γ-rays. The C-ion beam from the Superconducting Cyclotron facility at the INFN-Laboratori Nazionali del Sud (LNS, Catania, Italy) was used, with E ~ 45 MeV/u at the cell entrance, corresponding to LET (in water) ~ 49 keV/µm; γ-rays from the Cs-137 irradiator of the ISS were used as reference. Different end points have been investigated, namely: cell killing and lethal mutation, evaluated as early and delayed reproductive cell death, respectively; chromosome damage, as measured by micronuclei induction (MN) and DNA damage, in terms of H2AX phosphorylation/dephosphorylation kinetics. Linear dose-response relationships were found for cell killing and for induction of lethal mutations, with RBEs of about 1.4 for the former and of about 1.6 for the latter, indicating the presence of genomic instability that is greater in the progeny of C-ions irradiated cells. H2AX phosphorylation/dephosphorylation kinetics have shown a maximum foci number at 30 min after irradiation, higher for γ-rays than for C-ions. However, in the first 12 h the fraction of residual gamma-H2AX foci is higher for C-ions irradiated cells, indicating a lower removal rate possibly related to multiple/more complex damage along the particle track. MN induction, observed after 72 h from irradiation, was also greater for C-ions. Overall, these data indicate a more severe DNA damage induced by 45 MeV/u C-ions with respect to γ-rays, likely responsible of an increased cellular misrepair, leading to the greater observed levels of chromosome damage and, eventually, genomic instability; they give strong support to the idea that higher damage severity at molecular level, determined by the typical deposition pattern of densely ionizing radiation, is the earliest relevant factor for the more severe late effects at cellular level.

Speaker: Dr Emiliano Fratini (Centro studi e ricerche e museo storico della fisica “E. Fermi”, Rome, Italy)

15:45 Coffee break

Poster Session 3

179 Dielectric Relaxation Spectroscopy and Dynamic Light Scattering Studies of Exosomes Released from Ionizing Radiation Exposed MG-63 Osteosarcoma Spheroids

Exosomes are small membrane vesicles secreted by a variety of cell types into the extracellular environment. They have also been isolated from numerous body fluids such as plasma, serum and urine. Their presence in biological fluids suggests their involvement in diverse physiological and/or pathological events. In particular, it has been indicated that exosomes could be carriers for intercellular communications. In fact, they may shuttle lipids, proteins and mRNA or microRNA from exosome-producing cells to target cells. The cell-cell communication mediated by human cancer-derived exosomes seems to be involved in malignant progression and they are expected to play a role in the response of tumor cells to ionizing radiation (IR). Multicellular tumor spheroids are an in vitro model which mimic numerous aspects of in vivo human solid tumors. In fact, the cell-cell interactions and microenvironmental conditions make spheroids comparable to micrometastases and avascular tumors. Thus, they can be extremely useful in studying the effects of IR on cancer cells and exosomes secreted in vitro from tumor spheroids may resemble in a more realistic manner the exosomes secreted by in vivo tumors. The aim of the present study was to examine the effect of IR on the size and two electrical passive parameters of cell membrane (conductivity and permittivity) of exosomes secreted in vitro from MG-63 osteosarcoma spheroids. MG-63 spheroids were irradiated with 5 Gy and exosomes were isolated from spheroids at different times from irradiation. Dynamic light scattering technique and dielectric relaxation spectroscopy in the radiofrequency range were used to determine the size and the conductivity and permittivity of exosomes, respectively. The results obtained show that IR influence the size of exosomes. In fact, exosomes isolated from non-irradiated spheroids are larger in diameter than exosomes isolated from irradiated spheroids at all times examined. Dielectric relaxation measurements reveal that IR induce changes in both conductivity and permittivity of exosomes. Interestingly, exosomes show the same dielectric characteristics of irradiated MG-63 spheroids from which they are released. These data indicate that exosomes show a specific signature of the treatment at which tumor cells have been exposed. Exosomes could therefore represent an important tool to monitor the effects of anticancer treatments. This work is dedicated to the memory of our colleague Maria Teresa Santini.

Speaker: Dr Gabriella Rainaldi (Istituto Superiore di Sanità, Department of Hematology, Oncology and Molecular Medicine)

180 Innate Anti-tumour Reactions in Radioresistant and Radiosensitive Mice after Fractionated Low-level Irradiations with X-rays

BALB/c mice are more sensitive to ionizing radiation than C57BL/6 mice, the latter being regarded as the relatively radioresistant strain. Likewise, the incidence of cancer following absorption of medium doses of low-LET radiation is more frequent in the former compared to the latter mice. In this study we evaluated the effects of fractionated X-ray exposure of mice from the two strains on cytotoxic activities of cells involved in the innate anti-tumour defence and the development of the induced tumour colonies. NK cell-enriched splenocytes (NK cells) and peritoneal macrophages (Mφ) were collected from BALB/c and C57BL/6 mice pre-irradiated daily with X-rays at 0.01, 0.02, or 0.1 Gy (five days per week for 2 weeks; total absorbed doses of 0.1, 0.2, and 1.0 Gy, respectively). On the selected days after completion of the exposures cytotoxic activities of NK cells and Mφ and production of nitric oxide (NO) by Mφ were assayed. In addition, two hours after completion of the irradiations BALB/c or C57BL/6 mice were intravenously injected with syngeneic L1 sarcoma and Lewis Lung Carcinoma cells, respectively, and 14 days later the developed tumour colonies were counted on the surface of the lungs. NK cells collected from all the irradiated BALB/c or C57BL/6 mice demonstrated comparable up-regulation of their cytotoxic functions which were, for the most part, mediated by perforin and the Fas receptor ligand (FasL). Likewise, Mφ collected from both strains of the mice exhibited the similarly stimulated anti-tumour cytotoxicities and produced significatly more NO following the fractionated absorption of all the three total doses of X-rays. Finally, in both strains of the animals the repeated irradiations with X-rays significantly reduced the number of the induced tumour colonies in the lungs. The obtained results indicate that several low-level irradiations with X-rays comparably stimulate anti-tumour reactions in radiosensitive and radioresistant mice.

Speaker: Prof. Marek K. Janiak (Military Institute of Hygiene and Epidemiology)

181 Unraveling radon-related lung cancer mortality risks affected by smoking: a WISMUT cohort study

We have developed a technique to account for missing information on smoking habits in the quantification of lung-cancer risks related to the exposure to radon and its progeny. Such risk calculations are often based on epidemiological miner-cohort studies, but contrary to the exposures to radon, cohort-wide information on the tobacco consumption – a crucial factor in lung-cancer induction – is generally missing. As a proof of concept, we have applied our model to a large cohort of male uranium miners who were employed at the WISMUT Company in the former German Democratic Republic. The cohort comprises over 35,000 former employees among whom 461 died from lung cancer in the follow-up period 1955-1998. Most of them had been exposed to uranium ore dust, radon and its progeny, and to unknown quantities of tobacco smoke. The missing information on smoking has been partly retrieved in a case-control study. The relevant smoking parameters are assembled in so-called smoking-status spectra that are next projected onto the entire cohort using a Monte-Carlo sampling method. The randomly assigned smoking behavior serves as a proxy for the missing information and is used as an input for subsequent lung-cancer risk analysis by means of a two-mutation carcinogenesis (TMC) model. An ensemble of 200 independent smoking-projections and subsequent TMC calculations yields frequency-density spectra of the TMC-model parameters. These are found to vary not much more than a factor ~2 from their respective mean values. Based on these mean parameter values, separate lung-cancer risk calculations with respect to the exposure to radon, tobacco-smoke or a combination of both can be performed. The presented mapping technique hence enables us to unravel risks related to the delicate interplay between radon and tobacco-smoke exposure. In order to increase the statistical power of this study and to include higher radon exposures, we have also modeled an extended cohort of 58,987 former, male WISMUT employees with 3,016 lung-cancer deaths in the follow-up period 1946-2003. Preliminary results of this study seem to indicate some intriguing differences with respect to those of the smaller WISMUT cohort.

Speaker: Dr Teun van Dillen (National Institute for Public Health and the Environment (RIVM))

182 SIGNIFICANCE OF GENE P53 MUTATIVE CHANGES IN LUNG CANCER AMONG NUCLEAR WORKERS

Internal exposure to alpha-particle radiation from incorporated Pu-239 increases the risk of lung cancer. Studies including complex approaches on revelation gene alterations at different cancer stages are perspective. Tumor suppressor gene p53 is one of the most important gene regulating cellular cycle. Alterations of gene p53 play an important role in human carcinogenesis, and are observed approximately in 50 % of malignant neoplasms in human lung. In this research the results of studies on mechanisms of lung cancer in Mayak workers exposed to internal alpha-radiation from incorporated Pu-239 are presented. Biospecimens of unaltered lung tissue and cases of proliferative, pretumor, and tumor changes of lung epithelium of Mayak workers were selected. To reveal the protein altered immunohistochemical analysis was conducted using antibodies for protein p53 in cases selected. Cells with mutative protein p53 were found in all cases of proliferative, pretumor, and tumor changes of lung epithelium. Heterogeneity of protein p53 accumulation was observed in cells of lung tissue with different degree of degeneration. Based on registry, concentration of cells with mutative protein p53 increased with the degree of dedifferentiation. Maximum protein p53 accumulation was registered in tumor cells. Occurrence of cells with mutative protein p53 allowed considering mutations in gene p53 and loss of it’s main function as “a genome guard”. Studies on 5, 7, and 8 exons located in central region of gene p53 were conducted. These exons considered as high mutable because 90% of all mutations of gene p53 were revealed there. Searching for mutations in exons mentioned above was performed using TTGE and sequencing methods. In spite of the fact that protein p53 was revealed in all proliferative, pretumor cases, mutative changes were found only in 20% of them. Mutations were found in 40% of all biospecimens of tumor tissue of lung selected for the study, in which mutative protein was revealed by immunohistochemical analysis. All mutations were revealed in the central region of gene p53 (5, 7, and 8 exons). According to the data obtained, in addition to mutative alterations in gene p53 posttranscriptional events leading to mutations in protein p53, such as splicing mutations or posttranscriptional alterations of mRNA activity with microRNA participation might be observed.

Speaker: Dr Galina Rusinova (Southern Urals Biophysics Institute)

183 Gain of chromosome band 7q11 and overexpression of CLIP2 in radiation-associated papillary thyroid carcinomas

The main consequence of the Chernobyl accident has been an increase in childhood papillary thyroid carcinomas (PTC) in the contaminated areas. Consequently, we aimed to identify genomic radiation biomarkers. Within the EU-funded project GENRISK-T we analysed 80 PTC from patients that were matched for age at diagnosis, sex and residence. DNA and RNA from the tumours were provided by the Chernobyl Tissue Bank. The cohort consisted of patients exposed (born before the Chernobyl reactor accident) and not exposed (control group, born at least one year after the accident; median age 1.5 years at the time of exposure) to radioiodine fallout. Array CGH was performed to detect copy number alterations in the tumours. We found that gain of the chromosome band 7q11.22-11.23 was associated (FDR=0.035) with exposure to radioiodine fallout. 39% from the exposed group and none from the unexposed group showed the alteration. Since only a subgroup of cases in the exposed group exclusively showed gain of chromosome band 7q11, it is likely that different molecular subgroups and routes of radiation-induced carcinogenesis exist. Candidate genes (n=56) from the gained region showed enrichment of GO (Gene Ontology) terms associated with “DNA repair” (PMS2L3, PMS2L5), “response to DNA damage stimulus” (BAZ1B, PMS2L3, PMS2L5, RFC2), and “cell–cell adhesion” (CLDN3, CLDN4). The mRNA expression of the genes PMS2L11, PMS2L3, and STAG3L3 correlated with gain of 7q11.22-11.23. In particular, CLIP2 was shown to be specifically overexpressed in the radiation-exposed cases at the mRNA and protein level (IHC). In order to characterise the function of CLIP2 and its so far unknown role in radiation-induced carcinogenesis we aim to identify the functional network that interacts with the CLIP2 protein. Therefore a CLIP2 correlation network from global mRNA expression data from PTC was calculated. The resulting gene list was analysed using different biological databases. In order to reconstruct the CLIP2 interactome time-series data of global mRNA expression from an in vitro cell culture model of PTC after perturbation by siRNA knockdown of CLIP2 expression are currently carried out. This study shows that genomic radiation markers provide novel insights into the radiation-related carcinogenesis of young onset PTC. Research Unit of Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany;

Speaker: Ms Julia Hess (Research Unit of Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany)

184 Influence of the embryonic developmental stage on brain cancer-induction by ionizing radiation in Ptc1+/- mice

Cancer risk deriving from in utero exposure to ionizing radiation is an important and unresolved issue of radiation protection, as clear-cut conclusion on the estimated excess relative risk of childhood cancer has not been reached. Experimental studies conducted on sensitive models of radiation-induced pediatric tumors may be useful helping to improve mechanistic understanding and quantification of risk. The Patched1 heterozygous knockout mice (Ptc1+/-) provide a powerful model of medulloblastoma (MB), a frequent pediatric tumor that closely mimics a subset of the human disease. Irradiation of newborn Ptc1+/- mice dramatically increases the frequency and shortens the latency of MB. Origins of MB are related to development of the normal cerebellum. In mammalian embryos the cerebellum comprises two distinct germinal zones: (i) the ventricular zone, harboring multipotent stem cells that give rise to most of the neurons and glia, and (ii) the external granule layer (EGL), a secondary germinal zone of line-restricted granule cell precursors (GCPs). In the mouse, starting at embryonic day (E) 13-14, GCPs leave the rhombic lip and migrate over the cerebellar anlage to start forming an EGL (E15), where GCPs remain mitotically active until the second postnatal week. Proliferation in the EGL is regulated by Purkinje neurons via Sonic Hedgehog signaling. Although the cell of origin for human tumors has been often inferred based on expression of markers associated with particular cell types, changes in marker-expression during transformation and tumor heterogeneity with respect to expression of lineage makers may represent confounding factors. Here, we took advantage of a peculiarity of cerebellar neurogenesis, i.e., that distinct populations are born during temporal windows of specification. We therefore applied an oncogenic insult (X-rays) to embryos at E13.5, when the multipotent stem cells are specified but the EGL has not yet formed, or at E16.5, during the phase of expansion of the GCPs to form the EGL. The tumorigenic potential of irradiation in distinct pools of multipotent or fate-restricted progenitors is being evaluated. The results of these experiments will be discussed in light of their potential to shed light on MB origin, a matter of paramount importance having implication for development of targeted cellular therapies.

