Post on 02-Dec-2018
European Radiation Research 2018, August 21-25, Pécs, Hungary
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European Radiation Research
2018
The 44th Annual Meeting of the European
Radiation Research Society
Pécs, Hungary, August 21-25, 2018
Under the auspices of the European Radiation
Research Society and the Hungarian Biophysical
Society
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Dear Colleagues,
It is our great pleasure to welcome you to European Radiation Research 2018.
European Radiation Research 2018 (ERR2018) is the 44th Congress organized under
the auspices of the European Radiation Research Society (ERRS). One of the major
goals of ERRS is to encourage international cooperation and communication in all
fields of radiation research. To fulfill this goal ERR2018 brings together specialists
from all the disciplines involved in radiation science.
ERR2018 gives a unique occasion to highlight the most recent knowledge’s in
radiation research and to dialogue among radiation scientists from different countries
of the world. We are grateful that so many highly respected scientists have accepted
our invitation to lecture at this meeting. The submitted abstracts reflect the high
standard of radiation research in Europe and throughout the world. We are delighted
that we received a substantial number of contributions from other continents indicating
the international interest in ERRS meetings.
On the behalf of the Organizing Committee we wish you a pleasant stay in Pécs
and hope that you will enjoy all aspects of the congress.
Géza Sáfrány Katalin Lumniczky
Congress President Congress Secretary General
László Mátyus Lorenzo Manti
President President
Hungarian Biophysical Society European Radiation Research Society
European Radiation Research 2018, August 21-25, Pécs, Hungary
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European Radiation Research 2018 is organized under the auspices of the European
Radiation Research Society and the Hungarian Biophysical Society
Gold Sponsors
and
http://www.pxinc.com/ http://www.accela.eu/
The organizers also acknowledge the generous support of
University of Pécs, Medical School
http://aok.pte.hu/en
National Public Health Institute
https://www.oki.hu/
Hungarian Biophysical Society
http://www.mbft.hu/index-en.php
Radiobiology Foundation
Soft Flow Bioscience
http://www.softflowbio.com/
Frank Diagnosztika
http://www.frank-diagn.hu/
Self Publishing Editor: G. Sáfrány
Scientific abstracts in this book are summaries of oral and poster presentations given at ERR2018. No peer-review was performed. The copyright remains with the authors of the respective contributions
Printed in PTE-ÁOK Nyomda, Pécs, Hungary, 2018
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Committees
Organizing Committee
Sáfrány G. Congress President
Lumniczky K. Secretary General
Pusztai M. Treasurer
Jendrolovics K. Secretariat
International Scientific Committee
Manti L. President
Atkinson M.
Baatout S.
Benderitter M.
Benotmane A.
Borgmann K.
Bouffler, S.
Dörr W.
Haghdoost S.
Hammond E.
Hofer M.
Janiak JM.
Kadhim M.
Kraft G.
Lyng F.
Moreels M.
Mothersill C.
Sabatier L.
Sminia, P.
Tapio, S.
Hungarian Scientific Committee
Nyitrai M. President
Hideghéty K.
Jurányi Zs.
Lumniczky K.
Madas B.
Thuróczy Gy.
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Program Committee
Lumniczky K. President
Madas B.
Szatmári T.
Hargitai R.
Congress Secretariat Kitty Jendrolovics
Department of Radiobiology and Radiohygiene, Public Health Directorate,
National Public Health Institute
1221 Budapest,
Anna u. 5.
Hungary
Tel. +36-1-4822010
E-mail: info@err2018.hu
European Radiation Research 2018, August 21-25, Pécs, Hungary
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General information
Congress Venue
University of Pécs, Medical School (http://aok.pte.hu/about-the-school)
7624 Pécs, Szigeti út 12.
Badge
Participants are requested to wear their badge at all times during the conference.
The badge serves as an entrance ticket to the scientific sections, and to all social
events.
Insurance
The organizers do not accept liability for individual medical, travel or personal
insurance and participants are strongly advised to take out their own personal
insurance policies.
Language
The official language of the meeting is English. No simultaneous translations will
be provided.
Poster presentations
The size of the posters should be 90 cm (width) x 120 cm (height). All posters are
displayed through August 23, Thursday and 24, Friday. Posters should be
mounted on August 23, before 9:00 AM and removed on August 24 after 18:00.
The organizers are not responsible for loss of posters.
Projection facilities
Power point presentations should be used.
Slides must be presented at the projection desk at least 15 minutes before the start
of the corresponding section.
Social events
Welcome Reception will be organised on August 21, Tuesday at 19:00.
An Organ Concert is enjoyed at 19:30 on August 22, Wednesday at Cathedral of
Saint Peter and Saint Paul.
The Congress Dinner will be held on August 23, Thursday at 19:30 in Mokos
vineyard, Palkonya. Busses will depart from the Congress at 18.45.
The Congress Diner is open for all registered participants. Non-registered persons
can buy a ticket at the Congress Secretariat to attend the Dinner.
European Radiation Research 2018, August 21-25, Pécs, Hungary
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AWARDS
Bacq & Alexander Award offered by ERRS
Kevin M. Prise
Young Investigators Awards offered by ERRS
MICHAELIDESOVÁ, Anna
TSALKOVA, Yuliya
CRAENEN, Kai
PISCIOTTA, Pietro
TABASSO, Antonella
KIS, David
PETRINGA , Giada
WOZNY, Anne-Sophie
FABUSHAVA, Kseniya
O-BRIENNE, Grainne
FRENEAU, Amélie
VERMEULEN, Stephanie
BASELET, Bjorn
RAMADAN, Raghda
MEDIPALLY, Dinesh
PUSTOVALOVA, Margarita
BRUNNER, Szilvia
BALAZS, Katalin
SHAFAROST, Krystsina
SHAFAROST, Aliaksandr
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Program Overview
Tuesday, August 21, 2018
14:00-18:00 Registration
18:00-18:15 Opening Ceremony, Welcome Address Lecture Room III.
18:15-18:30 Gold Sponsor presentation Lecture Room III.
18:30-19:15 Opening lecture Lecture Room III.
19:30 Welcome Reception
Wednesday, August 22, 2018
09:00-09:45 Keynote presentation Lecture Room III.
10:00-11:10 Stem cell radiation biology Lecture Room III.
10:00-11:10 Multidisciplinary European Low Dose Initiative
Lecture Room IV.
11.10-11.40 Coffee Break
11:40-13:00 Effects of non-ionizing radiations Lecture Room III.
11:40-13:00 Multidisciplinary European Low Dose Initiative
Lecture Room IV.
13:00-14:00 Lunch
European Radiation Research 2018, August 21-25, Pécs, Hungary
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13:00-14:00 ERRS council meeting
14:00-15:00 Bacq & Alexander Award Lecture Lecture Room III.
15:00-15:30 Coffee Break
15:30-18:00 Radiation effects on the immune system Lecture Room III.
15:30-18:00 Laser driven ionizing radiation and biomedical application
Lecture Room IV.
19:30 Organ concert at the Cathedral of Saint Peter and Saint Paul
Thursday, August 23, 2018
09:00-09:45 Keynote presentation Lecture Room III.
10:00-11:20 Radiation and exosomes Lecture Room III.
10.00-11.20 Radioecology meets radiobiology Lecture Room IV.
11:20-11:50 Coffee Break
11:50-13:00 Radiation and exosomes Lecture Room III.
11:50-13:00 Pre-clinical and clinical high LET radiobiology
Lecture Room IV.
13:00-14:00 Lunch & Poster Session
14:00-15:00 Poster Session
15:00-15:45 Keynote presentation Lecture Room III.
European Radiation Research 2018, August 21-25, Pécs, Hungary
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15:45-16:15 Coffee Break
16:15-18:00 Radon as an environmental and a therapeutic agent
Lecture Room III.
16:15-18:00 Pre- and clinical research supporting human radiotherapy I.
Lecture Room IV.
19:30 Congress dinner at Mokos vineyard, Palkonya
Friday, August 24, 2018
09:00-09:45 Keynote presentation Lecture Room III.
10:00-11:20 Individual radiosensitivity and radiosusceptibility
Lecture Room III.
10.00-11.20 Radiation hormesis Lecture Room IV.
11:20-11:50 Coffee Break
11:50-13:00 Individual radiosensitivity and radiosusceptibility
Lecture Room III.
11:50-13:00 Non-targeted effects of ionising radiation
Lecture Room IV.
13:00-14:00 Lunch & Poster Session
14:00-15:00 Poster Session
European Radiation Research 2018, August 21-25, Pécs, Hungary
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15:00-15:45 Keynote presentation Lecture Room III.
15:45-16:15 Coffee Break
16:15-18:15 Radiation effects on the brain
Lecture Room III.
16:15-18:30 Pre- and clinical research supporting human radiotherapy II.
Lecture Room IV.
18:35-19:30 ERRS General Assembly Lecture Room III.
Saturday, August 25, 2018
09:00-10:00 Keynote presentation Lecture Room III.
10:00-11:20 Radiation-induced repair, inter- and intracellular signalling
Lecture Room III.
10.00-11.20 Radiation-induced carcinogenesis and senescence
Lecture Room IV.
11:20-11:50 Coffee Break
11:50-13:00 Radiation-induced repair, inter- and intracellular signalling
Lecture Room III.
11:50-13:00 Proton and high LET radiation effects Lecture Room IV.
13.15 Closing Ceremony Lecture Room III.
13:30-14:15 Lunch
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Detailed Program
Tuesday, August 21, 2018
14:00-18:00 Registration
18:00-18:15 Opening Ceremony, Welcome Addresses Lecture Room III.
18:15-18:30 Gold Sponsor presentation Lecture Room III.
Chairpersons: G. Sáfrány and L. Manti
18:15-18:30 Precision X-Ray Inc.: Small animal, pre-clinical X-Ray irradiation
systems
18:30-19:15 Opening lecture Lecture Room III.
Chairpersons: G. Sáfrány and L. Manti
18:30-19:15 Zs. Izsvák: Non-viral Sleeping beauty-based vector for Immunotherapy
to fight against cancer
19:30 Welcome Reception
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Wednesday, August 22, 2018
09:00-9:45 Keynote presentation Lecture Room III.
Chairperson: T. Szatmári
9:00-9:45 N. Alessio, D. Aprile, T. Squillaro, G. Di Bernardo and U. Galderisi:
Low dose radiation and aging: a study on mesenchymal stem cells
10:00-11:10 Stem cell radiation biology Lecture Room III.
Chairpersons: U. Galderisi and T. Szatmári
10:00-10:20 O. Arrizabalaga, E. Nasonova, IS. Schroeder and S. Ritter: Exposure to
ionizing radiation affects the characteristics of neural stem cells
10:20-10:35 M. Pustovalova, A. Grekhova, N Vorobyeva, AN. Osipov: Early and
delayed effects of low-dose x-ray exposure in human mesenchymal stem
cells: DNA double-strand breaks, proliferation, senescence.
10:35-10:50 J. Konířová, L. Cupal, M. Zíková, A. Michaelidesová, J. Vachelová and
M. Davídková: Response of neural stem cell to photon radiation
10:50-11:10 AS Wozny, G. Vares, G. Alphonse, C. Monini, N. Magné, C. Cuerq, A.
Fujimori, JC. Monboisse, M. Beuve, T. Nakajima and C. Rodriguez-
Lafrasse: Spatial ROS distribution contributes to the differences in the
invasion/migration processes of cancer stem cells in response to photons
and carbon ions.
10:00-11:10 Multidisciplinary European Low Dose Initiative
Lecture Room IV.
Chairpersons: S. Tapio and B. Madas
10:00-10:10 S. Tapio: Introduction to Multidisciplinary European Low Dose
Initiative (MELODI)
European Radiation Research 2018, August 21-25, Pécs, Hungary
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10:10-10:30 BG. Madas, A. Auvinen, E Cardis, Ma. Durante, M. Harms-Ringdahl,
JR. Jourdain, M. Kreuzer, A. Ottolenghi, S. Pazzaglia, KM. Prise, R.
Quintens, L. Sabatier and S. Bouffler: Research prioritisation in
MELODI
10:30-11:10 J. Hall: Radiation Biomarkers: fact or fiction?
11.10-11.40 Coffee Break
11:40-13:00 Effects of non-ionizing radiations Lecture Room III.
Chairpersons: Gy. Thuróczy and MO. Mattsson
11:40-12:05 MO. Mattsson and M. Simkó: Non-ionising electromagnetic fields in
medical applications
12:05-12:25 Gy. Thuróczy, N. Nagy, G. Jánossy and Zs Jakab: TransExpo:
International study of childhood leukemia and residences near electrical
transformer rooms
12:25-12:45 AM. Marjanovic Cermak, I. Pavicic and I. Trosic: Cellular response to
short-term 1800 MHz radiofrequency radiation exposure
12:45-13:00 I. Gresits, F. Simon, Gy. Thuróczy: Non-calorimetric determination of
absorption power during magnetic nanoparticle based hyperthermia
11:40-13:00 Multidisciplinary European Low Dose Initiative
Lecture Room IV.
Chairpersons: S. Tapio and B. Madas
11:40-12:20 R. Haylock and M. Gillies: INWORKS and an updated NRRW analysis
12:20-13:00 S. Tapio: Potential mechanisms contributing to radiation-induced heart
disease
13:00-14:00 Lunch
European Radiation Research 2018, August 21-25, Pécs, Hungary
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14:00-15:00 Bacq & Alexander Award Lecture Lecture Room III.
Chairpersons: L. Manti and G. Sáfrány
KM. Prise: From Radiation Tracks to Immune Signalling: a Bystander
Perspective
15:00-15:30 Coffee Break
15:30-18:00 Radiation effects on the immune system
Lecture Room III.
Chairpersons: US. Gaipl and K. Lumniczky
15:30-16:10 SM. Candéias: Radiation-induced bystander signals modulate immune
cell activation
16:10-16:50 B. Frey, M. Rückert, A. Derer, M Hader, R. Fietkau, US. Gaipl: Immune
modulation by radiation – impact for the design of
radioimmunotherapies
16:50-17:10 E. Persa, T. Szatmári, G. Sáfrány, K. Lumniczky: In vivo irradiation
effects on activation of dendritic cells in mice
17:10-17:30 F. Rapp, US. Gaipl, B. Frey, A. Donaubauer, I. Becker, L. Deloch, F.
Rödel, S. Hehlgans, S Ritter, K. Shreder, A. Maier, C. Hartel, G. Kraft
and C. Fournier: Radon exposure and its impact on the immune system
and genetic risks
17:30-17:45 M. Bugden, K. Thomas, D. Li and Y. Wang: Combining Low Dose
Radiation and Immune Checkpoint Therapy for Cancer Treatment
17:45-18:00 N.R.E.N. Impens: Is metabolic syndrome affecting the adverse outcomes
of low doses or dose rates of ionising radiation?
European Radiation Research 2018, August 21-25, Pécs, Hungary
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15:30-18:00 Laser driven ionizing radiation and biomedical application
Lecture Room IV.
Chairpersons: E. Beyreuther and K. Hideghéty
15:30-16:10 E. Beyreuther: Radiobiological characterization of laser driven particle
beams – Dresden activities
16:10-16:30 D. Papp and C. Kamperidis: Laser-based radiation sources for
biomedical applications
16:30-17:10 K. Hideghéty, R. Polanek, RE Szabó, Sz. Brunner, T. Tőkés: Biomedical
potential of laser-driven particle acceleration
17:10-17:30 R. Polanek, D. Papp, C. Kamperidis and K. Hideghéty: Electron beams
accelerated by the proposed SYLOS laser system at ELI-ALPS – A
Monte Carlo dosimetry characterization
17:30-17:45 ER. Szabó, T. Tőkés, R. Polanek, Sz. Brunner, Sz. Czifrus, A. Fenyvesi,
B. Biró, E. Beyreuther, J. Pawelke, K. Hideghéty: Simple vertebrate
model development for radiobiology research at ELI-ALPS on laser
driven hadron beams
17:45-18:00 Sz. Brunner, T. Tőkés, ER. Szabó, IZ. Szabó, R Polanek, E.
Beyreuther3, J. Pawelke, K. Hideghéty: Zebrafish model for
investigation on the biological effects of proton beam
19:30 Organ concert at the Cathedral of Saint Peter and Saint
Paul
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Thursday, August 23, 2018
09:00-09:45 Keynote presentation Lecture Room III.
Chairperson: M. Kadhim
9:00-9:45 V. Jendrossek: Linking radiation-induced DNA-damage to systemic
anti-tumor responses and normal tissue toxicity
10:00-11:20 Radiation and exosomes Lecture Room III.
Chairpersons: M. Kadhim and K. Lumniczky
10:00-10:40 R. AL-Abedi, A. Mayah, S. Brooks and M. Kadhim: Ionising radiation
enhance metastasis/invasiveness capacity of unirradiated cells: The role
of extracellular vesicles /exosomes
10:40-11:20 M. Rezvani: Treatment of Radiation Lesions with Secretome of Stem
Cells
10.00-11.20 Radioecology meets radiobiology Lecture Room IV.
Chairpersons: C. Mothersill and F. Bréchignac
10:00-10:20 C. Mothersill and C. Seymour: Genomic Instability and non-targeted
effects; are they important for environmental radiation protection?
10:20-10:40 F. Bréchignac: Neglecting the ecosystem concept in radiological
protection is prone to jeopardize the pertinence of environment
protection measures
10:40-11:00 PN. Schofield: A role for epigenetic mechanisms in the effects of low
dose radiation on ecosystems
11:00-11:20 N.R.E.N. Impens: Joint Roadmap for Radiation Protection Research:
Focusing on the radiobiology-radioecology interface.
11:20-11:50 Coffee Break
European Radiation Research 2018, August 21-25, Pécs, Hungary
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11:50-13:00 Radiation and exosomes Lecture Room III.
Chairpersons: M. Kadhim and K. Lumniczky
11:50-12:20 K. Lumniczky, T. Szatmári, E. Persa, D. Kis, N. Sándor, R. Hargitai, G.
Sáfrány: The role of extracellular vesicles in mediating or mitigating
ionizing radiation effects in the bone marrow
12:20-12:40 A. Abramowicz, M. Smolarz, L. Marczak, MD. Story, M. Pietrowska, P.
Widlak: Ionizing radiation affects the composition of the proteome of
exosomes released by head and neck carcinoma in vitro.
12:40-13:00 MJ. Atkinson, L. Mutschelknaus, M. Schneider, O. Azimzadeh, G.
Olatunji, R. Yentrapalli, N. Anastasov, VB. O'Leary, S. Tapio and S.
Moertl: Do radiation-induced changes in exosomal cargo predict
survival?
11:50-13:00 Pre-clinical and clinical high LET radiobiology
Lecture Room IV.
Chairpersons: L. Manti and T. Friedrich
11:50-12:10 L. Manti: Effect of normal-cell premature senescence on tumour-cell
proliferative potential following charged particle irradiations
12:10-12:25 J. Reindl, K. Ilicic, W. Friedland, T. Friedrich, S. Girst, C. Greubel1, M.
Sammer, B. Schwarz, C. Siebenwirth, D.W.M. Walsh, A.A. Friedl, T.E.
Schmid, M. Scholz and G. Dollinger: Understanding the enhanced
radiobiological effectiveness of high-LET particles utilizing ion
mircoirradiation and super resolution microscopy.
12:25-12:40 B. Baselet, O. Azimzadeh, N. Erbeldinger, MV Bakshi, T. Dettmering,
A. Janssen, S. Ktitareva, D. Lowe, A. Michaux, Q. Roel, K. Raj, M.
Durante, C. Fournier, MA Benotmane, S. Baatout, P. Sonveaux, S.
Tapio and A. Aerts: The cardiovascular system in space: differential
impact of single-dose Fe ion and X-ray irradiation on endothelial cells.
12:40-13:00 T. Friedrich and M. Scholz: Strategies for model validation in
radiobiology
13:00-14:00 Lunch & Poster Session
European Radiation Research 2018, August 21-25, Pécs, Hungary
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14:00-15:00 Poster Session
15:00-15:45 Keynote presentation Lecture Room III.
Chairperson: B. Madas
15:00-15:45 G. Kraft: Radiation Research and Society
15:45-16:15 Coffee Break
16:15-18:00 Radon as an environmental and a therapeutic agent
Lecture Room III.
Chairpersons: G. Kraft and B. Madas
16:15-16:40 J. Wiedemann, V. Grünebaum, M. Dornhecker, S. Lerchl and C.
Fournier: Establishment of a Psoriatic Skin Model for α-Irradiation
16:40-17:00 BG. Madas and EJ. Drozsdik: Searching for correlations between the
spatial and temporal aspects of the biological and health effects of
inhaled radon progeny.
17:00-17:20 M. Fejgl, K. Jílek, J. Lenk, A. Froňka and I. Hupka: Measurement of the
air exchange rate in buildings with elevated levels of indoor radon
concentration using VOC tracers
17:20-17:40 Zs. Homoki: Radiation levels in Hungarian buildings
17:40-18:00 A. Maier, F. Papenfuß, M. Schmitt, G. Kraft and C. Fournier: Radon
solubility in different materials
16:15-18:00 Pre- and clinical research supporting human radiotherapy I.
Lecture Room IV.
Chairpersons: C. Hoeschen and Zs. Jurányi
16:15-16:35 E. Beyreuther: Radiation quality influence on normal tissue cell
response
European Radiation Research 2018, August 21-25, Pécs, Hungary
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16:35-16:55 K. Hideghéty, ER. Szabó, R. Polanek, Sz. Brunner, T. Tőkés: Different
in vivo models for research on normal tissue toxicity due to ionizing
radiation
16:55-17:15 C. Hoeschen: How to take care of the largest man-made source for
radiation exposure – medical applications – by structured research and
harmonization of practices
17:15-17:30 T. Suckert, J. Müller, E. Beyreuther, M. Gotz, F. Tillner, M. Schürer, A.
Dietrich, R. Bütof, A. Lühr, C. von Neubeck, M. Krause: Normal tissue
reaction following proton irradiation of the mouse brain
17:30-17:45 G. Petringa, L. Manti, F. Cammarata, G. Cuttone, D. Margarone, A.
Picciotto, P. Pisciotta, L. Giuffrida, G. Russo1, V. Scuderi, and G.A.P.
Cirrone: The Proton Boron Capture Therapy: a new proton therapy
enhancement technique
17:45-18:00 A. Michaelidesová, J. Vachelová, K. Pachnerová Brabcová, J. Konířová,
V. Vondráček and M. Davídková: An in-vitro study of Boron Proton
Fusion Therapy
19:30 Congress dinner at Mokos vineyard, Palkonya
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Friday, August 24, 2018
09:00-09:45 Keynote presentation Lecture Room III.
Chairperson: FM. Lyng
9:00-9:45 M. Ricoul, T. Gnana-Sekaran, P. Brochard and L. Sabatier: Molecular
cytogenetics: from biological dosimetry to deciphering mechanistic
effects
10:00-11:20 Individual radiosensitivity and radiosusceptibility
Lecture Room III.
Chairpersons: L. Sabatier and FM. Lyng
10:00-10:30 N. Foray: Individual radio-sensitivity and susceptibility: towards a
classification of human syndromes with the ATM nucleoshuttling model
10:30-11:00 K.I. Jönsson: Extreme radiation tolerance: lessons from tardigrades
11:00-11:20 CJ Talbot, D Azria, T Burr, J Chang-Claude, A Dunning, C Herskind, D
De Ruysscher, R Elliott, S Gutiérrez-Enríquez, P Lambin, A Müller, T
Rancati, B Rosenstein, T Rattay, P Seibold, L Veldeman, A Vega, F
Wenz, R Valdagni, A Webb, C West: The REQUITE project:
integrating biomarkers and clinical predictors of radiotherapy side
effects
10.00-11.20 Radiation hormesis
Lecture Room IV.
Chairpersons: MK. Janiak and C. Seymour
10:00-10:20 MK. Janiak, M. Wincenciak, A. Cheda and EM. Nowosielska: Cancer
immunotherapy with low-level whole-body exposures to ionizing
radiation
10:20-10:35 C. Seymour and C. Mothersill: Hormesis, chronic doses and
bioaccumulation – some heretical thoughts!
European Radiation Research 2018, August 21-25, Pécs, Hungary
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10:35-10:50 Y. Socol, YY. Shaki and L. Dobrzyński: Damped-oscillator model of
hormesis and its consequences
10:50-11:05 Y. Socol, YY. Shaki and A. Vaiserman: Thyroid cancer: over-
radiosensitivity or overdiagnosis?
11:05-11:20 C. Rangacharyulu and K. Olshanoski: Radiation Hormesis - A Universal
Phenomenon or an Uncommon Antidote?
11:20-11:50 Coffee Break
11:50-13:00 Individual radiosensitivity and radiosusceptibility
Lecture Room III.
Chairpersons: L. Sabatier and FM. Lyng
11:50-12:10 P. Schofield, U. Kulka, M. Gruenberger, B. Grosche, G. Woloschak and
Balazs Madas: FAIRing and TOPing in radiation science
12:10-12:25 M. Majewski, V. Vainstein, L. Basile, Z. Gluzman-Poltorak, J. Tom, M.
Port and M. Abend: Pre-exposure Gene Expression as Marker of
Individual Radiosensitivity in Non-human Primates
12:25-12:45 D.K.R. Medipally, A. Maguire, J. Byrant, V. Untereiner, GD
Sockalingum, J. Armstrong, AD. Meade and FM Lyng: Assessment of
risk of radiation toxicity in prostate cancer patients using high
throughput FTIR spectroscopy of non-invasive blood plasma samples
12:45-13:00 Zs.S. Kocsis, P. Ágoston, Gy. Farkas, M. Kun-Gazda, G. Székely, T.
Major, D. Mihály, G. Stelczer, K. Jorgo, L. Gesztesi, Cs. Polgár, Zs.
Jurányi: Chromosome aberration biodosimetry to compare three kind of
prostate radiotherapy
11:50-13:00 Non-targeted effects of ionising radiation
Lecture Room IV.
Chairpersons: YE. Dubrova and D. Hoorelbeke
11:50-12:10 YE. Dubrova: Radiation-induced mutation in the mammalian germline:
Where are we now?
European Radiation Research 2018, August 21-25, Pécs, Hungary
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12:10-12:30 D. Hoorelbeke, E. Decrock, M. De Smet, M. De Bock, B. Descamps, V.
Van Haver, T. Delvaeye, H. Thierens, D. Krysko, C. Van Hove, L.
Leybaert: Connexin channels and Ca2+/ROS/NO signalling are key
players in the radiation-induced bystander effect in microvascular brain
endothelial cells.
12:30-12:45 R. Ramadan, E. Vromans, D.C. Anang, E. Decrock, S. Baatout, L.
Leybaert, A. Aerts: Blocking hemichannels protects against radiation-
induced endothelial cell damage.
12:45-13:00 H. Steel, C. Box, U. Oelfke, S. Bartzsch: A role for the bystander effect
in the in vitro response to microbeam radiation.
13:00-14:00 Lunch & Poster Session
14:00-15:00 Poster Session
15:00-15:45 Keynote presentation Lecture Room III.
Chairperson: MJ. Atkinson
15:00-15:45 D. Bazyka, N. Gudzenko, I. Dyagil, I. Ilienko, D. Belyi, V. Chumak, A.
Prysyazhnyuk: Health Effects of Chornobyl: from Epidemiology to
Molecular Quantification
15:45-16:15 Coffee Break
16:15-18:15 Radiation effects on the brain
Lecture Room III.
Chairpersons: R. Benotmane and MJ. Atkinson
16:15-16:45 R. Benotmane: Long-term cerebrovascular and cognitive impairments
following Childhood exposure to radiation
16:45-17:05 M. Verslegers, E. Coninx, P. Verstraelen, M. Neefs, W.H. De Vos, L.
Moons, R. Quintens, S. Baatout: Hippocampal aging following early-life
X-ray exposure.
European Radiation Research 2018, August 21-25, Pécs, Hungary
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17:05-17:25 T. Tőkés, I. Plangár, E.R. Szabó, I. Mán, K. Brinyiczki, M. Boros, K.
Hideghéty: Acute and late consequences of partial brain irradiation in
rats
17:25-17:45 L. Mouton, O. Etienne, F. Boumezbeur, F. Boussin and D. LeBihan:
Assessment of long-term grey matter alterations in a mouse model of in
utero irradiation using anatomical and diffusion MRI.
17:45-18:00 K. Craenen, M. Verslegers, L. Craeghs, J. Buset, R. D’Hooge, S.
Baatout, L. Moons and R. Benotmane: Folic acid as a protectant against
prenatal irradiation-induced birth defects and sensory impairment
18:00-18:15 A.C.M. Mfossa, S. Baatout, R. Benotmane, D. Huylebroeck and R.
Quintens: Role of the novel p53 target D630023F18Rik, in embryonic
brain development and the cellular response to DNA damage.
16:15-18:30 Pre- and clinical research supporting human radiotherapy II.
Lecture Room IV.
Chairpersons: P. Sminia and B. Mukherjee
16:15-16:35 P. Sminia, R.M. de Kruijff, A.J.G.M van der Meer, G. Becerril Aragon,
A. Gasol Garcia, R.S. Narayan, S.M.A. van der Pol, F. Bikhezar, H.E.
de Vries, B.J. Slotman and A.G. Denkova: Nanocarriers for the delivery
of radiosensitizing agents and radionuclides to brain tumours.
16:35-16:55 B. Mukherjee: Non-conventional approaches of drug targeting: some
findings related to non-radioactive and radioactive labelled therapeutics
16:55-17:15 I. Prlić, M. Surić Mihić, M. Justić, L. Pavelić, T. Meštrović, M.
Hajdinjak, Z. Cerovac: Contemporary radiation protection trends in IR
and IC – new electronic dosimetry developments - Do we need a new
type of digital personal dosemeters?
17:15-17:30 N. Belmans, S. Baatout, R. Jacobs, I. Lambrichts and M. Moreels:
Increased oxidative stress and an adaptive antioxidant response in saliva
after dental CBCT exposure in children.
17:30-17:45 B. Baselet, S. Baatout, P. Sonveaux and A. Aerts: Ionizing radiation and
metabolism: can mitochondrial drugs be used for radiation protection of
the vascular system?
European Radiation Research 2018, August 21-25, Pécs, Hungary
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17:45-18:00 T. Hülber, Zs.S. Kocsis, E. Kis, G. Sáfrány, Cs. Pesznyák: Overview of
the performance parameters and unique features of a recently developed
automatic micronucleus assay evaluation system
18:00-18:15 S. Rudigkeit, J. Reindl, N. Matejka, M. Sammer, B. Schwarz, G.
Dollinger, D.W.M. Walsh: Long term imaging of cells after targeted
irradiation of mitochondria.
18:15-18:30 K. Olshanoski and C. Rangacharyulu: Beta and Gamma ray Dose
Estimates of Alpha Emitters for Brachytherapy Uses
18:35- ERRS General Assembly Lecture Room III.
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Saturday, August 25, 2018
09:00-10:00 Keynote presentation Lecture Room III.
Chairperson: W. Dörr
9:00-9:45 G. Iliakis: Logic and necessities in the choice of repair pathways
processing radiation-induced DNA double strand breaks
10:00-11:20 Radiation-induced repair, inter- and intracellular signalling
Lecture Room III.
Chairpersons: G. Iliakis and W. Dörr
10:00-10:20 E. Mladenov and G. Iliakis: Repair of CRISPR/Cas9 generated DSB
clusters with increased complexity
10:20-10:40 M. Hausmann, JH. Lee, E. Bobkova, E. Maus, F. Schmidt-Kaler, M.
Eryilmaz, E. Wagner, M. Krufczik, A. Hofmann, D. Depes, S.
Schumann, F. Bestvater, C. Cremer6, DW. Heermann, H. Scherthan, I.
Falkova, M. Falk, G. Hildenbrand: Analysis of the nano-topology of
repair clusters: Towards understanding of its impact on repair pathway
decision
10:40-11:00 S. Stouten, SV. Lunel and F. Dekkers: Towards a better understanding
of dose and dose-rate effectiveness factors
11:00-11:20 V. Todorovic, A. Prevc, M. Niksic Zakelj, P. Strojan, M. Cemazar and
G. Sersa: Response of isogenic head and neck cancer cell sublines to
ionizing irradiation
10.00-11.20 Radiation-induced carcinogenesis and senescence
Lecture Room IV.
Chairpersons: D. Klokov and R. Hargitai
10:00-10:20 G. O’Brien, L. Cruz Garcia, N. Brown, R. Finnon, J. Zyla, J. Polanska,
C. Badie: Tracking Sfpi1/PU.1 and Kras mutations in murine acute
myeloid leukaemia: role in radiation leukaemogenesis
European Radiation Research 2018, August 21-25, Pécs, Hungary
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10:20-10:35 Y. Shimada, M Nishimura, T. Imaoka, Y. Shang, K. Toshiaki, K.
Ariyoshi, M. Sunaoshi, R. Takei and S. Kakinuma: Age dependence of
cancer risks links to tissue stem/progenitor cell response to ionizing
radiation.
10:35-10:50 S. Kakinuma, J. Xiaohai, H. Tachibana, T. Morioka, K. Daino, C.
Tsuruoka, Y. Shang, Y. Amasaki, Y. Shimada: Cancer risk after
exposure to space radiation by pathological and genomic analyses
10:50-11:05 M. Flegal, L. Bertrand, M. Moustaqil, A Sansaloni, F. Heather, D.
Golubeva, I. Velegzhaninov, E. Plyusnina, A. Moskalev, S. Sebastian,
D. Klokov: Effects of Low-Dose Gamma-Radiation on Senescence in
vitro and in vivo.
11:05-11:20 F. Soysouvanh, A. Benadjaoud, M. Dos Santos, M. Mondini, J. Lavigne,
V. Buard, G. Tarlet, S. Adnot, E. Deutsch, O. Guipaud, V. Paget, A.
François and F. Milliat: Ionizing radiation-induced endothelial
senescence and role in normal tissue injury.
11:20-11:50 Coffee Break
11:50-13:00 Radiation-induced repair, inter- and intracellular signalling
Lecture Room III.
Chairpersons: G. Iliakis and W. Dörr
11:50-12:10 J. Vanderstraeten, J. Buset, N. Ben Said, B. Baselet, MC. Many, S.
Baatout, AC. Gérard, H. Derradji: Oxidative stress and VEGF
expression post-irradiation are modulated by iodine deficiency in breast
cells.
12:10-12:30 A. Freneau, A. Vaurijoux, P. Voisin, L. Roy and G. Gruel: Comparative
study of molecular and cellular effects induced by different energy X-
rays.
12:30-12:45 M. Temelie, N. Moisoi and DI. Savu: Intracellular and intercellular
signaling following DNA damage is modulated by the mitochondrial
kinase PINK1
European Radiation Research 2018, August 21-25, Pécs, Hungary
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12:45-13:00 A. Kumar, B. Babu, P. Mukherjee, S. Chandna: Mediatory role of miR-
31 in radiation-induced tumour cell death may be influenced by
(dys)functional status of p53.
11:50-13:00 Proton and high LET radiation effects
Lecture Room IV.