Speaker: Dr Mirella Tanori (Laboratory of Radiation Biology and Biomedicine, Agenzia Nazionale per le Nuove Tecnologie, l'Energia e lo Sviluppo Economico Sostenibile (ENEA) CR-Casaccia)

185 Influence of nuclear fragmentation on microdosimetry spectra from 4He, 7Li and 12C beams in water

Presently protons and 12C nuclei are successfully used for treating cancer. Other projectiles, e.g. 4He and 7Li, differ in their biological action from 12C nuclei, but still have favourable beam divergence similar to 12C and thus can be considered as new treatment options. In view of possible applications of nuclei lighter than carbon in cancer therapy, the quality of their radiation fields has to be studied with accounting for nuclear fragmentation in collisions of projectiles with nuclei in tissues. With our Monte Carlo Model for Heavy-ion therapy (MCHIT) [2,3] based on the Geant4 toolkit [1] we study propagation of 4He, 7Li and 12C nuclei with similar ranges (~ 180 mm) in a water phantom and calculate corresponding microdosimetry quantities measured with a compact Tissue Equivalent Proportional Counter (TEPC) detector. The impact of beam fragmentation on microdosimetry spectra is investigated for all projectiles, and contributions from nuclear fragments of certain charge are identified with respect to the TEPC position in the phantom. MCHIT describes well microdosimetry spectra measured with 12C beams [4]. As found, about 55% of the carbon projectiles undergo fragmentation before reaching the Bragg peak depth. However, there is still room for improvements of Geant4 models describing propagation of 7Li nuclei in water, as certain deviations from measured microdosmetry spectra [4] are found. References [1] I. Pshenichnov et al., Phys. Med. Biol. 52 (2010) 7295 [2] I. Pshenichnov et al., Nucl. Inst. Meth. Phys. Research B 266 (2008) 1094; 268 (2010) 604 [3] J. Allison et al., IEEE Transact. Nucl. Sci. 53 (2006) 270 [4] G. Martino et al., Phys. Med. Biol. 55 (2010) 3441

Speaker: Mr Lucas Burigo (Frankfurt Institute for Advanced Studies, J.-W. Goethe-University, 60438 Frankfurt am Main, Germany)

186 Proton microchannel irradiation of a three-dimensional skin model reveals reduced side effects for tumor therapy

Purpose: We investigate the use of a microchannel proton irradiation, which is a matrixwise application of a proton microbeam for tumor therapy. It minimizes the risk of normal tissue damage in radiation therapy of tumors while keeping tumor control as for conventional broad beam irradiation due to the lateral scattering of the protons on their way to the tumor. Methods: Microbeam irradiation was performed in a 3D human skin model (EpidermFT, MatTek, USA), at the microprobe SNAKE of the 14 MV Munich tandem accelerator. A counted number of 20 MeV protons were applied either homogeneously or in 10 or 50 µm wide channels separated 500 µm to compare the influence of the irradiation mode on various damage endpoints. Results: Cellular viability measured by the MTT assay revealed only small changes in tissue viability when irradiating with proton microchannels in comparison to unirradiated controls. In contrast, homogeneously irradiated tissues had significantly lower tissue viability. Levels of Interleukin-6, TGF-Beta and MMP-1, which are produced at the site of inflammation, decreased significantly faster within 14 days after microchannel irradiation compared to homogeneous proton irradiation. Chromosome aberrations, measured as micronuclei induction in keratinocytes, showed normal radiobiological effectiveness (RBE) of 1.21±0.17 for homogeneously irradiated tissues while microchannel irradiation showed an effectively reduced RBE of 0.24±0.10 and 0.41±0.10 for 10 µm and 50 µm wide channels, respectively. Conclusion: We conclude that proton microchannel irradiation produces significantly less inflammation and DNA damage than homogeneous proton irradiation. This work has been supported by the DFG Cluster of Excellence: Munich-Centre for Advanced Photonics.

Speaker: Dr Thomas Ernst Schmid (Technische Universität München, Germany)

187 Unique regulation of proteins in response to different radiation qualities and doses

In the last decade studies have shown that unique gene and protein expressions are induced in response to low doses (in the range of 10 – 100 mGy) compared to high doses (1 to 4 Gy) of ionising radiation. Studies showed that different numbers of genes or proteins are altered in response to radiation and only a small number of genes or proteins were the same for low and high doses. We aimed to investigate this in more details by irradiating cells with two different doses in the low dose region and also study if the radical scavenger DMSO could modify the protein response. CHO AA8 cells were exposed to acute doses of 10, 100 or 1000 mGy γ-rays (dose rate 0.43 Gy/min) or α-particles in the absence or presence of DMSO. The proteins were extracted from cells 3 hours after irradiation and used for protein expression analysis by two-dimensional polyacrylamide gel electrophoresis. The results presented demonstrate that unique patterns of up or down regulated protein spots are produced in response to different radiation qualities as well as doses. Work is now in progress to identify the proteins involved and to provide a better understanding of the cellular processes induced by low doses of radiation from high or low LET radiation.

Speaker: Dr Ainars Bajinskis (Stockholm University)

188 Response of an experimental prostate tumor to single and fractionated doses of photons and carbon ions

Introduction: Carbon ion radiotherapy enables the highly conformal treatment of deep-seated tumors due to the finite range and the inverted depth dose profile of charged particles. This physical advantage goes along with a higher linear energy transfer (LET) in the Bragg-peak as compared to the beam entrance region and results in an increase of the relative biological effectiveness (RBE) towards the distal edge of the depth dose curve. RBE is a complex quantity, which depends on physical parameters such as dose, LET and particle type as well as on biological properties (e.g. tissue type and biological endpoint). Although RBE-values have been published for a variety of tumor cell lines, only few in-vivo data are currently available. Here we describe the response of a highly radio-resistant prostate tumor with special focus on RBE, dose-dependence and fractionation behavior. Material & Methods: Fresh pieces from the R3327-AT1 subline of the Dunning prostate tumor were transplanted s.c. into the distal thigh of anesthetized male young adult Copenhagen rats. Tumors were treated with 1, 2 and 6 fractions of either 12C-ions or photons. The target volume was positioned in a 2 cm SOBP of carbon ions. Local tumor control (LCT), which was the preferred biological endpoint to characterize treatment response was defined as no indication of tumor recurrence within an observation time of 300 days. Results: Dose escalation studies revealed that LCT can be achieved with both, 12C and photons in the Dunning prostate tumor system. Based on complete dose-response curves, the D50-values (dose at 50% complication probability) were 32.9 ± 0.9 Gy and 75.7 ± 1.6 Gy, for 12C and photons, respectively. The respective values for split doses were 38.0 ± 2.3 Gy and 90.6 ± 2.3 Gy. The corresponding RBE-values were 2.30 ± 0.08 for single and 2.38 ± 0.16 for split doses. For the 6 fraction study a clear dose-dependence was seen, while the maximum RBE remained below 3. Transient dry and moist desquamation of the skin occurred as most prominent side effect, independently of radiation quality. Conclusion: The increased effectiveness of carbon ions relative to photons is confirmed in an experimental tumor system. Exact positioning, a clear target verification and a well defined dosimetry enable a tumor conform treatment, which lead to only marginal side effects. Additional experiments are in progress to decipher possible mechanisms underlying the described differential radiation response.

Speaker: Dr Peter Peschke (Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany)

189 The effect of fractionated carbon-ion beams to tumor metastasis

[Background and Purpose] The fractionated irradiation is standard protocol for radiotherapy including carbon-ion beam (C-ions) therapy. There are big differences between single and fractionation, for example on DNA damage repair, reoxygenation of hypoxic region, etc. It is necessary to get the results using fractionated irradiation to know the effects in clinical. The purpose of this study is to examine the effects of fractionated irradiation for metastasis using C-ions and X-rays. [Materials and Methods] A mouse osteosarcoma cells, LM8 having highly metastatic potential were used. C-ions irradiation (290 MeV/u) was performed at the center position of 6cm-SOBP, and X-rays were used as reference beam. The interval among each fraction was 24 hours, and 1 to 5 fractions were used. [in vitro] The cytotoxic effects were examined using colony formation assay, and the anti-metastatic effects were examined by the inhibition of migration and invasion activities using Boyden chamber assay and Matrigel invasion assay, respectively. [in vivo] LM8 tumors inoculated into right hind leg of mice were irradiated at 12days after inoculation. Radiosensitivity for individual cell in a tumor was examined by in vivo-in vitro assay. The metastatic effects were analyzed with the spontaneous lung metastasis model. [Results] [in vitro] After X-rays fractionation, cell survival and metastatic potentials (migration and invasion activities) were increased depend on the fraction number, and the degree was significant at low dose region. On the other hand, the enhancement was not significant after C-ions fractionation. [in vivo] The survival of cells in local tumor and the number of lung metastatic nodules were increased with the increment of the fraction number after fractionated irradiation. The degree of increase was more remarkable by X-rays than C-ions. The RBE values of C-ions were increased with fraction number on both cell killing and suppression of metastasis, and the values were larger on metastasis suppression than on cell killing. [Conclusion] It is confirmed that the anti-metastatic effects showed the dependency of the fraction number as well as cell killing effects. The degree of the dependency was more significant for X-rays compared with C-ions with high LETs than X-rays with low LETs. It was suggested that C-ions therapy was superior to photon therapy not only on tumor cell killing but also on metastasis suppression even for clinical fractionated irradiation.

Speaker: Dr Yoshitaka Matsumoto (National Institute of Radiological Sciences)

190 Large RBE and small OER in heavy-ion induced biological effects mainly depend on direct action

The contribution of indirect action mediated by OH radicals in cell killing can be estimated from the experiment using an OH radical scavenger DMSO, which suppresses indirect action without affecting direct action. Exponentially growing Chinese hamster ovary cells under oxic and hypoxic conditions were exposed to X-rays and high-LET heavy ion radiations of 15 to 480 keV/micrometer in the presence or absence of DMSO, and their survival fractions were determined using a colony formation assay. The contribution of indirect action to cell killing decreased with increasing LET under both oxic and hypoxic conditions. The contributions under hypoxic condition were lower than that under oxic condition at each LET data point. The RBE and OER were determined at a survival level of 10%. The RBE values under both oxic and hypoxic conditions increased with LET, reached a peak at around 200 keV/micrometer, and then decreased with LET. The OER value started to decrease at around 50 keV/micrometer, and became below 2 at around 90 keV/micrometer, and then reached approximately 1 or slightly higher in the very high LET region. When the RBE and the OER were estimated separately for direct action (RBE-D and OER-D) and indirect action (RBE-I and OER-I), the RBE-D under both conditions were larger than RBE-I at 90-480 keV/micrometer. OER-D was smaller than OER-I at every LET data point. Thus, the direct action by heavy-ion beams gives a remarkably large RBE and small OER for cell killing in comparison to OH radical-mediated indirect action.

Speaker: Dr Ryoichi Hirayama (NIRS/National Institute of Radiological Sciences)

191 DE.TEC.TOR. - A Torino University spin-off company

Keywords: beam monitors, hadrontherapy, multilayer ionization chamber On October 2009 DE.TEC.TOR. s.r.l. (Devices & Technologies Torino) was founded as a spin-off company of University of Torino and it is in the process of becoming a spin-off of the Italian Institute of Nuclear Physics (INFN). The business of DE.TEC.TOR. is to produce beam delivery monitors (pixel and strip ionization chambers) to measure and verify position, shape and intensity of hadron beams. The devices are placed near-patient and they provides online measures during the treatment. At now the company collaborates with INFN, Torino University, the National oncological hadrontherapy Centre CNAO in Pavia, Italy, Paul Scherrer Institute (PSI) in Villigen, Switzerland and Centre de Protontherapie (CPO) in Orsay, France. New products: MLIC: a multilayer ionization chamber has been developed, in collaboration with PSI, to measure and verify proton beam Bragg peak curves with variable modulation of beam energy. MLIC detector consists of 128 parallel plate ionization chambers stacked in series and measured simultaneously. The total water equivalent depth is about 300 mm. Beam Diagnostic Monitor: to verify inter-spill position and intensity of hadron beam. It is developed in collaboration with CNAO.

Speaker: Mr Marco Lavagno (DE.TEC.TOR. SRL (a Torino University spin-off company))

192 Dose Delivery performances with clinical carbon beams

Purpose: Centro Nazionale di Adroterapia Oncologica (CNAO) is treating patients since September 2011, with a 3D active beam delivery system, which irradiates the tumor using a large number of beam spots. The first treatments were performed with protons while the commissioning phase is ongoing with carbon ion beam. In vitro and in vivo experiments with carbon have been performed and the clinical treatments are expected to start within the next months. A detailed analysis on Dose Delivery performances with carbon and proton beams will be shown: the results in terms of difference between required spots (number of particles and positions of each spot) and delivered ones will be presented. Methods: the CNAO facility is based on a synchrotron designed to accelerate and deliver proton or carbon ion beams optimized for clinical treatments. The delivery technique is the "quasi-discrete" active scanning where dedicated magnets are used to drive a pencil beam through the target and the beam is normally not switched off during the transition between adjacent spots. The DD is composed by five ionization chambers to check the spot position and the number of particles delivered during the treatment in order to achieve a dose distribution matching the prescription. The safety of the treatment relies on an interlock system able to interrupt immediately the beam in case of failure in the delivery process. Different sets of calibration for protons and carbon ions are used to correct detector sensitivity and to manage in accurate way the beam scanning. Results: the single spot position and fluence accuracies measured with protons will be compared with those obtained in the carbon deliveries. The operational characteristics of the scanning system for different beam energies and particles are measured and compared. The safety and reliability of the system will be presented analyzing the interlocks and failures occurred. The stability of the calibration curves will be shown. Conclusions: the Dose Delivery operational results measured at CNAO during the initial clinical experience with proton indicate a good accuracy, reliability and stability of the system. The commissioning phase of carbon beam will allow verifying if the same performances with clinical carbon beams can be achieved.

Speaker: Maria Adelaide Garella (Fondazione CNAO- INFN Sez. TO)

193 CYTOGENETIC EFFECTS of (D,T) NEUTRONS LOW DOSES AND LOW DOSE RATES IN HUMAN LYMPHOCYTES

Cytogenetic studies of a new neutron device ING-031, pulsed neutron generator with sealed tube, were carried out using G0 human lymphocytes as a test-system. The generator produced (D,T) neutrons with the energy of 14 MeV at the frequency of 1-100 Hz. Samples of whole blood were irradiated in stacked up glass Carrel flasks under proton equilibrium conditions. Thus, all samples were irradiated simultaneously, the dose and dose rate depending on the generator target to blood sample distance. Experiments were carried out at two frequencies, 5 and 50 Hz, in the dose range of 0.1-1.2 Gy. Doses were measured with fission foil detectors and calculated with Monte Carlo codes. Metaphase preparations of the 1st mitosis were conventionally stained. Aberrations recorded were dicentrics, centric and acentric rings, and excess acentric fragments. Conventional fitting of experimental data points for dicentrics and total aberrations produced linear regression curves with no difference between pulsed irradiation and continuous irradiation modes (previously obtained data) as well as between 5 and 50 Hz irradiation. However, thorough point by point inspection of chromosomal aberrations (CA) yield per unit neutron dose revealed the well-known, in photon radiobiology, pattern of hypersensitivity-induced radioresistance (HS-IR) dose-effect dependence: low CA yield, then sharp rise of CA at doses of 0.12-0.15 Gy (HS) followed by a (quasi)plateau (IR) up to 0.3-0.4 Gy, and finally transition to a regular linear-quadratic dependence. This is, perhaps, the first presentation of HS-IR phenomenon in neutron cytogenetic studies. When the data obtained were arranged as the CA yield per 1 Gy versus neutron dose rate at that point, another well-known dependence was observed: inverse dose rate effect. The CA yield (per 1 Gy) increased by a factor of 2.5 with neutron dose rate decrease from 60 mGy to 0.3 mGy. Earlier the inverse dose rate effect was reported by Hill et al. for C3H 10T1/2 cells transformation following fission neutron irradiation nearly at the same dose rate range. Since RBE>1 for cell transformation, it suggests that a certain type of CA (translocations?) was involved in the both studies. To conclude, two new phenomena were observed at low doses/low dose rates of 14 MeV neutrons: zones of hypersensitivity-induced radioresistance on the initial part of dose curves for CA induction in G0 human lymphocytes, and inverse dose rate effect for the same test-effect.

Speaker: Dr Ekaterina Koryakina (Medical Radiological Research Center)

194 Radiograaff, a proton irradiation facility for radiobiological studies at a 4MV Van de Graaff accelerator

A horizontal beam facility for radiobiological experiments with medium-energy protons has been setup at the 4MV Van de Graaff accelerator of the Institut de Physique Nucléaire de Lyon. At this energy, the Relative Biological Effectiveness (RBE) of protons may amount to 2-7 [Belli 1998], platform constitutes thus a tool to study the specific effects of high-LET radiations to cells. For such macroscopic-irradiation facilities, the dose distribution over the cell samples has to be uniform with accuracy better than ±5% and a controlled dose rate (2 Gy/min for clinical interest). The range of protons in the sample has to be controlled to ensure a track-segment irradiation protocol. A homogeneous irradiation field with a suitable proton flux is obtained by means of two collimators (various sizes) and two Au-scattering foils arrangements set 62 cm apart from each other. The last arrangement is set 120 cm upstream from the irradiation area. A monitoring chamber contains a movable Faraday cup, a movable quartz beam viewer for controlling the position and the intensity of the initial incident beam, four scintillating fibers for beam monitoring during the irradiation of the cell samples, and is ended by a thin aluminized mylar window (12 µm thick) for the beam extraction in air. The set-up was simulated by the GATE v6.1 Monte-Carlo platform. The facility performance were tested with 3.5 MeV protons using a silicon PIPS detector, placed in air in the same position as the biological samples to be irradiated, for proton energy measurement, fluence-homogeneity evaluation and flux calibration. With this double scattering system, a fluence heterogeneity of ±3% over a circular field of 20 mm diameter was obtained. A preliminary biological experiment was performed to test protocols with two Human Head and Neck Squamous cell lines carcinoma with quite different radiosensitivities. Cells were irradiated at 2Gy with a dose rate of 2Gy/min. DNA Double Strand Break induction and repair, were measured by scoring the γH2AX foci. Results will be presented and discussed. [Belli 1998] “RBE-LET relationships for cell inactivation and mutation induced by low energy protons in V 79 cells: further results at the LNL facility” M. Belli et al. , International Journal of Radiation Biology 74, no. 4 (1998): 501–509.

Speaker: Ms Julie Constanzo (IPNL, Université de Lyon, Université Lyon1, CNRS/IN2P3, 4, rue Enrico Fermi, 69622 Villeurbanne Cedex, France)

195 Combined Effects of Ionizing Radiation and Nanoparticles on Radiosensitive Yeast Cells

There are several lines of experimental proof that nanoparticles (NPs) have toxicity on various organisms. In general, NPs are defined as particles having one dimension below 100 nm. It has been reported that both TiO2 and ZnO NPs had toxicity to several prokaryotic cells such as and gram-negative Escherichia coli and gram-positive Basillus subtilis. In the meanwhile, ionizing radiation induces biological damage directly as a result of deposition of energy in cells or indirectly as a result of free radicals formation and oxidative damage. DNA, protein, lipid and other biomolecules can be denatured during exposure to high energy of ionizing radiation. This study was performed to evaluate the effects of combined treatment of TiO2 or ZnO NPs with gamma-rays on the growth of a radiosensitive strain Saccharomyces cerevisiae W303-1A which contains a mutant allele of YBP1, ybp1-1, encoding four amino acid substitutions which are responsible for an increased peroxide sensitivity, and consequently for an increased radiosensitivity. The yeast cells were treated with TiO2 and ZnO NPs and 10 Gy and 30 Gy gamma rays. The relative survival rate of the yeast cells after treatment with ZnO NPs decreased with increasing concentrations of NPs, while TiO2 NPs resulted in a slight increase in cell death. The combined treatment of NPs with gamma-rays did not significantly increase the death of the yeast cells. In conclusion, TiO2 is much less toxic to eukaryotic yeast cells than ZnO, while TiO2 is more toxic than ZnO to prokaryotic cells.

Speaker: Dr Jin Kyu Kim (Korea Atomic Energy Research Institute)

196 Proteomic analysis by SILAC and 2D-DIGE reveals radiation-induced endothelial response: Four key pathways

Epidemiological data show that ionising radiation increases the risk of cardiovascular disease. The endothelium is one of the main targets of radiation-induced damage. Rapid radiation-induced alterations in the biological processes were investigated after exposure to a clinically relevant radiation dose (2.5 Gy gamma radiation). The changes in protein expression were determined using the human endothelial cell line EA.hy926 as a model. Two complementary proteomic approaches, SILAC (Stable Isotope Labelling with Amino acids in Cell culture) and 2D-DIGE (Two Dimensional Difference-in-Gel-Electrophoresis) were used. The proteomes of the endothelial cells were analysed 4 h and 24 h after irradiation. Differentially expressed proteins were identified and quantified by MALDI-TOF/TOF and LTQ Orbitrap tandem mass spectrometry. The deregulated proteins were mainly categorised in four key pathways: (i) glycolysis/ gluconeogenesis and synthesis/degradation of ketone bodies, (ii) oxidative phosphorylation, (iii) Rho-mediated cell motility and (iv) non-homologous end joining. We suggest that these alterations facilitate the repair processes needed to overcome the stress caused by irradiation and are indicative of the vascular damage leading to radiation-induced cardio- and cerebrovascular impairment.

Speaker: Mrs Arundhathi Sriharshan (Helmholtz Center Munich)

197 Preliminary results from mechanistic modelling of X-ray promoted atherosclerosis in ApoE-/- mice

Mechanistic models may help to determine how ionising radiation promotes the development of atherosclerosis. For this purpose a model has been formulated that allows for radiation to contribute to the process of atherosclerosis in several ways. The model is based on what is known from the scientific literature on plaque formation and the possible influences of radiation on this process. The current formulation has been tailored to experiments carried out at the Dutch National Cancer Institute (NKI) with ApoE-/- mice. Plaque formation in this genetically modified mouse model is considered to partly mimic the process of atherosclerosis as it occurs in humans. At NKI the carotid arteries of groups of ApoE-/- mice were exposed to 0, 2, 8 or 14 Gy X-ray doses. After approximately half a year the arterial tree was removed from these mice and examined for plaques. It was observed that the number and surface area of plaques grew with increasing dose. Moreover, exposed mice demonstrated plaques of a more inflammatory phenotype that is prone to intraplaque haemorrhage. At the Dutch National Institute for Public Health and the Environment (RIVM) mechanistic modelling was therefore aimed at adequately predicting number, size and phenotype of the observed plaques. Preliminary results from this model fitting process will be shown. The fact that possible radiation effects are included in several steps of the model helps to indicate where radiation action is required to explain the data and where it is not. This is important for the more accurate determination of radiation risk for vascular disease, but it also constitutes significant input for new, tailored experiments aimed at advancing our knowledge on the influence of radiation on atherosclerosis.

Speaker: Dr Harmen Bijwaard (RIVM - National Institute for Public Health and the Environment)

198 IMPACT OF EXTERNAL AND INTERNAL LOW-DOSE IONISING RADIATION IN BIOCHEMICAL PARAMETERS OF MALE MICE

Twenty six years after the accident of the Chernobyl nuclear power plant, some populations are still exposed to chronic low dose of 137Cs, mainly through the diet. Moreover, people can also be contaminated with low doses of external ionising radiation. The health effects of a combination of internal and external exposure to low doses of radiation are still uncertain. The aim of this study was to evaluate acute and subacute biological effects of concomitant exposure assessing biochemical urinary parameters. We used six groups of adult male mice (n=20). Two groups were exposed to an acute internal low dose of 137Cs and administrated through the drinking water at radiation doses of 4000 and 8000 Bq/Kg. The same animals were external whole body irradiated with 0.3 Gy γ-raigs. Three other groups were exposed only to internal or external radiation. Finally, one group did not receive any radiation dose and was considered the control group. To evaluate acute and subacute effects, animals (half of each group) were euthanized at 15h and 1 week post-exposure respectively. Urine samples were collected for biochemical analysis, including the determination of isoprostane as an oxidative stress marker. Lactate dehydrogenase (LDH), γ-glutamil-transferase (GGT), and N-acetil-γ-D-glucosaminidase (NAG) activities were modified in groups exposed to internal and external radiation, mainly after 1 week post-exposure. Moreover, isoprostane levels increased following concomitant exposure. The present results suggest that coexposure to internal and external low doses of radiation caused renal damage evidenced by biochemical alterations, and oxidative damage. Keywords: ionising radiation, adult mice, oxidative stress, biochemical urinary parameters

Speaker: Dr Montserrat Bellés Mateu ("Rovira i Virgili" University)

199 COGNITIVE EFFECTS IN ADULT OFFSPRING INDUCED BY INTERNAL LOW-DOSE RADIATION EXPOSURE IN UTERO

In nuclear power plants accidents, such as Chernobyl or Fukushima, fission-product radioisotopes (137Cs, 90Sr, 239Pu and 131I) are released to the atmosphere. These radioactive compounds contaminate extended field areas and groundwater. As a result of these incidents, the population are exposed to external and internal ionising radiation. The brain is highly sensitive to ionising radiation during foetal and early post-natal period. That interval corresponds to the time of rapid proliferation and neuroblast migration from proliferative zones to the cerebral cortex. In previous studies, with the children exposed in utero to Chernobyl radiation and Hiroshima’s survivors, have been shown that the foetal exposure to high doses of radiation increased the risk of mental retardation, small head size and lower IQ. However, the cognitive effects of low dose radiations have not been yet defined. The aim of this study was to investigate the impact of internal low doses of 137Cs during pregnancy on the postnatal development and behaviour of the offspring. At the GD12, 137Cs was administrated through the drinking water at radiation doses of 0, 500, 1000, 2000, 4000 and 8000 Bq/Kg. The Functional Observation Battery (FOB) was assessed 1 and 3 months after birth. To evaluate the spontaneous behavior, learning, memory capacities and anxiety behavioural tests were conducted at the age of 4 months. In general terms, exposure of pregnant females to low doses of caesium, resulted in a dose dependent slight changes in behavioral patterns. Keywords: 137Cs, internal low dose radiation, behavioural tests, mice

Speaker: Ms Daisy Lafuente ("Rovira i Virgili" University)

200 Chronic low dose rate γ-irradiation of HUVECs: a high throughput gene expression analysis

Seeing the increasing use of nuclear power, planning of long term space missions, occupational and medical exposures, knowledge about health effects related to chronic low dose exposure are of importance. Recent epidemiological studies indicate an increased risk of cardiovascular diseases at low doses, however a comprehensive understanding of the underlying biological and molecular mechanisms is still lacking. The endothelium plays a pivotal role in normal vascular functioning. Therefore, within the DoReMi Network of Excellence (Task 7.3), we have chosen to assess the effect of chronic low dose rate γ-radiation exposure on Human Umbilical Vein Endothelial Cells (HUVECs) with a particular focus, in our laboratory, on the transcriptomic level by means of whole genome microarray analysis. The set-up of the irradiation experiments (performed in triplicate) is shown in the table: week 1 week 3 week 6 0 mGy/h 0 Gy 0 Gy 0 Gy 1.4 mGy/h 0.24 Gy 0.71 Gy 1.41 Gy 4.1 mGy/h 0.96 Gy 2.07 Gy 4.13 Gy Microarrays were performed in biological triplicates using the Affymetrix Human Gene 1.0 ST Arrays. Analysis of the obtained data was performed using the Partek Genomics Suite v6.5. Single gene analysis showed that the number of differentially expressed genes (P < 0.05 and FC > 1.5; < -1.5) between control and irradiated cells was in general quite small at all weeks suggesting that low dose rate exposure induces a rather subtle response. Therefore, Gene Set Enrichment Analysis (based on Kegg and Reactome databases) was used to see if predefined gene sets, instead of individual genes, were significantly enriched (FDR < 0.05). In general it was observed that after one week, both dose rates activate a broad range of cellular pathways including DNA repair, cell cycle and gene translation and transcription. Also, many gene sets related to cellular metabolism were significantly enriched after one week of exposure. Interestingly, after three weeks, enrichment of gene sets involved in inflammation and immune response was observed in the irradiated cells. After six weeks only a small number of gene sets were enriched in the irradiated compared to control cells. The complete data analysis has revealed interesting pathways that are specifically involved in the chronic low dose rate radiation response of HUVEC. Acknowledgements: This project is supported by the EU FP7 DoReMi Network of Excellence (grant agreement: 249689).

Speaker: Dr Louis de Saint-Georges (SCK-CEN)

201 Detection of acute subclinical cardiotoxicity after radiation exposure.

Radiotherapy (RTH) has a key role in breast cancer (BC) treatment. Studies have shown BC survivors have an excess risk of significant cardiovascular (CV) morbidity 10 years post-treatment, with similar features of an older unirradiated population. One of the underlying mechanisms is thought to be premature CV ageing. We aim to develop a novel biomarker to detect radiation exposure and cardiotoxicity post-RTH. We have studied whether acute subclinical cardiotoxicity can be detected by cardiac MRI, and if this correlates with in vitro radiosensitivity (RS) and biological ageing (BA) assays; (TRF length, comet assay, Sub G1 peak, genomic methylation) in peripheral blood lymphocytes (PBLs) of 10 left sided BC patients, at pre-RTH, six weeks and one year post-RTH. Cardiac MRI analysis showed no definitive acute or subacute changes post-RTH. The following changes were observed six weeks post-RTH; a reduction in the in vitro AR (P = 0.002) to irradiation, increase in comet tail DNA (P=0.033), increased levels of urinary 8-oxo-DG (P=0.031), no significant changes were found in genomic methylation or telomere length. When these assays were repeated one year post-RTH, they had returned to pre-treatment levels, suggesting no long term BA effect. To investigate whether the acute BA observed was due to a change in the PBL subpopulation or a possible bystander effect, these assays were repeated in a larger cohort (n=23). We successfully replicated the results of RS and BA assays. There was a significant lymphopenia six weeks post-RTH (P=0.008) however, no significant change in the overall distribution of the PBL subsets was found. Cytotoxic T cells correlated with apoptotic response (P=0.003) but with no other measure of RS or BA. MiRNA29c has been shown to be a novel biomarker of an unfit heart in humans and is involved in the modulation of apoptosis. We have carried out mouse models showing that cardiac tissue specific MiRNA29c levels are downregulated post-RTH in cardiac tissue (P=0.046) and plasma (P=0.02). We have shown that this reduction in MiRNA29c can also be detected in left-sided BCPs six weeks after radiotherapy commencement (P=0.002). These findings are important for the radiation community as the timing of in vitro RS and BA assays are key determinants of in vitro PBL radiosensitivity as there is a characteristic uniform response at six weeks which resolves within a year and miRNA29c may be an early indicator of cardiac tissue exposure to radiation.

Speaker: Ms Hussain Shahana (Department of Genetics, University of Leicester, Leicester LE1 7RH, UK)

202 Low-dose Irradiation Induced Mitochondrial Alterations in the Brain of Young Mice

Background: Central nervous system toxicity after high doses of irradiation is well documented. The biological effects of low doses are far less known. Accumulating data show, that low-dose irradiation might increase the incidence of ischemic cardiovascular diseases and the mechanisms by which this occurs is under intense investigation. However, much less attention is paid to low-dose radiation effects on the microvasculature of the brain and its microenvironment, although functional and/or morphological damage in these structures might be in great part responsible for the late non-ischemical neurological alterations after radiation. In our work we proposed to study low-dose irradiation induced mitochondrial damage in the whole brain and in the endothelial cells of the brain microvasculature. Methods: Since developing brain might be more susceptible to low-dose induced long-term alterations, we investigated 10-day old C57BL/6 mice. Animals were irradiated on their head with single doses of 0.1 or 2 Gy X-rays. 1, 3, 7 days and 1 month after irradiation mice were sacrificed, and single cell suspensions of the various brain regions made. Half of the brains were processed for biochemical measurements of enzyme activities of the respiratory chain in the mitochondrial membrane. The other halves were stained with Rhodamine 123 to evaluate alterations in the mitochondrial membrane potential and with Mitosox Red to determine the level of superoxide anions in the mitochondrial membrane. By costaining the cells with the endothelial specific marker CD31, we studied mitochondrial damage within the endothelial cells as well. Results: A reduction of cytochrome C oxidase activity was detected after irradiation with 2 Gy, while 0.1 Gy had only moderate effect compared to control. The other enzymes in the respiratory chain are under investigation. A moderate, dose-dependent increase was seen in the level of mitochondrial superoxide as well as in the level of mitochondrial membrane potential damage 3 days after irradiation, which normalized after 1 week. Mitochondrial damage in the CD31+ endothelial cells seems to be consistently higher than that found in the unfractionated brain cell suspension. Conclusions: Low-dose local irradiation induces moderate but detectable mitochondrial alterations both in the brain and in the endothelial cells of the microvascular compartment of the brain. This work was supported by the FP7 – CEREBRAD project.