Chairpersons: GE. Kraft and TE. Schmid
11:50-12:10 T.E. Schmid, A. Hunger, M. Sammer, E. Zahnbrecher, J. Reindl, K.
Ilicic, D. Walsh, C. Greubel, B. Schwarz, J.J. Wilkens, G. Dollinger,
S.E. Combs: Proton minibeam radiotherapy (pMBRT) as an innovative
radiotherapy approach.
12:10-12:30 P. Pisciotta, FP. Cammarata, L. Minafra, V. Bravatà, GI. Forte, V.
Marchese, R. Acquaviva, R. Tringali, P. Cirrone, G. Petringa, G.
Cuttone, L. Manti, G. Russo: Cell and molecular response to proton
radiation treatments in breast cancer: in vitro models and in vivo
applications.
12:30-12:45 L. Bruni, M. Manghi, W. Tinganelli and S. Croci: New biological target
of ionizing radiations damages.
12:45-13:00 A. Fogtman, G. Weerts and U. Straube: Radiation health risks of human
space flight beyond Low Earth Orbit (LEO).
13.15 Closing Ceremony Lecture Room III.
13:30-14:15 Lunch
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Poster session
Pre- and clinical research supporting human radiotherapy
1. Annique C. Hunger, Matthias Sammer, Jannis Schauer, Benjamin Schwarz,
Dietrich W.M. Walsh, Judith Reindl, Stephanie E. Combs, Günther Dollinger
and Thomas E. Schmid: Dose-dependent skin inflammation after
hypofractionated X-ray radiotherapy in an in vivo mouse ear model
2. Roser Esplugas, Noemí Serra, Montserrat Bellés, Victoria Linares, Meritxell
Arenas, Joan Carles Vallvé: Effect of 2 Gy-radiation on the expression of
miRNAs-146a, -155, -221 and -222 in HUVECs
3. Roser Esplugas, Noemí Serra, Montserrat Bellés, Victoria Linares, Meritxell
Arenas, Joan Carles Vallvé: Effect of radiotherapy, molecular parameters and
treatment on the expression of blood miRNAs-146a, -155, 221 and -222 in
women with breast cancer
4. Márta Sárközy, Renáta Gáspár, Ágnes Zvara, Laura Kiscsatári, Zoltán Varga,
Bence Kővári, Mónika G. Kovács, Gergő Szűcs, Gabriella Fábián, Gábor
Cserni, László G. Puskás, Thomas Thum, Zsuzsanna Kahán, Tamás Csont,
Sándor Bátkai: Selective heart irradiation induces cardiac overexpression of the
pro-hypertrophic miR-212.
5. Iryna Ilienko, Dimitry Bazyka: Search of potential gene expression biomarkers
of radiation injury at the late period after exposure
6. Daniel Cullen, Adrian Maguire, Jane Bryant, Dinesh Medipally, John
Armstrong, Mary Dunne, Aidan Meade, Orla Howe and Fiona Lyng: Raman
spectroscopy to predict radiation toxicity in prostate cancer patients
7. Gyöngyi Farkas, András Bajcsay, Gyula Ostoros, Zsolt Markóczy, Zsuzsa S.
Kocsis, Márta Kun-Gazda, Gábor Székely, Dalma Mihály, József Lövey, Csaba
Polgár, Zsolt Jurányi: Relationship between biodosimetry and respiratory
function values in lung stereotactic radiotherapy patients
European Radiation Research 2018, August 21-25, Pécs, Hungary
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8. Shinya Yokomizo, Mayumi Nishimura, Takamitsu Morioka, Yukiko Nishimura,
Chizuru Tsuruoka, Yi Shang, Kazumasa Inoue, Masahiro Fukushi, Shizuko
Kakinuma, Shimada Yoshiya: Environmental Enrichment Regulates Radiation-
Induced Apoptosis in The Mouse Intestinal Crypt
9. Minyoung Lee, Da Yeon Kim, Jae Youn Yi and Eun Ju Kim: When radiation
exposure: Energy metabolism mechanism of mitochondria by radiation in
skeletal muscle
10. Sridhar Muthusami, Prabakaran DS, Sivaraman T, Jae Ran Yu, Woo-Yoon
Park: FTS regulates ROS-induced Cu-Zn SOD expression in cervical cancer
cells
11. Yannick Saintigny, Vidhula Ahire, Marie Brocquehaye: Effect of different
radiation qualities on functional behavior of cancer cells using a bio-printed 3D
comprehensive model of chondrosarcoma embedded in healthy cartilage.
12. Roxana Cristina Popescu, Ecaterina Andronescu, Mihai Straticiuc, Cosmin
Mustaciosu, Mihaela Temelie, Laurentiu Mogoanta, George Dan Mogosanu,
Bogdan Vasile, Adina Boldeiu, Alexandru Mihai Grumezescu, Mihai Radu,
Marlon R. Veldwijk, Diana Savu: Low dose radiotherapy enhances iron oxide
nanoparticles internalizing and toxicity for MG-63 osteosarcoma cells
13. Rozina Aktar, Antje Dietrich, Anthony Chalmers, Michael Baumann,
Mechthild Krause, Rebecca Bütof: Establishment of an orthoptic glioblastoma
model by using G7-mCherry in small animals
14. Antoni Więdłocha, Ewa M. Nowosielska, Aneta Cheda, Marek K. Janiak:
Preclinical evaluation of whole-body irradiations with low doses of X-rays
combined with inhibition of immune checkpoints and a heat shock protein as a
novel therapy for lung cancer
15. Romuald Brzozowski, Bogusława Kucikowicz-Gleń, Tadeusz Strózik, Dorota
Wróblewska, Milena Zarębska: Quality control of the physical parameters of
medical radiological equipment
European Radiation Research 2018, August 21-25, Pécs, Hungary
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16. Ritter Zsombor, Balogh Péter,
Szöllősi Dávid, Schmidt Erzsébet, Michael
Irvine, David Tuch, Kunal Vyas, Horváth Ildikó, Máthé Domokos, Zámbó
Katalin: Application of CLI (Cherenkov Luminescence Imaging) and other
imaging modalities in the preclinical research
17. Mohammed Taqee Ansari: Herbal Medicine Today: Clinical and Research
Issues
18. Rita Hargitai, Sisko Salomaa, Päivi Roivainen and Katalin Lumniczky: Effect
of ionising radiation on the mitochondrial DNA damage in hair bulbs
19. Aneta Cheda, Ewa M. Nowosielska, Jerzy Gebicki, Andrzej Marcinek, Stefan
Chlopicki and Marek K. Janiak:Pyridinium compounds as new potential radio-
protective and radio-remedial agents – summary of the project
Stem cell radiation biology, immune system effects, exosomes
20. Tünde Szatmári, Rita Hargitai, Nikolett Sándor, Eszter Persa, Enikő Kis, Géza
Sáfrány and Katalin Lumniczky: Ionizing radiation induces AML related
changes in the cargo of extracellular vesicles from blood and bone marrow
21. Savneet Kaur Bains, Scott Bright, Eman Elbakrawy, Ammar Mayah, Edwin
Goodwin and Munira Kadhim: MV/Exosomes mediate cellular aging response
to ionising radiation
22. Dávid Kis, Eszter Persa, Tünde Szatmári, Nikolett Sándor, Rita Hargitai, Géza
Sáfrány and Katalin Lumniczky: Extracellular Vesicles mediate Radiation-
Induced Bystander Effects in the Bone Marrow
23. Katalin Balázs, Tünde Szatmári, Enikő Kis, Noémi E. Bogdándi , Piotr Widlak,
Géza Sáfrány, Katalin Lumniczky: Radiotherapy-induced changes in cellular
and soluble immunological markers of head and neck cancer patients
24. Al-Massarani Ghassan: Effect of acute whole-body gamma irradiation on
circulating microparticles levels in rats.
European Radiation Research 2018, August 21-25, Pécs, Hungary
32
25. Jeeyong Lee, Jiwon Ko, Hyuntaik Im, and Jae Youn Yi: Analyzing the radio-
resistance of Melanomas via the Secretome
26. Elizaveta Y. Moskaleva, Yulia P. Semochkina, Valentina.G. Shuvatova, Alla
V. Rodina, Anastasia P. Kuvyrchenkova: Regulation of tumors development by
intact and irradiated mesenchymal stem cells from different mouse tissues
27. Ewa M. Nowosielska, Aneta Cheda, Robert Zdanowski, Sławomir Lewicki,
Marek K. Janiak: Effect of internal contamination with tritiated water on the
neoplastic colonies in the lungs, innate anti-tumour reactions, cytokine profile,
and haematopoietic system in radioresistant and radiosensitive mice.
28. Lukasz Cheda, Krzysztof Kilian, Paulina Hamankiewicz, Weronika Wargocka,
Paulina Gapska, Natalia Rozwadowska, Maciej Kurpisz, Zbigniew Rogulski:
Monitoring of myocardial regeneration with stem cells using isotopic imaging
method
29. Paul Schofield, Michael Gruenberger, Mandy Birschwilks, Shin Saigusa, Bernd
Grosche, Gayle Woloschak, Soile Tapio, Nick Beresford, and Ulrike Kulka:
The STORE database; a platform for data and resource sharing in radiation
biology, radioecology and epidemiology
Effects of particle-radiations
30. Takahito Suzuki, Hu Jun, Yuki Morita, Chanis Pornnumpa, Yuki Tamakuma1,
Kazuki Iwaoka, Masahiro Hosoda and Shinji Tokonami: Calibration experiment
of Passive type radon-thoron discriminative monitor using radon and thoron
calibration facilities at Hirosaki University
31. Mihai Radu, Liviu Craciun, Mihaela Temelie, Mihaela Bacalum, Mihai
Straticiuc, Ana Chiriacescu, Tiberiu Esanu, Radu Vasilache and Diana Savu:
New radiobiology setup for proton irradiation adapted at the TR19 cyclotron of
IFIN-HH
32. Kristina Bannik, Sabine Zitzmann-Kolbe, Sabrina Jarke, Marco Jarzombek,
Andreas Sutter, Gerhard Siemeister, Dominik Mumberg: Radiobiological
effects of α-particle radiation from Ra-223 in cancer cells
European Radiation Research 2018, August 21-25, Pécs, Hungary
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33. Mihaela Bacalum, Simona Dîrleci, Mihai Straticiuc, Ion Burducea, Radu
Andrei, Decebal Iancu, Radu Vasilache, Diana Savu and Mihai Radu: Set-up
optimization of IFIN-HH 3 MV TandetronTM and proton beam dosimetry for
radiobiology experiments
Radiation-induced repair, inter- and intracellular signalling
34. Sharif Mortoga, Veronika Mladenova, Emil Mladenov and George Iliakis:
Regulated systems of I-SceI expression for in-depth studies of the biological
effects of DSBs and DSB-clusters
35. Alžběta Filipová, Lenka Zárybnická, Aleš Tichý, Anna Lierová, Marcela
Jeličová and Zuzana Šinkorová: Sodium orthovanadan inhibits p-53 mediated
apoptosis
36. Ioanna Karachristou, Antonio Pantelias, Gabriel Pantelias, Georgia Terzoudi:
The use of Centromeric/Telomeric PNA probes in prematurely condensed
peripheral blood lymphocyte chromosomes for partial body high dose
assessment
37. Stephanie Vermeulen, Anne Vral, Ans Baeyens: Receptor mediated
radiosensitivity and DNA repair capacity of breast cancer cell lines
38. Veronika Mladenova, Emil Mladenov and George Iliakis: Insights in the repair
of clustered double-strand breaks in human cells
39. Godoy, PRDV, Pour Khavari, A, Sakamoto-Hojo, ET, Saintigny Y, Haghdoost
S: Targeting cellular antioxidant system and DNA repair to sensitize glioma
stem cells to different LET radiation: low let chronic/acute dose rates (Cs137),
and high LET C-ions
40. Charlotte Webster, Ali Pour Khavari, Paulo Godoy and Siamak Haghdoost:
Analysis of DNA Repair Genes and Oxidative Stress in the Murine Brain
Following Acute and Chronic Ionizing Radiation Exposure
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Non-targeted effects of ionizing radiation
41. Kazumasa Sekihara, Kaori Saitoh, Haeun Yang, Takashi Miida, Keisuke Sasai
and Yoko Tabe: The direct and bystander effects of low-dose radiation on skin-
infiltrating model
42. Antonio Pantelias, Georgia Terzoudi, Ioanna Karachristou, Gabriel Pantelias:
Mechanistic insights into radiation induced complex chromosomal
rearrangements, genomic instability and chromothripsis
43. Marcus Unverricht-Yeboah, Ulrich Giesen, Ralf Kriehuber: Comparative gene
expression analysis after exposure to 123I-iododeoxyuridine, γ- and α-
irradiation – a possible tool for biodosimetry?
44. Tatsuhiko Imaoka, Yukiko Nishimura, Ken-ichi Kudo, Kiichi Kaminaga,
Akinari Yokoya, Noriko Usami, Mayumi Nishimura, Kazuhiro Daino, Daisuke
Iizuka, Yoshiya Shimada, Shizuko Kakinuma: Application of ex vivo and in
vivo models of rat mammary gland to study competition between irradiated and
non-irradiated cells
Radiation-induced carcinogenesis and senescence
45. Antonella FS Tabasso, Keyury Desai, Ioannis Lamprou, Donald JL Jones,
Salvador Macip & George DD Jones: Radiation-induced senescence: a possible
mechanism of resistance and a tumour-promoting pathway in NSCLC
46. Tamara V. Azizova, Gleb V Sychugov, Evgeniy L. Kazachkov, Evgeniya S.
Grigorieva and VS Revina: Plutonium-induced pulmonary fibrosis in nuclear
workers of the Mayak PA cohort exposed to internal alpha-radiation
47. Alla V. Rodina, Semen S. Arzumanov, Victor V. Safronov, Yulya P.
Semochkina, Marina Yu. Kopaeva, Elizaveta Yu. Moskaleva: Late
radiobiological effects in mice following combined low-dose extended gamma
and mixed gamma, neutron irradiation
48. Jin Xiaohai, Shizuko Kakinuma, Yutaka Yamada, Takamitsu Morioka,
Kazuhiro Daino, Yoshiya Shimada: Effect of age at exposure on the incidence
of thyroid lesions after γ-ray irradiation in mice
European Radiation Research 2018, August 21-25, Pécs, Hungary
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49. Fabusheva K. M., Veyalkina N.N., Sushko S.N: Carcinogenesis in the lungs of
mice line Af under the influence of radiation and chemical factors
Radioactivity measurement protocols
50. Vitold Filistovič, Rita Plukienė, Benedikta Lukšienė, Zita Žukauskaitė, Nikolaj
Tarasiuk, Evaldas Maceika, Laima Kazakevičiūtė: Analytical two component
kinetic sorption model for support of fixed-bed column experiments with
sophisticated sorbents
51. Ivan Hupka, Michal Fejgl, Miroslav Hýža: System development for
measurement of artificial radioactivity in surface water bodies
Effects of non-ionizing radiations
52. Péter Pál Necz, Noémi Nagy and György Thuróczy: Personal RF
microenvironmental exposimetry in kindergardens and nurseries
53. A.S. Shafarost: Determination of cluster DNA damage in testicular tissue after
chronic irradiation with EMR of 1800 MHz
54. Alena Tsukanava, Natalia Chueshova, Ihar Cheshyk, Natalia Veyalkina:
Combined effects of irradiation in a dose of 1.0 Gy and magnetic field of
industrial frequency (50Hz) on cells death sperm of rat
55. N.N. Veyalkina, K.N. Shafarost: Evaluation of the genotoxicity of UV
radiation by the micronucleus test on rats keratinocytes
56. Y. Shiroto, Y. Kitayama, R. Saga, H. Yoshino, S. Terashima, Y. Hosokawa,
and E. Tsuruga: Ultraviolet B degrades ciliary zonules in vitro
57. Mihaela Temelie, Mustaciosu Cosmin, Nicoleta Moisoi and Diana Savu: In
vitro neuronal differentiation leads to enhanced cellular vulnerability to
genotoxic stress induced by UV-irradiation
European Radiation Research 2018, August 21-25, Pécs, Hungary
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58. Zsuzsanna Németh, Györgyi Kubinyi, József Bakos, Brahim Selmaoui and
György Thuróczy: Effects of ultraviolet radiation on full thickness human skin
model in vitro (SKIN-RF project)
Late program change
1. Katrien Konings, Charlot Vandevoorde, Sofie Isebaert, Karin Haustermans,
Sarah Baatout and Marjan Moreels: X-rays versus protons: do they affect the
Hedgehog pathway and migration of cancer cells differently?
European Radiation Research 2018, August 21-25, Pécs, Hungary
37
Abstracts
The abstracts are in alphabetical order according to the first authors; the presenting
authors are shown in bold characters!
The abstracts are shown without any reformatting as submitted by the presenting
authors.
The meeting organizers take no responsibility for the content of the abstracts.
European Radiation Research 2018, August 21-25, Pécs, Hungary
38
Low dose radiation and aging: a study on mesenchymal stem cells
Nicola Alessio1, Domenico Aprile
1, Tiziana Squillaro
1, Giovanni Di Bernardo
1 and Umberto
Galderisi1,2
1 Luigi Vanvitelli Campania University, Department of Experimental Medicine, Naples, IT
2 Temple University, SBARRO Institute for Cancer Research and Molecular Medicine,
Philadelphia, PA, USA
Mesenchymal stromal/stem cells (MSCs) are pluripotent cells that reside in bone marrow, adipose tissue and
in the stroma of other organs and tissues. MSCs can differentiate into osteocytes, chondrocytes and
adipocytes. Furthermore, MSCs support hematopoiesis, contribute to the homeostasis of many tissues and
modulate inflammatory response.
The presence of MSCs in adipose tissue that is widespread distributed in our body increase the possibility that
they may receive genotoxic damage, such as ionizing radiation. Following damage, cells may accumulate
irreversible damages that trigger either senescence or apoptosis.
Senescence is a permanent cell cycle arrest that leads to loss of cellular functions. Several lines of evidence
correlate cellular senescence to aging.
We performed a preliminary in vitro investigation on environmental factors that can contribute MSC
senescence, given the key role that these cells play in our body. We demonstrated that low radiation exposure
of MSCs induced senescence that was associated with the presence of unrepaired DNA in cells’ nuclei.
Senescence of MSCs following radiation treatment induced changes in their secretome profiles. We evidenced
that senescent MSCs secreted several pro-aging factors that by paracrine action induced senescence of other
cells. Insulin growth factor binding proteins (IGFBPs) are among the most important pro-aging factors we
found in MSC secretome, since blocking their action greatly reduced the negative paracrine effects of
senescent MSCs.
This study prompted us to evaluate if environmental stress, such as those received with Computer tomography
(CT) analysis, can contribute to radiation gerontogen effect by inducing senescence of MSCs as well as of
other cells. These senescent cells can release pro-aging factors in serum, such as IGFBPs. Indeed, some
findings evidenced that IGFBP serum levels are associated with aging. In 25 patients we found a significant
increase of IGFBPs levels 48 hours after CT analysis.
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Establishment of an orthotopic glioblastoma model by using G7-mCherry in small animals
Rozina Aktar1,2
, Antje Dietrich1,2
, Anthony Chalmers6, Michael Baumann
1,2,3,4, Mechthild Krause
1,2,3,4,5,
Rebecca Bütof1,3,5
(1) ) OncoRay ̶ National Center for Radiation Research in Oncology, Faculty of Medicine and University
Hospital Carl Gustav Carus, Technische Universität Dresden, Helmholtz-Zentrum Dresden-Rossendorf,
Dresden, Germany (2) German Cancer Consortium (DKTK), Partner Site Dresden and German Cancer
Research Center (DKFZ), Heidelberg, Germany (3) Department of Radiotherapy and Radiation Oncology,
Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden,
Germany (4) Helmholtz-Zentrum Dresden – Rossendorf, Institute of Radiooncology ̶ OncoRay, Dresden,
Germany (5) ) National Center for Tumor Diseases (NCT), Partner Site Dresden, Germany: German Cancer
Research Center (DKFZ), Heidelberg, Germany; Faculty of Medicine and University Hospital Carl Gustav
Carus, Technische Universität Dresden, Dresden, Germany, and; Helmholtz Association / Helmholtz-Zentrum
Dresden - Rossendorf (HZDR), Dresden, Germany (6) University of Glasgow, England
Glioblastoma multiforme (GBM), accounting for 30% of adult brain tumors, is the most malignant and lethal
glial tumor with a median survival of less than 15 months. Therefore, animal models representing human
GBM in terms of initiation, progression and treatment resistance are needed for preclinical studies to predict
the clinical efficacy of newly developed treatment options. Orthotopic tumour models, i.e. transplantation of
tumour material into the appropriate neuroanatomical structures, are considered to better reflect the clinical
situation.
Here we have developed an orthotopic GBM model by transplanting the G7-mCherry cell line as cell
suspension with the help of a stereotactic system. Tumour development was imaged twice weekly by optical
imaging using in-Vivo Xtreme II and magnetic resonance imaging (MRI). Tumor histology was analyzed via
staining with hematoxylin and eosin and human origin of tumors was verified by a specific anti-human Ki-67
antibody.
Orthotopic tumour growth was observed in 66.6% of animals. Optical imaging was less sensitive (deep seated
tumours and autofluorescence from skin) to detect and monitor the tumour growth of the fluorescence
containing cell line, whereas MRI was shown to be more effective to determine anatomical location and
volume of the tumour. Moreover, fluorescence signal strength was unable to predict tumour volume.
Histological analysis confirmed development of an infiltrative glioblastoma tumour.
In the future, further cell lines will be orthotopically transplanted using the stereotactic technique. A
systematic comparison of radiobiological characteristics between these orthotopic tumours and their
subcutaneous counterparts is planned. The micromilieu parameters (e.g. vessels, perfusion, hypoxia) are of
major interest and results should contribute to an improvement in the evaluation of translational aspects of
radiooncological preclinical experiments.
European Radiation Research 2018, August 21-25, Pécs, Hungary
40
Ionising radiation enhance metastasis/invasiveness capacity of unirradiated cells: The role of extracellular vesicles /exosomes
Raheem AL-Abedi , Ammar Mayah , Susan Brooks, and Munira Kadhim
Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes
University, Oxford Objective, Metastasis is a multistep process, by which cancer cells dissociate from the initial site and travel to form new secondary tumours at distant sites. This process has been reported to require epithelial-mesenchymal transition (EMT), by which the epithelial cancer cells convert to a mesenchymal form, associated with increasing levels of vimentin and decrease in E-cadherin markers. Cancer metastasis can also associate with aberration in cells’ glycosylation. Ionising irradiation could have an effect on metastasis, by stimulating cancer cells to change from the epithelial to mesenchymal form. The cells behaviour could be mediated by production of extracellular vesicles / exosomes from irradiated cells, which may have an impact on un-irradiated cells. Methods, Human Breast Cancer (MCF7) cells were irradiated with 2 Gy x-ray and exosomes were purified from the media and transferred to unirradiated cells. All the groups were analysed for the induction of invasiveness markers in the recipient cells, these including Vimentin and E-cadherin as well as glycosylation (HPA). Results and Conclusion, High induction of invasiveness capacity was observed in the recipient cells due to the change in the level of EMT (Vimentin and E-cadherin) markers as well as glycosylation (HPA) compared to the cells treated with exosomes from unirradiated cells. Inhibiting both exosome cargo molecules (RNAs, and proteins) abrogated the ability of the exosomes derived from irradiated cells to increase the invasiveness behaviour or change the level of EMT markers and HPA. Data suggested that exosomes derived from irradiated cancer cells are frequently involved in the capacity of invasiveness and implicate a role for RNA and protein molecules of exosomes.
European Radiation Research 2018, August 21-25, Pécs, Hungary
41
Ionizing radiation affects the composition of the proteome of
exosomes released by head and neck carcinoma in vitro
Agata Abramowicz1, Mateusz Smolarz
1, Lukasz Marczak
2, Michael D. Story
3, Monika
Pietrowska1, Piotr Widlak
1
1 Maria Sklodowska-Curie Institute – Oncology Center, Gliwice, Poland
2 Institute of Bioorganic Chemistry, Poznań, Poland
2 UT Southwestern Medical Center, Dallas, USA
Objective: Exosomes are a subset of extracellular vesicles that form in a process involving endosomes and
multivesicular bodies and have a diameter in the 30-150 nm range. These vesicles are released into the
extracellular microenvironment by all types of cells in either physiological conditions or in response to stress
factors and participate in different aspects of cell-to-cell communication. Among stress factors known to
affect exosome-based communication is the exposure to ionizing radiation and exosomes are postulated to
participate in radiation-induced bystander effect. However, information on proteome component of exosome
cargo is rather limited in the context of radiation response. Here we aimed to use a comprehensive proteomics
approach to characterize proteome of exosomes released in vitro by irradiated cells and to identify proteins
and their associated biological functions upregulated by ionizing radiation.
Methods: UM-SCC6 cells derived from oral carcinoma were irradiated with 0, 2, 4, and 8 Gy dose than
incubated for another 24 hours (no substantial cell death was observed during this time). Exosomes were
purified from culture media by a (mini) size exclusion chromatography and characterized by the particle size
distribution (by a light scattering), electron microscopy, and protein markers. A fraction enriched in
(confirmed) exosomes was subjected to a shot-gun proteomics profiling using a high-resolution LC-MS/MS.
Biological functions associated with identified proteins were identified using the Gene Ontology tools.
Results: There were about 1200 proteins encoded by unique genes identified in either experimental condition,
which appeared exosome-specific (i.e., absent in culture medium not pre-conditioned with cells). The levels of
about 40% of identified proteins were affected by radiation (comparing to not irradiated control; 0 Gy),
including 425 IR-upregulated and 47 IR-downregulated species. Comparison of differences between
exosomes released by cells irradiated at different doses allowed to observe different dose-related patterns; the
most numerous group of exosome proteins affected by radiation consisted of 369 species, whose abundance in
secreted vesicles increased at all radiation doses (0<2~4~8). A few groups of Biological Processes associated
with IR-modulated proteins could be identified, including response to ionizing radiation and metabolism of
ROS, DNA repair, DNA packaging, and de novo protein folding. Moreover, proteins associated with
processes potentially involved in the bystander effect (exemplified by stress-activated MAPK cascade,
transcription in response to stress and apoptotic signaling) were detected only among IR-upregulated but not
among ID-downregulated species.
Conclusions: Proteins present in exosomes released by irradiated cells are associated functionally with key
elements of the cellular response to radiation and are involved in signal transmission between irradiated and
not irradiated cells.
European Radiation Research 2018, August 21-25, Pécs, Hungary
42
Herbal Medicine Today: Clinical and Research Issues
Mohammed Taqee Ansari
MAK College Of Pharmacy; http://www.makpharmacollege.ac.in/
Collaborate Company: Mak Labs Pvt Ltd
Demonstrate our indigenous medicines that are made by 1000 years old Herbal
& Ayurvedic medicines practicing field in the spectrum of finding remedies for
Asthma, Tuberculosis, Cancer and various other ailments.
Allopathic practitioners in India are outnumbered by practitioners of traditional
Indian medicine and homeopathy (TIMH), which is used by up to two-thirds of its
population to help meet primary health care needs, particularly in rural areas. India has
an estimated 2.5 million HIV infected persons. However, little is known about TIMH
use, safety or efficacy in HIV/AIDS management in India, which has one of the
largest indigenous medical systems in the world.
The purpose of this review was to assess the quality of peer-reviewed, published
literature on TIMH for HIV/AIDS care and treatment. Of 206 original articles
reviewed, 21 laboratory studies, 17 clinical studies, and 6 previous reviews of the
literature were identified that covered at least one system of TIMH, which
includes Ayurveda, Unani medicine, Siddha medicine, homeopathy, yoga and
naturopathy. Most studies examined either Ayurvedic or homeopathic treatments.
Only 4 of these studies were randomized controlled trials, and only 10 were published
in MEDLINE-indexed journals.
Overall, the studies reported positive effects and even "cure" and reversal of
HIV infection, but frequent methodological flaws call into question their internal and
external validity. Common reasons for poor quality included small sample sizes, high
drop-out rates, design flaws such as selection of inappropriate or weak outcome
measures, flaws in statistical analysis, and reporting flaws such as lack of details on
products and their standardization, poor or no description of randomization, and
incomplete reporting of study results.
European Radiation Research 2018, August 21-25, Pécs, Hungary
43
Exposure to ionizing radiation affects the characteristics of neural
stem cells
Onetsine Arrizabalaga1, Elena Nasonova
1,2, Insa S. Schroeder
1 and Sylvia Ritter
1
1 GSI Helmholtz Centre, Biophysics Dept., Darmstadt, Germany
2 JINR, Dubna, Russia
OBJECTIVE:
Ionizing radiation (IR) is a commonly used technique for diagnosis and tumour therapy, such as cranial
cancers. Cognitive dysfunctions and brain abnormalities that appear later in the life of patients cranially
irradiated at a young age, has led to an increased interest in deciphering the mechanisms underlying the
radiosensitivity of neural stem cells (NSC) and the impact on their functionality. The depletion of NSC
following irradiation as well as their inability to proliferate and replenish the pool of neurons and glial cells
are discussed as underlying mechanisms.
METHODS:
NSC derived from human embryonic stem cells (hESC, H9) were used as a model system, and their response
after exposure to therapeutically relevant doses of X-rays (0.5 - 2 Gy, 250 kV, 16 mA) was investigated.
Radiation-induced cell death (TUNEL assay) and chromosomal aberrations (mFISH-analysis) were examined
during the first days after exposure and at later times (two weeks after irradiation) in the progeny of the
irradiated cells. Additionally, the ability of NSC to form three-dimensional neurospheres (a particular NSC
assay) was analysed directly after exposure and after one and two days. This assay resembles the in vivo
situation of neural (re)generation better than two-dimensional cultures. Within the neurospheres, NSC
maintain the ability to proliferate, self-renew and differentiate. First experiments with C-ions (25mm SOBP,
106–147 MeV/u, 75 keV/µm LET) were performed.
RESULTS:
Exposure to IR resulted in a dose-dependent increase in the number of chromosomal aberrations in the first
post-irradiation metaphases. The damaged cells were removed from the culture through apoptosis. The
proportion of apoptotic cells was dose-dependently elevated up to three days after exposure and reached
control level after one week. Regarding genome integrity, 10% of non-irradiated NSC carried aberrations,
which are spontaneously formed and non-transmissible to the progeny. In addition, the progeny of irradiated
NSC carried transmissible aberrations (translocations) two weeks after exposure.
The ability of NSC to form neurospheres was hindered dose-dependently. The effect was reduced when the
neurosphere formation was initiated one or two days after irradiation due to the removal of damaged cells by
apoptosis. The size of neurospheres formed after exposure to IR also decreased dose-dependently. Iso-doses
of C-ions reduced more effectively the amount and size of the neurospheres formed in comparison to X-rays.
Differences in the composition of NS from control and irradiated NSC are currently being quantified by qPCR
and verified by IF.
CONCLUSION:
An important implication of exposing NSC to therapeutically relevant doses of IR is that a small fraction of
the progeny of irradiated cells carries stable aberrations that could lead to malfunctioning of NSC or their
differentiated counterparts, or even to tumour formation.
The timing of irradiation is also an essential factor since the brain of small children is still developing. Due to
a decreased ability to form neurospheres in vitro, the (re)generation of the irradiated brain could be
compromised. The composition of neurospheres formed from irradiated NSC might hint clues for the
underlying mechanisms.
Work supported by BMBF, Germany (02NUK034C)
European Radiation Research 2018, August 21-25, Pécs, Hungary
44
Do radiation-induced changes in exosomal cargo predict survival?
Michael J. Atkinson, Lisa Mutschelknaus, Michael Schneider, Omid Azimzadeh, Gege Olatunji, Ramesh
Yentrapalli, Natasa Anastasov, Valerie B. O'Leary, Soile Tapio and Simone Moertl
Institute of Radiation Biology, Helmholtz Zentrum München, Germany Extracellular vesicles (EVs) are membrane-bound subcellular bodies released from cells. Dependent upon preparation
techniques, the EVs released by cells can include exosomes, apoptotic bodies and microvesicles in different proportions.
Exosomes are a subclass of EVs defined by size (40-100nm diameter), surface markers, and endocytic pathway origin.
Once released into the local environment by their donor cells, exosomes are readily taken up by other (recipient) cells,
where both their external membrane and internal cargo may have an influence on cellular activity. As exosomes have
been shown to modify tissue responses to environmental stressors, it is tempting to suggest that exosomes could mediate
some of the non-targeted effects of irradiation. In the BHY squamous head and neck cancer cell line, we have observed a
radiation dose-dependent increase in the number of exosomes released at doses up to 3Gy (1), with both the lipid and
protein content of collected exosomes being transferred into recipient cells. At the same time, the capacity for a recipient
cell to take up exosomes was increased by prior irradiation (1).
Radiation-induced changes in exosomal cargo were first observed for the long non-coding RNA PARTICLE, a
transcriptional regulator coordinating epigenetic and genetic responses of stressed cells (2), and for highly selective
changes to the exosomal microRNA content (3). Most recently we have been able to demonstrate specific radiation-
induced changes in the exosomal proteome (4).
The functional consequences of the uptake of exosomes appear to centre on promoting wound healing, with improved
rate of DNA repair (1) and activation of AKT-mediated cell migration in treated cells (4). Such a protective effect of
exosomes would suggest a strategy to ameliorate radiation damage following exposure.
References
1 Mutschelknaus L. et al (2016) PLoSOne 11(3) e0152213
2 O'Leary V.B. et al (2015) Cell Reports 11(3) 474-485
3 Yentrapali R. et al (2017) Int J. Radiation Biology 93(6) 569-580
4 Mutschelknaus L. et al (2017) Scientific Reports 7:12423
European Radiation Research 2018, August 21-25, Pécs, Hungary
45
Plutonium-induced pulmonary fibrosis in nuclear workers of the
Mayak PA cohort exposed to internal alpha-radiation
Tamara V. Azizova1, Gleb V Sychugov
2 , Evgeniy L. Kazachkov
3, Evgeniya S. Grigorieva
1 and
VS Revina1
1 Southern Urals Biophysics Institute, Ozyorsk, Russia
2 State Educational Institution of Higher Vocational Education Southern Urals State Medical
University of the Ministry of Health Care of the Russian Federation, Chelyabinsk, Russia
Objective
To identify plutonium-induced pulmonary fibrosis (PuPn) in atomic industry workers occupationally exposed
to internal alpha-radiation and to define its morphological distinctive characteristics.
Methods
Immunochemistry was used to analyse biological samples of lung tissues (fixed in paraffin and stored as
paraffin tissue blocks) from 56 workers clinically diagnosed with PuPn, from 34 workers diagnosed with
pulmonary fibrosis of non-plutonium pathogenesis (as an outcome of chronic inflammatory lung diseases and
congestive heart failure) and from 35 workers free from any lung pathology aimed to identify morphologic
distinctive characteristics of PuPn.