Speaker: Katalin Lumniczky (National Research Institute for Radiobiology and Radiohygiene, Budapest, Hungary)

203 The tumor suppressor gene Patched1 protects the mouse lens against spontaneous and radiation-induced cataract

Cataract (the opacification of the ocular lens) is the most frequent cause of blindness worldwide. The eye is well known to be one of the most radiosensitive tissues in the body and is clearly recognized that cataracts can be induced by high doses (>2 Gy) of ionizing radiation. Although much work has been carried out in this area, the exact mechanisms of radiation cataractogenesis are still not fully understood. In particular, very urgent is to resolve the question of threshold dose for cataract development, because recent epidemiological data indicate that cataracts are also observed in populations exposed to lower doses. Thus, the establishment of an animal model of radiation cataract will help to elucidate the pathology underlying human cataractogenesis and to obtain more evidence concerning the existence of a threshold. The Patched heterozygous mice represent a significant radiation-induced cataract mouse model developing early lens opacity (4 weeks post-irradiation with a single dose of X-rays) with an incidence of 30%. We took advantage from the deep knowledge of this mouse model to investigate on the mechanisms responsible of cataract development with particular attention to other possible risk factors that cooperate to the etiology of this pathogenesis such as the age sensitivity to the radiation exposure and the gender dependence.

Speaker: Dr Mariateresa Mancuso (ENEA CR Casaccia)

204 Radioprotective and radiomitigative efficacies of CBLB502 are mediated through granulocyte-colony stimulating factor and interleukin 6.

It has been demonstrated previously that Salmonella flagellin derivative CBLB502 may serve as an excellent radiation countermeasure if administered in a protective regimen. Flagellin/CBLB502-elicited radioprotection required TLR5 and the TLR signaling adaptor MyD88. Radioprotection was, at least in part, mediated via direct effects CBLB502 on cells in bone marrow. It was well documented that flagellin is a particularly potent activator of proinflammatory mediators, mostly cytokines and chemokines, that induce immune cell trafficking and activation, including the recruitment of neutrophils and dendritic cells. It was observed that radiosensitive and radioresistant cells played distinct roles in the innate response to flagellin giving rise to different sets of cytokines. The purpose of this study was to elucidate the role of granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6) induced by CBLB502 in protection and mitigation of radiation injury. C57BL/6 mice were injected with different doses of CBLB502 either before (-30 min) or after (+24h) lethal (9.5 Gy) total body irradiation (TBI) and survival over 30 days was monitored. Two routes of administration – intravenous and subcutaneous – were evaluated evaluated for both prophylactic and treatment schedules. The levels of cytokine in serum for both schedules were determined as well. Here we demonstrated that the levels of G-CSF and IL-6 after CBLB502 administration reflect CBLB502 radioprotective and mitigative efficacy and may serve as biomarkers. The role of these two cytokines was confirmed using neutralizing antibodies during treatment of acute radiation syndrome (ARS) by CBLB502 administration after lethal TBI. Using the approach of generating bone marrow irradiation chimeras we also demonstrated that functional TLR5 expression in both nonhemopoietic (radioresistant) and the hemopoietic (radiosensitive) compartments is necessary to achieve CBLB502 radioprotection.

Speaker: Dr Alexander Shakhov (Cleveland BioLabs, Inc.)

205 Effects of post-irradiation administration of IB-MECA, an adenosine A3 receptor agonist, on hematopoiesis and survival of sublethally or lethally irradiated mice

IB-MECA, an adenosine receptor agonist selective for adenosine A3 receptors, was tested from the point of view of its abilities to influence hematopoiesis in sublethally gamma-irradiated mice and survival of lethally irradiated ones. The drug was administered in therapeutic (post-irradiation) treatment regimens. After a sublethal radiation dose of 4 Gy, IB-MECA was found to stimulate significantly important hematopoietic parameters of the bone marrow progenitor and precursor cells when given i.p. in two doses on days 1 and 2 after irradiation. These hematopoiesis-stimulating effects of IB-MECA manifested itself when the agonist was administered alone. as well as when given concommitantly with granulocyte colony-stimulating factor or an inhibitor of cyclooxygenase-2, meloxicam. However, following an exposure to lethal doses of gamma-rays, no modulation of survival of the experimental mice by IB-MECA was recoreded, even when various treatment regimens comprising both intraperitoneal and peroral administration routes were attempted. It follows from the findings that IB-MECA acts as a stimulator of hematopoiesis and, as such, it is maximally effective when administered after sublethal doses of radiation inducing the bone marrow radiation syndrome. After lethal radiation doses, seriously suppressing hematopoiesis and damaging significantly also other organismal systems, like the gastrointestinal one, IB-MECA is, consequently, not efficacious. Since, nevertheless, there are no signs of undesirable side effects of the treatment with IB-MECA in irradiated mice, activation of adenosine A3 receptors may be considered as a usable component in the spetrum of pharmacoloical means potentially utilizable in the treatment of the acute radiation disease. The work was supported by the Ministry of Defence of the Czech Republic (project Receptor, grant no. 1001 8 5090) and by the Grant Agency of the Czech Republic (grant numbers 305/08/0158 and P303/11/0128).

Speaker: Dr Michal Hofer (Laboratory of Experimental Hematology, Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic)

206 Effect of radiotherapy conditions on biological response of normal and cancer cells

Jacek Rogoliński1, Maria Konopacka1, Aleksandra Rusin 1, Krzysztof Ślosarek2 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland 1Center for Translational Research and Molecular Biology of Cancer 2 Department of Radiotherapy and Brachytherapy Treatment Planning Cancer radiotherapy regimens use radiation of varying dose rates (100 – 600 MU/min). We tested whether irradiation of cell cultures at the same dose but different dose rate, at different medium depths, in the beam axis or off the irradiation field will generate the same extent of damaged cells. The study was carried out using two cancer cell lines (A549 and HCT116) and one normal line (BEAS-2B). As a radiation source Cliniac 2300 accelerator was used, delivering photon radiation (6 MV). 5 Gy dose was used (at 100 and 600 MU/min dose rate); cells were placed in a water phantom at two depths (3 or 15 cm), either within or outside of the irradiation field. Biological damage was assessed as: micronuclei frequency, apoptosis induction, cell survival and cell senescence. Dose rate: The radiation, at the same dose, when delivered at a lower dose rate, induces a higher degree of biological damage than radiation of greater dose rate. This relationship is observed only within the beam field. Depth: At a greater depth more cytogenetic damage is observed for the same dose, as compared to smaller depths. Positioning: Cells placed outside of the irradiation field are damaged to the same extent irrespective of depth and dose rate. Cell survival: Cell survival determined by MTS assay after 48h elapsed from irradiation did not differ between treatments (cells placed at different depths: 3cm or 15 cm in a water phantom and irradiated with different dose rates: 100 Gy/min or 600 Gy/min) and was altered as compared to untreated control. Type of cells: These observations pertain to neoplastic and normal cell types. It was found that biological response of cells for the same dose depends on various exposure conditions of radiotherapy (dose rate, depth of medium, positioning with respect to axis). The observations presented herein can be used in the future for radiotherapy planning.

Speaker: Dr Jacek Rogolinski (Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology)

207 Preferential radioprotection of normal cell DNA by antioxidants.

In the presented study, electron radiation (22 MeV) dose distribution in a water phantom was compared with biological effects (formation of micronuclei and induction of apoptosis) in irradiated cells. The influence of antioxidants on radiation – induced genetic damage during radiation therapy was also tested. Experiments were performed using normal bronchial epithelial cells (BEAS-2B) and lung cancer cells (A549). Formation of micronuclei and apoptotic-like bodies were evaluated using micronucleus test. Measurements were performed for different phantom depths (3-16 cm). Irradiated cells were placed in the beam axis or outside the radiation field. We studied also the modulating effects of vitamin C, vitamin E and ferulic acid on clastogenic activity of ionizing radiation in cancer cells located in a beam and receiving a 5 Gy dose, and normal cells located outside of the radiation field and receiving 0.2 Gy of scattered radiation. Results indicate the discrepancy between the distribution of physical dose at different depths of the water phantom and biological effects. It is of special meaning in case of irradiation at bigger depths or placed outside the field during the exposure. Treatment with either ferulic acid or vitamin E reduced micronuclei frequency in normal cells irradiated outside the beam, while at the same time increased micronuclei frequency in directly irradiated cancer ones. The effect of vitamin C was concentration-dependent and did not vary between normal and cancer cells. The use of vitamin E and ferulic acid may augment the efficacy of radiation therapy by enhancing the response of cancer cells to the radiation and simultaneously these substances can protect normal cells exposed to low dose of scattered radiation outside the radiation field during radiotherapy. Our results suggest that healthy cells exposed outside the radiation field during radiotherapy can be damaged to a greater extent than can be predicted from the dosimetric curve of absorbed dose. The protection of normal cells against these distant effects appears to be an important element of radiotherapy.

Speaker: Dr Maria Konopacka (Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Center for Translational Research and Molecular Biology of Cancer)

208 Hydrogen peroxide enhances radiation-induced apoptosis in PC-3 human prostate cancer cell line under hypoxic conditions

Purpose: We previously reported that hydrogen peroxide (H2O2) strongly enhanced radiation-induced apoptosis in PC-3 human prostate cancer cell line, that this apoptosis was lysosome dependent, and that mitochondria existed downstream of lysosomes in the apoptotic pathway (IJROBP75: 449-454, 2009). In this study, we studied how H2O2 affected radiation-induced apoptosis in the PC-3 human prostate cancer cell line under hypoxic conditions. Materials and Methods: PC-3 cells were maintained in a humidified incubator at 37 degrees C under either hypoxic conditions (1% O2) or normoxic conditions (21% O2). Subsequently, the PC-3 cells were exposed to 0.1 mM H2O2 just before the irradiation of 10 Gy, which were administered with 10-MV X-rays. The percentage of apoptotic cells was determined by flow cytometry. Detection of apoptotic cells, ROS production, and morphemic changes of lysosomes and mitochondria was examined using a CCD camera-equipped fluorescence microscope. Results: Also under hypoxic conditions, H2O2 enhanced radiation-induced apoptosis in the PC-3 human prostate cancer cell line. In addition, the proportions of apoptotic cells with X-rays of 10 Gy and 0.1 mM H2O2 under hypoxic conditions were higher than the proportions of apoptotic cells only with X-rays of 10 Gy under normoxic conditions. As is the case with combination treatment of X-rays and H2O2 under normoxic conditions, long term production of ROS, lysosomal rupturing, and mitochondrial fragmentation were observed under hypoxic conditions. Immediately after administration of H2O2 into the medium, O2 partial pressure of the medium in which PC-3 cells were cultured under hypoxic conditions increased. However, the average O2 partial pressure increase in the medium after 7 minutes of the administration of 0.1 mM H2O2 was a mere 3.6 mmHg. Meanwhile, the activation of glutathione peroxidase in the PC-3 cells under hypoxic conditions decreased. Conclusion: H2O2 decreased the activation of glutathione peroxidase in the hypoxic PC-3 cells and may have led to prevent the removal of the hydroxyradical generated by irradiation, ruptured lysosome, etc.. In conclusion, it was suggested that these phenomena were some of the reasons why H2O2 enhanced radiation-induced apoptosis in the PC-3 human prostate cancer cell line under hypoxic conditions.

Speaker: Dr Shinji Kariya (Department of Diagnostic Radiology and Radiation Oncology, Kochi Medical School)

209 Difference in the Radioresistance of DU145 and PC3 Prostate Cancer Cells: Role of Reactive Oxygen Species, GSH and Nrf2

Understanding about the genes which govern tumor radiosensitivity, can help in better selection of patients for radiotherapy protocols, predicting prognosis, and perhaps in decreasing therapy-related side effects. In order to study the molecular determinants of tumor radiosensitivity, we have chosen two prostate tumor cells namely, PC-3 and DU145. These two tumor cell lines showed significant difference in radiosensitivity between them as reflected in clonogenic survival assay and neutral comet assay. Among the two, PC3 cells were found to be more radiosensitive (SF8 = 0.02) than that of DU145 cells (SF8 = 0.06). As these tumor cells have arisen from same tissue background, but exhibits difference in radiosensitivity, they will be ideal to study the differences at molecular level, which can be correlated with their radiosensitivity. When we measured the level of reactive oxygen species (ROS), both basal and inducible levels of ROS seen in PC3 cells were higher than that of DU145 cells. In mitochondrial ROS status also similar trend was observed. DU145 cells also showed significantly high level of basal GSH than that of PC3 cells. The ratio between GSH and GSSG was also higher in DU145 than PC3 cells. Since difference in the ROS and the antioxidant levels observed in these two cell lines, we investigated the role of redox sensitive transcription factors like NRF2 and Nf-κB and its dependent genes in determining the differences in radiosensitivity of these tumor cell lines. The level of Nrf2, Nrf2 dependent genes, and Nf-κB was analysed after irradiation, using real time PCR and electrophoretic mobility shift assay. Basal as well as inducible levels of these genes in DU145 were significantly higher than the PC3 cells. To prove this point further, we have checked the survival fraction of these tumor cells in the presence of inhibitors of Nrf2 and HO1. Presence of these inhibitors significantly reduced the survival fraction of these tumor cells against the radiation exposure further suggesting role of Nrf2 and associated genes in determining the radiosensitivity. This reduction in survival fraction was drastic in radioresistant DU145 cells than that of PC3 cells. This was further confirmed by knocking down the Nrf2 and HO1 expression and then checking the survival fraction. To conclude, the role of Nrf2 and its dependent genes in determining the radioresistance has been established in prostate cancer cells.

Speaker: Mr Sundarraj Jayakumar (Radiation Biology and Health Sciences Division, Bhbha Atomic Research Centre, Mumbai, India)

210 Phosphorylation of threonine 190 is essential for nuclear localization and endocytosis of the FTS (Fused Toes Homolog) protein

Fused Toes Homolog (FTS) is a member of a group of proteins termed as E2 variants and this group of proteins lacks an active cysteine residue that is required for ubiquitin transfer. We have identified the expression of this protein in early stages of cervical cancer and its translocation into nucleus from cytoplasm upon irradiation. Also nuclear localization of FTS upon irradiation is related with radiation resistance. Here we present that a threonine residue at position 190 is essential for its nucleocytoplasmic shuttling and function. Upon LMB treatment we found that FTS was located in the nucleus and it suggests that direct role of nuclear export signal (NES) is required for the binding to CRM1 and facilitates nuclear export. The threonine residue was phosphorylated and promoted the phosphorylation of EGFR, p38 and JNK facilitating vesicular trafficking of early to late endosomes. Mutational change of the threonine into alanine resulted in the cytoplasmic localization of FTS and failed to phosphorylate EGFR and its downstream effector proteins. In addition the mutation also reduced the number of early endosomes formed and also resulted in the clustering of late endosomes around the perinuclear region. These data suggest that threonine residue of FTS at position 190 is not only essential for its function but also for the formation, maturation and trafficking of early endosomes to late endosome/Lysosome, as well as we speculate that FTS may function at a connection point in the vesicle tethering.

Speaker: Prof. Woo-Yoon Park (Chungbuk National University, Cheongju, Korea)

211 Nicotynic Acid Derivatives as New Potential Radio-Protective and Radio-Remedial Agents

Disorders caused by the short-term absorption of moderate-to-high doses of ionizing radiation are often associated with suppressed heamatopoiesis as well as primary and secondary inflammation and thrombosis. Nicotinamide (NA), a derivative of nicotinic acid (NAc) and a precursor of nicotinamide adenine dinucleotide (NAD+), is metabolized in the body to 1-methylnicotinamide (MNA) which exerts both anti-thrombotic and anti-inflammatory activities based on its effects on the vascular endothelium. Also, 1,4-dimethylpyridine (1,4-DMP), a pyridinium salt formed during coffee roasting, and 1-methyl-3-acetylpyridine (1,3-MAP), a synthetic analogue of NAD+, may exert similar properties. Hence, the aim of the present study was to assess potential radio-protective and radio-remedial effects of NAc, NA, MNA, 1,4-DMP, and 1,3-MAP. The 30-day survival of BALB/c mice was assessed after whole body irradiation (WBI) with γ-rays at a dose of 7.5 Gy. NAc, NA, MNA, 1,4-DMP, and 1,3-MAP were given to the animals in drinking water at 100 mg/kg b.w. daily, starting 7 days before, on the day of, or 7 days after the exposure to ionizing radiation and continued until the animals’ death or the end of observation. Another group of mice was exposed (WBI) to X-rays at 6.5 Gy and on the selected days thereafter spleen and bone marrow cell as well as the peripheral blood leukocyte and thrombocyte counts were estimated. Application of the derivatives of NAc significantly increased the survival of mice from the groups in which administration of these compounds started 7 days before (NAc, MNA, 1,4-DMP), on the day of (1,4-DMP), and 7 days after (NAc, MNA, 1,4-DMP, 1,3-MAP) the irradiation. Inconsistent results obtained in mice exposed to X-rays indicate that stimulation of haematopoiesis does not seem to be the underlying mechanisms of the enhanced survival and other possibilities, such as reversal of the radiation-induced inflammatory and/or thrombotic reactions in association with the modulated endothelial function, need to be considered.