Results
Mayak Production Association (PA) is a Russian large-scale nuclear enterprise producing weapon-grade
plutonium which was set in operation in 1948. Equipment defects and irregularity of technology process, low
level of production mechanisation and automatization in early years of the enterprise operation caused high
levels of alpha-radioactivity in occupational air. As Mayak PA workers did not use individual protection mean
for organs of respiratory system, alpha-particles of plutonium-deposited in workers’ organisms via inhalation
and were accumulated in main organs of plutonium deposition (lung, liver, bones). The first plutonium-
induced pulmonary fibrosis (PuPn) was diagnosed in a polonium production plant worker in 1956 based on
clearly manifested clinical, functional and x-ray abnormalities. Some years after registering of the first clinical
diagnoses an experimental model of PuPn was presented.
Retrospective expert review verified 188 clinical cases of PuPn in Mayak PA workers registered in 1948 –
1960: 117 cases were diagnosed in males, 61 cases were diagnosed in females. PuPn incidence was
significantly associated with cumulative lung absorbed dose from internally deposited alpha-particles. A dose
threshold was found for PuPn in the Mayak PA worker cohort, namely, the cumulative lung absorbed dose
from internal alpha-particles of 0.6 Gy.
Conclusions
PuPn is a specific type of pulmonary fibrosis which may be distinguished from pulmonary fibrosis of non-
plutonium pathogenesis. PuPn is a tissue effect induced by internal alpha-radiation exposure.
European Radiation Research 2018, August 21-25, Pécs, Hungary
46
Set-up optimization of IFIN-HH 3 MV TandetronTM
and proton beam
dosimetry for radiobiology experiments
Mihaela Bacalum1, Simona Dîrleci
2, Mihai Straticiuc
3, Ion Burducea
3, Radu Andrei
4, Decebal
Iancu4, Radu Vasilache
5, Diana Savu
1 and Mihai Radu
1
1 Horia Hulubei National Institute of Physics and Nuclear Engineering, Department of Life and
Environmental Physics, Bucharest-Magurele, Romania 2 Faculty of Physics, University of Bucharest, Bucharest-Magurele, Romania
3 Horia Hulubei National Institute of Physics and Nuclear Engineering, Department of Applied
Nuclear Physics, Bucharest-Magurele, Romania 4 Horia Hulubei National Institute of Physics and Nuclear Engineering, Department of Tandem
Accelerators, Bucharest-Magurele, Romania 5 CANBERRA PACKARD S.R.L., Bucharest, Romania
Objective: The aim of the present study was to optimize the IFIN-HH existing facility - a 3MV TandetronTM
accelerator to be further used for proton irradiation of biological samples [1].
Methods: The proton beam is scattered by passing through an Au foil and extracted into air through a 1 μm
thick Si3N4 window. Irradiations were performed at 3 cm from the window with 2 MeV protons, with a
uniform distribution over a 30 mm2 area. Particle fluence was estimated and monitored using Rutherford
backscattering spectrometry (RBS). In-air, for relative dose measurements, a Markus ionization chamber and
a radiochromic film were used to estimate the quality of the irradiation spot. The chamber in which the cells
were grown prior to irradiation, was specially designed for the experimental set-up.
Results: Preliminary in vitro studies were performed on L929 and V79 fibroblast cells irradiated at different
doses between 0.5-3 Gy. Investigated endpoints were cell viability, colony formation, apoptosis and
generation of reactive oxygen species (ROS).
Conclusion: The results obtained show a similarity with data reported using other proton beam facilities.
Acknowledgments: This work was partially supported by grants from the Romanian National Authority for
Scientific Research, CNCS-UEFISCDI, projects number 23ELI/2017, 231PED/2017, PN 16420201, PN
16420203 and PN 18090202.
[1] I. Burducea et al., Nuclear Instruments and Methods in Physics Research B 359, 1219, 2015
European Radiation Research 2018, August 21-25, Pécs, Hungary
47
MV/Exosomes mediate cellular aging response to ionising radiation
Savneet Kaur Bains1, Scott Bright2, Eman Elbakrawy1, Ammar Mayah1, Edwin Goodwin3, and Munira Kadhim1
1 Department of biological and medical sciences, Oxford Brookes University, Oxford, UK 2Department of Radiation Physics, University of Texas MD Anderson Cancer Centre,
Houston, Texas, U.S.A, 3The New Mexico Consortium, 100 Entrada Drive, Los Alamos New Mexico 87544, USA
Objective, Cells/tissues have many stress responses allowing them to cope with various insults. Radiation is one such stressor as are the secretory phenotype of Senescent which include free radicals and inappropriate communication signals. Both stressors use some of the same communication molecules that are encapsulated in membrane-bound vesicles (exosomes). Shared molecular signalling connects radiation and senescence stress responses, meaning there is likely interplay between these responses when both are active e.g., if one stress response is active it may potentiate or inhibit another stress response to which it is connected. We hypothesized that exosome-encapsulated signals from senescent cells can either impair or promote radiation damage recovery. And signals from the radiation-damaged tissue will accelerate cellular senescence in other unirradiated/irradiated tissue. Methods, Normal human early passage (young) cells as well as late passage senescent cells were irradiated with 2 Gy and 10 Gy of x-rays and cultured until the young 10 Gy cells became senescent. Cells were analysed over the first 24 hours post radiation and after a latency period, which is particularly relevant to radiation-induced stress response. Cells were assayed for viability, senescence, DNA damage, H2AX, DNA methylation, and proteomic analysis Results and Conclusion, Initial results showed comprehensive radiation induced senescence in cells exposed to 10 Gy. Maximal senescence was observed at 10 days. Exosome transfer experiments indicated that exosomes can increase the number of cells undergoing senescence. We also explored DNA methylation as a marker of aging, this appears to be independent of radiation exposure and therefore cell-cycle dependent or chronological process indicating that stress-induced senescence is epigenetically different to natural aging. We may also see differences between naturally aged cell exosomes and radiation-stress-induced exosomes. Current and future experiments will explore late/deep senescence and consider the exosome contents that drive this response. This study is supported by: THE DUNHILL MEDICAL TRUST number RB07004
European Radiation Research 2018, August 21-25, Pécs, Hungary
48
Radiotherapy-induced changes in cellular and soluble immunological
markers of head and neck cancer patients
Katalin Balázs1, Tünde Szatmári
1, Enikő Kis
1, Noémi E. Bogdándi
1, Piotr Widlak
2, Géza
Sáfrány
1,
Katalin Lumniczky 1
1National Public Health Centre –
National Research Directorate for Radiobiology and
Radiohygiene, Budapest, Hungary, 2Maria Sklodowska-Curie Memorial Cancer Center and Institute
of Oncology, Gliwice Branch, Poland
Objectives: Tumour growth is very often associated with a progressively developing systemic
immune suppression. Various cell types are responsible for this immune suppression, among which
regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs) play a particularly
important role. Radiotherapy can modify not only the amount and function of these cells but also the
level of different soluble immunological markers in blood plasma. Thus radiotherapy-induced
changes might represent prognostic markers of therapy-response and our aim was to identify them.
Methods: We examined the phenotypical changes in peripheral blood mononuclear cells focusing on
CD4, Tregs, natural killer cells (NKs), dendritic cells (DCs) and MDSCs by flow cytometry and the
level of several plasma proteins investigated by multiplex protein array. Based on the results of
protein array the level of promising plasma proteins were validated with enzyme-linked
immunosorbent assay. Samples from 25 head and neck cancer patients were investigated before,
directly after and one month after radiotherapy. The results were compared with values of healthy
patients.
Results and Conclusions: The number of CD4+ cells in cancer patients before radiotherapy was
moderately higher compared with control values, but one month after the therapy returned to normal
values. The amount of Tregs and NK cells was less in cancer patients before treatment but one month
later the level of both cell types increased. CTLA-4 expressing Treg cells were elevated significantly
(p=0.039) directly after radiotherapy. This activation marker might serve as indicator of radiation
exposure and/or therapy-response. We also made correlation analysis between the changes of the
cellular markers and the clinical parameters of the cancer patients (response to treatment, rate of
radiotherapy-related side effects). The amount of CD11b+ DCs positively correlated with
dose/fraction directly after the radiotherapy which might relate to the therapy-induced inflammation
processes. The level of NK cells and PD-1+ Tregs positively correlated with Acute Mucosal
Reaction (AMR) directly after the radiotherapy. The increased NK and PD-1+ Treg numbers most
probably also reflect an acute inflammatory response. The amount of Tregs decreased significantly
(p=0.0356) one month after the radiotherapy in those patients receiving lower dose/fraction (1.6-2
Gy) in contrast with higher dose/fraction (3 Gy) patient group. CD11c+ DCs decreased significantly
(p=0.0332) one month after the therapy in good prognosed patient group.
Regarding soluble markers: the level of Adiponectin and B-cell activating factor (BAFF) increased
significantly (compared with values before radiotherapy) in all patient one month after the
radiotherapy so they might serve as late indicators of radiation exposure. Patients indicate radiation
exposure but they can not discriminate between patients receiving lower or higher total doses.
Further proteins identified on the array are analysing through ELISA.
The main conclusion of our work is that immunological markers are potential prognostic and/or
predictive markers of radiotherapy-response.
This work was funded by the CONCERT and OPERRA EU-FP7 projects and by the National
Research, Development and Innovation Office (grant agreement number: VKSZ_14-1-2015-0021).
European Radiation Research 2018, August 21-25, Pécs, Hungary
49
Radiobiological effects of α-particle radiation from Ra-223 in cancer
cells
Kristina Bannik1, Sabine Zitzmann-Kolbe1, Sabrina Jarke2, Marco Jarzombek2, Andreas Sutter2, Gerhard Siemeister1, Dominik Mumberg1
1 Bayer AG, R&D Pharmaceuticals, Therapeutic Research Groups Oncology II, Berlin, Germany
2 Bayer AG, R&D Pharmaceuticals, Toxicology, Berlin, Germany
Purpose: In recent years, the targeted alpha therapy is emerging as innovative approach to the treatment of advanced cancers. Although the therapeutic effect of α-particle treatment has been demonstrated preclinically in vivo and in vitro, and even in early clinical studies, the interpretation of biological effects of α-particle irradiation in cancer cells is still challenging because of the specific properties of α-particles. This study aimed to evaluate the time-dependent biological effects of α-particle irradiation in various cancer cell lines. Moreover we investigated the therapeutic efficacy of fractionated treatment and the combination of α-particle irradiation with DNA repair inhibitors. Methods: As a model we used lung (H460), ovarian (OVCAR-3, COV362, cov644, ES2) and prostate (22Rv1, LNCaP) cancer cell lines. The cells were irradiated with different activities and exposure times of Ra-223 in a Transwell® system. The exposure to α-particles was performed by mixing radium in ionic form (RaCl2, Xofigo™) with ethanol (1.3 kBq/cm2) for coating the bottom of a well. The cells were seeded on a membrane (10 µm) and irradiated with α-particle in the direction from the bottom of the wells through the membrane. Cell cycle distribution, cell death, and DNA damage response were measured by immunofluorescence staining and subsequent flow cytometric analysis. Cell survival, DNA repair kinetics, and genotoxicity were measured by colony formation, comet assay and micronucleus test. Inhibitors of DNA repair pathways ATM, ATR and DNA-PK were combined with α-particle irradiation. Results: The α-particle irradiation increased the initial number of 53BP1 foci in an activity and time-dependent manner in lung and ovarian cell lines. Moreover, the higher activity of 20 kBq induced more DNA damage during a short time (4 h) irradiation as compared to the lower activity of 5 kBq, but this difference was not observed after prolonged irradiation (16 h). The amount of unrepaired DNA damage after 4 h of α-particle irradiation remained in cells up to 3 days. The cell cycle was arrested at the G2 phase following a prolonged α-particle radiation. We did not observe a significant difference between single and fractionated α-particle irradiation. The combination of α-particle irradiation and DNA repair inhibitors yielded an additive effect in H460 cells. The frequency of micronuclei was increased in an activity and time-dependent manner after α-particle exposure at 5, 10, 20, and 40 kBq. The number of dead cells was significantly increased after 8 h exposure. Additionally, the cytotoxicity was increased more than 60% after 1 h-exposure at 5 kBq in TP53 wt H460 cells and after 8 h exposure in TP53 mutant 22Rv1 cells. Conclusion: This study demonstrates that α-particle irradiation is highly cytotoxic at low activity and short time exposure in different cancer cell lines. The reduction of cell survival may result from a G2 cell cycle arrest due to the inability to repair DNA double strand breaks and induction of cell death.
European Radiation Research 2018, August 21-25, Pécs, Hungary
50
THE CARDIOVASCULAR SYSTEM IN SPACE: DIFFERENTIAL
IMPACT OF SINGLE-DOSE FE ION AND
X-RAY IRRADIATION ON ENDOTHELIAL CELLS
Bjorn Baselet1,2
, Omid Azimzadeh3, Nadine Erbeldinger
4,5, Mayur V Bakshi
3, Till Dettmering
4, Ann Janssen
1,
Svetlana Ktitareva4, Donna Lowe
6, Arlette Michaux
1, Quintens Roel
1, Kenneth Raj
6, Marco Durante
4,5,
Claudia Fournier4, Mohammed A Benotmane
1, Sarah Baatout
1,7, Pierre Sonveaux
2, Soile Tapio
3 and An Aerts
1
1 Belgian Nuclear Research Centre (SCK•CEN), Radiobiology Unit, Mol, Belgium
2 Université catholique de Louvain, Pole of Pharmacology and Therapeutics, Brussels, Belgium
3 Helmholtz Zentrum Munich, Institute of Radiation Biology, Neuherberg, Germany
4 GSI Helmholtz Centre for Heavy Ion Research, Darmstadt, Germany
5 Technical University Darmstadt, Darmstadt, Germany
6 Public Health England, Radiation Effects Department, Didcot, UK
7 Ghent University, Department of Molecular Biotechnology, Ghent, Belgium.
Objective: When humans leave low Earth orbit, they are exposed to a unique and hostile space environment
which is quite different from that on Earth. They are continuously exposed to cosmic ionizing radiation and
microgravity, and they experience high levels of psychological stress due to confinement, sleep deprivation
and a high workload. Out of all these space stressors, exposure to cosmic ionizing radiation is the biggest
show stopper for missions to the moon, mars and beyond. Unfortunately, little is known about the biological
and molecular mechanisms which are altered after particle irradiation in healthy tissues. Endothelial cells
(EC), forming the inner layer of all vascular structures, are especially sensitive to ionizing radiation and, if
damaged, can contribute to radiation-induced cardiovascular disease.
Methods: Transcriptomics, proteomics and cytokine analyses were used to compare the response of ECs
irradiated with a single 2 Gy dose of X-rays or Fe ions measured one and 7 days post-irradiation. To support
the observed inflammatory effects, monocyte adhesion on ECs was assessed.
Results: Experimental data indicate time- and radiation quality-dependent changes of the EC response to
irradiation. The irradiation impact was more pronounced and longer lasting for Fe ions than for X-rays. Both
radiation qualities decreased the expression of genes involved in cell-cell adhesion and enhanced the
expression of proteins involved in caveolar mediated endocytosis signalling. Endothelial inflammation and
adhesiveness were increased with X-rays, but decreased after Fe ion exposure.
Conclusions: Fe ions induce pro-atherosclerotic processes in ECs that are different in nature and kinetics than
those induced by X-rays, highlighting radiation quality-dependent differences which can be linked to the
induction and progression of cardiovascular diseases (CVD). Our findings give a better understanding of the
underlying processes triggered by particle irradiation in ECs, a crucial aspect for the development of
protective measures for astronauts in space.
Acknowledgements: This work was funded by EU FP7 DoReMi network of excellence (grant #249689), EU
FP7 project ProCardio (grant #295823), the Belgian Federal Agency for Nuclear Control FANC-AFCN (grant
#CO-90-13-3289-00) and the Belgian Fonds National de la Recherche Scientifique (F.R.S.-FNRS). BB is
supported by a doctoral SCK•CEN grant. PS is a F.R.S.-FNRS Senior Research Associate.
European Radiation Research 2018, August 21-25, Pécs, Hungary
51
IONIZING RADIATION AND METABOLISM: CAN
MITOCHONDRIAL DRUGS BE USED FOR RADIATION
PROTECTION OF THE VASCULAR SYSTEM
Bjorn Baselet1,2
, Sarah Baatout1,3
, Pierre Sonveaux2 and An Aerts
1
1 Belgian Nuclear Research Centre (SCK•CEN), Radiobiology Unit, Mol, Belgium
2 Université catholique de Louvain, Pole of Pharmacology and Therapeutics, Brussels, Belgium
3 Ghent University, Department of Molecular Biotechnology, Ghent, Belgium.
Objective: Mitochondria are often regarded as the powerhouses of the cell because they supply
most of the cellular energy. But mitochondria are also involved in other tasks, such as oxidative
stress signalling, calcium regulation and apoptotic control. Consequently dysregulation of
mitochondrial functions can result in the promotion of apoptosis and senescence and in an increased
inflammatory status. Recently, ionizing radiation has been separately shown to promote
atherosclerosis-related processes in the cardiovascular system and to induce mitochondrial damage,
as seen in radiotherapy patients and in vitro cell models. Therefore we aimed to assess the effects of
the mitochondrial drug rosiglitazone as a radioprotective agent to counteract the radiation-induced
atherosclerosis-related processes in endothelial cells.
Methods: Assays for apoptosis, cellular metabolism, mitochondrial DNA content, mitochondrial
functionality and DNA damage were used to compare the response of control and rosiglitazone
treated ECs irradiated with a single 2 Gy dose of X-rays over multiple time points.
Results: Experimental data indicate that while ionizing radiation decreases the activity and
number of mitochondria in endothelial cells, rosiglitazone is able to rescue this phenotype. In
addition, it decrease the levels of apoptosis after radiation exposure and limits the release of reactive
oxygen species production inside the cells. Furthermore, rosiglitazone diminishes the amount of
DNA damage suggesting a decreased level of DNA damage after radiation exposure.
Conclusions: Rosiglitazone is able to protect mitochondria against the detrimental effects of
ionizing radiation exposure. Addition of the drug before irradiation decreases DNA damage
signalling, suggesting a radioprotective effect in endothelial cells. These findings indicate that
rosiglitazone is a potential drug to protect patients from developing radiation-induced cardiovascular
disease. Moreover, our study indicates that there is a need to continue research into the effects of
mitochondrial drugs in the field of radiation protection.
Acknowledgements: This work was funded by EU FP7 DoReMi network of excellence (grant
#249689), EU FP7 project ProCardio (grant #295823), the Belgian Federal Agency for Nuclear
Control FANC-AFCN (grant #CO-90-13-3289-00) and the Belgian Fonds National de la Recherche
Scientifique (F.R.S.-FNRS). BB is supported by a doctoral SCK•CEN grant. PS is a F.R.S.-FNRS
Senior Research Associate.
European Radiation Research 2018, August 21-25, Pécs, Hungary
52
Health Effects of Chornobyl: from Epidemiology to Molecular
Quantification
Dimitry Bazyka, Natalya Gudzenko, Iryna Dyagil, Iryna Ilienko, David Belyi, Vadim Chumak,
Anatoly Prysyazhnyuk
National Research Center for Radiation Medicine, Kyiv, Ukraine
Objective. The results are presented of long-term follow-up of cancer and noncancer effects in Ukrainian
population exposed to ionizing radiation after the Chornobyl accident.
Methods. Analytical case-control studies were carried out in a cohort of 110,645 accident cleanup workers
(Romanenko et al. 2008; Zablotska et al. 2013) who have been registered in the Chornobyl State Registry of
Ukraine (SRU) and residing in six regions of Ukraine (five regions, or oblasts and Kyiv city) for 1987-2014 .
The cohort was amended to 152,520 (60% of registered male cleanup workers). For search and diagnosis
confirmation of the incident studies of leukemia, thyroid cancer and multiple myeloma the cohort database
was linked to the National Cancer Registry of Ukraine (NCRU). Uniform reconstructive dosimetry was
performed for cases and controls using RADRUE method. For exposed to high doses a dicentric assay and
FISH WCP-1,2,4 data were applied along with official dose records. Molecular studies in cohort members
included gene expression and polymorphism, FISH, relative telomere length, immunophenotype, micronuclei
test, histone H2AX, TORCH infections, oxidative stress.
Results. The number of power plant employees and first responders with acute radiation syndrome under
follow-up by the National Research Center for Radiation Medicine decreased from 179 in 1986–1991 to 105
in 2011–2015. Cancers and leukemia (21) and cardiovascular diseases (21) were the main causes of deaths
among acute radiation syndrome survivors (57) during the postaccident period. Increased radiation risks of
leukemia in the Ukrainian cohort of cleanup workers exposed to low doses are comparable with those among
survivors of the A-bombing in Japan. Additionally, an excess of chronic lymphocytic leukemia was
demonstrated in the cleanup workers cohort for 26 yrs after the exposure. Analysis of 17 CLL cases from the
cohort showed more frequent mutations in telomere-maintenance pathway genes POT1 and ATM compared
with unexposed CLL patients from Ukraine (28) and Dana Farber Cancer Institute (100) (Ojha et al., 2018). A
significant excess of multiple myeloma incidence [standardized incidence rate (SIR) 1.61 %, 95% confidence
interval (CI) 1.01–2.21], thyroid cancer (SIR 4.18, 95% CI 3.76–4.59), female breast cancer (SIR1.57 CI
1.40–1.73), and all cancers combined (SIR 1.07; 95% CI 1.05–1.09) was registered. High prevalence was
demonstrated for cardio- and cerebrovascular diseases and mental health changes. Genome instability
including elevated micronuclei counts, gamma-H2AX expression, telomere length variability and TP53,
BCL2, SERPINB9, CDKN2A, STAT3, MCF2L genes expression changes could serve as background of low-
dose health effects.
Conclusion. Performed studies demonstrate possibility of understanding the nature of radiation-induced
effects after low-dose exposure. To connect possible late effects such as cardio- and cerebrovascular effects,
specific subtypes of radiogenic cancers with radiation exposure the analytical cohort and case-control studies
need to include biomarkers of dose and disease supplemented by an uniform dosimetry.
European Radiation Research 2018, August 21-25, Pécs, Hungary
53
Increased oxidative stress and an adaptive antioxidant response in
saliva after dental CBCT exposure in children
Niels Belmans1,2
, Sarah Baatout2, Reinhilde Jacobs
3, Ivo Lambrichts
1 and Marjan Moreels
2
1Faculty of Medicine and Life Sciences, BIOMED, Hasselt University, Hasselt, Belgium
2 Radiobiology Unit, Belgian Nuclear Research Centre, Mol, Belgium
3OMFS IMPATH Research Group, Department of Imaging and Pathology, Faculty of Medicine, University of
Leuven, University Hospitals Leuven, Leuven, Belgium
BACKGROUND
Currently, a lot of uncertainty still exists about the biological effects caused by exposure to low doses of
ionizing radiation (IR). This is of major concern for radiation protection agencies, as the amount of medical
imaging procedures (e.g. computed tomography (CT), X-rays, etc.) keeps increasing annually, both in adults
and children. This is of particular importance in children as they are more radiosensitive than adults. The
effects of exposure to CT examinations have been studied in recent years, but biological effects associated
with cone beam CT (CBCT) are much less investigated. CBCT is a radiographic tool for diagnosis, treatment
planning, follow-up and research in dentistry, mostly in pediatric orthodontics. IR doses typically used in
CBCT range from 10 to 1,100 µSievert, which is lower than doses used in CT.
AIM
This project investigates biological changes in saliva after CBCT exposure in children.
MATERIALS AND METHODS
Oxidative stress levels (8-OHdG, antioxidant enzym activity, and total antioxidant capacity) and inflammatory
markers (interleukin-6 and -8) were measured in saliva samples from children (n = 117) and adults (n=19).
Samples were collected before, 30 minutes and 60 minutes after dental CBCT exposure.
RESULTS
In contrast to adults samples, data from children show significantly increased salivary 8-OHdG levels after
CBCT examination. The salivary antioxidant capacity decreases significantly in adults, whereas a significant
increase is seen in children. This is confirmed by an increase in salivary catalase and superoxide dismutase
activity in children. Finally, changes in interleukin-6 and -8 levels were observed.
CONCLUSION
Children show increased salivary 8-OHdG levels after CBCT examination combined with an increased
antioxidant enzyme activity and total antioxidant capacity, whereas adults show a lesser increase in 8-OHdG
levels and a decreased total antioxidant capacity. These data not only indicate that exposure to very low doses
of IR leads to acute biological changes, they also indicate that adults and children react differently to CBCT
exposure. This is an important observation in the light of radiation protection guidelines.
European Radiation Research 2018, August 21-25, Pécs, Hungary
54
Long-term cerebrovascular and cognitive impairments following childhood exposure
to radiation
Rafi Benotmane
Rafi.benotmane@sckcen.be
Belgian Nuclear Research Centre SCK•CEN, Radiobiology Unit, Mol, Belgium
Exposure to radiation increases the risk of stroke and mental disabilities, and this is highly illustrated in adult
survivors of childhood cancer such as central nervous system ‘CNS’ tumors with a high risk for significant
persistent functional and health-related late effects. The severity and incidence of neurologic late effects
depends on the patient's age at diagnosis. Of note, large number of survivors are presenting severe disabilities
consisting of a decline in intelligence quotient or academic achievement as well as deficits in motor function,
memory, attention, and hand eye coordination. Moreover, increase in cerebrovascular disease and mortality
has been observed in large cohorts of childhood cancer survivors, and stroke risk has been evidenced in long-
term survivors of Hodgkin disease, leukemia, and brain tumors (El-Fayech. et al, 2017). Unfortunately,
present knowledge is insufficient to predict these long-term effects after radiation therapy (RT) or accidental
exposure at young age. To this end, an innovative strategy is under investigation within the H2020 project
MEDIRAD aiming at identifying in saliva predictive epigenetic biomarkers for brain tumor induction
following exposure to CT scan doses at childhood.
Several studies have been conducted with the aim to illustrate the underlying mechanisms of such long-term
effects, among which the EU FP7 project CEREBRAD (GA: 295552). In this project, rodent behavioral
testing together with magnetic resonance imaging ’MRI’ have proven to be valuable tool to evaluate radiation-
induced cognitive and structural defects (Verreet T. et al, 2016). On the other hand, molecular and cellular
assessments clearly indicate that neurogenesis and synaptogenesis are hampered in adult brain following early
postnatal exposure to radiation (Kempf S. et al, 2015) (Quintens R. et al, 2015). Nevertheless, additional
research focusing on the role of epigenetic and the immune response is highly needed to increase our
knowledge to uncover the role of radiation in modulating the late central nervous response. Finally, design of
countermeasure strategy to ameliorate the cognitive functions following exposure to radiation at young age
will improve the quality of life of such exposed children.
Acknowledgement: This research is partly supported by the EU H2020 project MEDIRAD (GA: 755523) References: 1: El-Fayech C, Haddy N, Allodji RS, Veres C, Diop F, Kahlouche A, Llanas D, Jackson A, Rubino C, Guibout C, Pacquement H, Oberlin O, Thomas-
Teinturier C, Scarabin PY, Chavaudra J, Lefkopoulos D, Giroud M, Bejot Y, Bernier V, Carrie C, Diallo I, de Vathaire F. Cerebrovascular Diseases in Childhood Cancer Survivors: Role of the Radiation Dose to Willis Circle Arteries. Int J Radiat Oncol Biol Phys. 2017 Feb 1;97(2):278-286. doi:
10.1016/j.ijrobp.2016.10.015.
2: Verreet T, Rangarajan JR, Quintens R, Verslegers M, Lo AC, Govaerts K, Neefs M, Leysen L, Baatout S, Maes F, Himmelreich U, D'Hooge R, Moons L, Benotmane MA. Persistent Impact of In utero Irradiation on Mouse Brain Structure and Function Characterized by MR Imaging and
Behavioral Analysis. Front Behav Neurosci. 2016 May 4;10:83. doi: 10.3389/fnbeh.2016.00083.
3: Kempf SJ, von Toerne C, Hauck SM, Atkinson MJ, Benotmane MA, Tapio S. Long-term consequences of in utero irradiated mice indicate proteomic changes in synaptic plasticity related signalling. Proteome Sci. 2015 Nov 16;13:26. doi: 10.1186/s12953-015-0083-4.
4: Quintens R, Verreet T, Janssen A, Neefs M, Leysen L, Michaux A, Verslegers M, Samari N, Pani G, Verheyde J, Baatout S, Benotmane MA.
Identification of novel radiation-induced p53-dependent transcripts extensively regulated during mouse brain development. Biol Open. 2015 Feb 13;4(3):331-44. doi: 10.1242/bio.20149969.
European Radiation Research 2018, August 21-25, Pécs, Hungary
55
Radiobiological characterization of laser driven particle beams – Dresden activities
Elke Beyreuther
1 Helmholtz-Zentrum Dresden – Rossendorf, Dresden, Germany
2 OncoRay – National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl
Gustav Carus, Technische Universität Dresden, Helmholtz-Zentrum Dresden - Rossendorf, Dresden, Germany
The long-term aim of developing laser based particle acceleration towards clinical application requires not
only substantial technological progress, but also new technical solutions for dose delivery and quality
assurance as well as comprehensive research on the radiobiological consequences of ultra-short radiation
pulses with high pulse dose.
During the last years the laser driven technology was developed at such a rate that cell samples and small
animals can be irradiated. Within the joint research project “onCOOPtics” extensive in vitro studies with
several human tumor and normal tissue cells were performed revealing comparable radiobiological effects of
laser driven and conventional electron and proton beams1,2
. Using the same cell lines, these results were
substantiated comparing the radiobiological response to ultra-short pulsed electron bunches (pulse dose rates
of ≤1012
Gy/min) and continuous electron delivery at the radiation source ELBE3.
In a second translational step, in vivo experiments were established. Although the experiments were motivated
by future proton trials, first attempts were performed with electrons at the laser system JETI4, since the
delivery of prescribed homogeneous doses to a 3D target volume is easier for electrons than for protons. A full
scale animal experiment was realized for the HNSCC FaDu grown on nude mice ear. The radiation induced
tumor growth delay was determined and compared to those obtained after similar treatment at a conventional
clinical LINAC. Again, no significant difference in the radiation response to both radiation qualities was
revealed, whereas the successful performance of such a comprehensive experiment campaign underlines the
stability and reproducibility of all implemented methods and setup components.
During this experiment campaign several limitations of the model were identified which were in the following
redressed by co-injection of LN229 glioblastoma tumor cells with Matrigel5. Results of this optimization
process and the status of the experiments with laser driven protons at the laser system DRACO will be
presented.
The work was supported by the German Government, Federal Ministry of Education and Research, grant nos.
03ZIK445 and 03Z1N511. 1Laschinsky L et al. (2012) J. Radiat. Res. 53(3): 395-403.
2Zeil K et al. (2012) Appl. Phys. B 110(4): 437-444.
3Beyreuther E et al. (2015) Int. J. Radiat. Biol. 91(8): 643-652.
4Oppelt M et al. (2015) Radiat. Environ. Biophys. 54(2): 155-166.
5Beyreuther E et al. (2017) PloS one 12.5 (2017): e0177428.
European Radiation Research 2018, August 21-25, Pécs, Hungary
56
“Radiation quality influence on normal tissue cell response”
Elke Beyreuther
1 Helmholtz-Zentrum Dresden – Rossendorf, Dresden, Germany
2 OncoRay – National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Helmholtz-Zentrum Dresden - Rossendorf, Dresden,
Germany
Objective: From the various factors that are known to influence the radiobiological response to
therapeutic beams, the radiation type and beam energy or LET (linear energy transfer), and the beam
pulsing and dose rate are object of comprehensive investigations. Alterations of these parameters
might result in altered damage pattern and consequently in a different radiobiological effectiveness,
for example for the FLASH, single pulse, irradiation regime [1] and the use of multiple, ultra-short
laser driven particle pulses [2] where therapeutic relevant doses are administered within the fraction
of a second, i.e. at high dose rate. To characterize the influence of radiation quality, i.e. beam energy,
dose rate and pulsing, systematic in vitro studies performed at different accelerators will be
summarized in the talk.
Methods and results: Two normal human cell lines were applied to study the response to photons in
the range of 10 kV to 34 MV, to conventional and laser driven electrons, and to continuous and
pulsed proton beams. By measurements of chromosomal aberrations and DNA double-strand breaks
(DSB) the inverse correlation of photon energy and biological damage was revealed, whereas for the
proton studies no clear influence of pulsing was found. Furthermore, no influence on clonogenic
survival was observed comparing laser driven electrons of ultra-high dose rate (109 Gy/s, multiple
electron pulses) and conventional, linac electrons (continuous dose rate 3 Gy/min). By contrast, a
trend towards less effectiveness of pulsed laser driven electrons was revealed by measurements of
residual DNA DSB. To investigate this finding in more detail, radiobiological experiments were
performed at the superconducting research electron linac ELBE, which is able to provide electron
beams with very variable pulse sequences and to mimic both laser driven and clinical linac electron
beams. Thereby, the DSB studies were complemented by DSB repair kinetics.
Conclusion:
Comprehensive in vitro studies of the effects of various radiation qualities revealed the influence of
beam energy and LET, but show no clear result with respect to pulse structure and dose rate.
References:
[1] Favaudon et al. Sci Transl Med. 2014;6(245):245ra93.
[2] Karsch et al. Acta Oncol. 2017;56(11):1359-1366.
European Radiation Research 2018, August 21-25, Pécs, Hungary
57
Neglecting the ecosystem concept in radiological protection is prone to
jeopardize the pertinence of environment protection measures
François Bréchignac1
1International Union of Radioecology (IUR) & Institute of Radioprotection and Nuclear Safety
(IRSN), Direction general, Center of Cadarache, 13115, St Paul-lez-Durance cedex, France
Objectives
There is still no consensus within the scientific community as to whether or not radioactive
environmental contamination, such as resulting from the Chernobyl or Fukushima disasters, is
promoting a deleterious ecological impact. This situation is critical as it is prone to favor unjustified
distrust from society with respect to the ability of authorities to take adequate measures for mastering
nuclear risk and protecting the environment. It is argued that one key challenge for radiation research
when facing this general context is to widen traditional radiation biology, focused on DNA and cells
of individual organisms, towards radiation ecology featuring an ecosystem-centered
conceptualization. If life is driven by processes that act at subsystem level, i.e. the molecular
engineering that founds the organisms’ physiology, it depends as well on processes that act at system
level, i.e. emergent properties of the ecosystem dimension such as life support, since both types of
processes have jointly emerged through evolution. Organisms and populations of species only exist
as embedded within an ecosystem featuring multispecies interactions.
Methods and Results
The scientific literature published on the ecological effects of radiation on non-human biota
essentially stem from two different methods of experimental investigations. The first involves
inference strategies in laboratory controlled conditions which are focused on individual organisms
and their biology (mutations, reproduction, mortality and morbidity). The second exploits field
studies and experimentations in contaminated territories which focus on species populations trends
and long-term ecosystem-related ecological fitness. Currently, these two methods often yield
diverging results, the interpretation of which is not yet fully understood. A few examples from recent
literature will be discussed to illustrate that the ecological impact of radiation (as well as of any
stressor) cannot easily be understood if not addressing the issue also from an ecocentric perspective.
Conclusion
Due to the above-mentioned considerations, environment radiological protection measures that are
developed exclusively from subsystem understanding (dose-response curves established for
individual organisms) for practical reasons, as in current radioprotection guidance, may actually miss
their protection objective which consensually sits at population and ecosystem level. Recently
reported discrepancies in assessing ecological impact most probably result from different processes
of response to stress when looked at populations and ecosystem levels or at organism, cellular and
molecular level.
European Radiation Research 2018, August 21-25, Pécs, Hungary
58
New biological target of ionizing radiations damages.
Luca Bruni1, Massimo Manghi
2,1, Walter Tinganelli
3 and Simonetta Croci
2,1
1 Centro Fermi - Enrico Fermi Historical Museum of Physics and Study and Research Centre,
Rome, Italy 2 University of Parma, Medical Surgery Department, Neuroscience Unit, Biophysics and Medical
Physics Section, Parma, Italy 3TIFPA – Trento Institute for Fundamentals Physics and Applications, Trento, Italy
OBJECTIVE In order to explain how ionizing radiations (IRs) work, radiobiologists have often pointed out on genomic
DNA, as main IRs biological target. Indeed a wide spectrum of DNA lesions has been pictured so far, such as
damages to nucleotide bases (base damages), DNA single-strand breaks (SSBs) and double-strand breaks
(DSBs). Beside the DNA lesions, the corresponding assays to detect them were developed. The aim of this
investigation is first at all the evaluation of cell cytoskeleton, as a new biological target for IRs, using new
quantitative parameters, such as Modified Directional Index (MDI)1. The experiment is done with Hs 578Bst
cells - non-cancer, non-immortalized, human, breast, epithelial cell line. It might be conceived as a cellular
model closest to a healthy tissue, neighbouring a cancer resected area.
METHODS
Hs 578Bst cell line is irradiated with protons (total dose delivered 8Gy), at the Proton Therapy Centre of
Trento (Italy) and with X-rays (total dose delivered 25Gy), at TIFPA (Trento-Italy). Both the irradiations are
performed 24h after cell seeding; the cells are seeded at the concentration of 8000cell/ml. After cells
irradiation the plasmatic membrane is removed by means a mild denaturation, which attempts to preserve very
nearly all the bigger cytoskeleton structures. Following the plasmatic membrane denaturation Hs 578Bst cells
are fixed using a biological cabinet airflow, so as to avoid the use of any chemicals reagents. Later on the cells
are scanned with Atomic Force Microscopy (AFM) (non-contact mode and on air). The parameters,
quantifying different linearity and integrity of the cytoskeletal structures, are obtained analysing the AFM
images
RESULTS
Quantitative parameter values calculated on the irradiated Hs 578Bst cells images are compared with control
ones. The cytoskeleton damage involves principally the smallest structures and not the bigger ones. Following
radiation experiments, Hs 578Bst cell line does not show neither remarkable morphology alterations nor
modifications. Furthermore it does not display any increase of mortality.
CONCLUSIONS
This research demonstrates the use of the cytoskeleton as IRs biological target, beside the most common
DNA. Following both careful and mild plasmatic membrane denaturation, the cells show some of the
damaged cytoskeleton structures.
Moreover the quantification of the cytoskeleton damages can be reached using parameters defined on
microscopy images. 1) Massimo Manghi, Luca Bruni and Simonetta Croci: MDI: integrity index of cytoskeletal fibres observed by
AFM. EPJ Plus, Volume 131, Issue 6, article id.213, 12 pp; 2016.
European Radiation Research 2018, August 21-25, Pécs, Hungary
59
Zebrafish model for investigation on the biological effects of proton beam
Szilvia Brunner1, Tünde Tőkés1, Emilia Rita Szabó1, Imre Zoltán Szabó1, Róbert Polanek1, Elke
Beyreuther2,3, Jörg Pawelke2,3, Katalin Hideghéty1,4
1ELI-ALPS, ELI-HU Non-Profit Ltd., Dugonics ter 13, Szeged 6720, Hungary
2Helmholtz-Zentrum Dresden – Rossendorf, Dresden, Germany
3OncoRay – National Center for Radiation Research in Oncology, Faculty of Medicine and
University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany
4Department of Oncotherapy, Szeged, Hungary
Purpose/Objective: Our aim was to optimize the in vivo zebrafish embryo model for radiobiological
studies on proton beams, in particular applicable for investigations on laserdriven protons (LDP)
characterized by ultra-high dose rate, ultra-short pulses. Material/methods: 3 times repeated
experiments were carried out at the University Proton Therapy Dresden varying the different
physical and biological parameters. 96 embryos (24 hpf)/group were irradiated at the plateau and at
the middle of Spread Out Bragg Peak of the medical proton beam, furthermore by the 6 MV photons
of the LINAC at 5, 10, 15, 20 and 30 Gy dose levels. Each days after irradiation the embryos were
observed. The morphological malformations (length of the embryos, the degree of the yolk sac
edema and the diameter of the eyes.) were photo-documented. These photos from the 3rd, 4th day
were analysed
quantitatively. In paralell histopathologic changes were evaluated. (changes of the small
intestine goblet cells, eye cellular layer, tissue necrosis). At 5 Gy dose level the irradiation
induced DNA double-strand breaks were measured (stained immunohistochemically, gammaH2AX )
Results: No changes could be detected morphologically at <10 Gy dose levels. At 15 Gy, 20
and 30 Gy, significant dose dependent increase of the yolk sac edema and decrease of the body
length and eyes were observed at both time points. In the following organs: central nervous system,
eye, muscle, liver, small intestine dose and linear energy transfer dependent changes were revealed
histologically. As well as the number of gamma-H2AX foci-s exhibited significant difference caused
by protons compared to the photon beam.
Conclusion: Complex analysis of different radiation qualities could be developed, by
quantification of. irradiation-caused macro-and micro morfological damages and molecular
changes using a vertebrate model. The examined parameters resulted in reliable basis for
radiobiological experiments with proton beams including later LDP.
Supported by: The ELI-ALPS project (GINOP-2.3.6-15-2015-00001) is supported by the
European Union and co-financed by the European Regional Development Fund. The project
has received funding from the European Union's Horizon 2020 research and innovation
programme under grant agreement no 654148 Laserlab-Europe and by the German BMBF,
grant no. 03Z1N511.
European Radiation Research 2018, August 21-25, Pécs, Hungary
60
Quality control of the physical parameters of medical radiological
equipment
Romuald Brzozowski1, Bogusława Kucikowicz-Gleń
1, Tadeusz Strózik
1, Dorota Wróblewska
1,
Milena Zarębska1
1 National Centre for Radiation Protection in Health Care, Quality Control Department, Lodz,
Poland
The main purpose of the quality control program is to ensure and maintain an appropriate quality level of
medical equipment that uses ionizing radiation, and makes it one of the fundamental elements of radiological
protection of the patient.
Based on Council Directive 2013/59/EURATOM of 5 December 2013, in Poland, was created a legal system
that allowed for the formation the network of laboratories accredited by the Polish Center for Accreditation
(PCA). As part of the quality control of radiological equipment, these laboratories perform specialist tests on
the basis of the criteria involved in annex no. 6 in regulation of the Minister of Health of 18 February 2011 on
the conditions for the safe use of ionising radiation for all types of medical exposure. Currently in Poland,
accreditation for specialist tests of radiological equipment has: 43 laboratories in the field of X-ray diagnostics
and interventional radiology, 2 laboratories in the field of nuclear medicine, and 1 laboratory in the field of
radiotherapy.
Quality Control Department of the National Center for Radiological Protection in Health Care (QCD of the
NCRPHC) performs specialist tests of radiological equipment since 2009. Initially, based on the authorization
of the State Sanitary Inspection, and then since 2012 on the basis of PCA accreditation. A quality management
system has been introduced, in accordance with the requirements of the ISO/IEC 17025. Test procedures were
created and supervision over measuring equipment (calibration, periodically check) was implemented. A very
important element related to the confirmation of the competence of the staff and the maintenance of the
quality of the performed tests is participation in internal (repeated measurements by staff) and external
projects (participation in interlaboratory comparisons) in which the staff regularly participates.
This study presents the results of specialist tests performed by QCD of the NCRPHC in the years 2009-2017
in the field of general radiography, fluoroscopy, dental, mammography, computed tomography, conventional
tomography, nuclear medicine and medical monitors.
The results show that the implemented quality control system enables to effectively identify devices that are
not compliant, including obsolete, requiring repairs or modernisation radiological equipment.
European Radiation Research 2018, August 21-25, Pécs, Hungary
61
Combining Low Dose Radiation and Immune Checkpoint Therapy
for Cancer Treatment
Michelle Bugden, Kiersten Thomas, Deyang Li and Yi Wang
Canadian Nuclear Laboratories, Chalk River, Ontario, Canada
Introduction: Immune checkpoint therapy, which removes the “brake” of the immune system to fight
cancer, is considered as one of the major breakthroughs in cancer treatments. Both pre-clinical
studies and clinical trials have demonstrated improved safety and impressive anti-tumor activity
compared to traditional treatments. However, the major obstacle of immune checkpoint therapy is its
low response rate (only 20-30%). To tackle this problem, one approach is to combine traditional
cancer therapies, especially radiation therapy (RT), with immune checkpoint therapy. Contrary to
high dose irradiation therapy which induces both immune stimulatory and suppression effects, low
dose whole body irradiation (WDI) only induces the immune activating effects. It has been reported
that low-dose irradiation (LDI) activates the immune system by altering tumor and immune cell
surface molecule expression, promoting T-cell-stimulatory capacities of DC, or an anti-tumor
macrophage phenotype. Low dose radiation also plays a significant role on vascular normalization
within tumors, which substantially enhances immunotherapeutic success as it reverses the hypoxic
microenvironment and enables immune effector cell infiltration. We are proposing that low dose
radiation is an adjunct therapy for immune checkpoint therapy, and the combined treatment enhances
the anti-tumor effects of immune checkpoint therapy.
Methods: CT26 (colon adenocarcinoma) cell line were injected s.c. into the right flank of six-week
old female BALB/c mice. Both PD-L1 and gamma beam whole body radiation treatments were
started on the first day after the tumor injection (Day1). The animal was treated with total body
irradiation (TBI) at 10mGy (low dose) or 100 mGy (medium dose), twice a week, and for 5 weeks -
a total dose of 100 mGy or 1 Gy respectively. Tumor volumes were measured with a digital caliper
three times a week and calculated as tumor volume = a*b*b/2, where a is the longest dimension and
b is the perpendicular dimension.
Results: We examined whether low dose radiation could improve anti-tumor effects of anti-PD-L1
treatment in vivo. Compared to the control group, tumor growth suppression was not significant in
the anti-PD-L1 alone group until day 18 (P>0.05) but was significant in the LDR+PD-L1 combined
treatment group (P<0.05). We also found that LDR only treatment slightly reduced the tumor
growth (P<0.05), and MDR has no effects on tumor growth (P>0.05).
Conclusions: Low dose radiation significantly improves the anti-tumor effects of PD-L1 immune
checkpoint therapy, by reducing the tumor volume as well as shortening the response time of PD-L1
immune checkpoint therapy alone.
European Radiation Research 2018, August 21-25, Pécs, Hungary
62
Radiation-induced bystander signals modulate immune cell
activation
Serge M. Candéias
Proteomics, Metals and Differentiation,
Laboratory of Chemistry and Biology of Metals,
UMR5249 CEA-CNRS-UGA
CEA-Grenoble. France
The immune system plays a central role in the protection of the organism against infectious and non-infectious
trauma and participates in the restoration of tissue homeostasis and function after physical insults. These
protective functions critically rely on the ability of cells of the innate and adaptive arms of the immune system
to be recruited and activated to the site of damage through the production of inflammatory mediators and
danger signals.by injured cells.
Ionizing radiation exposure generates cell and tissue damage that may ultimately activate the immune system.
Indeed, although the cellular response to ionizing radiation is by and large dominated by the activation of
mechanisms aimed at avoiding the transmission of potentially mutagenic lesions, radiation exposure also
induces a systemic response to restore tissue homeostasis, structure and function. Inflammation may play a
role in this process, after the recruitment and activation of innate immune cells by soluble factors
released/produced by irradiated cells. This phenomenon is thought to be critically important after exposure to
low doses of radiation. As a first step to understand how irradiated cells are able to communicate with and
activate immune cells, we analysed and compared the response of primary human fibroblasts and
keratinocytes to low and high doses of radiation, and investigated whether the bystander signals produced by
these irradiated cells are able to modulate the functionality of immune cells.
serge.candeias@cea.fr
European Radiation Research 2018, August 21-25, Pécs, Hungary
63
Pyridinium compounds as new potential radio-protective and radio-
remedial agents – summary of the project
Aneta Cheda1, Ewa M. Nowosielska
1, Jerzy Gebicki
2, Andrzej Marcinek
2, Stefan Chlopicki
3 and
Marek K. Janiak1
1Military Institute of Hygiene and Epidemiology, Department of Radiobiology and Radiation
Protection, Warsaw, Poland 2Technical University of Lodz, Institute of the Applied Radiation Chemistry, Lodz, Poland
3Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Crakow Poland.
Objective: Ever since the harmful effects of ionizing radiation were recognized, a quest has been set
out for effective radio-protectors, i.e., compounds designed to reduce radiation-induced damage in normal tissues. Pyridinium salts can protect against ionizing radiation-induced damage through both anti-thrombotic and anti-inflammatory activities based on their effects on the vascular endothelium. Thus, the aim of the present study was to assess mechanisms of the increased post-irradiation survival of mice treated with pyridinium salts (nicotinic acid, NAc; nicotinamide, NA; 1-methylnicotinamide, MNA; 1-methyl-3-acetylpyridine, 1,3-MAP; 1,4-dimethylpyridine, 1,4-DMP).
Methods: Male BALB/c mice were exposed to whole-body irradiation (WBI) with 60Co source at 6.5 Gy/h mean dose rate to obtain the absorbed doses of 6.5, 7.0 or 7.5 Gy per mouse. For the experiments pyridine compounds: NAc, NA, MNA, 1,3-MAP, or 1,4-DMP were dissolved in drinking water and given to the animals at concentrations assuring their daily consumption by a mouse at 100 mg/kg body mass (b.m.). Applications of the compounds started 7 days before, on the day of, or 7 days after WBI. The 30-day survival was estimated and numbers of the spleen, bone marrow, and peripheral blood cells were counted. Levels of IL-1beta, IL-6, IL-8, TNF-alfa, tromboxane, and prostacyclin (PGI2) were assayed by the ELISA methodology.
Results: We demonstrated that single exposure of BALB/c mice to WBI at 6.5 to 7.5 Gy led to the decrease in the 30-day mortality rate when the animals were fed the following pyridinium compounds: a) MNA from the 7th day before or 7th day after WBI, b) NAc from the day of WBI, c) 1,3-MAP from the 7th day after WBI. The effect was most pronounced in case of MNA administered from the 7th day after WBI with 7.5 Gy – mortality rate decreased from 81% to 53%. The obtained data suggest that stimulation of haematopoiesis by the tested pyridinium compounds is not the likely explanation of the enhanced survival. Acute WBI of mice at 6.5 or 7.0 Gy γ-rays resulted in the significant and proportional to the dose decrease in production of PGI2 until the 14th day after the irradiation. The level of PGI2 tended to increase in irradiated mice fed MNA; this tendency was most pronounced when the animals that were given MNA from the 7th day after WBI. Other mechanisms (e.g., mitigation of radiation-induced inflammation, thrombosis, and/or depressed endothelial function) of the prolonged survival after application of NAc derivatives or 1,4-DMP and exposure to 6.5 to 7.5 Gy γ rays need to be considered. Conclusion: The results obtained in mice exposed to sublehtal doses of gamma-rays suggest that the enhanced survival of the animals may be related to modulated inflammatory and/or thrombotic reactions, but not to stimulation of haematopoiesis. This study was supported by the grant No. DEC-2011/01/B/NZ7/05406 from the Polish National Science Centre.
European Radiation Research 2018, August 21-25, Pécs, Hungary
64
Monitoring of myocardial regeneration with stem cells using isotopic
imaging method
Łukasz Cheda1, Krzysztof Kilian
2, Paulina Hamankiewicz
1, Weronika Wargocka
1,
Paulina Gapska3, Natalia Rozwadowska
3, Maciej Kurpisz
3, Zbigniew Rogulski
1
1Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw,
101 Żwirki i Wigury Str., 02-089 Warsaw, Poland
2Heavy Ion Laboratory, University of Warsaw, 5a Pasteur Str., 02-093 Warsaw, Poland
3Institute of Human Genetics, Polish Academy of Sciences, 32 Strzeszyńska Str.,
60-479 Poznań, Poland
Cardiovascular disease (CVD) is a major cause of morbidity and mortality, especially in highly
developed countries, and is commonly associated with myocardial infarction (MI). According to the
World Health Organization, myocardial infarction and coronary artery disease lead
to 29% of deaths worldwide.
One of the method of heart treatment after MI is reconstruction the loss of myocardium with new
functional cardiac cells. The experimental studies and early clinical trials conducted
to overcome these limitations focused on identifying perfect cell stem candidates for cellular therapy,
the best method of cell delivery, optimal cell dose and timing of its administration.
One of the method of determination of effectiveness of cell therapy is the application
of radioisotopes. In this study we present method of monitoring the biodistribution
of mesenchymal and myoblasts stem cells radiolabeled with [99m
Tc]HMPAO. The application of
radiolabeled compounds allow us to short-term monitoring of administration
and biodistribution of stem cell, injected to the myocardium using the SPECT (Single Photon
Emission Computed Tomography) imaging technique.
The obtained results confirmed that injected stem cell remain at the place of administration after 24
hours post injection to myocardium.
Acknowledgements
This work was supported by The.National Centre for Research and Development (NCBiR): PBS1
Research Grant, Contract No. PBS3/A7/27/2015.
The study was carried out at the Biological and Chemical Research Center, University
of Warsaw, established within the project co-financed by European Union from the European
Regional Development Fund, under the Operational Program Innovative Economy,
2007–2013.
European Radiation Research 2018, August 21-25, Pécs, Hungary
65
Folic acid as a protectant against prenatal irradiation-induced birth
defects and sensory impairment
Kai Craenen1,2
, Mieke Verslegers1, Livine Craeghs
3, Jasmine Buset
1, Rudi D’Hooge
3, Sarah
Baatout1, Lieve Moons
2 and Rafi Benotmane
1
1 Belgian Nuclear Research Centre SCK•CEN, Radiobiology Unit, Mol, Belgium
2 University of Leuven (KUL), Neural Circuit Development and Regeneration, Leuven, Belgium
3 University of Leuven (KUL), Laboratory of Biological Psychology, Leuven, Belgium
Objective - Ionizing radiation has become an intrinsic part of modern society and medicine. However, the
aftermath of nuclear disasters and the atomic bombings have illustrated the potential detrimental health effects
as well. For example, congenital anomalies such as neural tube defects (e.g. exencephaly) and eye defects
(e.g. microphthalmos/anophthalmos) appear more prevalent in regions severely contaminated by radioisotopes
originating from the Chernobyl disaster. This increase is attributed to the high radiosensitivity of the unborn
child, with the resulting phenotypes being dependent on the dose and time of irradiation. Indeed, in previously
published work, we could identify mouse embryonic day (E) 7.5 (early neurulation) as the most radiosensitive
stage for inducing exencephaly and microphthalmos/anophthalmos with a high (1.0 Gy) X-ray dose.
Continuing, in the past folic acid (FA) has been identified as an antiteratogen with the capacity to prevent
malformations such as neural tube defects, cleft palate and heart defects. FA is an antioxidant and a one-
carbon donor with a crucial role in DNA synthesis/repair. As such, the mode-of-action of FA may be crucial
in counteracting many of the cellular consequences of ionizing radiation exposure (e.g. increased oxidative
stress and DNA damage). In this project, we first investigated the effect of FA food fortification on the
prevention of severe radiation-induced congenital defects in mice. Secondly, to complement the limited
amount of data on behaviour and cognition following X-ray exposure at the time of neurulation, we evaluated
the long-term consequences of sub-lethal X-irradiation at E7.5 and the potential beneficial effect of FA.
Methods – For the study of congenital defects at E18, dams were fed FA fortified diets (3.5 mg/kg (control),
10 mg/kg or 20 mg/kg FA) starting one week before coupling, and were exposed to 1.0 Gy of X-rays at E7.5.
For the long-term functional tests, dams and progeny were fed 3.5 mg/kg or 10 mg/kg FA diets, and exposed
to 0.1 Gy or 0.5 Gy at E7.5. Between 5-14 weeks of age, the prenatally irradiated mice were subjected to a
behavioural test battery and in vivo optical coherence tomography (OCT) imaging.
Results – 10 and 20 mg/kg FA fortification resulted in a significant decrease in radiation-induced
anophthalmos, exencephaly and agnathia. However, no marked dose response could be observed with the
double 20 mg/kg fortification, suggesting that elevating the FA dose beyond the suggested dose in mice (10
mg/kg) holds no added value. In terms of long-term radiation-induced defects, we revealed a decrease in
visual acuity (optomotor test) from 0.1 Gy onward, and a decreased thickness of the retinal ganglion cell layer
after 0.5 Gy. Continuing, we observed aberrant olfactory habituation/dis-habituation and anosmia after 0.5 Gy
irradiation. Notably, FA food fortification (10 mg/kg) increased the dose threshold from 0.1 to 0.5 Gy to
develop visual acuity defects and ameliorated the radiation-induced olfactory anomalies.
Conclusions - In all, we demonstrated the radioprotective effect of FA for various X-ray-induced birth-
defects. Furthermore, we offered a more in-depth screening of behavioural anomalies following low and
moderate X-ray doses at E7.5 and could unveil a protective effect of FA for radiation-induced visual and
olfactory decline. From a radiation protection viewpoint, our study thus supports food fortification with FA to
partially prevent high-dose induced congenital defects and adult behavioural anomalies following low and
moderate doses.
European Radiation Research 2018, August 21-25, Pécs, Hungary
66
Raman spectroscopy to predict radiation toxicity in prostate cancer
patients
Daniel Cullen1,2
, Adrian Maguire1,2
, Jane Bryant1,2
, Dinesh Medipally1,2
, John Armstrong3, Mary
Dunne3, Aidan Meade
1,2, Orla howe
1,4 and Fiona Lyng
1,2
1 Radiation and Environmental Science Centre, FOCAS Research Institute, Camden Row, Dublin 8, Ireland
2 School of Physics, DIT, Kevin Street, Dublin 8, Ireland
3Department of Radiation Oncology, Saint Luke's Radiation Oncology Network, St Luke's Hospital, Dublin,
Ireland
4 School of Biological Sciences, DIT, Kevin Street, Dublin 8, Ireland
Objective: The success of radiation therapy in tumour control depends on the total dose given but the
tolerance of the normal tissues surrounding the tumour limits this dose. It is not known why some patients
develop radiation toxicity, and currently, it is impossible to predict before treatment which patients will
experience adverse effects as a result of radiotherapy. An assay to predict risk of radiation toxicity would
guide the selection of treatment modality to reduce this risk in high risk patients or allow dose escalation in
low risk patients to improve tumour control. This study aimed to evaluate Raman spectroscopy for
identification of cancer patients at risk of severe radiation toxicity following radiotherapy.
Methods: Forty patients were enrolled prospectively on a radiotherapy trial and were followed up for at least
8 months following radiotherapy and toxicity was recorded using the National Cancer Institute Common
Terminology Criteria for Adverse Events (NCI-CTCAE) grading system. Blood samples were acquired at
baseline (ie. before treatment commenced) and cultured in-vitro. Cultures were irradiated to 0.05 Gy and 0.5
Gy. Raman spectra were acquired from lymphocytes. Radiosensitivity was assessed in parallel using the
H2AX assay and the G2 chromosomal radiosensitivity assay.
Results: Differences in spectroscopic profiles were observed between patients who showed either severe late
toxicity or no/minimal late toxicity at follow-up. Differences in H2AX fluorescence intensity and G2
radiosensitivity scores were also observed between the patients who showed severe late toxicity and those
showing no/minimal toxicity at follow up.
Conclusion: These results suggest that Raman spectroscopy may have potential in predicting radiation
toxicity outcome in prostate cancer patients which could provide a more personalised approach to
radiotherapy treatment.
European Radiation Research 2018, August 21-25, Pécs, Hungary
67
Radiation-induced mutation in the mammalian germline: Where are
we now?
Yuri E. Dubrova1
1 Department of Genetics and Genome Biology, University of Leicester, UK
The effort to predict the genetic consequences of exposure to mutagens, including ionising radiation has
certainly been one of the most important issues of human genetics in the past fifty years. Given that the
majority of chromosome aberrations and many gene mutations lead to inherited diseases, the analysis of
radiation-induced changes in germline mutation rates could provide important data on the genetic risk of
human exposure to ionising radiation. However, despite numerous studies, experimental evidence for the
effects of radiation exposure on mutation induction in the human germline still remains highly controversial.
It should be stressed that the lack of reliable approaches for monitoring germline mutation currently presents
the main obstacle to evaluating the genetic risk of human exposure to mutagens. Recent advances in genetic
technologies have provided new microarray-based and next generation sequencing-based tools for the
genome-wide analysis of genetic variation, which have the potential for characterizing germline mutation in
humans and mice. Using microarray-based comparative genomic hybridisation and whole genome sequencing
we have recently analysed the genome-wide effects of ionizing radiation on germline mutation in mice. We
found that the frequency of de novo Copy Number Variants (CNVs) and insertion/deletion events indels was
significantly elevated in offspring of exposed fathers. We also showed that the spectrum of induced de novo
SNVs is strikingly different; with clustered mutations being significantly over-represented in the offspring of
irradiated males. Our study highlights the specific classes of radiation-induced DNA lesions that evade repair
and result in germline mutation and paves the way for similarly comprehensive characterizations of other
germline mutagens. Applying this strategy to characterise germline mutation induction in humans a should
lead to a step change in our understanding of the sensitivity of the mammalian germline to exogenous
mutagens.
European Radiation Research 2018, August 21-25, Pécs, Hungary
68
Effect of radiotherapy, molecular parameters and treatment on the
expression of blood miRNAs-146a, -155, -221 and -222 in women with
breast cancer
Roser Esplugas1,2
; Noemí Serra2; Montserrat Bellés
1,2; Victoria Linares
1,2; Meritxell Arenas
3; Joan Carles
Vallvé4*
1Physiology Unit, School of Medicine, IISPV, Rovira i Virgili University, Reus, Spain
2Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV, Rovira i Virgili University,
Reus, Spain 3Radiation Oncology Department, Sant Joan University Hospital, IISPV, Rovira i Virgili University, Reus,
Spain 4 Research Unit on Lipids and Atherosclerosis, Sant Joan University Hospital, IISPV, Rovira i Virgili
University, Reus, Spain *Corresponding author. Tel.: +34 977759367. E-mail address: jc.vallve@urv.cat (J.C. Vallvé)
Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer death in women.
Overall survival rate is 90 % with 5 years, and many of them receive radiotherapy (RT). BC survivors have a
high risk of cardiovascular disease (CVD) caused mainly by the effect of RT affecting heart and blood
vessels. In the past decade, microRNAs (miRNAs) has emerged as a major regulator of several pathologies
including CVD. As inherent gene regulators, miRNAs could offer new predictive and therapeutic possibilities.
Recent data supported the role of miRNAs-146a, -155, -221, and -222 in the progression of CVD, mainly by
regulating inflammation, oxidative stress, apoptosis, and angiogenesis in atherosclerotic plaque.
The aim of our study was to evaluate the modulation on RT-induced expression of these miRNAs in blood of
women with BC, as well as to assess the effects of molecular parameters and treatment on their levels.
Blood samples of 136 women with BC were collected pre- and post-RT. After miRNA isolation and Reverse
Transcription, the levels of miRNAs-146a, -155, -221 and -222 were measured at these two time-points by
real-time PCR. Then, change in miRNA expression was calculated for each sample.
Results exhibited a significantly positive correlation between changes of miRNAs expression (Pearson
correlation coefficient was above 0.809 for all bivariate correlations). Furthermore, we showed significantly
inverse correlation between expression change of miRNAs-146a and -155 and age at diagnose. Finally,
differences of all miRNA expression and associations of change in miRNA expression with BC-related
molecular parameters such as Ki-67 index and HER2, as well as with chemotherapy and targeted therapy
treatments were observed. Patients HER2 positive, whose expression decreased post-RT, had significant
higher change of all miRNAs than negative ones whose miRNAs levels increased post-RT. Moreover, change
of all miRNAs was statistically higher in patients with 15-50% Ki67 index than those negative for Ki67; in
both groups expression increased post-RT. Similarly, expression of miRNA-221 increased post-RT, and its
change was significantly higher in patients who received chemotherapy in comparison to non-chemotherapy
treated. Finally, women treated with targeted therapy had statistical enhanced change of all miRNAs than
other ones; and while expression in targeted therapy-treated decreased post-RT, it was enhanced in non-
treated with targeted therapy.
In conclusion, we observed that expression and change in expression of miRNAs-146a, -155, -221 and -222
were modulated by RT as well as by BC-related molecular parameters and received treatment. This
modulation together with other risk factors might be associated with the development of future cardiovascular
pathologies.
European Radiation Research 2018, August 21-25, Pécs, Hungary
69
Effect of 2 Gy-radiation on the expression of miRNAs-146a, -155, -
221 and -222 in HUVECs
Roser Esplugas1,2
; Noemí Serra2; Montserrat Bellés
1,2; Victoria Linares
1,2; Meritxell Arenas
3; Joan Carles
Vallvé4*
1Physiology Unit, School of Medicine, IISPV, Rovira i Virgili University, Reus, Spain
2Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV, Rovira i Virgili University,
Reus, Spain 3Radiation Oncology Department, Sant Joan University Hospital, IISPV, Rovira i Virgili University, Reus,
Spain 4 Research Unit on Lipids and Atherosclerosis, Sant Joan University Hospital, IISPV, Rovira i Virgili
University, Reus, Spain *Corresponding author. Tel.: +34 977759367. E-mail address: jc.vallve@urv.cat (J.C. Vallvé)
Breast cancer (BC) is the most frequent cancer in women, causing about half a million total deaths each year
worldwide. Radiotherapy (RT) is given to reduce the risk of recurrence and mortality of this cancer. However,
RT causes both early and late side effects. The main non-cancer cause of death in these patients is cardiac one.
RT-induced endothelium damage promotes atherosclerosis progression which lead to cardiovascular disease
(CVD) development. In the course of atherosclerosis, chemokines form a complicated network by promoting
specific cellular interactions. IL-8 is a pro-inflammatory cytokine highly expressed in atherosclerotic plaques
whose one of the main sources are endothelial cells. Moreover, recent evidence suggest that microRNAs
(miRNAs) are implicated in CVD processes connected with inflammation, oxidative stress, apoptosis, and
angiogenesis in atherosclerotic plaques; as well as in the radiation response. Previous studies supported the
association of miRNAs-146a, -155, -221, and -222 with endothelial dysfunction and adhesion, and also with
infiltration of inflammatory cells into the endothelial space.
Our purpose was to assess the modulation on the expression of these miRNAs as well as IL-8 release after
radiation exposure of human umbilical vein endothelial cells (HUVECs).
We determined radiation dose and time-points by assessing cytotoxicity (as % LDH release) and viability (as
% ATP release) of both 2 Gy and 10 Gy (doses rates of 4.5 Gy/min) from 1 to 48 h. Then, with selected dose
and time-points, we measured the expression of miRNAs-146a, -155, -221 and -222 by real-time PCR, as well
as IL-8 release.
Results indicated that either irradiations did not exhibit cytotoxic or impairing-viability effects on HUVECs at
measured times. After 2 h irradiation, levels of miRNAs-221 and -222 were significantly lower in 2 Gy-
irradiated group than sham-irradiated, whereas miRNA-155 was overexpressed. Furthermore, we observed
that the expression of selected miRNAs, except miRNA-221, was statistically downregulated at 24 h post-
irradiation in 2 Gy-irradiated groups in comparison to sham-irradiated. Finally, results showed that IL-8
release was not modulated by 2 Gy-radiation.
In conclusion, 2 Gy-radiation at HUVECs modulated the expression of miRNAs-146a, -155, -221 and -222,
which are involved in atherosclerosis. This modulation could be related to the progression of CVD that
women with BC usually develop after RT treatment.
European Radiation Research 2018, August 21-25, Pécs, Hungary
70
Carcinogenesis in the lungs of mice line Af under the influence of radiation
and chemical factors
Fabusheva K. M1., Veyalkina N.N
1., Sushko S.N
1.
1SSI «Institute of Radiobiology of the National Academy of Sciences of Belarus», Gomel, Belarus
The study of the development of lung neoplasms in experimental models on laboratory
animals is relevant in connection with the need to develop new approaches of diagnosis, treatment
and prevention of lung cancer. The high-cancer line Af of mice is characterized that in the process of
development spontaneous lung tumors appear in intact mice, the number of which increases under
the influence of mutagenic and carcinogenic factors.
The aim of the research was to study chemical- and radio-induced carcinogenesis in lungs
of mice line Af in vivo.
Materials and methods. The experiments were performed on white laboratory mice line Af
of both sexes, at the age of 2.5-3 months. There were formed the following groups of animals: 1 −
intact control (9 females, 10 males), 2 − irradiation (10 females, 9 males) and 3 − inhalation priming
(9 females, 10 males). Irradiation of mice was performed on a gamma-installation «IGUR» (137
Cs
source) at a power of 46 cGy/min in a dose of 1 Gy. The inhalation effect of sulfur dioxide (SO2) and
ammonia (NH3) was carried out on the UIN-2M for an hour. The concentrations of gases in the
chamber were: CNH3 = 1 mg/m3, CSO2 = 5 mg/m
3.
The removal of animals from the experiment conducted in 5 months after exposure. Lungs of
animals were fixed in 10 % formalin and then studied with a binocular microscope. The frequency of
appearance of tumors was estimated (% of mice with adenomas). For histological analysis from the
paraffin blocks were made sections with thickness of 5-7 microns, which were stained with
hematoxylin-eosin and studied under a light microscope.
Statistical processing of data was performed using software packages IBM SPSS Statistics
21.
Results. Studied the induced tumorigenesis in the lungs of linear mice after irradiation at a
dose of 1 Gy or inhalation of a mixture of ammonia and sulphur dioxide. At the end of the
experiment the death of animals in the experiment is not observed. The proportion of animals with
lung adenomas in the age control group was 5.3%. In animals with adenomas in the lungs fixed no
more than one tumor which was a small size in diameter of 0.5-1.7 mm.
In the groups subjected to irradiation or in halation priming, the proportion of animals with
lung adenomas were significantly increased compared to the control group and amounted to 36.8%
(p= 0.042) and 38.9% (p=0.019), respectively. No statistically significant differences inside the
groups depending on sex of animals.