Speaker: Dr Aneta Cheda (Military Institute of Hygiene and Epidemiology)

212 High-mobility group box 2 (HMGB2) modulates radioresponse in colorectal cancer cell

Objectives: To improve the chemoradiotherapy efficacy in cancer patients by investigating the biological function of HMGB2 with respect to radiation response regulation. Methods: HMGB2 gene knockdown cells were constructed by infecting shRNA expressing lentivirus. mRNA expression was measured by realtime qRT-PCR and protein level or phosphorylation was by Western blot analysis. Clonogenic assay was performed to count the radiosensitivity. Immunofluorescence staining of gamma-H2AX was examined under confocal microscope. Results: HMGB2 knockdown sensitized HCT-116 and HT-29 colorectal cancer cells to radiation. This may be due to increase of DNA damage and delayed DNA damage repair in HMGB2 knockdown cells. Interestingly, HMGB2 expression was downregulated by ionizing radiation in colorectal cancer cells with functional TP53 gene and it was confirmed that p53 activation inhibited HMGB2 transcription. Conclusions: Because HMGB2 was necessary to protect colorectal cancer cells from ionizing radiation, radiotherapy outcome could be enhanced by targeting HMGB2. p53-mediated downregulation of HMGB2 is an important mechanism in modulation of HMGB2 expression and may be applied to predict therapeutic efficacy.

Speakers: Dr Jae-Hoon Jeong (Korea Institute of Radiological and Medical Sciences), Dr Young-Joo Shin (Inje University Sanggye Paik Hospital)

213 The potential impact of low dose ionizing γ-radiation on Ikaros-mediated immune activation in B lymphoblasts

The biological effects of low dose ionizing radiation (LDIR) remain insufficiently understood. In our previous study, Ikaros was phosphorylated in response to LDIR in IM-9 B lymphoblasts cell line. Ikaros encodes a zinc finger transcription factor that is an important regulator of a hematopoietic stem cells (HSCs) progression in the B lymphoid lineage development, differentiation and proliferation. This study showed the biological effects of LDIR in radiation-sensitive immune cells. We observed that almost 20% of cell proliferation was enhanced by LDIR (0.05 Gy) in IM-9 B lymphocytes. We found that the phosphorylation of Ikaros at Ser304 and Ser306 residues was up-regulated through CK2/AKT pathway in LDIR irradiated IM-9 B lymphocytes, decreasing its DNA binding activity. We also revealed that Ikaros protein was translocated from the nucleus to the cytoplasm after LDIR irradiation, and then binded with Autotaxin (ENPP2, ATX) protein. In addition, we found that the lysoPLD activity of ATX was increased by the interaction between Ikaros and ATX protein. The lysoPLD activity of ATX converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) which is a bioactive phospholipid involved in proliferation, differentiation, and migration of immune cells. Taken together, these results indicate that Ikaros may be an important regulator of immune activation through the interaction with Autotaxin. Therefore, we suggest that low dose ionizing radiation can show a beneficial effect stimulating the immune activity in IM-9 B lymphoblasts.

Speaker: Dr Kwang Hee Yang (Korea Hydro & Nuclear Power co. LTD.)

214 Identification and functional analysis of radiation-induced microRNA changes in endothelial cells

Due to the damage incurred by normal tissue, especially the endothelium, radiation doses used in tumour therapy are limited. A better understanding of the processes governing the radiosensitivity of endothelial cells is therefore desirable for improved radiation therapy. We have previously demonstrated that the sensitivity of endothelial cells to ionizing radiation depends on microRNA-regulated gene expression (Kraemer et al.). We now report the analysis of individual microRNAs (miRNAs), this reveals involvement of both indirect and direct protein targets. By siRNA-mediated knockdown of two miRNA-processing enzymes (Ago2 and Dicer) we showed that miRNA-regulated gene expression has a prosurvival function in endothelial cells after radiation exposure. Specific miRNAs responding to ionizing radiation were identified. The effects of individually upregulated miRNAs on survival and cell cycle regulation after radiation were measured by transfection with either specific precursor- or anti-miRs. Out of 7 analyzed miRNAs, 3 miRNAs (miR-216a, miR-518d-5p and miR-525-3p) had an impact on the cellular response to irradiation. All three enhanced apoptosis and reduced survival, as predicted from the inhibtion of miRNA processing. We were able to identify proteins targeted of these miRNAs through proteome analysis of anti-miR transfected cells using two-dimensional difference gel electrophoresis and mass spectrometry (2D-DIGE-MS). Gene Ontology (GO Term) analysis assigned the majority of the changed proteins to cell cycle regulation, apoptosis and DNA repair. Ingenuity pathway analysis categorized the deregulated proteins to biological networks of cell death, lipid metabolism and biochemistry of small molecules. Using sequence analysis to identify miRNA-target sequences in the 3´-UTRs, and luciferase reporter assays of the miRNA-UTR target interaction we validated the detected target proteins as direct (presence of miRNA binding site and regulation of lucifease stability by the miRNA) or indirect targets of the investigated miRNAs. Our data suggest that miRNA-mediated gene silencing has an essential function in the radiation response. In the future, targeting these miRNAs or their target proteins could be an important tool to sensitize tumors during radiation therapy and to protect normal tissue. The research leading to these results is supported by the BMBF Grant NUK007E (KVSF). Kraemer et al. Radiat Res. 2011 Nov; 176(5):575-86

Speaker: Dr Anne Kraemer (Institute of Radiation Biology, Helmholtz Center Munich, Germany)

215 Ascorbic acid and AHCC suppress fibrosis of lung and kidney caused by irradiation

Irradiation in radiation therapy or bone marrow transplantation is known to cause damage to normal tissues. Although some substances are reported to suppress the damage(=radioprotectors), most of them have strong side effects and are not applicable in vivo. However, ascorbic acid and AHCC (Active Hexose Correlated Compound) are known as safe radioprotectors with antioxidant activity. While examining some radioprotectors using mice, we found that ascorbic acid and AHCC suppress fibrosis of lung and kidney caused by irradiation. Materials and Methods: C57BL/6J mice,4-6weeks old were exposed to X-ray radiation in the following conditions; (1) 40Gy in 20 equal fractions, (2) 10Gy (single doze), (3)15 Gy (single doze), (4)20Gy (single doze) (In groups (1)-(4), exposure area was limited to the thorax and upper abdomen.), (5) 4Gy (single doze, total body irradiation). Ascorbic acid, AHCC or sterile saline was administrated to each mouse by subcutaneous or intraperitoneal injection prior to irradiation. We measured the Sircol level of lung and kidney and the Cystatin-C level of blood and urine of each mouse. Results: Ascorbic acid and AHCC suppressed all the Sircol level of lung and kidney and Cystatin-C level of blood and urine in groups (1)(male), (2)(female) and (3), they suppressed the Sircol level of lung in groups (1)(female) and (4), and they also suppressed the Sircol level of lung and kidney in group (5). Conclusions: Fibrosis of lung and kidney after irradiation was suppressed by ascorbic acid and AHCC. We might be able to prevent tissue impairment after irradiation by injecting ascorbic acid and/or AHCC.

Speaker: Dr Tadashi Hongyo (Osaka university)

216 Radiation sensitivity markers in HNSCC

Besides surgery and chemotherapy, radiotherapy is one of the main treatment options of head and neck squamous cell carcinoma (HNSCC). Radiation resistance of some tumours causes local recurrences that go along with unfavourable prognosis. Therefore, the characterisation of the radiation response of tumour cells is a major issue for the development of personalised therapy strategies. The identification of biomarkers predicting radiation sensitivity of HNSCC would allow the development of personalised therapy approaches that spare patients unnecessary radiation exposures by adapting and optimising the applied doses during radiation therapy. We identified cytogenetic markers (gains on chromosome bands 1q43 and 16q23-24) by CGH in a set of 117 HNSCC that correlated to a reduced loco-regional-progression-(LPR)-free survival after radiotherapy. Since the analysed tumours were exclusively treated by radiotherapy, it can be assumed that reduced LPR-free survival is caused by radioresistance of tumours that do not respond adequately to radiation therapy. Array CGH and FISH indicated 16q24.3 to be the region of interest on chromosome 16, which contains an interesting candidate gene, FancA. FancA is a member of the Fanconi Anaemia (FA)/BRCA pathway, a pathway disrupted in patients suffering from the disease FA. Interestingly, FA patients have a high risk in developing HNSCC which suggests a role of this pathway in HNSCC development. Quantitative RT-PCR confirmed increased FancA expression on RNA-level in tumours with gain on 16q24.3. In order to determine the effect of FancA on radiation sensitivity of cells, we stably transfected a cell line (OKF6/TERT1) with a FancA over-expressing vector and measured various endpoints like overall survival (by colony forming ability) and chromosomal aberrations (rate of dicentric chromosomes and translocations) after applying different doses of γ-radiation in comparison to non-transfected cells and vector control cells. The results suggest reduced radiation sensitivity in FancA over-expressing cells. The findings of our study demonstrate that chromosomal gains on 1q and 16q may represent prognostic markers in HNSCC and that these alterations may explain to some extent the unfavourable prognosis of a subgroup of patients after radiotherapy, by affecting candidate genes like FancA.

Speaker: Dr Verena Zangen (Helmholtz Zentrum München, German Research Centre for Environmentel Health, Research Unit of Radiation Cytogenetics)

217 ANTIOXIDANT AND ANTIAPOPTOTIC ACTIVITIES OF COPPER COMPLEXES WITH SCHIFF-BASE DERIVATIVES OF L-HISTIDINE UPON IRRADIATION

L.G. Zhamharyan, T.Zh. Hovsepyan, E.A. Arakelova, V.A. Ayvazyan, A.S. Boyajyan Institute of Molecular Biology, National Academy of Science of the Republic of Armenia Numerous data have demonstrated the induction of apoptosis by ionizing radiation (IR) through activation of Bax signaling pathway. It has been also shown the key role of antioxidant enzymes, namely superoxide dismutase (SOD) and catalase, in protection of cells from aberrant apoptosis stimulated by reactive oxygen species (ROS) upon IR. The purpose of this study was to determine whether copper complexes of Schiff-base L-histidine derivatives, early shown to increase viability of the experimental animals after IR, may affect the defense capabilities of the antioxidant system and prevent the upregulated apoptosis in radiation conditions. Among targeted compounds were: Cu(II)(picolinyl-L-histidine)2, Cu(II)(nicotinyl-L-histidine)2 and Cu(II)(isonicotinyl-L- histidine)2. Experiments were performed using white male rats with the weight of 160-180 g. The animals were divided into the following groups: 1. intact animals; 2. irradiated animals (5 Gy; control); 3. animals treated subcutaneously (s/c) with radioprotectors 1 hour prior to irradiation in dose of 10 mg/kg. Blood samples were collected on days 3-28 after IR exposure. Each sample was divided into two aliquots: one was used to obtain serum for determination of Bax levels by ELISA; another was immediately used to obtain hemolyzed erythrocytes for further measurement of SOD and catalase activities by spectrophotometric assay. Experimental data revealed that all tested compounds have SOD-mimetic effects differed from each other by duration. The detected increase in SOD activity was as much as 2-3-fold compared to the activity of this enzyme in control group, which was 2-3-fold lower than in norm. In addition, all tested compounds decreased IR-induced high BAX levels up to norm. The most pronounced effects were observed in case of Cu(II)(Isonicotinyl-L-histidine)2, which also increase the activity of catalase. On the base of the result obtained we concluded that Cu(II)(Isonicotinyl-L-histidine)2, may be recommended as multifunctional radioprotective agent. The study was supported by ISTC #1764 grant. Key words: Schiff-base derivatives, L-histidine, copper complexes, ionizing irradiation, superoxide dismutase, catalase, apoptosis, Bax.

Speaker: Lusine Zhamharyan (dr)

218 Frequency of CD34+ Hematopoietic Stem Cells in Peripheral Blood is Prognostic Factor of Larynx Cancer Response to Radiation Therapy

CD34+ hematopoietic stem cells (HSC) are known to migrate from bone marrow into peripheral blood and tumor tissue under the influence of variety of factors produced by the last one. One can suppose that HSC are capable to render considerable influence on cancer progression and efficiency of treatment through inhibition of intratumoral T-lymphocytes, differentiation into dendritic and endothelial cells and participation in formation of pre-metastatic niches. The purpose of the work was to estimate significance of circulating HSC for prognosis of radiosensitivity of larynx cancer in connection with possible participation of these cells in formation of tumor vasculature. Frequency of СD34+CD45low HSC was determined in peripheral blood of 92 cancer patients before treatment and 83 control healthy persons by flow cytometry. The patients were treated with conventional regime of radiotherapy. Regression degree was assessed after the first stage of radiotherapy (30-40 Gy) as an indicator of short-term response to radiotherapy. Frequency of circulating HSC did not significantly differ between total group of cancer patients and age-matched healthy persons. However patients with regional lymph nodes involvement demonstrated increase of HSC frequency, especially expressed at early stages (Т1+Т2) of disease: mean frequencies (±SE) of HSC in N+ and N0 patients were (18.8±5.8)x10^-4 and (8.2±1.2)x 10^-4, accordingly (р=0.02). There was not found significant association between quantity of circulating HSC and such conventional prognostic factors as primary tumor extent (stage T) or histological type. At the same time pretreatment frequency of these cells was associated with short-term clinical outcome: high degree of cancer regression (> 50 %) was observed in 74% of patients with relatively high frequency of circulating HSC (>6.0x10^-4) and only in 31% of patients with low frequency of these cells. The difference was highly significant in group of patients with stages Т3+Т4 (р=0.001). The correlation can be explained by known ability HSC to differentiate into endothelial cells or stimulate formation of vessels and thereby to increase vascularization of tumor tissue, oxygenation and, hence, radiosensitivity. Future investigation is necessary to estimate prognostic value of HSC frequency with respect to long-term results of radiotherapy. This work was supported by Ministry of Education and Science of the Russian Federation (grant #14.740.11.0180).