Neoplasms in the lungs of mice exposed to radiation exposure or inhalation priming had a
similar structure and were classified as highly-differentiated adenocarcinoma. Tumors were located
in the surface and the edge zones of the lung and went beyond the surface of the organ. The
connective tissue capsule around the tumor nodes was not visible. The tumor parenchyma was
formed by large lobules of hyperplastic glandular tissue, consisted of numerous densely located cells.
The nuclei of cells were round and oval shapes with a distinct karyolemma and had well-visible
nucleoli. Mitosis figures were rarely found. Inside the tumor tissue were separate vessels filled with
formed elements of the blood. Directly to the tumor nodes were the well-preserved alveoli.
Conclusion. In the groups subjected to radiation or inhalation exposure, the proportion of
animals with lung adenomas were significantly increased compared to the control group. The role of
irradiation or chemical inhalation influence on carcinogenesis in the lungs of mice line Af is
discussed.
European Radiation Research 2018, August 21-25, Pécs, Hungary
71
Relationship between biodosimetry and respiratory function values
in lung stereotactic radiotherapy patients
Gyöngyi Farkas1, András Bajcsay
2, Gyula Ostoros
3, Zsolt Markóczy
3, Zsuzsa S. Kocsis
1, Márta
Kun-Gazda1, Gábor Székely
1, Dalma Mihály
2, József Lövey
2, Csaba Polgár
2, Zsolt Jurányi
1
1 National Institute of Oncology, Center of Radiotherapy, Department of Radiobiology and Diagnostic Onco-Cytogenetics, , Budapest, Hungary
2 National Institute of Oncology, Center of Radiotherapy, Budapest, Hungary
3 National Koranyi Institute of Pulmonology, Budapest, Hungary
Objective: As stereotactic therapy causes more lung tissue irradiation with small doses hence pneumonitis and extensive lung fibrosis is possible, we wanted to survey respiratory function (FVC, DLCO) and the relationship between DLCO, FVC and biodosimetry values in such patients. Methods: 36 patients were recruited and blood was taken before radiotherapy (8 x 7.5 Gy), immediately after and 3, 6, 9, 12 months later. Respiratory function measurements (FVC, DLCO) were performed at the same time points. The null point blood was irradiated in vitro in a water phantom with 3, 6 Gy at the same conditions (1400 MU/min, 6 FFF) as the patients. Chromosomes were prepared and at least a hundred cell divisions were analysed in a sample: aneuploidy, chromatid deletion and chromosome fragment, dicentric, ring, translocation, total aberration, aberrant cell number were scored. V1Gy and V54Gy isodose volumes were recorded. Data analysis was performed with Statistica 7 and Origin 8.6 softwares and Pearson correlation analysis. We obtained ethical approval for our study. Results: The dicentric + ring (1.0±0.3 /100 cells before therapy) and total aberrations (3.9±0.6 /100 cells before therapy) increased in trend due to the radiation therapy in the lung stereotaxic patients culminating three month after radiotherapy (8.1±1.8 dicentrics/100 cells and 19.2±3.2 total aberrations/100 cells). The average DLCO value (indicator of respiratory function) increased slightly, reaching its maximum at nine month after radiotherapy (47.5±3.9% to 48.7±3.9%). On the other hand individual patients’ DLCO before therapy varied greatly, the actual/predicted values varied between ten to hundred percent. Furthermore the individual change in DLCO between timepoints could be even 18% in both directions. The aberrant cell number of the in vitro irradiated (6 Gy) blood weakly correlated (Pearson coefficient 0.47 for aberrant cell number/V1%) with the chromosome aberrations (divided by the isodose surface volumes with or without) six month after radiotherapy of the patients. The relative chromosome aberration values did not correlate better than the absolute values. On the other hand, if chromosome aberrations were divided by the isodose volumes this resulted in a better correlation with the respiratory data than without division. (e.g. dicentrics+rings of 6 Gy in vitro irradiation did not correlated with total aberrations of the 6th month or 6th month-zero total aberrations, but the coefficient of total aberrations of the 6th month/V54 was 0.44). Conclusion: The high variability of DLCO data is due to COPD, but we cannot definitely explain why there are both negative and positive changes in DLCO after the radiotherapy. We propose the radiotherapy may have an inflammatory effect, which increases blood supply of the lung, increasing DLCO, or other immune system-linked explanations are also possible. We found, that despite the fact that the small dose volumes of stereotactic radiotherapy are much higher than in any other kind of radiotherapy it did not cause any detectable avarage DLCO decrease of the population during the first year follow-up after radiotherapy. Furthermore we found that the chromosome aberrations in the 3 and 6 Gy in vitro irradiated blood were also able to predict the FVC value decrease after radiotherapy, hence their value appear to be a promising biomarker.
European Radiation Research 2018, August 21-25, Pécs, Hungary
72
Measurement of the air exchange rate in buildings with elevated
levels of indoor radon concentration using VOC tracers
Michal Fejgl1, Karel Jílek
2, Jan Lenk
1, Aleš Froňka
2, Ivan Hupka
1
1National Radiation Protection Institute (NRPI), Section of Monitoring, Prague, Czech Republic
2NRPI, Section of Natural Radioactivity, Prague, Czech Republic
The Czech Republic has one of the highest values of the mean indoor radon activity by air volume in
buildings in the world (118 Bq/m3) mainly due to the geological bedrock rich in uranium. In more than 3 % of
family houses it was found that the activity is higher than reference level for indoor radon activity in buildings
with a living room (300 Bq/m3) [1]. With such values, the risk of developing lung cancer is elevated.
Therefore, the Czech legislation recommends utilizing the air exchange rate measurements as a diagnostic tool
facilitating the assessment of radon exposure and, consequently, enabling the initiation of remedial measures
implementation or further intervention to reduce the exposure.
The fundamental objective of this project is to develop a detection method of air exchange rate in residential
buildings, schools or workplaces using volatile organic compounds (VOC) as tracers and sorption tubes (ST)
as integral detectors.
For this purpose, there is a
VOC generator – ST and thermal desorption-gas chromatography (TD-GC) system that enables the
calculation of integral concentration of analytes adsorbed on tubes throughout the exposure time. Using
five different fluorine-based perfluoroalkylcycloalcanes (PFC), it is possible to measure up to 5-compartment
buildings and to determine the air exchange even between these compartments. The advantage of chosen
components is the possibility of performing the experiments in the presence of dwellers in the building under
normal conditions and during different time periods (days, weeks or months). After the exposure in the
aforementioned buildings, ST are analysed by a TD-GC system using electron capture detector. The resulting
concentration of the compounds is then exploited to calculate the air exchange rate.
In present experiments, three different types of sorbents (Chromosorb SKC 102, Carbopack B and Tenax TA
80/100) were used. A 220l barrel with controlled air flow, temperature and humidity as well as several VOC
generators with known compound flow served to simulate the dwelling conditions. The exposure time varied
from one day up to three weeks. Additionally, another measurement was completed in a sealed room with
similar parametric control as in the barrel. The range of evaporation speed for utilized generators was 1-3
mg/day for barrel and 5-30 mg/day. As the evaluated concentration in ST depends on the exposure time, its
calculated range was 1-1000 ng/tube. The detection limit of this method is ~ 0,1 ng/tube. The average uptake
rate for the three sorbents is 2,1 ng/(ppm.min).
In conclusion, the purpose of the project is to allow general public to have a possibility of air exchange rate
testing through parcel delivery (VOC – ST system) with subsequent evaluation at NRPI. Eventually, this
method is being prepared to be accredited in 2019.
References:
[1] State Office of Nuclear Safety Regulation no. 422/2016 Sb.
European Radiation Research 2018, August 21-25, Pécs, Hungary
73
Sodium orthovanadan inhibits p-53 mediated apoptosis
Alžběta Filipová1, Lenka Zárybnická
1, Aleš Tichý
1, Anna Lierová
1, Marcela Jeličová
1 and
Zuzana Šinkorová1
1 Faculty of Military Health Sciences in Hradec Kralove, University of Defence in Brno, Czech
Republic
Objective: The interest in chemical radioprotective substances (RRs) able to minimize the effects of
ionizing irradiation is linked with the extraordinary progress of biomedical analysis revealing its
negative effects on living organisms. Radioprotective effects based on substances inhibiting
apoptosis are characteristic of late generation radioprotective drugs; therefore, p53 inhibitors have
been proposed in this endeavor. P53 acts as a transcriptional factor regulating the transactivation of
multiple proteins involved in growth arrest, DNA repair, and apoptosis. In this regard, sodium
orthovanadate (OV) is known to inhibit p53 mediated apoptosis and is used in this mouse
experimental model.
Methods: OV was applied intraperitoneally either 5 minutes before or 5 minutes after whole body
gamma irradiation of C57Bl6 mice (7 Gy). Animal survival was monitored daily. Total cell count
analysis in peripheral blood and flow cytometry analysis of the lymphocyte population was
performed.
Results: The LD50/30 confirmed that sodium orthovanadate applied intraperitoneally into mice has
only a partial mitigation effect, significantly reducing the LD50 value observed at 30 days post-
irradiation. The cellular composition in bone marrow did not change significantly; however, we
observed some changes in the lymphocyte population during the first month after irradiation.
Conclusion: Our findings on lethally irradiated mice indicated that OV effectively suppresses p53
mediated apoptosis when applied before whole body irradiation.
This study was supported by project GAČR – 17-13541S.
European Radiation Research 2018, August 21-25, Pécs, Hungary
74
Analytical two component kinetic sorption model for support of
fixed-bed column experiments with sophisticated sorbents
Vitold Filistovič1, Rita Plukienė
1, Benedikta Lukšienė
1, Zita Žukauskaitė
1, Nikolaj Tarasiuk
1,
Evaldas Maceika1, Laima Kazakevičiūtė
1
1 State research institute Center for Physical Sciences and Technology, Vilnius, Lithuania
The most dangerous radioactive waste produced in nuclear power plants is spent nuclear fuel (SNF) and high
level radioactive waste (HLRW). From the point of view of safe handling and disposal of high level nuclear
waste many countries propose deep argillaceous formations. Scenarios of barrier failure and radionuclide
release to the near-surface environment are important to consider safety assessment exercises. In these and
other applications involving contaminant transport by groundwater, solid/liquid distribution coefficients (Kd)
are widely used to describe the retention of contaminants, i.e., sorption and other processes retarding their
migration.
The movement of water and chemicals in soils or sediments is generally better described with multi-
componential non-equilibrium kinetic sorption models than with more commonly used one-componential
kinetic sorption and has advantages in determining of sorption parameters. There is presents analytical and a
semi-analytical solution for non-equilibrium solute transport during steady unidirectional flow in a finite
porous media in the case when diffusion is neglected, and for a semi-infinite porous media when diffusion
processes are relevant and cannot be neglected. The solutions can be used to model transport in porous media
where the solute sorption is governed by two a first-order rate chemical non-equilibrium processes, then the
each adsorbed molecule can be attached to the two different active sites (Type-1 and Type-2) of the sorbent.
The transport equation incorporates conditions characterized by advection, dispersion, and first-order
degradation or decay.
As a special case the raw sorption process in a non-equilibrium active species transport was examined.
General solutions were derived for the third-type (Cauchy type) boundary value problem (BVP) condition,
used to prescribe the concentration flux through the upper boundary layer and for initial value problem (IVP)
condition, used to prescribe the initial constant concentrations values. Solution was derived with the help of
Laplace transformations.
European Radiation Research 2018, August 21-25, Pécs, Hungary
75
Effects of Low-Dose Gamma-Radiation on Senescence in vitro and in
vivo
Mathew Flegal
1, Lindsey Bertrand
1, Mehdi Moustaqil
2, Audrey Sansaloni
2, Frances Heather
3,
Diana Golubeva 4, Ilya Velegzhaninov
4, Ekaterina Plyusnina
5, Alexey Moskalev
5, Soji Sebastian
1,
Dmitry Klokov 1,6,
*
1, Canadian Nuclear Laboratories, Chalk River, Ontario, Canada
2, University of Montpellier, Montpellier, France
3, University of Waterloo, Waterloo, Ontario, Canada
4, McMaster University, Hamilton, Ontario, Canada
5, Institute of Biology, Komi Science Center of Russian Academy of Sciences, Syktyvkar, Russia
6, University of Ottawa, Department of Biochemistry Microbiology and Immunology, Ottawa,
Ontario, Canada
*, Email: dmitry.klokov@cnl.ca
Radioprotection standards are based on the Linear-No-Threshold hypothesis; however, cellular and
molecular responses to low-dose radiation (LDR) exposures often deviate from linearity. This
continuing controversy sparks heated debates over the adequacy of regulatory radioprotection
standards. Cancer is a main readout of human health risk upon radiation exposures. Age is the largest
contributing factor to cancer risk. Our earlier studies showed that LDR exposures may lead to a delay
in tumorigenesis in mice in vivo, presumably by activating DNA repair pathways and immune
defence systems. More recently, we found that 10 or 100 mGy of gamma-radiation suppressed
replicative senescence and associated gene expression changes in human fibroblasts in vitro. This
was accompanied by substantial alterations in the aging-related miRNA profiles. In contrast, LDR
exposure did not affect stress-induced premature senescence. A striking reversal of aging-related
functional decline of mouse myoblasts due to exposure to LDR in vitro was observed, which may be
associated with epigenetic chromatin alterations. Lastly, partial suppression of senescence markers
was found in the kidneys of aging mice (up to 26 months) if animals were exposed to LDR at the age
of 2 or 13 months. Our results suggest that cellular senescence responses to LDR are qualitatively
and quantitatively different from those elicited to high-dose radiation. Studies to investigate the
relevance of these effects to tumorigenesis, and therefore to cancer risk prediction, are warranted.
European Radiation Research 2018, August 21-25, Pécs, Hungary
76
Radiation health risks of human space flight beyond Low Earth Orbit
(LEO).
Anna Fogtman1, Guillaume Weerts
1 and Ulrich Straube
1
1 European Astronaut Centre, European Space Agency, Cologne, Germany.
Almost 60 years now humans have been flying to space, successfully completing missions in order to expand our knowledge about the universe. Over those years we also have gained a lot of experience about the risks of human space flight. In the forthcoming perspective of the end of the International Space Station (ISS) service, international space agencies urge to plan the next step of human space exploration beyond LEO. They are aware of additional health risks of exploration class missions to the Moon and beyond. The European Astronaut Centre (EAC) of the European Space Agency (ESA) is the home base for all European Astronauts. It’s core mandate is to select, train and protect astronauts before, during and after space missions. The unique role of the Space Medicine Team (SMT), within EAC, is to provide medical support and protect astronauts’ health throughout their entire careers. One of the major challenges for SMT is to understand, predict and reduce potential hazards for astronauts during deep space missions, from which space radiation is the most significant one. Currently, the international space agencies have different approaches to radiation health risk assessment, which are tailored for ISS operations in LEO. ESA adopted dose limits for ionizing radiation to Blood Forming Organs (BFO), as a multilateral consensus for ISS missions. NASA uses in addition a sophisticated model, deriving the limits from a 3% lifetime excess cancer mortality rate. Also other space agencies apply a variety of risk assessment tools, however current standards are not feasible beyond LEO. In order to safeguard European Astronauts through their deep space missions, there is a necessity to better understand the effects of space radiation to the human body, estimate the health risk and predict the consequences of long-duration space flight. Here, we present an exemplary introduction to current knowledge on biological effects to the human body, that may occur due to ionizing radiation in the deep space environment. We show current strategies for health risk assessment and the challenges for risk modelling of long-duration deep space missions. Finally, we discuss the initial collaborative steps which have been taken by the ESA Space Medicine Team and international partners towards a framework for radiation risk management under the Radiation Protection Initiative (RPI). We acknowledge, that only international joint effort between space agencies and commercial entities will facilitate the development of a standardized approach to risk management. Therefore, it will enable safe human-based exploration class missions to the Moon and Mars.
European Radiation Research 2018, August 21-25, Pécs, Hungary
77
Individual radio-sensitivity and susceptibility : towards a classification
of human syndromes with the ATM nucleoshuttling model
Nicolas Foray1
1 INSERM, UMR1052 Unit, Radiobiology Group, Lyon, France
Accumulated data on individual factors have suggested that the individual response to ionizing radiation (IR)
cannot be ignored and raises some clinical and societal issues. Recently, to avoid any confusion in the terms
related to IR radiation, we proposed the following definitions:
- “radiosensitivity” as any clinical and cellular consequences of IR attributable to cell death (e.g., tissue
reactions, clonogenic cell survival);
- “radiosusceptibility” as the RI cancers or any feature that is attributable to cell transformation;
Recent advances suggest that the delay in the nucleo-shuttling of ATM proteins may be reliable parameters
for predicting any level of individual radiosensitivity. However, the model of the nucleo-shuttling of ATM
also helps in deciphering the carcinogenesis linked to radiation. I will present further investigations suggesting
that some pathways specifically link to cell death and some other that specifically link to DNA damage
misrepair in response to IR.
European Radiation Research 2018, August 21-25, Pécs, Hungary
78
Comparative study of molecular and cellular effects induced by different
energy X-rays
Amelie Freneau, Aurelie Vaurijoux, Pascale Voisin, Laurence Roy and Gaetan Gruel
Institut de Radioprotection et de Sureté Nucléaire (IRSN), Laboratoire de Dosimétrie Biologique, BP
17, 92262 Fontenay aux Roses cedex, France.
During a radiological examination, dose delivered to the patient's organs varies from a few tenths to
a few tens of mGy. These low doses accumulate throughout the life of an individual, some patients
being subjected to dozens of examinations in the same year. The risks associated with such
exposures remain to be identified, understood and evaluated. Depending on the purpose of the
examination, the type of radiation used is not the same. In a radiological examination (interventional
radiology or mammography), low-energy X-radiation is used (< 100 keV). For other diagnosis
procedures, the energy used is several MeV. It is currently considered that photons irrespective of
their energy have the same radiation weighting factor. But, several studies have shown an increase in
the relative biological effect of photons when their energy decreases, in particular an increase in the
frequency of chromosomal aberrations and a decrease in clonogenic survival. In theory, these
differences in biological efficacy could have their origin in the topological differences at the
nanoscale of X-ray energy deposition as a function of their energy spectrum. Indeed, as the photon
energy decreases, the nature of their interactions with living matter changes. To study this difference,
an experimental strategy was spread out to study the early response as well as the late response after
exposure to three different radiation qualities of X-ray. We evaluated signaling of DNA damage by
monitoring γH2A.X and foci after exposure of G0/G1-phase synchronized human primary
endothelial cells to 0.25, 0.5, 1 2 and 5 Gy of 40 kV, 220 kV and 4 MV X-rays. A large number of
γH2A.X foci were analyzed from 30 min to 5 h using immunofluorescence labels and an automated
detection of nuclei and foci was conducted. Different characteristics, such as number and spatial
distribution of foci among others were explored. For the same dose of X-rays, at early times, some
differences in the kinetics of the number of γH2A.X foci per nucleus was shown among the three
radiation qualities studied. The spatial distribution of foci among others seems similar. However,
simultaneously, the comparative study of cell behavior after irradiation at 40 kV and 4 MV was
performed by video-microscopy and do not revealed a significant difference in the ability of cells to
divide after irradiation. Furthermore, to complete the comparative study, we counted the frequency
of micronuclei and/or nucleoplasmic bridges in mono- and binucleated cells and did not show a
significant difference between the two radiation qualities.
European Radiation Research 2018, August 21-25, Pécs, Hungary
79
Immune modulation by radiation – impact for the design of
radioimmunotherapies
Benjamin Frey1, Michael Rückert
1, Anja Derer
1, Michael Hader
1, Rainer Fietkau
1, Udo S. Gaipl
1
1 Department of Radiation Oncology, Universitätsklinikum Erlangen, Friedrich-Alexander-
Universität Erlangen-Nürnberg, Erlangen, Germany
Radiotherapy (RT) is a common treatment for cancer and about 60% of all cancer patients will receive it
during their course of illness. RT primarily aims to achieve local tumor control. The induction of DNA
damage, tumor cell death and the modulation of the tumor microenvironment are the main effects of ionizing
irradiation to reduce tumor masses, but also to modulate the immune system. RT might act as an in situ cancer
vaccine under certain microenvironmental conditions. However, RT also fosters the upregulation of immune
suppressive molecules such as the programmed cell death receptor ligand 1 (PD-L1, CD274). Ionizing
radiation in general has been demonstrated to impact on the immune system and in dependence on the
radiation dose particular immune modulations take place. The presentation will focus on how local irradiation
changes the tumor cell phenotype and the tumor microenvironment and consecutively does impact on local
and in particular on systemic changes in immune cell compositions. The dynamics of immune changes, the
radiosensitivity of distinct immune cells as well as biological basis for reasonable combination of RT with
immune stimulation will be discussed in detail, as well as how radiation-induced immune suppression can be
overcome. Regarding the latter, the impact of radiotherapy and chemotherapy on immune checkpoint
molecule expression will be outlined and possible mechanisms for that will be discussed. Based on the pre-
clinical knowledge, innovative clinical study concepts of radio-immune treatments will be presented. We
conclude that knowledge on immune modulations induced by ionizing radiation is important to optimize
multimodal cancer therapies aiming to achieve local and systemic tumor control and to define immune-related
biomarkers of radiation exposure for prognosis and prediction.
European Radiation Research 2018, August 21-25, Pécs, Hungary
80
Strategies for model validation in radiobiology
Thomas Friedrich1 and Michael Scholz
1
1 GSI Helmholtz Centre for Heavy Ion Research, Department of Biophysics, Darmstadt, Germany
Objective: As a complement to experimental studies, mathematical models play an important role
in the understanding of radiation action in cells, tissues and organisms. In the light of increased use
of particle therapy, there is a current renaissance in modelling the relative biological effectiveness
(RBE). The presentation provides a compilation of basic strategies which we follow for validation
and testing of the Local Effect Model (LEM) for predicting effects of ion radiation. These strategies
go beyond simply comparing theoretic predictions against experimental data obtained in individual
experiments and are helpful to investigate the model's predictive capability as well as its limitations,
and also shed light on the validity of underlying assumptions.
Methods: In particular we report on testing model predictions against a large ensemble of
experimental data. For that we compiled in-vitro cell survival data from more than 1000 experiments
in a data collection (Particle Irradiation Data Ensemble, available under www.gsi.de/bio-pide), each
of which provides experimental RBE information. Furthermore, we address the model outreach, i.e.
its performance in extended applications which allow to describe (i) the radiation action of other
radiation qualities than ions (e.g. ultrasoft or orthovoltage X-rays) within the same model formalism,
(ii) dose response modifications for different exposure situations (e.g. protracted irradiation or cell
cycle specific irradiations) and (iii) different endpoints than cell survival (e.g. double strand break
repair kinetics, clinical endpoints or carcinogenesis). Finally, we report on model robustness tests by
means of a sensitivity analysis of all parameters involved, where the main strategy is to avoid free
input parameters in order to keep the model truly predictive instead of only being capable of fitting
experimental data.
Results: The presented strategies allow a deeper insight into model performances and limitations,
and therefore facilitate a critical and more comprehensive assessment of the underlying model
assumptions, that would hardly be possible with a classical model comparison against individual sets
of data.
Conclusions: The application of enhanced strategies of model validation is supportive in
investigating model properties and assessing the model prediction reliability.
European Radiation Research 2018, August 21-25, Pécs, Hungary
81
Effect of acute whole-body gamma irradiation on circulating
microparticles levels in rats.
AL-MASSARANI GHASSAN
Biomarkers Laboratory, Department of Radiation Medicine , Atomic Energy Commission of Syria
(AECS)
E-mail: ascientific2@aec.org.sy
Background: Damage to cellular membrane and disruption of the cytoskeleton is a well recognized
complication of the irradiation. The disorganization of the cytoskeleton leads to form membrane
blebs, which is called microparticles (MP). Our objective was to determine the gamma-irradiation
effect on the circulating MP as biomarker of cellular membrane damage in blood of rats.
Materials and methods: The Wistar rats were divided into six groups: a control group and 5 groups
of rats receiving a different dose of irradiation (0.5, 1, 2, 4 and 8 Gy) for different times (24h, 72h
and 1 week). MP in blood of rats were counted by flow cytometer after 24h, 72 h and one week post-
irradiation.
Results: Quantified MP showed that there was increased in MP count in irradiated rats compared to
control group, (p<0.05) at all the time points and in a non-dose dependant manner, Whereas at one
week post-irradiation the increase in MP levels is clearly less pronounced with lethal dose (8 Gy,
p=0.09). After one day of irradiation, the levels of MP in rats irradiated with 4-8 Gy was
significantly lower than those in rats irradiated with doses of 1-2 Gy, without reaching their values in
controls.
However, we observed a significant decrease in the number of MP (72h and one week) post
irradiation at all doses except for 0.05 Gy compared to those found 24h after irradiation. It seem that
there a partial restoration in MP levels with time elapsed from the exposure to gamma irradiation.
Conclusion: The number of MP in rats exposed to whole-body gamma irradiation was increased in a
dose-dependent manner and it partly recovered during the 72h interval after irradiation. We suggest
that MP count may be an early indicator of the membrane damage induced by ionizing radiation.
European Radiation Research 2018, August 21-25, Pécs, Hungary
82
Targeting cellular antioxidant system and DNA repair to sensitize
glioma stem cells to different LET radiation: low let chronic/acute dose
rates (Cs137), and high LET C-ions
Godoy, PRDV3, Pour Khavari, A
3, Sakamoto-Hojo, ET
1, 2, Saintigny Y
4, Haghdoost S
3,4,
1Department of Biology, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto,
2Department of
Genetics, Faculty of Medicine of Ribeirão Preto; Brazil, 3Department of Molecular Biosciences, The Wenner-
Gren Institute, Stockholm University, Sweden, 4LARIA – CIMAP, University of Caen Normandie, France
Objective: Glioblastoma (GBM) is a malignant brain tumor with poor outcome and is resistant to several
type of treatments. Glioma stem cell (GSC) presents an enhanced antioxidant defense compared to their
serum-differentiated counterparts and is resistant to chemo- and radio- therapy. There are strong evidences
that carbon ion irradiation could be an approach to overcome GSCs. In this context, our aim is to reduce
GBM radioresistance by applying strategies of gene silencing for genes playing roles in antioxidant defense
mechanisms, thus causing an increase in the generation of radiation-induced ROS. The increase in the
induction of oxidative damage is expected to lead to glioma stem cell differentiation and reduction in the
self-renewal capacity, thus ultimately reducing the GBM malignancy. To achieve this, we aim to compare the
expression of antioxidant proteins, BER, nucleotide pool clearance and stem cell markers (Western Blot,
WB), oxidative stress marker extracellular 8-oxo-dG, and self-renewal (sphere formation assay) in GBM
CSC exposed to low LET chronic and acute dose rates (Cs137) and high LET (C) ions.
Methods: GSCs were isolated from the U87MG GBM cell line using neural stem cell medium (DMEM F12,
b27 without antioxidants, EGF and FGF2). We used low LET (Cs137) chronic versus acute dose rate, 24
mGy/h and 0.39 Gy/min respectively, and high LET Carbon ions (approximately 2 Gy/min) irradiation. For
neurosphere formation, cells were exposed to doses ranged from 0.5 to 8 Gy gamma and 0.5 to 4 Gy carbon
ions radiation, and spheres were conted 6 days after irradiation. For protein expression, the exposed cells
were lysed 3, 6 and 24h after irradiation.
Results: Preliminary results showed that GBM stem cell is significantly more sensitive (p≥0.05) to carbon
ions (IC50=1.3±0.4 Gy) than gamma under acute (IC50=2.6±0.2 Gy) and chronic dose rate (IC50=11±3 Gy),
considering the neurosphere assay. The chronic irradiation increased 8-oxo-dG induction over the time, with
the peak at 20 days of exposure, induced cell differentiation (analyzed as decreased MUSASHI1 expression)
and showed an tendency of downregulation of NRF2 (Nuclear Factor, Erythroid 2 Like 2) GSS (glutathione
synthetize), hMTH1 (8-oxo-dGTP-ase), PRDX2 (Peroxiredoxin-2), GSTO1 (glutathione S-Transferase
Omega 1), APE1 (APEX nuclease (multifunctional DNA repair enzyme) 1) and SOD1 (superoxide dismutase
1) mostly after 20 days of continuous irradiation (11.5 Gy). For the acute dose rate, there was an clear
increase tendency of most of the proteins 6h after IR for all doses tested, and a clear downregulation after 24h
of exposure, mostly observed for the higher doses of 7.5 and 11.5 Gy. The analysis pf the proteins after
Carbon ions exposure is ongoing.
Conclusion: Downregulation of proteins involved in ROS response under acute and chronic dose rates
needs to be verified. Some of the proteins analyzed in this project will be chosen for silencing in the GSC.
We expect that the present study will provide interesting results regarding glioma radiosensitization.
European Radiation Research 2018, August 21-25, Pécs, Hungary
83
Non-calorimetric determination of absorption power during
magnetic nanoparticle based hyperthermia
Gresits I1,2
., Simon F1., Thuróczy Gy
2.
1
Department of Physics, Budapest University of Technology and Economics, Budapest, Hungary 2
Department of Non-Ionizing Radiation, National Public Health Institute, Budapest, Hungary
Cancer is one of the major diseases worldwide. Despite the recent technical advancements in cancer
treatment such hyperthermia, the need for better efficient method for cancer therapy remains. One of
the recent methods in the cancer treatment is called nanomagnetic (nanofluid) hyperthermia (NMH).
The NMH is intensively studied in the last decade as a new prospect of cancer therapy. It involves
raising the temperature locally in the tumour with the intact of normal tissues. A major challenge is
to determine the heat absorbed during in vivo conditions and conventional methods are either
invasive or inaccurate. Herein, we present a method which allows determining the power absorbed
during nanomagnetic hyperthermia. Furthermore, our method needs low RF power with less robust
coils via reflection measurements. We validated this approach by a comparison of the absorbed
power with the theoretically expected values obtained from the result with well-known calorimetric
approach. This method allows determining directly the absorbed power for magnetite samples.
The present method is based on the monitoring of the quality factor (Q) of resonant coils in empty
case and even if it is filled with nanomagnetic material. The absorbed power can be obtained from
these quality factors using Eq. (1).
(1)
We studied two kinds of resonant circuits: a conventional tank circuit which consists of a solenoid
for the test-tube studies and a so-called birdcage coil which is employed in magnetic resonance
imaging . Quality factors of resonant circuits were determined from reflectometry. We validated by
comparing it with a conventional calorimetric measurement in for a test tube with high RF input
power esing Eq. (2):
(2)
where csample denotes the specific heat of the sample being assumed that
We got an excellent agreement between the data points and the calculation. Furthermore, we
could improv the accuracy of the quality factor using pulsed excitation.
European Radiation Research 2018, August 21-25, Pécs, Hungary
84
Radiation Biomarkers: fact or fiction?
Janet Hall
Centre de recherche en cancérologie de Lyon, INSERM 1052, CNRS 5286, Université Claude
Bernard Lyon 1, Lyon, 69424, France
Objectives: The integration of ionising radiation (IR) biomarkers into epidemiological studies to substantiate
the radiation causality of health risks associated with low dose and low dose rate IR exposures and, in
particular, to address the differences in radiation sensitivity between individuals, or groups, was highlighted
by the HLEG on European Low Dose Risk Research and has been incorporated into the Melodi SRA since the
outset. A number of key considerations including the choice of population to be investigated, the study design,
ethical considerations, the logistics of biological sample collection, processing and storing, the limitations of
and thus the choice of the biomarker or bioassay to be used, as well as potential confounding factors were
reviewed by the DoReMi consortium (Pernot et al., Mutation Res. 751 (2012) 258-286). This review
concluded that at that time there was no ideal biomarker for assessing exposure, effect or susceptibility of low
dose radiation exposure in population studies, although some good candidates did exist. Possible strategies for
the integration of biomarkers into molecular epidemiology studies addressing cardiovascular disease have also
been reviewed by DoReMi (Kreuzer et al., Mutat. Res. Rev., 764 (2015) 90–100) and for the effects of
uranium exposure by the concerted action CURE (Concerted Uranium Research in Europe) (Laurent et al., J.
Radiol. Prot., 36 (2016) 319–345). Given the increased mechanistic understanding of responses to low dose
radiation an update of the Pernot et al. paper was undertaken (Hall et al., Mutat Res., 771 (2017) 59-84).
Methods: A panel of experts* reviewed technical and analytical biomarker advances, particularly in the
“omics” fields, the findings from in vitro and in vivo animal models that had used low radiation doses (below
1 Gy) and pilot biomarker studies in several populations.
Results and Conclusions: A key issue identified was deciding which biomarkers to carry forward from
discovery to implementation. A roadmap was drawn-up to aid in this decision process of when to progress or
drop a biomarker that took into account critical issues such as assay reproducibility, sensitivity and specificity.
This roadmap was then used to summarise the current status of proposed biomarkers for epidemiological
studies investigating low dose health effects. Based on this evaluation it was concluded that most potential
biomarkers remain at the discovery stage and for some there is sufficient evidence that further development
for use in low dose radiation molecular epidemiology studies is not warranted. One biomarker identified in the
final stages of development, and as a priority for further research, is radiation specific mRNA transcript
profiles. The challenge will now be moving this forward and validating its use in prospective studies before
application in molecular epidemiological studies.
*Janet Hall, Penny A. Jeggo, Catharine West, Maria Gomolka, Roel Quintens, Christophe Badie, Olivier
Laurent, An Aerts, Nataša Anastasov, Omid Azimzadeh, Tamara Azizova, Sarah Baatout, Bjorn Baselet,
Mohamed Abderrafi Benotmane, Eric Blanchardon, Yann Guéguen, Siamak Haghdoost, Mats Harms-
Ringhdahl, Julia Hess Michaela Kreuzer, Dominique Laurier, Ellina Macaeva, Grainne Manning, Eileen
Pernot, Jean-Luc Ravanat, Laure Sabatier, Karine Tack, Soile Tapio, Horst Zitzelsberger, Elisabeth
Cardis
European Radiation Research 2018, August 21-25, Pécs, Hungary
85
Effect of ionising radiation on the mitochondrial DNA damage in hair bulbs
Rita Hargitai1, Sisko Salomaa
2, Päivi Roivainen
2, Katalin Lumniczky
1
1Department of Radiation Medicine, National Public Health Institute - Division of Radiobiology and
Radiohygiene, Budapest, Hungary
2Department of Environmental and Biological Sciences, University of Eastern Finland, Kuopio,
Finland
Ionising radiation affects macromolecules in the cells directly and also indirectly, through the
elevated amount of reactive oxygen species (ROS). Mitochondrial DNA (mtDNA) can be found in
large amounts in the cells. MtDNA is highly sensitive to the damaging effects of radiation and ROS,
because it’s repairing mechanisms are weaker than those of the genomic DNA. Therefore, mutations
appear at a higher rate and they persist for a longer time in the mtDNA. It has been demostrated in
several types of tissues that specific deletions appear in the mtDNA after exposure to ionising
radiation. In this study, our aim was to find a biological, non-invasive marker that is able to detect
local exposure to ionising radiation. We analysed hair bulbs, because hair is present on the whole
body surface, and it can be collected in a minimally invasive way. We hypothesized that such a
marker would be suitable for biodosimetry measurements, or to assess the exposure to radiation of
the skin in the case of radiotherapy or interventional radiography. We collected hair samples from
patients with prostate cancer, who were treated with fractionated stereotactic radiotherapy. Hair
samples were collected before the beginning of the treatment and after the last fraction from the
treated body area (pubic hair) and from a control area (scalp hair). DNA was isolated from hair
samples and mtDNA deletions and lesions were examined with real-time PCR method. Our results
showed that the ratio of mtDNA deletions in hair bulbs did not change significantly after radiation,
and there was no significant difference in the amount of mtDNA lesions between pre-treatment and
post-treatment time, either. Our results suggest that mtDNA deletions and lesions in hair bulbs
appear not to be suitable biomarkers of exposure to ionising radiation.