Speaker: Prof. Irina Zamulaeva (Medical Radiological Research Center of the Ministry of Health of the Russian Federation)

219 Sensitivity of Melanoma B16 Side Population Cells to Ionising Radiation and Methotrexate

Cancer stem cells (CSC), detected in variety of tumor types and cancer cell lines, were shown to be more resistant to the low-LET radiation in comparison to the other cancer cells. CSC are supposed to determine long-term efficiency of cancer therapy. Therefore, exploration of regularities and mechanisms of CSC radio- and chemosensitivity is of great interest at present. The aim of this investigation is to compare CSC and the other cancer cells sensitivity to the low (60Со gamma-rays) radiation and methotrexate. To identify putative CSC we used flow cytometry-based side population (SP) technique, founded on high capacity of CSC to efflux the vital dye Hoechst 33342. SP and non SP cells were sorted and exposed to radiation at doses of 1-10 Gy. Colony-formation test was applied to evaluate SP and non SP survival rate after the irradiation. To study cytotoxic effect of methotrexate 40% confluent melanoma B-16 culture was treated with the drug at final concentrations 1µg/ml or 5µg/ml during 2 days. Cell number and percent of SP were assessed at the end of the incubation. Proportion of SP was 0.9±0.2% in control 70% confluent cultures. Radiosensitivity of SP cells was below in comparison to that of the other cells: D0 average values (±SE) were 2.3±0.3 Gy and 1.4±0.2 Gy, accordingly (р=0.047). Total number of cells incubated with methotrexate was approximately 20 times less than that in control probes. However percent of SP was significantly increased up to 5.7±2.1 and 2.8±0.7 after methotrexate treatment (1µg/ml or 5µg/ml, accordingly) (p<0.01 as compared with control). The results indicate that cytotoxic effect of methotrexate extends more to non SP than to SP cells. In unexposed samples only 14.5 % of SP cells were in S+G2+M phases in comparison with 61.6 % in non SP (p<0.01), as it was shown by flow cytometry of sorted cells after standard staining with propidium iodide. Therefore one of the possible reasons for different sensitivity of examined cell populations to low-LET radiation and methotrexate is distinction in proliferative activity. According to multiple world-wide investigations, quiescent state appears to be necessary for preserving the self-renewal of stem cells and is a critical factor in the resistance of CSC to radio-, chemotherapy and targeted therapies. Our results confirm this thesis for SP of melanoma B16 cells. This work was supported by the Foundation for Assistance to Small Innovation Enterprises (FASIE), Russia.

Speaker: Ms Olga Matchuk (Medical Radiological Research Center of the Ministry of Health of the Russian Federation)

220 Modification of Immunity in Individuals with Late Radiation-Induced Neutropenia Using Hemopoietic Cells of Fetal Liver

Modification of Immunity in Individuals with Late Radiation-Induced Neutropenia Using Hemopoietic Cells of Fetal Liver A. Akleyev, I. Dolgushin, G. Dimov The objective of the study was to assess the effect of hemopoietic cells of fetal liver (HC FL) on the key characteristics of innate immunity in persons who developed neutropenia at late time after a long-term chronic radiation exposure. The study group was composed of 12 residents of Techa riverside villages, including 10 women and 2 men aged 49-80 years, who were exposed to combined external gamma-raditaion and internal radiation, mostly due to 90Sr. Mean radiation dose to red bone marrow amounted to 0.92±0.46 Gy. To alleviate late radiation-induced neutropenia, HC FL were administered in the ratio of 1.4•106 cells per kg of the patient’s body weight. Immunological examinations were performed before the treatment was started, and then were repeated over the 18 months after the course of treatment (at month 6, 12 and 18). The number of neutrophils was constantly increasing over the whole period of observation, and it reached statistically significant differences from the initial values after 2 months, or later. The most substantial changes in the immune status following treatment with HC FL were manifested by the innate immunity. Attention is attracted to the progressive, in dynamics, suppression of innate immunity factors (reduced number of NK cells, phagocytic activity and values of intracellular oxygen-dependent metabolism of neutrophils and monocytes) playing a leading role in the maintenance of anti-tumor immunity. Patients treated with HC FL were also noted to have increased levels of proinflammatory cytokines while the levels of antiinflammatory cytokines were diminished.

Speaker: Mr Andrey Akleyev (Urals Research Center for Radiation Medicine)

221 IDENTIFICATION OF TRANSCRIPTION FACTORS ASSOCIATED TO DIFFERENTIALLY EXPRESSED GENES IN IRRADIATED GLIOBLASTOMA CELL LINES

Glioblastoma multiforme (GBM) is among the most lethal of all human tumors, and radiotherapy remains one of the main treatment, besides surgery and chemotherapy. New strategies and Improvements in the therapies are required to overcome the cellular resistance of GBM. The identification of transcription factors (TFs) associated with significantly modulated genes in irradiated GBM could give clues for searching interesting molecular targets. In the present work, we studied four GBM cell lines (T98G, U251MG, U343MG-a and U87MG) under irradiation condition (gamma-rays), and characterized cellular and molecular signaling pathways involved in radiation responses. Gene expression profiles analyzed by the cDNA microarray method indicated a list of differentially expressed genes in irradiated cells (8 Gy), which was used as a data basis to search TFs that were associated to the significant differentially expressed genes, using an in silico approach (FatiGO+). Several TFs were correlated with the lists of modulated genes, whose functions were related to apoptosis, cell cycle, cell adhesion, and DNA repair. The results indicated the following TFs: AP-1 (T98G and U87MG), SREBP1 and CEBPG (U87MG), and HEB (T98G, U87MG and U251). We also found that HEB was up-regulated in U87MG, T98G and U251 cells, as confirmed by the Real Time qPCR analysis. Western Blot also confirmed HEB up-regulation in irradiated and sham-irradiated cells. HEB is a member of the basic-helix-loophelix (B-HLH) domain proteins, being involved in the nervous system development. The knockdown of HEB by siRNA achieved 70% of inhibition (72h after transfection), as confirmed by Western Blot, but the down-regulation of HEB did not cause any significant effect on clonogenic survival, cell proliferation, and cell viability in U87MG irradiated cells (0.5 Gy/min, 2, 4, and 8 Gy) analyzed between 48 and 72h after siRNA HEB transfection. The results of the present work can pinpoint some potential TFs that may possibly be associated to the resistance of GBM cells to radiotherapy, but other TFs should be investigated as molecular targets for therapies to increase cell death in glioma cells. (Supported by FAPESP Proc. Nº 2009/10925-6; CNPq; CAPES).

Speaker: Elza Sakamoto-Hojo (Department of Genetics, Faculty of Medicine of Ribeirão Preto-USP)

Modulation of Radiosensitivity

222 Hypoxia gene expression classifier predicts benefit from radiosensitizers

Hypoxic tumours are associated with increased resistance to radiotherapy. In head and neck squamous cell carcinomas (HNSCC), this resistance can be counteracted via addition of hypoxic modification to the radiotherapy. Tumours are heterogeneous with respect to the degree and extend of hypoxia, and several methods have been developed to detect relevant tumour hypoxia. These include direct measurements in the tumour by an oxygen electrode, infusion and detection of exogenous hypoxia tracers, or quantification of endogenous hypoxia markers expressed by the tumour cells under hypoxic conditions. While there are extensive studies in relation to prognostic impact, less is known about the relevance of hypoxia assessing methods in relation to the prediction of response to hypoxic modification of radiotherapy. A recently developed 15-gene hypoxia classifier has been shown to have predictive impact for identification of patients responding to hypoxic modification of radiotherapy with Nimorazole. Tumours from 323 HNSCC patients included in the randomized, double blinded DAHANCA 5 trial were classified using the 15-gene hypoxia as being “more” or “less” hypoxic. 114 (35%) were classified as “more” hypoxic. The prognostic impact of the classifier was demonstrated in the placebo arm of the trial in terms of loco-regional tumour control, LRC (5-year actuarial values 44% vs 18%, p = 0.004), and disease specific survival, DSS (51% vs 30%, p = 0.04). Importantly, only patients with “more” hypoxic tumours had a significant benefit of hypoxic modification with Nimorazole compared with placebo (LRC: 49% vs 18%, p = 0.001; DSS: 48% vs 30%, p = 0.04). “Less” hypoxic tumours had no significant effect of hypoxic modification and the outcome was similar to “more” hypoxic tumours treated with Nimorazole. When stratified for HPV-status, the benefit of Nimorazole was restricted to HPV-negative and “more” hypoxic patients. Patients with HPV-positive tumours had a generally improved outcome, irrespective of their hypoxic status and whether or not the patients received Nimorazole. Studies are currently ongoing to implement the hypoxia classifier in clinical practice in Denmark, and to include the hypoxia classifier in international clinical trials on Nimorazole. Other ongoing studies are addressing the relevance of hypoxia in HPV-positive tumours and the correlations with other hypoxia assessing methods. Finally, the hypoxia classifier is being evaluated for other tumour sites.

Speaker: Jan Alsner (Aarhus University Hospital)

223 Immunological aspects of the modulation of the radiosensitivity by hyperthermia

Hyperthermia (HT) has been shown in various cell culture, preclinical animal and clinical models to be a very potent radio- and chemosensitizer. Recent research suggests that local heating (41-43°C for 1 hour) of the tumour may lead to abscopal, systemic and immune stimulatory effects, since cancer cells can be rendered visible to the immune system by standard therapies either alone or in combination with (further) immune stimulation. The tumour microenvironment that is present after therapy strongly determines which immune cells get recruited and triggers the activation or suppression of dendritic cells (DCs). The latter take up and cross-present tumor peptide-antigens, a prerequisite for the initiation of MHC class I dependent cytotoxic T cell (CTL) anti-tumor responses. Danger signals like high-mobility group box 1 (HMGB1) protein and heat shock protein 70 (Hsp70) are discussed to be potent stimulators of DCs when they get released by therapy-stressed tumour cells. The immunogenic potential of HT when applied in combination with RT will be presented and focus will be set on the modulation of the innate and adaptive immune system by either RT alone or in combination with HT treatment. Acknowledgement: The German Research Foundation (GA 1507/1-1 and DFG -Graduiertenkolleg 1660: Key signals of the adaptive immune response), the German Federal Ministry of Education and Research (BMBF; m4 Cluster, 01EX1021R and GREWIS, 02NUK017G), and the European Commissions (DoReMi, European Network of Excellence, contract number 249689) supported this work.

Speaker: Prof. Udo Gaipl (Department of Radiation Oncology, University Hospital Erlangen)

224 The role of TGFbeta3 and induced nitric oxide activity in connection with induced resistance against low dose hyper-radiosensitivity

We have previously found that protracting a priming dose of 0.3 Gy over 1 hour resulted in permanent elimination of low dose hyper radio-sensitivity (HRS) instead of the transient elimination by an acute priming with the same dose. The cells exposed to the low dose-rate priming secreted a factor into the medium, which removed HRS transiently in recipient cells. The factor could also be induced by low dose-rate irradiation of cell conditioned medium but only when serum was present in the medium during conditioning. We have now identified the serum factor as interleukin-13 and the factor secreted by the low dose-rate primed cells as TGFβ3 and propose a model for a self-sustaining molecular mechanism responsible for permanent elimination HRS transmitted to the progeny. Two cell lines known to display HRS were used, T-47D breast cancer cells and T98G glioblastoma cells. The change to a HRS-negative phenotype was found to be transiently induced by extracellular TGFβ3, which could be activated through iNOS activity by low dose-rate irradiation (0.2-0.3 Gy/h for 1 h) of cell conditioned medium. However, direct cell irradiation at low dose-rate induced a permanent elimination of HRS by activation of a self-sustaining mechanism found to depend on iNOS activity and resulting in continuously elevated cytoplasmic levels of TGFβ3. The HRS-negative phenotypes of T-47D and T98G were reversed by iNOS inhibitor 1400W. Interestingly, HRS was induced by 1400W in the HRS-negative cervix cancer cell line NHIK 3025. The effect of low dose-rate irradiation could be mimicked by high dose-rate irradiation or reoxygenation after hypoxia in combination with NO. The data show how resistance against low levels of DNA-damage can be turned on (by TGFβ3) or off (by iNOS inhibition) in cells. In addition, the data contributes to the understanding of the importance of distinguishing between dose-rates in relation to radiation protection issues and indicate that the effects induced by low dose-rate irradiation are related to NO production and independent of DNA-damage. Preliminary results confirm the findings in a mouse model.

Speaker: Dr Nina F. Jeppesen Edin (University of Oslo)

225 Association between XRCC1 polymorphisms and acute side effects induced by radiotherapy in breast cancer patients

Clarice Patrono1, Laura Verna2, Silvia Sterpone3, Valentina Palma1, Renata Cozzi3, Antonella Testa1 1ENEA Casaccia, Rome, Italy; 2S. Pietro Hospital, Radiotherapy Unit, Rome Italy; 3Department of Biology, “Roma Tre” University, Rome, Italy Therapeutic exposure to ionizing radiation can induce normal tissue side effects which consistently differ among individuals suggesting a possible genetic control. Single Nucleotide Polymorphisms (SNPs) in genes involved in DNA repair affecting protein function, could modulate DNA repair capacity and influence the individual radiosensitivity. The aim of the present prospective study was to evaluate the association of polymorphisms in XRCC1, a gene involved in base excision and single-strand breaks repair, with the risk of radiotherapy-induced acute side effects in BC patients. We genotyped for XRCC1 -77 T>C, Arg194Trp, Arg399Gln variant alleles a cohort of 78 Italian BC patients, receiving a standard radiotherapy regimen after breast-conserving surgery. The severity of acute skin adverse reactions was assessed according to Radiation Morbidity Scoring Scheme (EORT/RTOG). We found that the development of acute side effects (grade > 2) was significantly lower (p<0.05) in BC patients bearing XRCC1 399 variant allele. We also evaluated the association of XRCC1 variants and BC susceptibility, comparing the genotype distribution of patients to matched healthy subjects. We found a significant association between BC occurrence and XRCC1 -77 T>C and Arg399Gln variant alleles; a significant decreased risk of developing BC was found associated with XRCC1 haplotype H4, containing the three wild type alleles. Our results suggest that XRCC1 Arg399Gln SNP could be protective against early severe side reactions after radiotherapy in BC patients. Moreover, XRCC1 SNPs may be implicated in the occurrence of BC as previously reported.

Speaker: Dr Clarice Patrono (ENEA C.R. CASACCIA, Rome, Italy)

226 Telomere profiling : toward glioblastoma personalized medicine

Despite a standard of care combining surgery, radiotherapy (RT) and Temozolomide chemotherapy, average. Glioblastoma patient survival is still less than one year. However, recent advances in molecular signalization profiling in glioblastoma have provided new targets for personalized medicine development and new irradiation techniques, such as carbon ion Hadrontherapy, are now available. The latter technology leads to a higher biological response, while minimizing adverse effects on healthy tissues in comparison with RT. As carbon ion Hadrontherapy access is restricted to RT resistant patient, photon irradiation resistance biomarkers are needed. Long telomeres and high TA have widely been associated with photon radio-resistance in other cancers. Moreover, telomere protection, function and length also depend on the Shelterin protein complex (TRF1, TRF2, TPP1, POT1, TIN2, hRAP1). We thus decided to evaluate an enlarged telomeric status (TL, telomerase catalytic subunit (hTERT) and the shelterin component expression level as a potential radioresistance biomarker in a panel of 11 glioblastoma-derived cell lines. In addition, nothing was known about the role of telomeres in carbon ion response. We have thus evaluated this telomeric status after both types of irradiations. We report a significant correlation between TL, basal POT1 expression level and photon radioresistance and a significant variation in TERT, TERF1 and POT1 expression after photon irradiation. Strikingly, all of these correlations were lost when considering carbon irradiation. We thus propose (i) a model of telomeric damage implications in cell response to both irradiations, (ii) to assess POT1 expression level or TL on tumor biopsy to identify radioresistant patients.

Speaker: Mr Sylvain FERRANDON (EMR3738-Cellular and Molecular Radiobiology Laboratory)

Non-Cancer Effects

227 Effects of Exposure to Space Radiation on Brain and Behavior

On exploratory class missions, such as a mission to Mars, astronauts will be exposed to types and doses of radiation (cosmic rays) that are not experienced in low earth orbit where the space shuttle and International Space Station operate. Cosmic rays are composed of alpha particles, protons and particles of high energy and charge (HZE particles), such as 56Fe and 16O. Exposure to HZE particles and protons produces oxidative stress and inflammatory responses in the central nervous system leading to a disruption of neuronal function and, in turn, to a disruption of behavioral performance. The changes in neuronal function include loss of hippocampal neurogenesis, changes in signaling molecules and dopaminergic neurotransmission in the striatum, and disruption of autophagy, the process by which damaged proteins are sequestered and removed from the cell. Changes in cognitive/behavioral performance include changes in baseline anxiety (emotion), measured using the elevated plus-maze; in spatial and non-spatial learning and memory, measured using the Morris water maze and novel object recognition task; and in the responsiveness of the organism to changes in environmental contingencies, measured using operant responding on an ascending fixed-ratio schedule. While exposure to non-lethal levels of all types of HZE particles and protons can produce changes in neural and behavioral endpoints, there are differences in the effectiveness with which different HZE particles disrupt neuronal function and cognitive performance. The factors influencing the effectiveness of HZE particles in disrupting neuronal function and cognitive performance include the characteristics of the specific particle (its energy and linear energy transfer) and the characteristics of the individual (age and gender). The interaction between the characteristics of the HZE particles to which astronauts will be exposed and the characteristics of the individual astronaut will affect the probability that exposure to space radiation will disrupt neuronal function and, consequently, the probability that there will be deficits in cognitive performance which have the potential to affect the ability of astronauts to successfully meet mission requirements. Supported by NASA Grant NNX08AM66G

Speaker: Bernard Rabin (Psychology/UMBC)

228 Risk of radiation-induced cataract for interventional cardiologists

Interventional cardiologists are exposed to X-rays during their occupational activity and may be at risk to develop early cataracts known as radiation-induced eye lens opacities. The O'CLOC study (Occupational Cataracts and Lens opacities in interventional Cardiology) was performed in France to quantify this risk. O’CLOC study is a cross-sectional multicenter study including an exposed group of interventional cardiologists – ICs – and a comparable unexposed group of non medical workers. Individual information, including risk factors of cataract were collected. A specific part of the questionnaire focused on occupational history in cardiology and procedures description (kind, frequency, use of radiation protection tools) in order to retrospectively assess cumulated eye exposure of ICs. All participants had a clinical eye examination based on the international standard lens opacities classification – LOCS III – that allowed screening of type (nuclear, cortical or posterior subcapsular) and stage of cataracts. The study included 106 ICs (mean age=51±7 yrs.) and 99 unexposed people (mean age =50±7 yrs.). For ICs, mean duration of activity was 21 years. Based on retrospective assessment combining information from occupational questionnaire and doses per procedures observed in European countries (ORAMED project), eye lens dose cumulated during occupational life in cardiology ranged from 25 mSv to 1650 mSv (mean=454 ± 369 mSv). About 20% of them had cumulated more than 500mSv corresponding to the ICRP eye lens dose threshold and more than 60% of ICs had been exposed at least once to an annual eye lens dose above 20mSv which corresponds to the ICRP recommended annual limit. Regarding nuclear and cortical lens opacities stage ≥1, no significant difference was observed between both groups. In contrast, posterior subcapsular lens opacities (stage≥1) were significantly more frequent among interventional cardiologists (17% vs. 5%, p = 0.006), corresponding to an adjusted OR= 3.8 [1.3 – 11.4]. This risk increased with duration of activity but no clear relationship with workload was observed. However, the risk appeared lower for regular users of protective lead glasses (OR=2.2 [0.4–12.8]). Interventional cardiologists are at high risk of posterior subcapsular cataracts. Use of protective equipment against X-rays, in particular lead glasses, can limit this risk.

Speaker: Ms Sophie Jacob (IRSN, PRP-HOM/SRBE/LEPID)

229 Differential Effects of Full Body Single Low Dose Proton and Iron Radiation on Acute Myocardial Infarct Recovery in Adult Mice

Astronauts are exposed to space radiation (IR). It is important to evaluate the effect of cosmic radiation on cardiovascular (CV) system. Approximately 41% of space radiation is predicted to be HZE particles with 13% being from 56Fe particles. We hypothesized that (1) low-dose space IR-induced biological responses may be long-lasting and are IR type-dependent; and (2) IR may increase CV risks in the aging heart (IR+A) and affect the processes of CV repair after acute myocardial infarct (AMI) (IR+A+AMI). Nine months C57BL6 male mice were irradiated once with proton 0.5Gy/1GeV/n or 56Fe 0.15Gy/1GeV/n. Cardiac function was assessed by echocardiography and hemodynamic measurements. AMI was induced by ligation of left anterior descending (LAD) coronary artery 1-3 months post IR. Cardiac inflammation, apoptosis and fibrosis were evaluated post AMI. In IR+A group, cardiac function was not different among control, proton and 56Fe irradiated mice 1-3 months post-IR. In IR+A+AMI group, mortality was not different among the groups. Compared to control-AMI mice, inflammation was increased similarly in both proton-AMI and 56Fe-AMI mice up to day 7. Between days 7 and 14, inflammation remained the same in proton-AMI mice, whereas it was sharply decreased in the 56Fe-AMI mice. Compared to control-AMI mice, there was a significant (p<0.01) increase in apoptosis in both proton-AMI and 56Fe-AMI mice by 14 days, and was significantly (p<0.03) higher in 56Fe-AMI vs. proton-AMI mice. Four weeks post AMI, proton-AMI mice had better cardiac functional recovery compared to control-AMI and 56Fe-AMI mice; whereas ejection fraction % (EF%) was most decreased in 56Fe-AMI mice (56Fe-AMI vs. proton-AMI: 18 vs. 48 %, p<0.007, vs.  65-70% pre-AMI EF% for all groups). There was a 2-4-fold increase in left ventricular end diastolic pressure (LVEDP - an indicator that heart is not pumping blood well) in 56Fe-AMI vs. Proton-AMI (p<0.04), suggesting that 56Fe-AMI hearts developed cardiac de-compensation. AMI led to small transmural infarct in control-AMI mice, large transmural infarct in 56Fe-AMI and small superficial infarct in proton AMI mice. Our results revealed that low dose full body single proton or 56Fe IR do not affect cardiac function under normal conditions. Further, a single proton IR three months prior to AMI is beneficial, whereas 56Fe IR is deleterious, for AMI recovery, suggesting that low dose HZE particle radiation have long-lasting negative effect on CV risks such as AMI.

Speaker: Dr David Goukassian (Tufts University School of Medicine)

230 The role of miRNAs in the response of human keratinocytes to ionizing radiation

Understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive. Recent publications describe microRNAs (miRNAs), a group of short non-coding RNAs that negatively regulate gene expression, as potential modulators of cellular response to ionizing radiation (IR), both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNAs machinery in the cellular response to IR remains to be clarified. To address this question, we settled up an expression study of miRNAs in primary human keratinocytes. We analyzed the results of global miRNA profiling, performed by microfluidic system of qPCR assay, which permit to assess the expression of almost 800 annotated miRNAs. The keratinocytes derived from 3 or 4 independent donors were cultured to a proliferative or a differentiated state mimicking basal and suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. We found that proliferative cells irradiated at 6 Gy display a global fall of miRNA expression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. To test if those global effects could be due to a specific regulation of the miRNA biosynthesis by IR, we investigated expression of DICER1 or EIF2C2, two crucial components of the miRNA pathway. We didn’t observe any significant modulation of these proteins expression after irradiation. Using a bilateral paired t-test, we identified miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells by pre-miR transfection: we observed a significant decrease of cell viability 72h after irradiation. Altogether, our results indicate that the miRNAs play a weak but significant role in response to ionizing irradiation to ensure the short-term survival of irradiated human skin primary keratinocytes.

Speaker: Mr Nicolas Joly-Tonetti (Université Claude-Bernard Lyon1, Université de Lyon, CNRS UMR5534)

231 CHRONIC BRONCHITIS INCIDENCE AMONG MAYAK NUCLEAR WORKERS

Chronic respiratory diseases are widely spread among the general population. In recent decades, there has been a steady growth of chronic obstructive pulmonary disease worldwide. The World Health Organization predicts that this disease will become the third most common cause of death by 2020. The epidemiological studies of the Japanese A-bomb survivors, patients medically exposed to radiation for diagnostic and therapeutic procedures and occupational exposures of nuclear workers revealed a statistically significant relationship between radiation exposure and mortality from respiratory diseases although other studies did not [UNSCEAR 2008]. Chronic bronchitis incidence (491 ICD-9 code) has been analyzed in the cohort of 12,210 workers (29% females) first employed at one of the main plant (reactors, radiochemical or plutonium) of Mayak Production Association during 1948-1958 and followed-up to the end of 2005. Work histories and dose estimates from the Doses-2005 dosimetry system formed the basis for this study. Retrospective diagnosis verification confirmed 1,175 cases of chronic bronchitis in the study cohort. Preliminary analysis of chronic bronchitis incidence in relation to total absorbed external gamma-ray lung dose for chronic bronchitis incidence did not reveal a statistically significant excess risk after adjusting for gender, attained age, calendar period, plant, smoking, total absorbed lung dose from internal alpha-particle radiation due to incorporated plutonium. In contrast, preliminary analysis in relation to internal alpha-particle lung dose revealed statistically significant increased risk of chronic bronchitis incidence after adjusting for non-radiation factors and external gamma-ray lung dose. The study was performed with the financial support of the RF Federal Medical Biological Agency and European Commission within the framework of grant agreement No. 249675 “Epidemiological Studies of Exposed Southern Urals Populations” (SOLO).

Speaker: Dr Tamara Azizova (Southern Urals Biophysics Institute)

232 Characterization of the cellular response of HUVECs and EA.hy926 cells following exposure to low dose acute X-irradiation

High radiation doses (> 5 Gy) are known to increase the risk of cardiovascular diseases. In recent years, epidemiological data support the fact that lower radiation doses increase the risk of cardiovascular diseases as well (cfr. Atomic bomb survivors). However, lack of statistical power in the epidemiological studies requests a better understanding of the underlying biological and molecular mechanisms for an accurate low dose risk assessment. The endothelium is believed to be a critical target in the development of radiation-related cardiovascular diseases. Hence, we used primary Human Umbilical Vein Endothelial Cell (HUVEC) and the immortalized derivative endothelial cell line EA.hy926 as models to characterize the endothelial response to acute low and medium doses (0.05 - 5 Gy) of X-irradiation (250 keV, 15mA, 1mm Cu). The dose rate was 0.25 ± 0.01 Gy/min. Investigated endpoints included DNA damage, cell cycle changes and associated apoptosis. In addition, the production of reactive oxygen species (ROS), an important player in both cardiovascular diseases and cellular radiation response, was assessed. DNA damage and repair were studied by means of immunostaining for γH2AX-foci and quantitative fluorescence microscopy respectively 30 min and 24 h post irradiation (p.i.). Apoptosis, cell cycle changes and the production of ROS was assessed by flow cytometry. Apoptosis was investigated 24, 48 and 72 h p.i. using Annexin-V/propidium iodide (PI) costaining. Cell cycle changes were studied 8, 24, 48 and 72 h p.i. via PI staining. Production of ROS was examined 30 min p.i. using the ROS sensitive dye CM-H2DCFDA. Our results indicate that both in HUVECs and EA.hy926 cells, fundamental cell cycle changes due to G2 arrest is limited to higher dose irradiation (5 Gy). A significant increase of apoptotic cells was observed after 0.1 Gy and higher, but results were not consistent over multiple experiments. The production of ROS was significantly increased after exposure to 0.1 Gy and higher. More subtle, but significant effects such as DNA damage could be observed down to the lowest tested dose of 0.05 Gy. In conclusion, HUVECs elicited in general a less pronounced response compared to EA.hy926 cells as shown by smaller γH2AX-foci number, smaller percentage of apoptotic cells, and a less pronounced G2 arrest. Acknowledgements: This project is supported by the EU FP7 DoReMi Network of Excellence (grant agreement: 249689).

Speaker: Ms Charlotte Rombouts (SCK-CEN/Ghent University)

20:00 Social dinner

Friday, 19 October

Keynote Lecture 4

233 CANCER STEM CELLS

Cancer stem cells (CSCs) are a subset of tumor cells that are thought to be responsible for tumor initiation, maintenance and spreading. These cells are largely resistant to conventional therapies, which target preferentially their differentiated non-tumorigenic progeny. We developed a technology that allowed us to isolate and expand in vitro CSCs from several solid tumors, including glioblastoma, prostate, breast, lung, colon, thyroid and ovary cancer. Orthotopic injection of these tumorigenic cells in immunocompromised mice recapitulate the behaviour of human cancer and provide reliable preclinical models that can reproduce unlimited times the tumors of single patients. The use of a medium-throughput platform that quantifies relevant proteins and post-translational modifications of signaling proteins involved in cell survival, coupled with a functional screening of pathway inhibitors, allowed us to map the pathways active in CSCs to be targeted for effective therapies. By unsupervised clustering analysis, we were able to identify different subclasses of tumors that respond differently to targeted therapy and radiation. Notably, in vitro testing of therapeutic compounds was able to predict the in vivo sensitivity in preclinical models. More importantly, the first clinical data showing a possible predictive value of in vitro CSC analysis are emerging in metastatic patients that failed multiple line of treatment. Thus, although the identification of CSCs is relatively recent, this research area appears extremely promising as it is likely that CSC targeting may significantly contribute to the rational design of novel targeted therapies for cancer.

Speaker: Prof. Ruggero De Maria (Regina Elena National Cancer Institute)

Internal Emitters

234 Radioactivity from within: Using radionuclides to track health and disease

Molecules appropriately equipped with signal-transmitting (radioactive) tags are critical tools for our ability to non-invasively track the molecular processes that are fundamental for maintenance of life, the development of disease and the efficacy of the interventions designed to stop, reverse or stabilize those disease processes. The radionuclide chosen for this molecular imaging is important since it can affect the distribution and retention in the body, the quality of the images obtained and the radiation doses to the subject. The molecules used to track the processes may visualize either by means of their passive, compartmental distributions or through their active distribution and metabolism and/or binding interactions with specific targets such as receptors, integrins, adhesion molecules. The successful use of imaging molecules as pharmacodynamic markers of the effects of drugs on the body is highly dependent on the diversity of molecules being developed and on the depth of the characterizations from bench to bedside being performed. It is important to study the quantitative pharmacokinetics of what happens to the radioactive molecules in vivo during the relevant observation period (i.e. their uptake, localization and elimination) at least during the development stage before clinical implementation. Examples of some implications of these evaluations from preclinical to clinical positron emission tomography (PET) will be presented here.

Speaker: Dr Sharon Stone-Elander (Karolinska Institute, Stockholm, Sweden)

10:30 Coffee break

235 Application of the Comet (SCGE) and Micronucleus (MN) Assay to Estimate Health Risk due to Exposure at Low and High Doses of 131I

Iodine-131 is used in nuclear medicine both diagnostically and therapeutically to improve health conditions by early disease detection and restore health by slowing an overactive thyroid or even killing cancer cells. An emerge of the 131I among emission of various radioactive isotopes, might be one of the first sign of misfortunate accidental exposures from various radiation sources, including nuclear power plants, that even if infrequently, continue to occur. The aim of our study was to investigate the influence Iodine-131, from the diagnostic and therapeutic treatment exposures, on cellular DNA repair efficiency at molecular (SCGE) and cellular (MN) levels, in order to estimate a potentially associated health risk at low and high dose regions. Study group consisted of 41 individuals diagnostically exposed to low dose of the 131I (in the range 1.85~4.45 MBq, Aśr = 2.96 ± 0.82 MBq) and 37 persons therapeutically exposed to high dose of the 131I (in the range 300~650 MBq, Aśr = 497.3 ± 88.1 MBq). A reference group consisted of 30 healthy individuals. Blood samples that were collected for cellular studies underwent immediately cytogenetic procedures according to standard protocols. For the DNA damage detection the alkaline version of the single cell gel electrophoresis (SCGE) assay was applied in cells before irradiation, immediately after, and again after a one hour of post irradiation repair incubation. Strong variation between cellular responses of thyroid diseases patients to both low and high doses of 131I was observed. A diagnostic (very low) dose of 131I caused in 84% of the patients a visible decrease of the micronuclei formation in the cells, below the mean level of the control. Results of the DNA repair competence assay also have shown higher level of residual damage in control group when compared to patients diagnosed with the low 131I dose. There was a significant increase in baseline level of DNA damage, which suggests that not all DNA damages caused by radioisotope had been repaired during 5 weeks from a therapeutic applications of Iodine-131. Variability in the observed responses in both situation after low and high doses of 131I, implies a strong necessity of personalized investigation of the individual susceptibility to IR exposure. Acknowledgments Work partly supported by NCBiR Research Task No. SP/J/6/143339/11 and grants: MN i SW 0296/B/P01/2008/35, IAEA Research Contract No. 17101/R0, 2012.