European Radiation Research 2018, August 21-25, Pécs, Hungary
86
Analysis of the nano-topology of repair clusters: Towards
understanding of its impact on repair pathway decision
Michael Hausmann1, Jin-Ho Lee
1, Elizaveta Bobkova
1, Emanuel Maus
1, Franz Schmidt-Kaler
1,
Marion Eryilmaz1, Emma Wagner
1, Matthias Krufczik
1, Andreas Hofmann
2, Daniel Depes
3, Sarah
Schumann4, Felix Bestvater
5, Christoph Cremer
6, Dieter W. Heermann
2, Harry Scherthan
7, Iva
Falkova3, Martin Falk
3, Georg Hildenbrand
1,8
1University of Heidelberg, Kirchhoff-Institute for Physics, Heidelberg, Germany
2 University of Heidelberg, Institute for Theoretical Physics, Heidelberg, Germany
3 Czech Academy of Sciences, Institute of Biophysics, Brno, Czech Republic
4 University of Würzburg, Dept.Nuclear Medicine, Würzburg, Germany
5 German Cancer Research Center, Heidelberg, Germany
6 Institute for Molecular Biology, Mainz, Germany
7 Bundeswehr Institute for Radiobiology, Munich, Germany
8University of Heidelberg, Universitätsmedizin Mannheim, Dept. Radiation Oncology, Mannheim,
Germany
Objectives: Several investigations of chromatin repair processes after cell exposure to ionizing radiation have
revealed the importance of 3D-chromatin and repair foci architecture for further processing DNA repair and
protein recruitment. In order to better understand how nano-topologies are correlated to repair process
decision and outcome, investigations were performed by light microscopic, super-resolution techniques which
enable optical resolution down to about 10 nm even in 3D conserved cell nuclei.
Methods: Localization microscopy is based on the concept of using fluorescent labels that can be switched
between two different spectral states (e.g. off/on) to achieve temporal isolation and thus spatial separation of
molecular signals leading to pointillist images and quantitative structural parameters. For multi-colour
localization microscopy, molecular labelling with specific antibodies against γH2AX, MRE11, 53BP1, pATM
and heterochromatin (H3K9me3) in combination with specific uniquely binding oligonucleotides for ALU
sequences was applied. Mathematical procedures based on Ripley´s point-to-point distance distributions and
topology analysis of persistence homologies, were used for data evaluation and parameter determination.
Results: Molecular (re-)arrangements were investigated after exposure to ionizing radiation (photons, α-
particles, ions) and during repair processes. These experiments included the quantitative studies of
conformational changes of heterochromatin, repair foci formation, and recruitment of repair proteins under
different radiation conditions. After specific labelling by antibodies against heterochromatin or oligo-
nucleotide nano-probing against ALU-repeats, network-like chromatin structures were detected and
characteristic re-arrangements were elucidated. The data indicate dose and repair process-dependent re-
organisation of chromatin addressed. The recruitment of repair proteins and foci formation at DNA damage
sites revealed differently structured γH2AX clusters with repair protein embedded. The sub-organisation along
particle tracks was quantified and correlated to different pathways of repair.
Conclusion: The investigations demonstrate the broad potential of localization microscopy for understanding
of mechanisms of radiation and repair processes in cell nuclei. Cell type dependent spatial re-arrangements of
molecular complexes and mechanisms behind such radiation and repair response on the single cell level may
be used for biological dosimetry and individual medical treatment control.
European Radiation Research 2018, August 21-25, Pécs, Hungary
87
INWORKS and an updated NRRW analysis
Richard Haylock1 and Michael Gillies
1
1Public Health England, Centre for Radiation, Chemical, Environmental Hazards, Didcot,
Oxford, UK
INWORKS:
Objectives: We report on an analysis of non-cancer mortality and in particular circulatory diseases in a large
pooled cohort of externally-exposed nuclear industry radiation workers from the UK, France and USA.
Methods: The cohort of 308,297 workers was analysed using Poisson regression methods to investigate
whether cumulative external radiation dose was associated with non-cancer mortality and particular,
circulatory diseases.
Results: In total 22% of the cohort were deceased with 46,029 deaths attributed to non-cancer outcomes
including 27,848 circulatory disease deaths. The average cumulative equivalent dose (Hp(10)) was 25.2 mSv.
A positive association between cumulative radiation dose and all non-cancer causes of death was observed
(excess relative risk (ERR) per sievert =0.19; 90%CI: 0.07; 0.30). This was largely driven by the result for
circulatory diseases (ERR/Sv=0.22; 90%CI: 0.08; 0.37).
Conclusions Evidence that non-cancer disease risks may be increased by occupational external radiation
exposure particularly for ischemic heart disease and cerebrovascular disease was found. However,
heterogeneity in the results was observed that warrants further investigation.
National Registry for Radiation Workers (NRRW) update:
Objectives: The NRRW provides direct evidence of cancer risk from low dose and dose rate occupational
external radiation exposure in the UK. We report here on an update to the third analysis using 10 additional
years follow-up.
Methods: Poisson regression was used to investigate the association between mortality and incidence of
malignant neoplasms excluding leukaemia as a single group and cumulative external radiation exposure. A
range of individual cancer types were also examined.
Results: Almost 35,000 deaths and 25,000 cancer incidences were examined among 174,541 persons, an
increase of 49% over the third analysis. Mortality and incidence risk estimates for the group of all malignant
neoplasms excluding leukaemia were significantly raised (ERR/Sv mortality = 0.28; 90%CI: 0.06, 0.53 and
ERR/Sv Incidence = 0.32; 90%CI: 0.14, 0.51) and confidence bounds that were narrower compared with the
third analysis reflecting the additional statistical power available. The linear trends in relative risk for both
mortality and incidence of these cancers remained statistically significantly raised when information relating
to cumulative doses above 100mSv was excluded.
Conclusions: The analysis strengthens the precision of the overall cancer risk estimates in the third analysis of
this important occupational cohort. The results remain consistent with those based on atomic bomb survivors
that form the basis of the current ICRP recommendations.
European Radiation Research 2018, August 21-25, Pécs, Hungary
88
Different in vivo models for research on normal tissue toxicity due to
ionizing radiation
Katalin Hideghéty1,2
, Emilia Rita Szabó1, Róbert Polanek
1, Szilvia Brunner
1, Tünde Tőkés
1,3
1ELI-HU Non-profit Ltd., Szeged, Hungary
2University of Szeged, Department of Oncotherapy, Szeged, Hungary
3University of Szeged, Institute of Surgical Research, Szeged, Hungary
Abstract
Purpose: of this talk is to provide an overview on the different in vivo systems established for radiation
toxicity evaluation related to therapeutic use of ionizing radiation. Additionally special vertebrate models
developed by our group for complex radiobiological effect assessment will be presented.
Methods: Review of literature data on standard models, on novel experimental techniques and on own
methods for complex evaluation on focal brain irradiation (by stereotactic and using small photon, electron
fields in rodents) and whole organism (fish embryo) radiation.
Results: The challenges of radiobiology research on novel RT methods such as synchrotron generated
Micro/Mini photon/proton Beam Therapy (MBT), laser driven particle acceleration, and binary approaches of
Boron Neutron Capture Therapy (BNCT), Boron Proton Capture Enhanced Proton Therapy (BPCEPT),
furthermore combinations by radiation modifiers will be presented. Well-established rodent models include
the murine acute upper intestinal crypt regeneration test, lung pneumonitis assay, oral mucositis analysis,
delayed skin reaction scoring and rat spinal cord damage (assessed by hind leg motion depletion), heart, liver
and kidney function and morphology for assessment and modification of the radiotherapy (RT) induced early
reactions, and late normal tissue complications. Several experiments (by special-frame stereotactic RT, by
shielded photon and self-developed electron insert collimated small field) led to definition of the optimal focal
brain irradiation technique and parameters for investigation of radiation injuries and its potential mitigation.
Irradiation of 40-Gy resulted in neurological deficit both at the level of cognitive function and morphology
after 4 months of irradiation. The Morris water maze test was found to be a highly sensitive tool for the
detection of neuro-functional impairment. Semi-quatitative histopathological evaluation revealed strong dose
dependent cellular changes in macrophage density, reactive gliosis, calcification and extent of demyelination
and necrosis. In the focus of our research stands the development of fish embryo model for radiobiology
investigations with numerous advantages. Zebrafish lines proved to be a tolerant, useful in vivo system
providing various quantitative end points from viability detection to molecular analysis through macro-and
micro morphological and organ function deteriorations for dynamic assessment of dose-and linear energy
transfer dependent effects of ionizing radiation, definition of relative biological effectiveness and to validate
radiation modifiers.
Conclusions: Divers in vivo experimental models and methods had been tested for investigation of normal
tissue injuries at morphologic, functional and molecular level. Each of them has its special advantages and
limitations. The zebrafish embryo system fills an important gap between the in vitro and in vivo models, and
proved to be a reliable biological tool for comparative radiobiology research. Our results warrant for further
work on refinement of the endpoints, in particular for development of late radiation sequel assessment.
The ELI-ALPS project (GINOP-2.3.6-15-2015-00001) is supported by the European Union and co-financed
by the European Regional Development Fund. The project has received funding from the European Union's
Horizon 2020 research and innovation programme under grant agreement no 654148 Laserlab-Europe.
European Radiation Research 2018, August 21-25, Pécs, Hungary
89
Biomedical potential of laser-driven particle acceleration
Katalin Hideghéty1,2
, Róbert Polanek1, Rita Emilia Szabó
1, Szilvia Brunner
1, Tünde Tőkés
1,3
1ELI-HU Non-profit Ltd., Szeged, Hungary
2University of Szeged, Department of Oncotherapy, Szeged, Hungary
3University of Szeged, Institute of Surgical Research, Szeged, Hungary
Abstract
Objective Recent advances in radiation oncology have increased the therapeutic index remarkably by more
selective dose delivery enabling dose escalation in the target and enhanced sparing of the normal tissues.
However, there is still a huge need for further improvement in order to increase the local and loco-regional
tumor control and survival.
Method Intensive development of laser-based techniques may result in relevant progress in radiation
oncology. There has been a vast development of laser-driven particle acceleration (LDPA) using high power
lasers. The main characteristics of LDPA are the ultra-high beam intensity, small beam size and the potential
particle and energy range selection in contrast to conventional accelerators generated single particle, quasi
mono-energetic beams.
Results There is a growing number of studies on the potential advantages and applications of Energy
Modulated X-ray Radiotherapy in the range of 2-10 MV with relative fast energy switching of the new
generation linacs due to the lack of appropriate technology to modulate photon energy. Furthermore, the
ultrahigh space and/or time resolution of super-intense beams are under intensive investigation at synchrotrons
(Micro-Beam Therapy) with growing evidence of significant improvement of the therapeutic index. The
development of laser-based thermal- epithermal neutron beams with as high as 1010
fluence rate could
enhance the research activity on Boron Neutron Capture (BNC) Therapy, an advanced cell targeted binary
treatment modality, using the high linear energy transfer (LET) of the two particles (7Li and
4He) released by
BNC reaction. Boron Proton Capture (BPC) Enhanced Proton Therapy is another binary approach, where
similar, but 11boron-enriched compounds can be used. Due to the 3 alpha particles release during the BPC
reaction additional high LET dose could be achieved at a certain energy level of the proton beam. The
biomedical application group at ELI-ALPS is preparing biological systems and endpoints (cell cultures,
zebrafish embryos and small animals) for the comparison of the effect of LDPA using conventional photon
and electron beams as reference. Current model development for in vitro and in vivo preclinical research on
both healthy tissues and diverse tumors will address the key biological questions concerning the LDPA with
large variety of particles, energies and intensity.
Conclusion The planned LDPA (photons, very high energy electrons, protons, carbon ions) at ELI facilities
have the unique property of ultra-high dose rate (>Gy/s-10
), short pulses, and at ELI-ALPS high repetition rate.
The clinical potential of the promising laser based approaches will be presented.
The ELI-ALPS project (GINOP-2.3.6-15-2015-00001) is supported by the European Union and co-financed
by the European Regional Development Fund. The project has received funding from the European Union's
Horizon 2020 research and innovation programme under grant agreement no 654148 Laserlab-Europe.
European Radiation Research 2018, August 21-25, Pécs, Hungary
90
How to take care of the largest man-made source for radiation exposure –
medical applications – by structured research and harmonization of practices
Christoph Hoeschen
Otto-von-Guericke University, Magdeburg, Germany
On behalf of
EURAMED scientific committee
Medical application of ionizing radiation is highly beneficial for many patients by providing efficient
diagnostic and therapeutic tools. However, it is the largest man-made source for radiation exposure
of the population in most countries. Within this talk, there will be a description of the dimension of
this radiation exposure with a focus on the European situation. For describing the exposure situation
most often the effective dose is used. This approach will be discussed briefly for medical exposures.
In addition, the importance of a benefit – risk evaluation as a major component of the justification
process, will be highlighted.
After this introduction, it will be shown, that there are two key components for effective radiation
protection in medical applications:
One is to foster research which is meaningful for achieving better radiation protection especially of
the patients on an individual patient basis.
The other one is to achieve a harmonization of practices to ensure the same level of benefit of all
kinds of procedures where ever the procedure is performed.
These are key components of the approach of EURAMED as the European platform for medical
radiation protection research. This platform had been launched as a fifth European platform for
radiation protection matters after MELODI, ALLIANCE, EURADOS and NERIS in 2016 as a Joint
Initiative of EIBIR and is a legal entity since 2017. EURAMED is providing a Strategic Research
Agenda (SRA) to give guidance on topics of meaningful research in terms of radiation protection as
well is preparing its own Roadmap to highlight a possible approach for most useful structured
research approaches to help solving the questions as addressed in the SRA. The five main topics
identified range from measurement of relevant parameters including uncertainty to biological
fundamental understanding of corresponding effects including e.g. individual susceptibility or
sensitivity, to optimization and harmonization of practices to justification and infrastructures. Both
the SRA approach as the roadmap approach will be explained. In both documents the necessity to
transfer the research results into clinical practice and to harmonize practices for the clinical practice
is highlighted. Therefore also dedicated training programs will be essential.
European Radiation Research 2018, August 21-25, Pécs, Hungary
91
RADIATION LEVELS IN HUNGARIAN BUILDINGS
Zsolt Homoki
National Public Health Institute
Public Health Directorate
Department of Radiobiology and Radiohygiene
Radiohygiene examinations were carried out by NPHI in 731 buildings (mostly homes) between 1995 and
2018. Gamma dose rate level was measured in 658 buildings and indoor radon concentration was detected in
670 buildings. The measurements were taken by passive and active detectors. Additionally, information was
recorded about the building characteristics and the type of building materials. Personal, detailed survey was
made in 289 buildings from the 731 using active devices. During these surveys, the indoor gamma dose rate
was usually measured in all bigger rooms at different heights and near to the surface of the walls and the
radon concentration was detected for several days in one or two rooms.
General statistical analyse was made from the results and separated groups was created concerning the
building characteristics to make special analyse. According to our results, the average indoor gamma dose rate
was 163 nSv/h in the examined Hungarian buildings. The minimum and maximum values were 58 and 465
nSv/h, respectively. For comparison, the average background gamma radiation was 104 nSv/h based on the
results of 435 measuring point. The values are expressed in environmental dose equivalent unit - H*(10).
According to the recorded building characteristic, slag was built-in into the floor space and/or some of the
walls were made from slag concrete in 60% of the examined dwellings. A separated analyse was made from
the result of these buildings to check the effluence of higher radioactivity of slag to the indoor radiation level.
The average indoor gamma dose rate was 186 nSv/h for buildings containing slag and 123 nSv/h for buildings
without slag.
The indoor radon concentration was detected in 452 buildings for at least 1 year period. Using these results we
calculated the average radon concentration and the distribution of values. The average radon concentration
was 108 Bq/m3 and the highest measured value was 781 Bq/m
3. The average radon level was higher than 300
Bq/m3 (reference level in Hungary) in 4.6% of the total sampling points.
European Radiation Research 2018, August 21-25, Pécs, Hungary
92
Connexin channels and Ca2+
/ROS/NO signalling are key players in the
radiation-induced bystander effect in microvascular brain endothelial
cells.
Delphine Hoorelbeke1, Elke Decrock
1, Maarten De Smet
1, Marijke De Bock
1, Bénédicte Descamps
2, Valérie
Van Haver1, Tinneke Delvaeye
1,3, Hubert Thierens
4, Dmitri Krysko
5, Christian Van Hove
2, Luc Leybaert
1
1.Physiology group, Dept. Of Basic Medical Sciences, UGent, Ghent
2. INFINITY-MEDISIP-iMINDS, UGent,
Ghent 3.
Inflammation Research Center, VIB/UGent, Ghent 4. Medical Physics-Dept. Basic Medical Sciences,
UGent, Ghent 5.Anatomy and Embryology group, Dept. Of Basic Medical Sciences, UGent, Ghent, Belgium
Objective:
The effectiveness of brain tumor radiotherapy is limited by the sensitivity of normal brain tissue to ionizing
radiation (IR). Evidence highlights a critical contribution of brain microvascular endothelial cells (BMEC) to
radiation-induced normal tissue damage. This damage includes, but is not limited to, double strand breaks
(DSBs), apoptosis, endothelial cell activation and senescence. DSBs are considered as early events,
responsible for the latter outcomes. Intercellular pathways can propagate these effects from directly irradiated
to adjacent non-exposed bystander cells, a phenomenon known as the radiation induced bystander effect
(RIBE). Intercellular communication can be mediated by channels composed of connexin (Cx) proteins: gap
junction channels (GJs) that connect the cytoplasm of neighboring cells, and hemichannels (HCs) that form a
pore in the plasma membrane and contribute to paracrine signaling. In physiological conditions GJs are open
and HCs are normally closed but may open in response to cell stress. Reactive Oxygen Species (ROS) are
frequently mentioned as important messengers in RIBE, however due to their limited life time they cannot
diffuse over long distances. In this project we aimed to explore the role of Cxs in RIBE and to unravel the
responsible messengers in this process.
Methods
We optimized an in vitro set-up in which BMEC were focally irradiated by X-rays (1 and 20 Gy, 230 kV, 13
mA) by using the SARRP (Small Animal Radiation Research Platform) device. As such, we were able to
investigate the effect in the bystander zone and the irradiated zone separately. By using different blockers and
KO strategies we determined the role of the Ca2+
/ROS/NO axis and Cx channels in RIBE.
Results
A spreading of γ-H2AX foci, i.e. a marker for DSBs, was detected following exposure to IR. By using a Ca2+
-
chelator, a ROS scavenger, a NO scavenger, a purinergic receptor blocker, an inhibitor of IP3-mediated Ca2+
-
release and Cx43-mimetic peptides (TAT-Gap19 and Gap26) this spreading was inhibited. TAT-Gap19
allowed us to selectively block Cx43 HCs without affecting GJs, thereby confirming their role in RIBE. Next
to this, we also detected an acute opening of these HCs within 5 min following irradiation which resulted in
ATP release and an increase in Ca2+
oscillations in directly irradiated cells together with an increase in
cytoplasmic Ca2+
concentration in bystander cells. ATP release was decreased when adding a Ca2+
-chelator,
ROS or NO scavenger. We thus show that ROS, NO as well as Ca2+
, IP3, extracellular ATP and HCs are key
players in RIBE.
Conclusions
We conclude that intercellular Ca2+
signalling via HCs and GJs with a predominant role for HCs, acts as a
feed-forward propagation mechanism of ROS/NO regeneration underlying the RIBE in BMEC. Further
unravelling these mechanisms can contribute to the development of protective strategies against radiation-
induced EC damage and the resulting healthy tissue damage.
European Radiation Research 2018, August 21-25, Pécs, Hungary
93
Overview of the performance parameters and unique features of a
recently developed automatic micronucleus assay evaluation system
Timea Hülber1,5
, Zs. S. Kocsis2, E. Kis
3, G. Sáfrány
3, Cs. Pesznyák
1,4
1 Institute of Nuclear Techniques, Budapest University of Technology and Economics, Budapest,
Hungary 2 National Institute of Oncology, Centre of Radiotherapy, Department of Radiobiology and
Diagnostic Onco-Cytogenetics 3 National Public Health Centre - National Research Directorate for Radiobiology and
Radiohygiene 4 National Institute of Oncology, Centre of Radiotherapy
5 Radosys Ltd.
Objective Cytokinesis-blocked micronucleus assay (CBMN) is a well-accepted, widely used biodosimetric assay. The
speed of the MN scoring has utmost importance in case of nuclear emergency cases, to which the
automatisation of the method is a beneficial contribution. It also could eliminate the subjectiveness of the
result obtained by a human scorer, as a consequence, makes the results more comparative.
In this paper we present the first phase of the validation of a recently developed compact and cost effective
automatic system designed specifically for the evaluation of GIEMSA stained CBMN slides.
Methods The speciality of the Radosys Radometer MN Series automated microscopy system is that instead of applying
a commercially available digital microscope a dedicated one is used. This allows the integration of hardware
control and image processing to a high extent. This approach made possible the development of a non-
standard autofocusing method, which leads to an optimized artefact elimination such as GIEMSA stain
residues. The access to the direct control of the illumination simultaneously with the image scanning is
capable of compensating the non-homogeneously stained compartments.
Results The performance parameters of the system are determined by the comparison of the results by automatically,
semi-automatically and manually scored dose-effect curve. We constructed a preliminary calibration curve in
the range of 0-6 Gy for gamma irradiation. The detection rate of the binucleated cells (BN) is 69±6%
independently from the staining variability. The MN detection rate in true positive BN cells on the reference
sample set is 74±6%.
Conclusion.
It is demonstrated that the parallel software-hardware development approach gives unique features to the
system which leads to a robust evaluation of the MN assay. According to the preliminary dose-effect curve the
doses above 1 Gy can be assessed automatically within a ±0.7 Gy. This performance is suitable for triage
purposes. For the full verification the increase of the number of the samples is needed.
European Radiation Research 2018, August 21-25, Pécs, Hungary
94
Dose-dependent skin inflammation after hypofractionated X-ray
radiotherapy in an in vivo mouse ear model
Annique C. Hunger1,2
, Matthias Sammer3, Jannis Schauer
3, Benjamin Schwarz
3, Dietrich W.M.
Walsh3, Judith Reindl
3, Stephanie E. Combs
1,2, Günther Dollinger
3 and Thomas E. Schmid
1,2
(1) Institute of Innovative Radiotherapy, Helmholtz Zentrum München, Neuherberg, Germany
(2) Department of Radiation Oncology, Technical University of Munich, Klinikum rechts der Isar, Munich,
Germany
(3) Institut für angewandte Physik und Messtechnik, Universität der Bundeswehr München, Neubiberg, Germany
Objective: A common side effect of radiation therapy of cancer patients is the damage of the healthy tissue.
Normal tissues can be spared if the radiation dose is delivered in fractions over several days. Hypofractionated
radiation therapy gives a smaller number of radiation fractions with an increased dose per fraction compared
to hyperfractionated regimes. The damage of the healthy tissue depends on the dose delivered per fraction and
the dosing schedule. Therefore, our aim was to investigate the dose-dependent acute response of the skin in an
in-vivo mouse ear model after hypofractionated X-ray radiotherapy.
Methods: The right ear of female Balb/c mice was irradiated with a total of four fractions at a RS225 (Xstrahl
GmbH) using 70 kV X-rays. Each group was irradiated with a daily fraction of either 5, 10, 20, 30 or 40 Gy.
The left ear of every mouse served as a control. The acute skin toxicities ear swelling, erythema and
desquamation were observed for 90 days after irradiation and compared to sham-irradiated mice. Ear swelling
was determined by measuring the ear thickness of the irradiated right ear and control left ear using a specially
adapted electronic external measuring gauge (C1X079, Kröplin GmbH, Germany) with measuring contacts 6
mm in diameter [Girst et al., 2016]. Desquamation and erythema were scored in four gradings: no, dry, crust,
moist desquamation for desquamation and no, mild, definite, severe for erythema and both were summed up
to one score.
Results: A small increase in ear thickness of about 40 µm was measured at day 21 after 5 Gy per fraction,
compared to the unirradiated left ear with a thickness of 220 µm. After 10 Gy per fraction the maximum
thickness of 460 µm was detected at day 22. The ear thickness of 30 Gy and 20 Gy irradiated mice peaked at
day 18 with a thickness of 580 µm and 520 µm, respectively. Upon irradiation with 40 Gy per fraction the ear
thickness increased up to 700 µm after 14 days. The skin score peaked at day 14-16 after 40 Gy, day 18 after
30 Gy, day 16 after 20 Gy, and at day 21-22 after 10 Gy and 5 Gy per fraction. The maximum reaction
decreases from 3.7, 3.5, 2.7, 2.2 to 1.08 with decreasing dose from 40 to 5 Gy. The fixation of the right ear for
irradiation leads to an increase in the skin score and ear thickness at day 3 and day 4 after the first fraction.
Conclusion: Our results show that the ear thickness and the skin score increase with increasing dose per
fraction. The smaller the dose per fraction the later the thickness and skin score reach its maximum. There is a
significant temporal correlation between the maximum in ear thickness and skin score. The thickness of the
unirradiated left ear was unchanged over a period of 90 days. Finally, irradiation with smaller doses than 20
Gy per fraction results in reduced adverse effects and can spare the healthy skin after fractionated
radiotherapy. In future, we want to examine the acute response of the skin after hypofractionated X-ray
radiotherapy using enlarged time intervals between fractions.
European Radiation Research 2018, August 21-25, Pécs, Hungary
95
System development for measurement of artificial radioactivity in
surface water bodies
Ivan Hupka, Michal Fejgl, Miroslav Hýža
National Radiation Protection Institute, Section of Monitoring, Prague, Czech Republic
Email: ivan.hupka@suro.cz
The main objective of this contribution is to present a research project concerning the system
development for continuous in situ measurement of artificial radioactivity in surface water bodies
enabling rapid determination of water contamination.
As of 2016, surface water bodies cover 51,7 % of drinking water sources in the Czech Republic [1].
In case of radiation accident surface water bodies are vulnerable in particular. They are susceptible to
be contaminated and, therefore, it makes them an important object of continuous measurement of
radioactivity helping state authorities to expedite their decision-making process.
The initial stage of this project was dedicated to the analysis of the Czech nuclear power plants
radiation accident scenarios. It was focused particularly on the identification of radionuclides that are
crucial in rapid accident recognition and on the comparison of their expected activity and its
detection limits required by the regulations. Based on obtained inference a monitoring station is
being developed at the moment. It is designed for continuous measurement of artificial radioactivity
in surface water bodies enabling rapid determination of water contamination. The outcome of this
project will be the construction of a “monitoring network” (i.e., several autonomous monitoring
stations placed in crucial spots incorporated in governmental Radiation Monitoring Network).
Construction of the monitoring system will be described in this task. Other important part of this
contribution is a long-term spectra deconvolution and analyses using Principal Component
Regression method allowing suppression of influence of the fluctuating natural radionuclides activity
in river water. This method enabled the detection limits of the 137
Cs in natural surface water to be
decreased by factor of ~ 8. A comparison between reached detection limits and the limits required by
regulations will also be presented.
This contribution is supported by research program BV VI2VS/575
References:
[1] Fousová, E.; Reidinger, J., Ministry of Agriculture of the Czech Republic, ISBN 978-80-7434-
377-3, Prague, 2017
European Radiation Research 2018, August 21-25, Pécs, Hungary
96
Logic and necessities in the choice of repair pathways processing radiation-
induced DNA double strand breaks
George Iliakis
Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School,
45122 Essen, Germany
Eukaryotic cells respond to DNA damage by activating a network of biochemical pathways
recognizing the damage and initiating responses leading to repair, apoptosis/autophagy or
senescence. This network of responses is commonly referred to as the “DNA damage response”
(DDR). Among the plethora of lesions generated in the DNA as a result of cell exposure to various
physical and chemical agents, DNA double strand breaks (DSBs) are the most severe inducing strong
DDR. This strong response is evolutionarily explained by the documented fact that DSBs bear high
risk for cell death or genomic instability leading to genomic alterations ultimately causing cancer.
Cells of higher eukaryotes process DSBs utilizing four distinct repair pathways, homologous
recombination repair (HRR), DNA-PK-dependent, classical non-homologous end-joining (c-NHEJ),
alternative endjoining pathway (alt-EJ), and single strand annealing (SSA). HRR relies on a sister
chromatid as template to restore integrity in the DNA during the S- or G2-phases of the cell cycle,
and is error-free on every count. c-NHEJ mediates fast joining of broken DNA ends to ensure
chromosome integrity through a highly efficient and well-coordinated molecular machine, albeit
without ensuring the restoration of DNA sequence at the DSB, or the joining of the original ends.
Alt-EJ is slower than c-NHEJ and carries higher probability than c-NHEJ for sequence alterations at
the junction and for the joining of unrelated DNA ends to generate chromosomal translocations. SSA
is inherently mutagenic due to the deletions associated with its function. Thus, in a hierarchical
categorization of the DSB repair pathways, alt-EJ and SSA will have the highest propensity for
errors and HRR the lowest; therefore the four distinct repair pathways are not equivalent options for
the processing of a given DSB. This raises critical questions regarding the “logic” of repair pathway
choice and makes selection on stochastic grounds unlikely. We will summarize results suggesting
that cells evolved mechanisms allowing them to select error-free repair pathways as first instance in
the repair of DSBs and opt for lower fidelity pathways on necessity grounds.
Work supported by grants from BMBF, BMWi and the DFG.
European Radiation Research 2018, August 21-25, Pécs, Hungary
97
Search of potential gene expression biomarkers of radiation injury at
the late period after exposure
1 Iryna Ilienko,
1 Dimitry Bazyka
1 State Institution “National Research Centre for Radiation Medicine of National Academy of Medical
Sciences of Ukraine”, Institute for Clinical Radiology, Department of Clinical Immunology, Laboratory of
immunocytology, Kyiv, Ukraine
Objective. The experience that was gained after the nuclear accidents in Chernobyl and Fukushima showed a
critical need in relevant molecular biomarkers to assess a personal dose of radiation that may be extremely
necessary not only for appropriate medical / clinical care but also for predicting slow stochastic effects such as
genomic instability and cancer.
Methods. Study group included 235 Chernobyl clean-up male workers 30 years after exposure (doses of
external exposure 419,48 ± 654,60 (M ± SD) mSv; age 58,34 ± 6,57 (M ± SD) yrs). A control group consisted
of 45 non-exposed subjects aged 41 to 67 years (mean 50.60 ± 5.37 (M ± SD)). Immune cell subsets
(CD45/14, CD3/19, CD4/8, CD3/HLADR, CD3/16/56, TCRγδ) was analysed by flow cytometry
(FACSCalibur, BD, USA). Relative quantification of gene expression (BCL2, TP53, CDKN2A, CCND1,
CLSTN2, GSTM1, IFNG, IL1B, MCF2L, SERPINB9, STAT3, TERF1, TERF2, TERT, TNF) was performed by
real time polymerase chain reaction using 7900 HT Fast Real-Time PCR System and TagMan technology
(Applied Biosystems, USA).
Results. Gene expression changes were shown with an increase of positive regulatory signals of apoptosis and
suppression of anti-apoptotic mechanisms. Dose-dependent decrease of BCL2 gene expression, as well as
SERPINB9, CDKN2A and STAT3 genes were found, with a maximum decrease at doses above 1 000 mSv.
The opposite were the results of MCF2L gene expression, which were determined аs a dose-dependent
overexpression of this positive regulator of apoptosis. The highest level of TP53 gene expression was found in
the subgroup of clean-up workers, irradiated in low doses from 0 to 100 mSv.
Investigations show the activation of immune inflammation genes. IL1B gene expression was the highest in
dose interval from 0 to 100 mSv, with a linear tendency to lower RQ IL1B with decreasing dose. Negative
correlation was established between RQ IL1B and the number of CD19+3- and CD3-HLADR+ B-
lymphocytes (respectively r = -0.83 and -0.94); the number of CD4+8- (r = -0.72) and the ratio of
CD4+/CD8+ cells (r = -0.84). Overexpression of gene IFNG was determined at irradiated at doses above 1000
mSv. TNF gene expression was the highest in the subgroup of clean-up workers, irradiated in the dose 100-
1000 mSv, while TNF gene suppression was detected at doses greater than 1000 mSv. The regulatory
overexpression of TNF gene was confirmed by a correlation with the number of CD3+16+56+ cytotoxic
lymphocytes, CD4-25+ and CD4+25+ lymphocytes and expression of TCRγδ associated with mitogen-
dependent activation lymphocytes. The activation of the immune inflammation genes was accompanied by
inhibition of CLTN2 expression - the positive regulator of adhesion. Overexpression of TERT gene expression
together with TERF1 and TERF2 inhibition, manifested in the distant period after irradiation and may initiate
the activation of mechanisms of premature aging of leukocytes and create conditions for the launch of
carcinogenic processes.
Conclusion. System violation of expression of genes, that regulate the key homeostatic cell systems such as
apoptosis, cell cycle, cellular adhesion, senescence and immune response offer as genetic biomarkers of
radiation damage in the remote period after exposure and the basis for dynamically monitoring of state health
of the victims of the Chernobyl disaster for the purpose of prevention and early diagnostics of
immunodeficiency states (including oncological and lymphoproliferative diseases).
European Radiation Research 2018, August 21-25, Pécs, Hungary
98
Application of ex vivo and in vivo models of rat mammary gland to
study competition between irradiated and non-irradiated cells
Tatsuhiko Imaoka1, Yukiko Nishimura
1, Ken-ichi Kudo
1, Kiichi Kaminaga
2,
Akinari Yokoya2, Noriko Usami
3, Mayumi Nishimura
1, Kazuhiro Daino
1,
Daisuke Iizuka1, Yoshiya Shimada
1, Shizuko Kakinuma
1
1 Department of Radiation Effects Research, NIRS, QST, Chiba, Japan
2 Tokai Quantum Beam Science Center, QuBS, QST, Tokai, Japan
3 Photon Factory, KEK, Tsukuba, Japan
Objective. Because of the quantum nature of ionizing radiation, its exposure at low doses is characterized by
physically inhomogeneous distribution of energy deposition in a tissue. Therein, only a minor fraction of cells
are affected, with others intact and surrounding the affected cells. Cell competition is a universally conserved
mechanism to eliminate cells that are less fit than their neighbors. Competition between radiation-affected and
unaffected cells, if there is any, can lead to elimination of affected cells, and hence, smaller cancer risk than
predicted from the linear-no-threshold theory. In the present study, we aimed to establish ex vivo and in vivo
experimental systems related with a rat model of radiation-induced mammary carcinogenesis.
Methods. Dispersed mammary epithelial cells were obtained from transgenic rats of enhanced green
fluorescent protein (EGFP) or Discosoma red fluorescent protein (DsRed) by chopping followed by enzymatic
digestion. The cells were cultured to form spherical colonies on a basement membrane gel. Cells were
irradiated with a soft X-ray microbeam generated at KEK Photon Factory BL-27B, followed by time-lapse
fluorescent microscopy. In transplantation, mammary fat pads of recipient rats were surgically cleared of
epithelial rudiments and injected with mammary cells.
Results. Regarding the ex vivo model, we established a method to form mosaic colonies of EGFP- and
DsRed-tagged cells, which allowed differential microbeam irradiation and subsequent monitoring up to a
week. For the in vivo model, appropriate choice of the recipient strain and the number of cells transplanted
enabled generation of a mosaic mammary gland in rats, where irradiated and sham-irradiated cells can be
differentially fluorescent-tagged with EGFP and DsRed. We successfully applied optical tissue clearing and
confocal imaging to rat mammary tissue, facilitating observation of the mosaic tissue. We will further present
preliminary observations using these techniques.
Conclusion. Ex vivo and in vivo experimental systems have been established to study whether cell
competition exists between irradiated and non-irradiated rat mammary epithelial cells.
This study is funded in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan
(“Nuclear Energy Science and Technology and Human Resource Development Project through Concentrating
Wisdom” and Grants-in-aid for Scientific Research (B) [15H02824 and 16KT0079]).
European Radiation Research 2018, August 21-25, Pécs, Hungary
99
Is metabolic syndrome affecting the adverse outcomes of low doses or dose rates of ionising radiation?
Nathalie R.E.N. Impens
1 Belgian National Nuclear Research Institute SCK•CEN,
Institute of Environment, Health and Safety, Mol, Belgium Objective In the framework of the development of the Joint Roadmap for Radiation Protection Research, the author has investigated a potential confounding factor that may affect the effects of low doses or low dose rates of ionising radiation. The Metabolic Syndrome (MS) was selected as potential confounding factor because of its increasing global prevalence and dramatic increase in the Western World. MS results amongst others in an increased prevalence of various cardiovascular diseases (CVD), Diabetes Mellitus Type 2 (DMT2), and increased “Low grade inflammation”, the latter considered as a trigger of autoimmune diseases such as Rheumatoid Arthritis. On the other hand, low doses and/or low dose rates of ionising radiation (in short referred to as ”IR”), used in medicine - accounting for nearly half of the total average IR exposure of the Western-European and US population - may result in damaged cells/tissues, inflammation and/or a triggered innate immune system. Adverse outcomes linked to low doses or dose rates of ionising radiation in common with the Metabolic Syndrome are various cardiovascular diseases and maybe also DMT2. The dose- effect relationship in the low-dose region has not been resolved. The more, next to adverse outcomes, IR may also reduce inflammation in particular cases, and is sometimes used to treat patients with Rheumatoid Arthritis. The aim is to investigate the potential interplay of exposure to IR and the MS. Methods Unraveling the adverse effects attributable to IR, MS and the combination of both will need epidemiological studies taking into account state-of-the-art factors influencing MS and well-documented doses/dose-rates of ionising radiation. A model including these factors as well as confounding factors will have to be set up and tested using epidemiological data. Mechanistic studies including identification of common pathways will be needed to model the combined effect of MS and IR. Results This presentation will include a review on factors influencing the progression of MS, the challenges to create a model combining IR and MS, and how IR and MS may influence each other’s adverse outcomes from a mechanistic point of view. A proposal how to approach the elucidation of the combined effects will be presented. Conclusion As IR is increasingly applied in medicine and the prevalence of MS is raising it might be worthwhile to initiate projects including experts in both domains. Such projects should be based on epidemiological and mechanistic approaches. Understanding the role of the MS in patients treated or diagnosed using IR should lead to preventive measures to reduce secondary effects to their treatments. Acknowledgements The CONCERT EJP receives funding from EURATOM research and training programme 2014-2018 uner Grant Agreement N°662287
European Radiation Research 2018, August 21-25, Pécs, Hungary
100
Joint Roadmap for Radiation Protection Research: Focusing on the
radiobiology-radioecology interface
Nathalie R.E.N. Impens
1 Belgian National Nuclear Research Institute SCK•CEN,
Institute of Environment, Health and Safety, Mol, Belgium
Objective
The primary purpose of radiation protection is human health. Exposures to low doses of ionising
radiation (LDIR) for which radiation protection may be required can originate from natural or
anthropogenic sources. Understanding the human health effects attributable to LDIR resulting from
realistic exposure scenarios are the research objective of MELODI.
Many of the exposure scenarios listed in the Joint Roadmap for Radiation Protection Research
prepared in the CONCERT EJP project result from human activities and generate human and
environmental exposure. For example, legacy sites originating from the time that the actual
legislation was not in place, or accidents such as Chernobyl and Fukushima affect both humans and
the environment. It is therefore our responsibility (towards our and next generations) to study the
effects not only on humans but also on the environment. Research related to exposure assessment
and effects on non-human biota are within the scope of the Radioecology ALLIANCE.
Methods
To investigate biological consequences of LDIR exposure on humans and non-human biota we
need to identify the primary mechanisms at molecular level up to the individual level. At this level,
many analogies can be found between research in MELODI and ALLIANCE.
Epidemiological studies may validate the understanding of the exposure-effect relationship in
humans, subject thorough consideration of confounding factors such as other stressors, lifestyle,
genetic predisposition etc. Population studies on key species playing central roles in the ecosystem
are needed as well for the sake of ecosystem preservation. Furthermore non-human fast-reproducing
biota are suitable to reveal genetic and epigenetic transgenerational changes, some of them might be
expected to be similar in humans and therefore useful to human LDIR research.
When studying the effects of LDIR, and more importantly if the exposure is protracted, there is a
need to take into account effects of other stressors. The concept of combining the exposome and
Adverse Outcome Pathways (AOP) that are induced by multiple stressors, are probably important for
both MELODI and ALLIANCE, and may require collaboration to optimize efficiency.
The reported difference in species’ sensitivities observed in lab and field studies are not well
understood. An ecosystem approach may reveal the underlying mechanisms.
Conclusion
This paper encourages a stronger integration of some aspects of human low dose research and
radioecology.
Acknowledgements & Disclaimer: The CONCERT EJP receives funding from EURATOM
research and training programme 2014-2018 uner Grant Agreement N°662287. This publication
reflects only the author's view. Responsibility for the information and views expressed therein lies
entirely with the author. The European Commission is not responsible for any use that may be made
of the information it contains
European Radiation Research 2018, August 21-25, Pécs, Hungary
101
Cancer immunotherapy with low-level whole-body exposures to
ionizing radiation
Marek K. Janiak1, Marta Wincenciak
1, Aneta Cheda
1 and Ewa M. Nowosielska
1
1Military Institute of Hygiene and Epidemiology, Department of Radiobiology and Radiation Protection
Warsaw, Poland
Abstract
Evidence amassed over the last several decades from epidemiological, experimental, and clinical
studies indicates that whole-body exposures at low doses (i.e., ≤100 mGy delivered over a short
time) of low-LET ionizing radiation inhibit the development and/or progression of various
neoplasms. The primary mechanism of such effects is thought to be stimulation of both the innate
and adaptive arms of anti-cancer immunity.
The recently redefined role of the immune system in anti-neoplastic surveillance assumes that the
system protects the host against the incipient cancer at the early stages of carcinogenesis, but later
‘edits’ the immunogenicity of the extant neoplastic cells and supports remodelling of the tumour
microenvironment towards the immunosuppressive and pro-neoplastic state.
The presentation will review immunosuppressive mechanisms induced by growing tumours as
well as immunomodulatory effects of whole-body low-dose exposures to X or gamma rays directly
or likely associated with cancer-inhibiting outcomes of such exposures. Suggestions will be provided
how such exposures can possibly restore and/or stimulate effective anti-neoplastic immunity during
the more advanced stages of carcinogenesis. Finally, we will postulate that, based on the
accumulated epidemiological and experimental data, whole or half-body exposures to low-dose low-
LET radiation should be viewed and further examined as a viable immunotherapeutic treatment
option for patients with systemic and metastatic cancer.
European Radiation Research 2018, August 21-25, Pécs, Hungary
102
Linking radiation-induced DNA-damage to systemic anti-tumor responses and normal tissue
toxicity
Verena Jendrossek
Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Essen,
Germany Fractionated radiotherapy (RT) is part of standard treatment for lung cancer patients but disease progression is common. Though RT is highly efficient in reducing tumor growth biological factors such as high intrinsic or microenvironment-mediated radiation resistance of the cancer cells and tumor immune escape limit successful RT. Moreover, adverse late effects in the highly radiosensitive normal lung preclude the use of curative doses leading to decreased quality of life or - as a consequence of treatment with suboptimal radiation doses - to fatal outcomes by local recurrence or metastatic disease. The use of RT has historically been based on its ability to locally damage cellular macromolecules, particularly the DNA and thereby effectively inducing growth arrest and cell death in irradiated tumor cells. However, the documented ability of RT to elicit systemic effects that impact treatment outcome led to a paradigm change in our current understanding of the biologic effects of ionizing radiation. It is assumed that radiation-induced DNA damage activates molecular and cellular processes that promote the recruitment of immune effector cells to normal and tumor tissues, and even elicit tumor-specific systemic immune responses promoting the regression of tumor lesions locally and at tumor sites outside the radiation field (abscopal effects). Moreover, RT was shown to help in overcoming immunosuppressive barriers within the tumor microenvironment (TME) that limit the antineoplastic effects of radiotherapy, immunotherapy or combinatorial treatments. We speculate that radiation-induced damage to the DNA of resident cells initiates a similar cascade of events that can lead within weeks to a pronounced lung inflammation (pneumonitis) or after months to excessive deposition of extracellular matrix molecules and tissue scarring (lung fibrosis). The presentation will highlight the current view on the molecular and cellular processes that link the initial damage to the DNA induced by ionizing radiation in normal and tumor tissues to the activation of the innate and adaptive immune systems. The presentation will also introduce a novel concept developed from our preclinical investigations in murine models to enhance RT-induced local and abscopal effects in malignant tumors, attenuate pathologic signals in the disease-promoting microenvironment in irradiated lungs or both, by targeting the CD73/adenosine checkpoint. The importance of these findings will also be discussed in view of the increasing interest in combining RT with immune checkpoint blockade to enhance the immune response against the tumor.
European Radiation Research 2018, August 21-25, Pécs, Hungary
103
Effect of age at exposure on the incidence of thyroid lesions after γ-ray
irradiation in mice.
Jin Xiaohai, Shizuko Kakinuma, Yutaka Yamada, Takamitsu Morioka, Kazuhiro Daino, Yoshiya
Shimada
Department of Radiation Effects Research, National Institute of Radiological Sciences
National Institutes for Quantum and Radiological Science and Technology
4-9-1, Anagawa, Inage-ku, Chiba, 263-8555, Japan
Purpose: Since the nuclear accident in Fukushima Daiichi nuclear power plants, long-term low dose
rate radiation exposure is concerned about health effects including cancer. There is an increasing
interest in the radiation exposure effects of children with particularly high radiation susceptibility.
Currently, highest incidence of thyroid cancer by ultrasound thyroid screening has become a serious
problem in Fukushima. However, it is not clear whether thyroid carcinogenesis is associated with
radiation exposure. The purpose of this study is to clarify the thyroid cancer risk after exposure at
childhood using experimental mouse model.
Since the nuclear accident in Fukushima, long-term low dose rate radiation exposure is concerned
about health effects including cancer. Specially radiation susceptible children have been worried.
Currently, ultrasound thyroid screening has revealed increase of thyroid cancer. However, it is not
clear whether the thyroid carcinogenesis is associated with radiation exposure. The purpose of this
study is to clarify the thyroid cancer risk after exposure at childhood using experimental mouse
model.
Method: B6C3F1 male and female mice were irradiated at 1 or 7 weeks of age with 0, 0.2Gy ,2Gy or
4Gy γ-ray doses. All mice were sacrificed when they became terminal condition or died. The
pathological specimens of all organs and tumors were archived electronically as digital data. We
performed the pathological diagnosis of thyroid lesions using archived samples then calculated the
risks of thyroid lesion after exposure to radiation, such as dose-response relationship and effect of
age at exposure.
Results and Conclusions:
Histopathological examination of thyroid lesions revealed follicular cell hyperplasia, follicular or
papillary adenoma or carcinoma. The incidence of follicular cell hyperplasia was highest than that of
adenoma and carcinoma. In mice after irradiation at 1 week of age incidence of tumor (adenoma and
carcinoma) shown an increasing tendency compared with mice after irradiated at 7 weeks of age. We
will discuss the relation between the risks of each thyroid lesion and dose of radiation or age at
exposure.
European Radiation Research 2018, August 21-25, Pécs, Hungary
104
Extreme radiation tolerance: lessons from tardigrades
K. Ingemar Jönsson
Kristianstad University, Department of Environmental Science and Bioscience, Kristianstad,
Sweden
Tardigrades represent a phylum of very small aquatic animals in which some species have evolved adaptations to survive
under extreme environmental conditions, mainly desiccation and freezing. The tolerance that these animals show against
environmental agents also include radiation. Following a seminal study in the mid-1960s, studies on several species have
now documented that tardigrades are among the most radiation-tolerant animals on Earth, surviving doses of ionizing
radiation at the level of kilo-gray. The tolerance includes both low-LET and high-LET radiation. The presentation will give
an overview of the tolerances of tardigrades, with an emphasis on our current knowledge on radiation tolerance, and patterns
of dose-response in different species will be reviewed. Recent studies have also shown that tardigrade embryos have
considerably lower tolerance to gamma radiation compared to adults, and embryos in the early stage of development are
clearly more sensitive to radiation than those in the late developmental stage. The molecular mechanisms behind radiation
tolerance in tardigrades are still largely unknown, and whether mechanisms related to both avoidance of DNA damage and
repair of damage are involved is still unclear. The potential of tardigrades to provide knowledge of importance for medical
sciences have long been recognized, and in a recent study tardigrade genes associating with DNA was transferred to human
cells and shown to improve their tolerance to radiation. With the recent sequencing of the tardigrade genome more similar
studies applying tardigrade genomics/proteomics to relevant aspects of human medicine are expected. In particular, the
fields of cancer and aging research have potential to learn from studies on tardigrades about mechanisms to deal with
environmental stress that challenge DNA integrity.
European Radiation Research 2018, August 21-25, Pécs, Hungary
105
Cancer risk after exposure to space radiation by pathological and
genomic analyses
Shizuko Kakinuma, Jin Xiaohai, Hirotaka Tachibana, Takamitsu Morioka, Kazuhiro Daino,
Chizuru Tsuruoka, Yi Shang, Yoshiko Amasaki, Yoshiya Shimada
Department of Radiation Effects Research, NIRS, QST, Chiba, Japan
Space astronauts are exposed to a variety of LET cosmic radiation, including high energy heavy particles such
as iron ions. The high LET radiation creates dense and complex DNA lesions that can be hardly repaired, and
leads to greater biological effectiveness and thus greater cancer risk than those of low LET radiation.
Although the biological effects of low LET radiation on cancer risks have been demonstrated by
epidemiological studies on atomic bomb survivors and patients with medical exposures, there are little data
for cosmic radiations.
In this study, we examined the results of lifespan study of B6C3F1 mice after exposure to iron, silicon or
argon ions at 1-week or 7-week old. The mice exposed at 1-week-old were more susceptible than adult
exposure. Hazard risks after exposure to these ions were higher by more than 10 folds than exposure to
gamma-ray and carbon ions. We show the spectrum and malignancy of tumours of mice exposed to these
ions, and genomic mutations in these tumours. We discuss the differences between high LET heavy ions and
low LET gamma rays in terms of cancer risk and the molecular mechanisms of carcinogenesis.
European Radiation Research 2018, August 21-25, Pécs, Hungary
106
The use of Centromeric/Telomeric PNA probes in prematurely
condensed peripheral blood lymphocyte chromosomes for partial body
high dose assessment
Ioanna Karachristou, Antonio Pantelias, Gabriel Pantelias, Georgia Terzoudi
Laboratory of Health Physics, Radiobiology & Cytogenetics
Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety
National Center for Scientific Research "Demokritos", Athens, Greece
In case of partial body high dose exposure, the use of the conventional dicentric assay does not
provide an accurate dose assessment, mainly due to the severe delay of the irradiated fraction of
blood lymphocytes as they proceed to mitosis. In fact, absorbed dose estimates are based on the
lymphocytes that succeed to proceed to their first post-exposure mitosis and are essentially restricted
to doses up to 5 Gy. To overcome these shortcomings, the use of Premature Chromosome
Condensation (PCC) methodology is an important alternative approach for biodosimetry purposes.
Towards this goal, the quantification of an exposure by means of the PCC method using chemical
inhibitors of protein phosphatase 1 and 2A such as Calyculin A, is currently based only on the
analysis of rings and fragments in Giemsa stained G2-lymphocyte chromosomes. Indeed, using this
approach the centromeres cannot be visualized in G2-chromosomes, and thus the scoring of
dicentrics is not feasible.
In the present study, the chemically induced PCC methodology using Calyculin A is combined with
the fluorescence in situ hybridization (FISH) technique using centromeric/telomeric peptide nucleid
acid (C/T PNA) probes to obtain the dicentric and centric ring frequency in G2/M PCC lymphocytes,
and compared to dose assessment based on M-phase cells only. Towards this goal partial body
exposure simulation experiments were performed where whole blood samples are irradiated and
mixed with non-irradiated to obtain fractions of 5%, 10%, 20%, 50% and 100% of irradiated blood.
Alternatively, partial body dose estimates were compared to those obtained using co-treatment with
caffeine and colcemid 3 hours before lymphocyte harvesting using conventional metaphase analysis.
The co-treatment with caffeine and colcemid before cell harvesting, will enable the severely
damaged blocked cells in G2-phase to be included in the analysis. The results obtained by the
different approaches in terms of their accuracy for partial body dose assessments will be presented
and discussed.
European Radiation Research 2018, August 21-25, Pécs, Hungary
107
Mediatory role of miR-31 in radiation-induced tumour cell death may
be influenced by (dys)functional status of p53
Ashish Kumar2, Bincy Babu
1, Prabuddho Mukherjee
1, Sudhir Chandna
1,*
1Division of Natural Radiation Response Mechanisms, Institute of Nuclear Medicine & Allied Sciences(INMAS), Brig. S.K. Mazumdar Road, Delhi 110054, India
2Department of Genetics, Texas Biomedical Research Institute, 7620 NW Loop 410, San
Antonio, Texas, 78227, USA *Corresponding author. E-mail address: sudhirchandna@yahoo.com (S. Chandna). Based on our recent findings in higher eukaryotic models, we investigated the possible presence of interplay
between p53 and the pro-apoptotic potential of miR-31, especially in the clinically relevant context of radiation-
induced cell death in normal versus cancerous cells.
Radiation responses of cell lines with functional versus dysfunctional/mutated p53 were compared following
ectopic manipulations with miR-31 mimics and inhibitors. Real-time PCR based miRNA expression analysis
as well as western blotting of p53 and various cell death associated proteins was used to assess the pro-
apoptotic role of miR-31, while colony forming assay and cell cycle analysis were used for cellular responses.
Binding of p53 with miR-31 promoter was investigated by ChIP assay combined with in silico binding site
prediction.
In primary (normal) or tumour cells with functional (radiation-responsive) p53, altering miR-31 expression did
not show any effect on the radiation-induced cell cycle perturbations, reproductive cell death or apoptosis.
However, tumour cells with dysfunctional p53 exhibited significant protection against radiation-induced cell
death following miR-31 suppression. Additionally, in cells with non-functional/mutated p53, miR-31
overexpression in itself was sufficient to induce significant cell death, indicative of a pro-apoptotic role in the
absence of p53 activity. Although, we failed to find unequivocal evidence towards direct interaction between
human/mammalian p53 and miR-31, in silico analysis points toward interesting crosstalk potential. MiR-31
inhibition further increased the migratory proficiency of tumour cells having mutated p53.
Collectively, these findings suggest a pro-apoptotic role of miR-31 selectively in tumour cells with
dysfunctional p53. Since nearly half of the tumours are known to carry inactivated p53, our study may help in
designing improved therapeutic strategies and in assessing miR-31 as a marker for radiosensitivity.
European Radiation Research 2018, August 21-25, Pécs, Hungary
108
Extracellular Vesicles mediate Radiation-Induced Bystander Effects in
the Bone Marrow
Dávid Kis, Eszter Persa, Tünde Szatmári, Nikolett Sándor, Rita Hargitai, Géza Sáfrány
and Katalin Lumniczky
National Public Health Institute, Division of Radiobiology and Radiohygiene,
Department of Radiation Medicine, Budapest, Hungary
Introduction: Radiation induced bystander effects (RIBE) refer to the induction of biological changes in cells
not directly hit by radiation. Due to RIBE the number of cells affected by radiation is larger than the actual
number of irradiated cells. This has important consequences in radiation protection and in radiation induced
health effect. The role of extracellular vesicles (EVs) in mediating RIBE has been raised by recent in vitro
studies, but in vivo investigations are still lacking.
Objectives: The main task of our work was to analyse cellular damage and phenotypical changes in mouse
bone marrow after direct irradiation and after in vivo injection of EVs isolated from irradiated animals.
Material and Methods: Here I present an in vivo study investigating the role of EVs in RIBE. 9-12 week-old
male C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2 Gy). 4 and 24 hours later EVs were
isolated from the bone marrow and were intravenously injected into unirradiated bystander animals. The
effects of EVs on the bone marrow cells of these bystander mice were compared to radiation effects in the
directly irradiated animals. The cellular damage were analysed by TUNEL assay and anti--H2AX (phospho-
Ser139) antibody by flow cytometry. The following cell types were measured in the bone marrow stem and
progenitor cell compartments by flow cytometry: hematopoietic stem cells (HSC) (Lin-Sca1+cKit+),
mesenchymal stem cells (MSC) (Lin-Sca1+, CD44+), lymphoid progenitors (L.PROG.) (CD45+CD90.2+),
granulocyte and monocyte precurzors (Gr1+, CD11b+), megakaryocytes and megakaryocyte progenitors
(CD41+CD61+) and erythroid precursors (Ter119+CD71+).
Results: Due to direct irradiation, the proportion of apoptotic cells and cells with DNA double strandbreaks
increased dose-dependently. Consequently, 24 hours after irradiation the number of radiosensitive L.PROG,
HSC and MSC in the bone marrow significantly decreased.
The effect of the EVs is confirmed by the significant changes in the bystander mice. Increased rate of
apoptosis was detected in the HSC and L.PROG populations 4 hours after injection of EVs isolated from mice
irradiated with 2 Gy. The number of HSC in the bone marrow significantly decreased 24 hours after injection
of EVs from mice irradiated with 0,25 ; 2 Gy, and MSC significantly decreased 24 hours after injection of
EVs from mice irradiated with 0,1 ; 0,25 ; 2 Gy. -H2AX positive cells were not detectable in the bone
marrow of bystander mice.
Conclusion: our results indicate that EVs can mediate radiation-induction bystander effects in the bone
marrow of non-irradiated bystander animals. These effects were not dose-dependent and manifested on certain
cell subpopulations only.
This work was supported by the DOREMI: EU-FP7 and CONCERT: LEU-TRACK projects.
European Radiation Research 2018, August 21-25, Pécs, Hungary
109
Chromosome aberration biodosimetry to compare three kind of
prostate radiotherapy
Zsuzsa S. Kocsis1, Péter Ágoston
2, Gyöngyi Farkas
1, Márta Kun-Gazda
1, Gábor Székely
1, Tibor
Major2, Dalma Mihály
2, Gábor Stelczer
2, Kliton Jorgo
2, László Gesztesi
2, Csaba Polgár
2, Zsolt
Jurányi1
1 National Institute of Oncology, Center of Radiotherapy, Department of Radiobiology and Diagnostic Onco-
Cytogenetics, Budapest, Hungary 2 National Institute of Oncology, Center of Radiotherapy, Budapest, Hungary
Objective: We aimed to compare the biodosimetrical values of three different radiotherapeutical modalities of
prostate adenocarcinoma. We wanted to investigate the relationship of the chromosome aberrations and the
side effects of radiotherapy, with the exclusion of the effect of irradiated volumes.
Methods: High dose rate (HDR, 19-21 Gy, 27 patients) and low dose rate (seed, 145 Gy, 55 patients)
brachyterapy and teletherapy (70-78 Gy, 44 patients) were administered to our patients. Side effects were
recorded and graded by the physicians and also by the patients (IPSS and QoL questionnaires) before
radiotherapy took place, immediately after, then 3, 6, 9, 12, 24 months later. Heparinised blood was taken at
the same visits, and lymphocytes were induced and cultured in cell-culture medium for 48 hours. After
arresting cell division, lymphocytes went through hypotonic swelling and their chromosomes were
precipitated and microscopically counted.
Results: The dicentric+ring chromosome (8.0±1.5 /100 cells) and total aberration values (17.7±2.3 /100 cells)
in teletherapy-treated patients were the highest, culminating after the therapy. The aberration values of seed-
treated patients were slightly higher (8.8±0.8 /100 cells total ab.) than that of HDR patients (5.0±0.8 /100 cells
total ab. at 3rd
month) culminating at the 3rd
and 6th months. The physician-graded genitourinary toxicities
were the highest in the case of seed patients (1.7±0.1 in avarage). There was only a few gastrointestinal side
effects in the whole cohort (one grade 2 after 24 month in 73 patient). The lowest IPSS values were obtained
in the HDR arm (6.3±1,2 in avarage), a compelling difference between the used modalities was seen right
after the therapy.
After the correlation analysis of the isodose surface volumes we found, that the small dose volumes (V1%,
V1Gy, V10%) of the HDR patients from Oncentra treatment planning system were correlated with the aberration
values seen at the 3rd
-month control (and only with this time point). The volumes, which were theoretically
calculated from total reference air kerma were also well correlated with the chromosome aberration values at
the 3rd
-month. The teletherapy isodose volume surfaces correlated with the chromosome aberration values
(dicentric+ring, aneuploidy, total aberrations, aberrant cell number) at every time point.
We found correlations between chromosome aberration values and severity of side effects in both treatment
groups. For example, terminal deletion, total aberration and aberrant cell numbers correlated moderately
and/or strongly with genitourinary toxicities at 3rd
, 9th months in HDR therapy. Furthermore dicentrics, rings,
aberrant cell and total aberration value correlated weakly with early genitourinary toxicities.
The Pearson correlation coefficient of the normalised values was higher when chromosome aberration values
were divided with the V1% isodose surface volumes of tele- or HDR therapy.
Conclusion: We concluded, that HDR brachytherapy caused the less chromosome aberrations and side
effects in our prostate adenocarcinoma patient cohort, although further recruitment and follow up is needed.
We also found that in the case of tele- and HDR therapy small isodose surface volumes from treatment
planning systems are eligible to normalise biodosimetric values providing better personal radiation sensitivity
parameters correlating with side effects. Therefore chromosome aberration method could be a useful tool for
radiotherapy personalisation.
European Radiation Research 2018, August 21-25, Pécs, Hungary
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X-rays versus protons: do they affect the Hedgehog pathway and
migration of cancer cells differently?
Katrien Konings1,3
, Charlot Vandevoorde2, Sofie Isebaert
3, Karin Haustermans
3, Sarah Baatout
1
and Marjan Moreels1
1
Radiobiology Unit, Belgian Nuclear Research Centre (SCK•CEN), Mol, Belgium 2 Radiation Biophysics Division, NRF iThemba LABS, Faure, South-Africa
3 Laboratory of Experimental Radiotherapy, Department of Oncology, KU Leuven, Leuven,
Belgium
In recent years an increase is observed in the use of proton therapy for the treatment of cancer
patients. This is in part because of the physical advantages protons offer compared to X-rays,
resulting in a more precise targeting of the tumor and an improved sparing of surrounding healthy
tissues. Metastasis is still an important cause of mortality in cancer patients and evidence shows that
conventional radiotherapy could actually increase the formation of metastasizing cells. An important
pathway implicated in the process of metastasis is the Hedgehog (Hh) signaling pathway. Recent
studies demonstrated that activation of this pathway in response to X-ray exposure can lead to
increased invasive and migratory capability of cancer cells. In addition, the Hh pathway is also
involved in radioresistance. Currently, the impact of particle radiation on the Hh signaling pathway is
unknown. Therefore the aim of this study was to investigate the effect of proton irradiation on the
migratory capacity of cancer cells and the expression of the Hh signaling pathway.
In the present study the effect of photons (X-rays) and protons was investigated in prostate cancer
cells (PC3) and paediatric medulloblastoma cells (DAOY). More specifically, cell survival,
migration and expression of genes involved in the Hh pathway was studied after exposure to both
radiation qualities, w/o addition of the Hh pathway inhibitor GANT61. Experiments with X-rays
were performed at SCK•CEN (Mol, Belgium), whereas experiments with a clinical proton beam (200
MeV, LET: 3.62 keV/µm) were performed at the iThemba LABS facility in South-Africa.
The Hedgehog inhibitor GANT61 did not have a radiosensitizing effect in PC3 cells for either
radiation quality. However, a radiosensitizing effect of GANT61 could be observed in the DAOY
cells after proton irradiation. Preliminary RT-qPCR results demonstrated that genes involved in the
Hh pathway were differentially affected after X-ray and proton irradiation in both cell lines. Results
from the migration assay revealed that the migration capacity of DAOY cells decreased in a radiation
dose dependent manner, with a more pronounced decrease of migration after proton irradiation
compared to X-ray irradiation. For PC3 cells, a similar dose-dependent decrease was observed after
proton as after X-ray irradiation.
In conclusion, exposure to X-ray and proton radiation induces cell line-dependent changes in the
gene expression of several components of the Hh pathway as well as the migration capacity.
Radiosensitization by GANT61 was only observed in DAOY cells. Future experiments will address
whether the migration capacity of cancer cells will be differentially affected after exposure to
different radiation qualities in combination with GANT61 .
Acknowledgements. This work is partly supported by the Federal Public Service in the context of the feasibility study ‘Application of hadrontherapy in Belgium’, which is part of action 30 of the Belgian cancer plan (CO-90-2088-01). Katrien Konings is a beneficiary of a SCK•CEN-KUL PhD grant. Karin Haustermans is a clinical research fellow of the Research Foundation Flanders.
European Radiation Research 2018, August 21-25, Pécs, Hungary
111
Response of neural stem cell to photon radiation
Jana Konířová1, 2
, Lukáš Cupal3, Martina Zíková
2, Anna Michaelidesová
1, 3, Jana Vachelová
1, and
Marie Davídková1
1 Czech Academy of Sciences, Nuclear Physics Institute, Prague, Czech Republic
2 Czech Academy of Sciences, Institute of Molecular Genetics, Prague, Czech Republic
3 Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering,
Prague, Czech Republic
In the brain, the neural stem cells are mainly located in two specific areas, namely in the
subventricular zone and in the subgranular zone of the hippocampus. Neural stem cells are defined
by their capability to self-renew and their capacity to differentiate into the basic types of neural cells
- neurons, astrocytes, and oligodendrocytes. Regulation of the balance between proliferation and
differentiation of neural stem cells is crucial for the development of the central nervous system and
its subsequent proper functioning. Disruption of this balance may lead to the development of various
pathologies including brain tumors.
Cranial irradiation is in adults and pediatric patients a frequently used tool for the therapeutic
management of primary and metastatic brain tumors. Despite this, remarkably little is known about
the impact of ionizing radiation on neural stem cells. Characterization of cell response to radiation is
crucial to better understand the brain tissue regeneration and late effects which follow radiotherapy.
The aim of our project is to describe the effect of radiation on neural stem cells in vitro. The
irradiation was performed a clinical 4 MV X-ray beam by doses of 0 to 8 Gy. Cell survival,
proliferation, induction of apoptosis, spontaneous differentiation and gene expression were
subsequently analyzed.
After irradiation we observed dose-dependent reduction in the cell number with a prolongation of
the growing disability. The number of apoptotic cells increased with the increasing radiation dose
and simultaneously the expression of genes involved in apoptosis was elevated. Moreover, radiation
promotes spontaneous differentiation of the neural stem cells into neuronal and astrocytic cell types.
European Radiation Research 2018, August 21-25, Pécs, Hungary
112
Radiation Research and Society
Gerhard Kraft
Biophysics Department of GSI, Darmstadt, Germany
When compared to other scientific areas, radiation research has a rather low reputation in the public opinion.
This due on one side to the connection of our topic to the hazards and accidents of nuclear power industries
(Chernobyl, Fukushima) and on the fact that ionizing radiation is not noticed directly by our normal senses:
ionizing radiation is frequently experienced as an invisible threat that could cause cancer and maybe other
diseases.
In general, the knowledge about ionizing radiation is rather low in the public and the fear of it seems to
originate rather by ignorance than by a real evaluation of the radiation risqué compared to its benefits. In this
situation, clarification of the radiation physics and biology and the transfer of this knowledge to public seems
to be the only way to obtain a better understanding of the real problems and to improve our public reputation.
This is also an important aspect concerning governmental funding.
An important issue in this information process is to illustrate the role of ionizing radiation in our daily life.
This includes not only the dangerous aspects of radioactivity in nutrients and from other natural sources but
also the benefit due to medical but also other applications. In medicine, the diagnostic power of x-ray imaging
in two and more dimensions like CT scanning is nowadays a very basic tool in all medical facilities. In
functional medicine, radioactive tracer techniques were pioneering for a quantitative understanding of the
organ regulations and are still used to test for malfunction for instance in the myocardia. In addition, radiation
therapy together with surgery plays a major role in tumor therapy where more than half of all patients receive
radiation with palliative but mostly with a curative goal. These treatments are very successful at relatively low
side effects.
In the complete scenario of artificial exposure to radiation, the risk has to be balanced versus the expected
benefit. There, the consequences of the natural exposure by ionizing radiation has to be explained and the fact
that natural and artificial radiation have the same risk factors when delivered at the same dose. However, the
fact of our permanent exposure to radiation has led to the development of a sophisticated intracellular repair
system, which help us also to counteract the effects of artificial radiation exposure.
Several years ago, these considerations have been the starting point to prepare an exhibition focused on the
topic: “Radiation Research” in a collaboration within the German competence center for radiation research.
There, in 26 posters important topics of radiation research are illustrated mostly in a very basic manner. For
deeper information, a booklet with the posters at one side and a more detailed text at the opposite page gives a
more detailed information.