Speaker: Prof. Antonina Cebulska-Wasilewska (Institute of Nuclear Physics PAN, Kraków, Poland)

236 Radiation-induced DNA Damage and Repair in Normal Thyroid Epithelial Cells: Comparison of Cell Cycle Stage

The thyroid is considered to be among the most radiosensitive organs. However, the biological nature of thyroid radiosensitivity as compared to other organs is largely unknown. The interest of using high-LET radiation in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. The high-LET alpha particle Astatine-211 (211At), that is concentrated in the thyroid by the same mechanism as 131I (NIS-mediated), has been proposed to be used as an alternative to 131I for therapeutic use. Of interest, 211At induces two to three times more DNA DSBs than low-LET irradiation in fibroblast cultures, making 211At an attractive source of internal radiation for comparative studies. As a result, we here study the radiation response, i.e. DSB repair, cell cycle checkpoint arrest and chromosomal aberrations in normal cycling versus stationary cells exposed to different radiation qualities. Both cycling and stationary primary pig thyrocytes cultured on petri dishes or chamber slides were exposed to low-LET (60Co) or high-LET (211At) radiation to absorbed doses of 0-3 Gy. Repair of DNA damages and cell cycle arrest were studied by detection of phosphorylated H2AX (gamma-H2AX) and phosphorylated Chk2 (pChk2 Thr68) with flow cytometry and western blotting. Micronuclei assay was used to detect chromosomal aberrations. The levels of gamma-H2AX and pChk2 (Thr68) decreases during the first 24 h in cycling cultures exposed to 60Co, whereas there are increasing levels in cycling cultures exposed to 211At. The decreasing levels in response to low-LET radiation might indicate DNA repair while the increasing levels in response to high-LET radiation possibly signify poor repair of DNA damage or formation of de novo DSBs during replication and DNA repair processing. The micronuclei assay shows that cycling cells irradiated with 211At (0-2 Gy) have an increasing ratio of micronuclei per cell nuclei up to 1 Gy. Doses above that tend to result in lower levels of micronuclei per cell, probably due to an efficient cell cycle arrest. Interestingly, cycling cells exposed to 1 Gy 211At get 9 times higher levels of micronuclei than cycling cells exposed to 1 Gy 60Co, giving a RBE of 9. The result indicates that thyrocytes might be a lot more sensitive to high-LET radiation than fibroblasts where a RBE of 3 has been found.

Speaker: Dr Madeleine Nordén Lyckesvärd (Institute of Clinical Sciences, Dept of Oncology, Sahlgrenska Academy, University of Gothenburg)

237 Influence of I-125 labeled TFO in SCL-II cells on cell survival, cell cycle and DSB induction

Triplex-forming oligonucleotides (TFO) are able to bind DNA in a sequence specific manner and are a promising tool to manipulate genes or gene regulatory units in a cellular environment. TFO posses a therapeutic potential e.g. as a carrier molecule for Alpha- or Auger-Electron-Emitter (AEE) to target specific DNA sequences in tumour cells. We established a method for the effective labeling of TFO with the AEE Iodine-125 (I-125) and studied the influence of labeled TFO in SCL-II cells with regard to cell survival, appearance of DNA Double-Strand-Breaks (DSB) and the induction of cell cycle arrest. The TFO employed in this study were two multi-binding TFO with several thousand binding sites each in the human genome and a single binding site TFO, specific for GAPDH. TFO labeling with I-125 was performed using the primer extension method. Cell survival and DNA DSB frequency in I-125-TFO transfected SCL-II cells were analyzed with the Colony-Forming-Assay and the 53BP1 Assay. Analysis of cell cycle was done after 7-AAD staining by flowcytometry. I-125-labeled TFO were shown to induce a pronounced decrease in cell survival and an increase of DSB. TFO targeting multiple sites differing in the total target number showed a significant different cell killing per decay that was in good accordance with the observed induction of DSB per decay. Single gene targeting I-125-labeled TFO significantly decreased cell survival and induced DSB as well. All three investigated TFO induced a significant cell cycle arrest in G2/M phase 8 h post-transfection. I-125-labeled TFO with a single binding site as well as TFO with multiple binding sites cause massive cell killing and increase substantially the DSB frequency in SCL-II cells. All investigated TFO induce a pronounced G2/M arrest at rather low numbers of accumulated decays. I-125-labeled TFO might be a very useful tool for basic DNA repair research. Funded by the Bundesministerium für Bildung und Forschung (BMBF), Grant number 02NUK005A

Speaker: Mr Volker Dahmen (Radiation Biology Group, Department of Safety and Radiation Protection, Forschungszentrum Jülich, 52425 Jülich, Germany)

Stem Cells Radiobiology

238 Genomic stability in human skin stem cells: repair or no repair?

Skin is a well-known target of ionizing radiation as both complications in normal tissues and radiation-induced cancers have been documented, mainly developing from epidermis. Human epidermis is constantly renewed over a cycle of 28 days. This short-term constant renewal is due to a specific type of cells called the keratinocyte progenitors. They are located in the deepest layer of epidermis, or basal layer. These progenitors are highly sensitive to ionizing radiation (1), thus responsible for the short-term effects of radiation, such as radiodermatitis, dry and moist desquamation. Moreover, the progenitors that have resisted to radiation exposure exhibit a reduced capacity of DNA damage repair (2) and a long standing genomic instability. Keratinocyte progenitors appear thus to be a major target for cancer formation in skin. The long-term maintenance of skin homeostasis is due to rare (0.2%) and dormant stem cells, also located in the basal layer of epidermis. We demonstrated that keratinocyte stem cells are radioresistant (1) and possess a high capacity of repair for all types of DNA damage (2). Moreover, stem cells have developed specific mechanisms of protection against genotoxic stress, including modification of their niche through the FGF2 growth factor (2; 3). Thus stem cell response to radiation appears to favour long-term tissue maintenance. However, as the error prone repair pathway NHEJ might be the main repair mechanisms in stem cells, tissue maintenance might be at the expense of genomic stability and overall stem cell response favour cancer formation (4). In skin, the fact that all basal cells appear to be direct targets of genotoxic stresses might explain why skin carcinoma is one of the most frequent tumour types in humans. To further explore this high cancer susceptibility, we are currently investigating how the mechanisms of genomic stability maintenance differ between stem cells from normal individuals versus radiosensitive patients. 1- Rachidi W et al. Radiotherapy and Oncology, 2007, 83, 267-276. 2- Harfouche G et al. Stem Cells, 2010, 28:1639–1648. 3- Marie M et al., Int J Radiat Biol, 2012, in press. 4- Harfouche G et al. Mutation Research Review, 2010, 704: 167–174.

Speaker: Dr Michele Martin (Laboratory of Genomics and Radiobiology of Keratinopoiesis, CEA, Evry, France)

10:30 Coffee break

239 Cancer Stem Cells in Radiation Oncology: From Biology to the Patient

The field of cancer stem cell biology has a very short steep career from its infancy into the stage of clinical impact. Radiation oncologists are among the first to have recognised the importance of this field linking locoregional control with effective treatment of cancer stem cells with radiation. Better understanding of cancer stem cell biology in solid tumours is hypothesised to lead to a higher proportion of curative treatments. The challenges of the cancer stem cell concepts and current hot topics, e.g. phenotypic plasticity of stemness and impact and dynamics of microenvironmental niches will be discussed. This will be taken to project the pathway for future applications of stem cell biology and clinic.

Speaker: Dr Thomas Brunner (Gray Institute, Oxford, UK)

240 Overcoming the resistance of head and neck squamous cell carcinoma to photon and carbon ion irradiation by targeting cancer stem cells

Although hadrontherapy is a very promising alternative to conventional radiotherapy in a large variety of cancers, recent clinical trials have shown that local control of head-and-neck squamous cell carcinoma (HNSCC) is worse than expected due to loco-regional recurrence of the tumor. In order to obtain more insight into the mechanisms of tumour escape, we have investigated the role of cancer stem-like cells (CSlC) obtained from the SQ20B radioresistant HNSCC cell line (SQ20B). According to the literature, we have shown that the resistance of CSlC may result from an imbalance between exacerbated self-renewal and proliferative capacities and the decrease in apoptotic cell death triggering. Attempts to modulate these processes seem therefore to be promising therapeutic strategies. Concerning the self-renewal pathway, a fundamental role of Bmi-1 was demonstrated in SQ20B-CSlC for which its inhibition significantly enhanced sensitivity to high and low LET radiation by triggering apoptotic cell death. We have also demonstrated that the radioresistance of CSlC cells could result from their high proliferative capacity related to high aldehyde deshydrogenase (ALDH) activity. Inhibition of ALDH using all-trans retinoic acid induced differentiation of CSlC associated with a significant decrease in cell survival after either carbon or photon irradiation. Regarding apoptosis, since irradiated CSlC do not undergo early apoptosis because of a transient arrest in G2/M followed by mitotic catastrophe, treatment of CSlC with the inhibitor of the G2/M arrest UCN-01 triggered early apoptosis thus leading to radiosensitisation after photon and carbon ion exposure. The combination of ATRA and UCN-01 treatment with irradiation drastically decreased the survival fraction at 2 Gy (SF2) of SQ20B-CSlC from 0.85 after photon irradiation to 0.38. Furthermore, SF2 decreased from 0.45 in response to carbon ions to 0.21 when associated with ATRA and UCN-01. In conclusion, whatever the pharmacological strategies used, an important radiosensitisation of CSlC was obtained. Adjuvant treatments targeting either the inhibition of survival/self-renewal pathways or the triggering of apoptosis should improve the results for patients treated with radio- or hadron- therapy.

Speaker: Dr Gersende Alphonse (Department of Cellular and Molecular Radiobiology EMR3738)

241 Tocopherol succinate-mobilized progenitors mitigate radiation injury and improve intestinal integrity after whole body irradiation

The goal of this study was to elucidate the role of α-tocopherol succinate (TS)- and AMD3100-mobilized progenitors in mitigating the ionizing radiation-induced gastrointestinal (GI) syndrome in mice. We demonstrate the efficacy of a bridging therapy which will allow the lymphohematopoietic system of severely immunocompromised victims exposed to ionizing radiation to recover from high doses of radiation. CD2F1 mice were irradiated with a dose of radiation causing GI injury (11 Gy, cobalt-60 gamma-radiation) and then transfused intravenously (retro-orbital sinus) with whole blood or peripheral blood mononuclear cells (PBMC) from TS- and AMD3100-injected mice 2, 24, or 48 h post-irradiation and monitored for 30-day survival. Jejunum sections were analyzed for tissue area, surviving crypts, villi, mitotic figures, basal lamina enterocytes, and bacterial translocation. Our results demonstrate that infusion of whole blood or PBMC from TS- and AMD3100-injected mice significantly improved survival of mice receiving a high dose of radiation. Histopathology and immunostaining of jejunum from irradiated and TS- and AMD3100-mobilized PBMC-transfused mice reveal significant protection of GI tissue from radiation injury. We observed that the infusion of PBMC from TS- and AMD3100-injected mice significantly inhibited bacterial translocation to various organs compared to mice receiving cells from vehicle-mobilized cells. We demonstrate that TS and AMD3100 mobilize progenitors into peripheral circulation and that the infusion of mobilized progenitor-containing blood or PBMC acts as a bridging therapy for immune system recovery in mice exposed to potentially fatal doses of ionizing radiation. With further preclinical investigation in large animals, we may approach a simpler, improved protocol for the clinical management of individuals suffering from high ionizing radiation doses causing GI syndrome.

Speaker: Dr Vijay Singh (Armed Forces Radiobiology Research Institute, USUHS, Bethesda, MD 20889, USA)

242 Radiation-induced cancer cell possibility to differentiate into cancer stem cell theory verification

Ionizing radiation (IR) has been proven to be a powerful medical treatment in cancer therapy. Rational and effective use of its killing power depends on understanding IR-mediated responses at the molecular, cellular and tissue levels. Increasing evidence supports that cancer stem-like cells (CSCs) play an important role in tumor regrowth and spread post radiotherapy, for they are resistant to various therapy methods including radiation. Researchers* at UCLA's Jonsson Comprehensive Cancer Center Department of Oncology found that that ionizing radiation reprogrammed differentiated breast cancer cells into induced breast cancer stem cells (BCSCs). This team found that these cells had more than 30-fold increased ability to form tumors compared with non-irradiated breast cancer cells. It is very important to verify in what dose the cancer cells may differentiate to cancer stem cells for the better prevention and effective medical treatment. In this investigation it was checked if cancer cell after irradiation with various dose (1-30Gy) of proton radiation may differentiate into cancer stem cells. It was noticed that cancer cells after irradiation (1Gy) changed their morphology . The survival factor was 67%. Evidence show that cancer cells are expressing different surface markers compared with cancer stem cells, it is also known that these cells have clearly different viability mechanism. To identify cancer stem cells from cancer cells the surface markers was used. The cell proliferation assay kit was used to measure proliferation. Viability was evaluated in two different methods: first with trypan blue (0,4%) using hemocytometer, second with propidium iodide and calcein using flow cytometer. With flow cytometer it was also analyzed cell cycle using Hoechst 33342 dye. For the survival fraction calculations all cells after irradiation were cultured into 6-well cell culture plate and analyzed by the colony clonogenic method. * Lagadec C and et al., Radiation-induced reprogramming of breast cancer cells. Stem Cells. 2012 May;30(5):833-44

Speaker: Ms Aušra Liubavičiūtė (Vilnius university Center for Physical Sciences and Technology)

243 Glioblastoma stem cells and radiation: metabolism and radiation sensitivity

Among brain cancers, glioblastoma multiforme (GBM) is the most aggressive form with a very poor prognosis. Surgery is the first-line treatment for GBM patients, followed by combined radio- and chemotherapy. In the most recent models of cancer, the presence of cells with stem-like properties is deemed to contribute to high recurrence rate and failure of conventional treatments. Literature data reports that gamma irradiation of GBM cells produced an increased fraction of stem-like cells in the residual tumour population compared with that in non-irradiated tumours. This was attributed to activation of DNA damage checkpoint response and increase in DNA repair capacity. Recent studies on gliomas based on microarray expression profiling as well as gene analysis have identified molecular subtypes associated with tumour grade, progression, and patient survival. Although these studies could not reach conclusive statements nor were able to predict more precise clinical outcomes for GBM patients, their relevance consists in revealing that gliomas can be divided into subtypes of different prognostic significance. In this framework, we have identified and characterized the “metabolite phenotypes” of thirteen glioblastoma stem cell (GSC) lines by 1H NMR spectroscopy through unsupervised analysis of spectral features. This study yielded two clusters with six lines belonging to a more “neural-like” and seven to a more “glioma-like” metabolic phenotype. Two GSC lines belonging to the two different metabolic clusters identified by 1H NMR, namely line 1 and line 83, were then examined after gamma irradiation with a Cs-137 source. Viability and cell death were analyzed in the dose range 1-10 Gy by colorimetric methods and cytotoxicity assays while apoptosis was evaluated in parallel by flow cytometry measurements using Propidium Iodine and Annexin-V. The overall results have shown that line 83 cells are much more radioresistant than line 1. Moreover, cell metabolism monitored by 1H NMR 48 hr after 10 Gy irradiation showed changes in mobile lipid signals and in signals of glu-gln metabolic pathway related to cell cycle check points and to oxidative stress response, respectively, for line 1 while no differences between irradiated and control cells appeared for line 83. These preliminary results suggest the presence of different mechanisms of radioresistance acting in the two GSC lines studied.

Speaker: Dr Antonella Rosi (Departement Technology and Health, Istituto Superiore di Sanità, Rome, Italy; INFN, Sez. Roma1-gruppo collegato Sanità, Rome, Italy)

12:30 Closing