This exhibition was shown at many schools and was well received by more than 20 000 scholars and by most
governmental officials.
European Radiation Research 2018, August 21-25, Pécs, Hungary
113
When radiation exposure: Energy metabolism mechanism of
mitochondria by radiation in skeletal muscle
Minyoung Lee1, Da Yeon Kim
2, Jae Youn Yi
2 and Eun Ju Kim
2
Korea Institute of Radiological & Medical Sciences, 1
Division of Radiation Research
Infrastructure Operation and 2
Division of Radiation Biomedical Research, Seoul, Korea
Objective: This work seeks to explore effects of treatment with ionizing radiation on mitochondrial and
energy metabolism in skeletal muscle. This work identifies for the first time the effects that IR has on
mitochondria content and cell metabolism in muscle cells
Methods: Peroxisome proliferator-activated receptor coactivator 1, an important stimulator of mitochondrial
biosynthesis, was quantified using quantitative reverse transcriptase polymerase chain reaction. Mitochondrial
content was measured using flow cytometry confirmed with confocal microscopy. Glycolytic metabolism was
quantified by measuring extracellular acidification rate and oxidative metabolism was quantified by
measuring oxygen consumption rate.
Results: Treatment of C2C12 myotube with radiation induced proliferator-activated receptor coactivator 1
leading to significantly increased mitochondrial content. Glycolytic and oxidative capacities were also
significantly increased following treatment with radiation.
Conclusion: We show here that nuclear proliferator-activated receptor coactivator 1 stimulates mitochondrial
biogenesis and respiration in muscle cells through induction of carnitine palmitoyl transferase 1, uncoupling
protein 2 and through regulation of the adenosine monophosphate-activated kinase.
European Radiation Research 2018, August 21-25, Pécs, Hungary
114
Analyzing the radio-resistance of Melanomas via the Secretome
Jeeyong Lee, Jiwon Ko, Hyuntaik Im and Jae Youn Yi
Division of Radiation Biomedical Research, Korea Institute of Radiological and Medical Sciences,
Seoul, 01812, Republic of Korea
Abstract
Melanoma is hard to treat because it shows strong radio-resistance. To improve the treatment of melanoma
patients, we try to sensitize melanoma against radiation therapy. Cancer cells secrete various proteins
coordinating neighboring cells. These secreted proteins could play a role in resisting radiation therapy. In this
research, we analyzed melanoma secretome and tried to identify proteins responsible for radio-resistance. We
quantified the radio-resistance of melanoma cell lines and performed microarray analysis in the presence or
absence of irradiation. By comparing the differentially expressed secretome, we selected candidates
responsible for radio-resistance in melanoma secretome. qRT-PCRs were also performed in order to verify the
integrity of microarray data. We observed that the addition of candidates in secretome enhanced the radio-
sensitivity using MTT assays. We are currently investigating the relationship between the candidates and
radio-resistance.
European Radiation Research 2018, August 21-25, Pécs, Hungary
115
The role of extracellular vesicles in mediating or mitigating ionizing
radiation effects in the bone marrow
Katalin Lumniczky, Tünde Szatmári, Eszter Persa, Dávid Kis, Nikolett Sándor, Rita Hargitai,
Géza Sáfrány
Department of Radiation Medicine, National Public Health Institute, Budapest, Hungary
Extracellular vesicles (EVs) are membrane-bound structures released by the cells in the
extracellular compartment. EVs are very heterogeneous in size and their internal cargo. Based on
their biogenesis they can be subgrouped into exosomes, microvesicles and apoptotic bodies. The
major role of EVs is intercellular communication, which is achieved by the diverse pool of molecular
mediators carried by EVs able to modify the function and/or fate of recipient cells. These mediators
can be nucleic acids (mRNAs and diverse miRNAs as well as other small or long non-coding
RNAs), proteins, lipids and small molecular weight metabolites, which by being protected by the
double membrane layer preserve their integrity and biological activity in the extracellular space. This
complex cargo allows for the transmission of “information packets”, which is a much more
economical and efficient way of signalling between cells than transmission of individual
“information units”.
Bone marrow is a particularly radiosensitive organ, where radiation damage of the stem cell
compartment can be significantly modulated by signals received from the microenvironment. Thus,
intercellular signalling is a key mechanism in modulating radiation damage in the bone marrow. A
growing number of evidence shows that EVs play an important role in the manifestation of ionizing
radiation-induced bone marrow damage. This presentation aims to give an overview on the
heterogeneity of bone marrow-derived EVs, and how radiation influences this heterogeneity both in
terms of phenotype and cargo. We will further review how various EV subpopulations can either
transmit radiation-induced signals to non-targeted cells within the hematopoietic system increasing
the level of radiation damage, or in contrary, are able to mitigate certain aspects of radiation damage.
The work described in this abstract has been supported by the European Commission, within the
CONCERT project. This project has received funding from the Euratom research and training
programme 2014-2018 under grant agreement No 662287 and from the National Research,
Development and Innovation Office (grant agreement number: VKSZ_14-1-2015-0021).
European Radiation Research 2018, August 21-25, Pécs, Hungary
116
Searching for correlations between the spatial and temporal aspects
of the biological and health effects of inhaled radon progeny
Balázs G Madas1 and Emese J Drozsdik
1, 2
1Environmental Physics Department, MTA Centre for Energy Research, Budapest,
Hungary 2Doctoral School of Physics, ELTE Eötvös Loránd University, Budapest, Hungary,
Objective: Exposure to radon progeny results in highly heterogeneous dose distributions within the bronchial
airways, while the risk of its main health consequence, i.e. lung cancer, is characterized by inverse exposure
rate effect. The objective of the present study is to identify tissue responses which may provide a link between
the spatial and temporal aspects of the biological and health effects of radon exposure.
Methods: Numerical modelling methods were applied to quantify the radiation burden in the deposition hot
spots of the bronchial airways, where local tissue doses are hundred times higher than the average over the
airway generation. Based on histological findings as well as theoretical considerations, basal and goblet cell
hyperplasia as well as increase in mucus layer thickness are supposed to occur in these regions as response to
the cell killing effect of alpha-particles emitted by inhaled radon progeny. Distribution of alpha particle hits
and cell nucleus doses were quantified by an own-developed Monte-Carlo code as the function of exposure in
the epithelium in normal and hyperplastic conditions with different number of progenitor cells. Surviving
fractions were estimated based on the number of cell nucleus hits, while cell division rates were computed
supposing equilibrium between cell death and cell division.
Results: Results show that both mucus discharge and the induction of hyperplasia reduce the radiation burden
of progenitor cells, such as average number of hits and average doses in cell nuclei. Hit and dose distributions
reveal that the induction of hyperplasia may result in a basal cell pool which is shielded from alpha-particles
(1) in particular in case of goblet cell hyperplasia. As compared to basal cell hyperplasia, goblet cell
hyperplasia is more effective in reducing the cell death rate in deposition hot spots for a given macroscopic
exposure (2).
Discussion and conclusions: Based on theoretical considerations, it is expected that the measure of
hyperplasia increases by exposure rate. As it has been shown, the increase in the measure of hyperplasia
results in lower tissue dose rates. Therefore a given total exposure can cause more genetic damage in the
tissue and increase the clonal expansion rate of mutated cells more effectively (3) if the duration is longer and
the exposure rate is smaller. In this way, the induction of hyperplasia can provide an explanation for inverse
exposure rate effect. For such changes in tissue architecture, however, high dose rates may be required which
occur only in the deposition hot spots. Therefore, the spatial dose distribution upon radon exposure may play
an important role in inverse exposure rate effect, and also in the observation that heterogeneous exposures
results in lower risk compared to homogeneous exposures as it can be seen in case of hot particles.
References
1. Madas BG. Radon induced hyperplasia: effective adaptation reducing the local doses in the bronchial
epithelium. J Radiol Prot. 2016 Sep;36(3):653–66.
2. Madas BG, Drozsdik EJ. Effects of mucus thickness and goblet cell hyperplasia on microdosimetric
quantities characterizing the bronchial epithelium upon radon exposure. Submitted.
3. Drozsdik EJ, Madas BG. Quantitative analysis of the potential role of basal cell hyperplasia in the
relationship between clonal expansion and radon concentration. Submitted. Available from:
https://arxiv.org/abs/1803.02630
European Radiation Research 2018, August 21-25, Pécs, Hungary
117
Research prioritisation in MELODI
Balázs G. Madas1, Anssi Auvinen
2, Elisabeth Cardis
3, Marco Durante
4, Mats Harms-Ringdahl
5,
Jean-René Jourdain6, Michaela Kreuzer
7, Andrea Ottolenghi
8, Simonetta Pazzaglia
9, Kevin M.
Prise10
, Roel Quintens11
, Laure Sabatier12
and Simon Bouffler13
1MTA Centre for Energy Research, Budapest, Hungary,
2University of Tampere, Tampere & STUK,
Helsinki, Finland, 3Barcelona Institute for Global Health, ISGlobal, Barcelona, Spain,
4Institute for
Fundamental Physics and Applications, TIFPA, Trento, Italy, 5Stockholm University, Stockholm,
Sweden, 6Institute for Radiological Protection and Nuclear Safety, IRSN, Fontenay-aux-roses,
France, 7Federal Office for Radiation Protection, BfS, Neuherberg, Germany,
8University of Pavia,
Pavia, Italy, 9Italian National Agency for New Technologies, Energy and Sustainable Economic
Development, ENEA, Rome, Italy, 10
Queens University Belfast, Belfast, UK, 11
Belgian Nuclear
Research Centre, SCK-CEN, Mol, Belgium, 12
French Atomic Energy Commission, CEA, Paris,
France, 13
Public Health England, PHE, Chilton, UK
MELODI (Multidisciplinary European Low Dose Initiative) is a European radiation protection research
platform with focus on research on health risks after exposure to low-dose/low dose-rate ionising radiation. A
major activity of MELODI is the continuous development of a long-term European Strategic Research
Agenda (SRA) on low-dose risk for radiation protection. The SRA is intended to identify priorities for
national and European radiation protection research programs as a basis for the preparation of competitive
calls at the European level. Recently, the EURATOM call of NFRP-2018-8 specifically indicated that a ‘Gap
analysis’ will be required for each proposal and NFRP-2018-9 could be usefully informed by such an analysis.
The SRA Working Group of MELODI consequently has undertaken a review of relevant EURATOM
research undertaken (or underway) in Framework programmes 6 and 7 (FP6, FP7) and Horizon 2020 (H2020)
identifying their relevance to the six key areas of research identified in the MELODI SRA and roadmap: (1)
To explore the shape of the dose-response relationship for radiation-induced health effects; (2) To understand
the potential impact of individual susceptibility on radiation-induced health effects; (3) To identify, develop
and validate biomarkers for exposure, early and late effects for cancer or/and non-cancer diseases; (4) To
explore and define the role of epigenetic modifications in radiation-induced health effects; (5) To explore the
roles of specific target cells for radiation-induced late developing health effects; (6) To understand the health
effects of inhomogeneous dose distributions, radiation quality and internal emitters. Although a mature
reflection and identification of knowledge gaps has not been possible in all cases as some projects have yet to
come to completion, it is clear that there are evidence gaps in all six areas that remain and areas of research
that have not been fully considered in the past. Besides the gaps identified, MELODI also encourages
education and training in disciplines to maintain, develop and improve skills amongst the low dose health risk
research community. In this regard it is important to encourage training by those in relevant more fundamental
sciences. The skills amongst the MELODI community in data management, data mining and bioinformatics
are judged to be suitable for further development. In terms of infrastructures for research, MELODI
encourages, where appropriate, (1) the use of archived biological materials from prior research, (2) the
integration of experienced laboratory networks improving the robustness of results via intercomparisons, (3)
the integration of expertise from outside the conventional fields of radiation research, (4) the use of the wider
EU scientific infrastructures for, amongst other things genomics, microscopy, structural biology, computing
where relevant, (5) the provision of access to the wider community of researchers, where new infrastructures
are proposed or developed.
1. Kreuzer, M. et al. Multidisciplinary European Low Dose Initiative (MELODI): strategic research agenda
for low dose radiation risk research. Radiat. Environ. Biophys. 57, 5–15 (2018).
European Radiation Research 2018, August 21-25, Pécs, Hungary
118
Radon solubility in different materials
Andreas Maier1, Franziska Papenfuß
1, Michaela Schmitt
1,2 Gerhard Kraft
1 and Claudia Fournier
1
1 GSI Helmholtz Centre, Biophysics Department, Darmstadt, Germany
2 Goethe University Frankfurt, Frankfurt am Main, Germany
Radon is a radioactive noble gas, which is known to be the second leading cause for lung cancer after smoking
but it is also used for the therapy of inflammatory diseases like rheumatoid arthritis. To better quantify the risk
and the anti-inflammatory effect of low concentrations of radon and subsequent low dose irradiation, we are
investigating the distribution of radon in different types of tissues and the deposited dose. To understand these
processes, the solubility and therefore the distribution of radon in different materials is important.
In the experiments, samples such as isotonic water solutions or oleic acids were exposed in a special designed
radon chamber for one hour. After this time, a saturation and subsequently a homogenous distribution of
radon in the sample is established. Following exposure, the samples are transferred into non-contaminated
container and hermetically sealed. Around four hours later, the radon concentration is in equilibrium with the
activities of the γ-emitting daughter nuclides 214
Pb and 214
Bi. The decay of their activity parallels the decay of
the primary radon and can be measured using a Ge detector. Back-extrapolation yields then the initial
concentration of radon. By knowledge of the radon activity concentration during exposure, the solubility
coefficient of the specific material can be calculated.
The selected samples include pristine substances like oleic acid or linoleic acid, which are the most abundant
fatty acids in the human body, or isotonic saline solution. In addition, tissue samples from pig like fat, muscle
or bone were measured.
The experiments show that the solubility of radon in fatty acids is about 50 times higher than in isotonic saline
solution. A similar behavior was measured for fatty tissue in comparison to muscle tissue. For other tissues,
experiments are currently performed, as well as experiments with a voluntary patient exposed in a radon
gallery under therapy conditions.
In our experiments, we could confirm an inhomogeneous distribution of radon in different materials. Here we
want to present the methods and first results, yielding indications for a better understanding of radon
distribution and deposited dose in the human body.
This work is supported by the German Federal Ministry of Education and Research (BMBF) under project
funding reference number 02NUK050A.
European Radiation Research 2018, August 21-25, Pécs, Hungary
119
Pre-exposure Gene Expression as Marker of Individual
Radiosensitivity in Non-human Primates
Matthäus Majewski1, Vladimir Vainstein
2, Lena Basile
2, Zoya Gluzman-Poltorak
2, Jamie Tom
2,
Matthias Port1
and Michael Abend1
1 Bundeswehr Institute of Radiobiology, Munich, Germany
2 Neumedicines Inc., Pasadena, USA
Objective:
The effect of an individual response to ionising radiation is known since the early 20th century. Also gender-
specific responses to radiation have been observed for decades. Despite this long time period, the biological
mechanisms are still poorly understood.
In our study we aimed to identify pre-exposure, gender-specific gene expression changes in a non-human
primate model to better understand individual radiosensitivity, using survival as the biological endpoint.
Methods:
56 rhesus macaques (Macaca mulatta) (28 males and 28 females) were exposed to a dose of 700-740 cGy
corresponding to a LD 70/60. Whole blood samples in PAXgene Blood RNA tubes (BD Diagnostics,
PreAnalytiX GmbH, Hombrechtikon, Switzerland) were taken before irradiation. After the isolation of the
total RNA we performed a two stage study design, where stage I was a whole genome screening (Illumina
NextSeq500 next generation sequencing system; Illumina, San Diego, USA) using 20 of the samples.
Differential gene expression (p-value ≤ 0.05; FC > |2.0|) was calculated between the measurements at death
relative to the pre-exposure expression values. Stage II is currently being performed as an independent
methodical validation, using the remaining 36 samples on a qRT-PCR based platform.
Results
37 of 56 (males: 17, females: 20) animals died within 10-21 days (the remaining ones were sacrificed 50-60
after exposure). For males/females we identified 106/237 differentially up- and 126/190 downregulated genes.
22 gender-overlapping up- and 14 downregulated genes were found. Altogether 58 candidate genes were
selected for independent validation and the results will be presented at the conference.
Conclusion
Whole genome mRNA screening allowed the identification of many gender dependent/independent candidate
genes which are currently validated. Results will be presented at the conference.
European Radiation Research 2018, August 21-25, Pécs, Hungary
120
Effect of normal-cell premature senescence on tumour-cell
proliferative potential following charged particle irradiations
Lorenzo Manti1
1 University of Naples Federico II, Physics Department & Istituto Nazionale di Fisica Nucleare
(INFN), Naples Section, Naples, Italy
The growing adoption of accelerated charged particle beams in cancer radiotherapy
(hadrontherapy) urgently requires dispelling several uncertainties that still affect particle
radiobiology [1]. Cellular response to charged particles qualitatively and quantitatively differs from
that observed after photons, encompassing complex and subtly orchestrated pathways, which are
strongly influenced by both physical (e.g. radiation quality) and biological (e.g. cell signalling)
factors. This confers charged particles of varying LET peculiar properties of potential clinical
relevance [2,3]. As an example, the observed specific immune response modulation with beneficial
systemic response by protons [4] adds as an attractive bonus to the reduced risk of secondary cancers
and late toxicity granted by their inverted dose-depth profile. Conversely, lack of precise knowledge
of the impact by radiation on the heterogeneous tumour milieu dynamically interplaying with healthy
cells via molecular signalling [5,6] strengthens the need for investigating the effect of particle
radiation-induced normal-cell premature senescence via SASP (Senescence-Associated Secretory
Phenotype) on tumours [7,8]. To this end, data from medium-transfer experiments will be presented
that have been obtained within the framework of the INFN-funded project ETHICS (pre-clinical
Experimental and THeoretical studies to Improve treatment and protection by Charged particleS) to
ascertain whether normal cells prematurely entering senescence following particle irradiations
influence the proliferative behaviour of irradiated tumour cells. Results will be discussed on the
effect of senescing fibroblast and HUVEC cells on Saos-2 osteosarcoma cells exposed to 12
C ions 16
microvascular endothelial cells (HMVEC) on breast metastatic MDA-MB cells after proton
irra
Superconducting Cyclotron of INFN-LNS, Catania, and Tandem-Alpi accelerator of INFN-LNL,
-galactosidase expression
at three time points post-irradiation (5, 16 and 23 days) in normal cell cultures; at the same times, the
proliferative ability of irradiated cancer cells that had been conditioned for 24 h with medium from
such cultures was measured by clonogenic assay. Preliminary ex-vivo data will be also presented
obtained by conditioning MDA-MB cells, which had been irradiated in a murine model (BALB/c
nude mice), with medium from in vitro irradiated HMVEC cells. 1. Durante M et al., Charged-Particle Therapy in Cancer: Clinical Uses and Future Perspectives, Nat Rev Clin Oncol,
14, 483-495 (2017)
2. Tommasino F and Durante M, Proton radiobiology. Cancers, 7,353-381 (2015)
3. Held KD et al., Effects of Charged Particles on Human Tumor Cells. Front. Oncol. 6:23 (2016)
4. Gameiro SR et al., Tumor cells surviving exposure to proton or photon radiation share a common
immunogenic modulation signature, rendering them more sensitive to T cell-mediated killing, Int J Radiat Oncol
Biol Phys, 95, 120-130 (2016)
5. Fujita M et al., Irradiation induces diverse changes in invasive potential in cancer cell lines. Semin Cancer Biol, 35,
45-52 (2015)
6. Luo X et al., Stromal-Initiated Changes in the Bone Promote Metastatic Niche Development. Cell Rep.14:82-
92 (2016)
7. Pribluda A et al., A senescence-inflammatory switch from cancer-inhibitory to cancer-promoting mechanism.
Cancer Cell, 24, 242- 256 (2013)
8. Mellone et al., Induction of fibroblast senescence generates a non-fibrogenic myofibroblast phenotype that
differentially impacts on cancer prognosis. Aging, 9, 114-132 (2016)
European Radiation Research 2018, August 21-25, Pécs, Hungary
121
Cellular response to short-term 1800 MHz radiofrequency radiation
exposure
Ana Marija Marjanovic Cermak1, Ivan Pavicic
1 and Ivancica Trosic
1
1 Institute for Medical Research and Occupational Health, Radiation Dosimetry and
Radiobiology Unit, Zagreb, Croatia
Objective
Rapid progress in the field of telecommunications has raised concern about possible hazardous effect of
radiofrequency (RF) radiation on living organism. Although potential health impacts of RF exposure have
been extensively studied, there are many uncertainties and questions regarding biological effects that occur at
the non-thermal level. One of the important mechanisms that should be considered is connected to redox
imbalance and development of oxidative stress. The aim of this study was to measure and compare the
biological effect of short-term 1800 MHz RF exposure on two morphological and functional distinct cell
culture types.
Methods
Chinese hamster lung fibroblast cells (V79) and human neuroblastoma cells (SH-SY5Y) were exposed to
1800 MHz modulated RF radiation at field strength of 30 V/m. The cell samples were irradiated inside of the
Gigahertz Transverse Electromagnetic Cell (GTEM) for 10, 30 and 60 minutes. Cellular viability was
measured by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide assay (MTT) and level of
reactive oxygen species (ROS) was determined by fluorescent probe 2′, 7′-dichlorofluorescein diacetate
(DCFH-DA). Enzyme-linked immunosorbent assay (ELISA) was used to measure concentrations of oxidised
proteins and malondialdehyde (MDA) was used to assess the level of lipid oxidative damage.
Results
Average specific absorption rate (SAR) was calculated to be 1.6 W/kg. The viability of radiation exposed cells
remained within normal physiological values throughout the measurement period. In V79 cells 10-minute
radiation caused significant increase in the level of ROS, while higher ROS values in SH-SY5Y cells were
observed for every radiation exposure time. After 60-minute exposure there was significant protein and lipid
oxidative damage in SH-SY5Y cells.
Conclusion
Modulated radiofrequency radiation at non-thermal level induces disbalance of cellular redox equilibrium.
Applied radiation caused transient changes in V79 cells, suggesting activation of adaptation mechanisms,
while due to its neural origin, SH-SY5Y cells appeared to be more sensitive to radiation exposure.
European Radiation Research 2018, August 21-25, Pécs, Hungary
122
Non-ionising electromagnetic fields in medical applications
Mats-Olof Mattsson1 and Myrtill Simkó
1
1 SciProof International AB, Östersund, Sweden
Non-ionising electromagnetic fields (EMF) ranging from static electric and magnetic fields, via low
frequency fields, to high frequency fields all the way up to the THz region have been studied for
decades regarding possible effects on health. To a large part, these studies have been dealing with the
potential to cause adverse health effects. However, in parallel the potential for various EMF to be
used for diagnostic or therapeutic purposes has been investigated as well.
A number of well-established and very useful tools based on various physical principles are available
for both diagnostic and therapeutic purposes. These include X-ray, MRI, acoustic millimeter waves
(ultrasound), and CT among others. Each of the techniques have annual world markets amounting to
billions of €. Nevertheless, each of the techniques also have several drawbacks regarding e.g.
appropriateness for certain body locations, resolution, tissue-type preference and costs. They may
also cause serious side effects in the therapeutic situation.
It is thus still necessary to develop additional instruments that can improve on diagnosis and/or
therapy, and at the same time being affordable to purchase, easy to use, and have reasonable
running costs. From that perspective, significant interest has appeared for developing approaches
that are based on different parts of EMF spectrum. Before such approaches will have any
meaningful clinical impact, a number of requisites have to be fulfilled. These include to understand
the interactions between different EMF on one hand and biological structures on the other, to
understand how biological effects occur and which they are if a therapeutic use is intended, to have
a relevant data base of experimental studies for design of further studies, and finally to perform
controlled clinical studies of sufficient size.
This presentation will provide an overview of recognized and possible biological interaction
mechanisms that are relevant for medical use of different EMF. Furthermore, different medical uses
of EMF will be described and discussed regarding potential and relevance. Finally, current and near-
future concerted research efforts on the international level will be briefly presented.
European Radiation Research 2018, August 21-25, Pécs, Hungary
123
Advances in Pre-Clinical Image Guided Radiation Therapy Systems
William E. McLaughlin
Precision X-ray Inc. 15 Commerce Dr, Unit 1. North Branford, CT 06471. USA
Small animal, pre-clinical X-Ray irradiation systems have progressed significantly in the last few years with
advances in multi-modal image-guided targeting in both 2D and 3D systems as well as advanced collimation
and improved dosimetry capabilities for these research based systems. These advances are allowing
researchers to much more closely emulate the radiation therapy settings and treatment regimens that are
utilized in the Clinic to allow more relevant testing in cells and small animals. We will discuss the highlights
of these advances and some of the applications that have been performed with these new pre-clinical IGRT X-
Ray systems. www.pxinc.com
European Radiation Research 2018, August 21-25, Pécs, Hungary
124
Role of the novel p53 target D630023F18Rik, in embryonic brain
development and the cellular response to DNA damage
André C.M. Mfossa1,2
, Sarah Baatout1, Rafi Benotmane
1, Danny Huylebroeck
2,3 and Roel
Quintens1
1 Radiobiology Unit, Institute of Environment, Health and Safety, Belgian Nuclear Research Centre, Mol, Belgium
2 Laboratory of Molecular Biology, Department of Development and Regeneration, KU Leuven, Belgium
3 Department of Cell Biology, Erasmus MC, The Netherlands
Objective
Prenatal exposure to moderate to high doses of ionizing radiation (IR) during neurogenesis activates a fast p53-
mediated transcriptional response in the embryonic brain. We have previously identified several novel
uncharacterized p53 targets, among which D630023F18Rik (human ortholog C2ORF80). This gene is highly and
dose-dependently induced after irradiation via binding of p53 to an alternative promoter of the gene.
Furthermore, expression of D630023F18Rik is highly enriched during normal mouse brain development and
during primary neuron maturation, suggesting it has an important function in these processes. The purpose of this
study is to gain more insight into the function of D630023F18Rik in normal brain development and the response
to ionizing radiation.
Methods
Novel transcript variants were identified using 5’- and 3’-Rapid Amplification of cDNA Ends. Quantitative
reverse transcription PCR using transcript-specific primers was used to quantify mRNA expression.
Spatiotemporal D630023F18Rik protein expression was assessed by immunostaining in brains of mice at
embryonic days 11, 13, 15,18 as well as in mouse primary cortical neurons at different days (1, 3, 7, 10 and 14)
of culture.
Results Different new splice variants were identified that are highly enriched in the brain compared to most other tissues.
Among these, several short transcripts expressing a novel exon were found to be induced by radiation, both in
vivo and in vitro. Interestingly, the stability of these short transcripts was enhanced after irradiation. Western
blotting indicates the existence of proteins of different sizes with different temporal expression profiles, although
the short, radiation-induced forms seem to be non-coding. Immunostaining analysis in the developing brain
shows cytoplasmic expression, mainly in brain regions related to the thalamocortical circuitry and migrating
interneurons. In the maturing neurons, D630023F18Rik was found to be expressed both in the cell body and the
nucleus.
Conclusion D630023F18Rik may play an essential role in the developing brain as exemplified by its particular
spatiotemporal expression patterns. Using in vitro and in vivo overexpression and knockdown strategies
complemented with protein interaction assays we are currently further investigating its potential functions.
Acknowledgments: This work is supported by the Research Foundation - Flanders (FWO-V G.0A31.16N).
André Claude Mbouombouo Mfossa is a recipient of a SCK•CEN-KU Leuven PhD grant.
European Radiation Research 2018, August 21-25, Pécs, Hungary
125
Assessment of risk of radiation toxicity in prostate cancer patients
using high throughput FTIR spectroscopy of non-invasive blood
plasma samples
Dinesh K.R. Medipally
1, 2, Adrian Maguire
1,2, Jane Byrant
2, Valerie Untereiner
4, Ganesh D
Sockalingum 3, John Armstrong
5, Aidan D Meade
1,2 and Fiona M Lyng
1,2.
1. School of Physics, DIT, Ireland.
2. DIT Centre for Radiation and Environmental Science, DIT, Ireland.
3. Université de Reims Champagne-Ardenne, MéDIAN-Biophotonique et Technologies pour la
Santé, France.
4. Plateforme en imagerie cellulaire et tissulaire (PICT), Université de Reims Champagne-Ardenne,
France.
5. Department of Radiation Oncology, St Luke's Hospital, Ireland.
Radiation therapy (RT) is used to treat approximately 50% of all cancer patients. However, radiation
therapy can fail to achieve tumour control in at least 25% of patients and at least 5-10% of cancer
patients can go on to suffer from adverse late effects which can seriously affect their quality of life.
Therefore, it is very important to identify the radiosensitive patients prior to radiotherapy. Currently,
there are no predictive assays in clinical use to identify patients at risk of radiation toxicity. Development of such a predictive assay could lead to individualised patient treatment and avoidance
of radiotherapy in radiosensitive patients. Fourier transform infrared (FTIR) spectroscopy can
provide a rapid, label-free and non-destructive measurement of the biochemical content of cells,
tissues and biofluids. The aim of this study was to investigate the potential of FTIR spectroscopy for
assessment of risk of radiation toxicity.
In this prospective study, blood plasma samples were acquired from 40 prostate cancer patients at
diagnosis. The patients were followed up for at least 8 months following hormone and radiotherapy
and toxicity was recorded using the National Cancer Institute Common Terminology Criteria for
Adverse Events (NCI-CTCAE) grading system. Spectra were recorded from each patient’s plasma
samples using high throughput (HT)-FTIR spectroscopy and the data was analysed in the R
environment. Significant differences were observed between infrared spectra of plasma samples from
prostate cancer patients at baseline and 2 months follow up, and at baseline and 8 months follow up,
as well as between spectra from patients showing acute and late toxicity and grade 0-1 and grade 2+
toxicity. Multivariate classification models (PCA-LDA, SVM and Random forest) were built and
tested on the infrared spectra from the prostate cancer patients. The classifiers were able to
discriminate between prostate cancer patients at baseline and 2 months follow up, and at baseline and
8 months follow up, as well as between patients showing acute and late toxicity and grade 0-1 and
grade 2+ toxicity with sensitivity and specificity rates ranging from 80% to 100%. This technology
may have potential to predict radiotherapeutic response in prostate cancer patients and could lead to
individualised patient radiotherapy.
European Radiation Research 2018, August 21-25, Pécs, Hungary
126
An in-vitro study of Boron Proton Fusion Therapy
Anna Michaelidesová1,2
, Jana Vachelová1,2
, Kateřína Pachnerová Brabcová2, Jana Konířová
1,2,
Vladimír Vondráček1 and Marie Davídková
2
1 Proton Therapy Center Czech, Department of Medical Physics, Prague, Czech Republic
2 Czech Academy of Sciences, Nuclear Physics Institute, Department of Radiation Dosimetry,
Prague, Czech Republic
The proton boron fusion reaction was introduced in 1960. During the reaction of
11B with a low energy
proton, three alpha particles are emitted and can damage tumour cells, just as in the case of alpha particles in
boron neutron capture therapy. Very recently biological experiments showed that this reaction can really
increase the biological effectiveness of proton therapy. The aim of this study was to investigate the
effectiveness of the boron in case of its use in proton therapy using a glioblastoma in-vitro model.
U87 MG (Human glioblastoma astrocytoma) cells were used for irradiations at the Proton
Therapy Center Czech by a monoenergetic pencil scanned beam of 190.6 MeV corresponding to
range in water of 23.9 cm. Cells were cultivated with sodium mercaptododecaborate (BSH), which is
an agent commonly used during boron neutron capture therapy. After the incubation, cell monolayers
in tissue flasks were irradiated in two positions of the Bragg curve (proximal and peak position) by
doses of 1, 2, 3, and 4 Gy.
Just after the irradiation cells were counted and reseeded for cell survival assay in 6-well plates.
Ten days later, cell colonies were coloured and counted. In comparison to the control samples, not
incubated with BSH, no cytotoxicity was found. There was not observed any effect of the BSH at the
proximal position of the Bragg curve. On the other hand, an increased effect of the proton radiation
was observed at the peak position, where the energy of protons is low enough to make the reaction of 11
B and protons possible according to the reactions cross section. Therefore, the use of BSH in clinics could increase the tumour control probability and decrease the secondary
unwanted effects on healthy tissues, due to the possibility to reduce the prescribed doses. Further experiments
are needed, but it seems that our results confirm the applicability of BSH in clinics. The newest results will be
presented.
European Radiation Research 2018, August 21-25, Pécs, Hungary
127
Insights in the repair of clustered double-strand breaks in human cells
Repair of CRISPR/Cas9 generated DSB clusters with increased
complexity
Emil Mladenov1 and George Iliakis
1
1 Institute of Medical Radiation Biology, University Duisburg-Essen, Medical School, Essen,
Germany
From the variety of DNA lesions induced by ionizing radiation, double-strand breaks (DSBs), are known to
provoke the most spectacular DNA damage responses affecting almost every aspect of the cellular
metabolism. DSBs emerge through diverse mechanisms, and are classified in subgroups of different
complexity. According to this classification, the simplest form is induced enzymatically, whereas the most
complex comprises DSB clusters, which are expected to be induced mainly by high LET radiation. Moreover,
DSB complexity is invoked to explain the increased efficacy of high LET radiation. Complexity is usually
defined as presence of additional lesions in the immediate vicinity of the DSB. DSB-clusters represent a
different level of complexity that can jeopardize processing by destabilizing chromatin in the vicinity of the
cluster.
Erroneous processing of DSBs severely affect cellular viability and result in increased genome instability. To
reduce such risks, cells of higher eukaryotes have evolved several DSB repair mechanisms differentiated by
their diverse efficiency and discrete repair accuracy. Indeed, DNA-PKcs dependent non-homologous end
joining, indicated as classical or canonical (c-NHEJ), homologous recombination mediated repair (HRR), and
alternative end-joining pathway (alt-EJ), operating as a backup (B-NHEJ), function in parallel to coordinate
the repair of DSBs throughout the cell cycle and in different stages of cellular growth. Despite a large amount
of data, revealing a role for all three repair pathways in elimination of simple DSBs, there is limited
information, of how DSB clusters are repaired in the contest of chromatin and what the consequences of their
improper processing are.
However, direct test of such hypothesis requires specific biological systems, which allows specific
investigation of DSB clusters. In the following study a genome editing technology, represented by the
CRISPR/Cas9 system, was modified to generate DSB clusters of different complexity within the Exon 3 of
human HPRT locus and the fluctuations in mutation frequency at the corresponding locus were assessed as a
function of increased DSB cluster complexity. Moreover, to determine the repair machineries involved in the
processing of DSB clusters, the effect of small molecular inhibitors, reducing the activity of key DSB repair
proteins (ATM, ATR, DNA-PKcs, PARP-1 and Rad51), was additionally validated. In addition to the
chemical inhibition of DSB repair pathway knock-out cells lines, deficient in DNA-PKcs, ATM and PARP1
were generated and tested in the above experimental setup. Our results clearly demonstrated that the increased
DSB cluster complexity significantly increase mutations formations, which is dramatically reduced in PARP1
deficient cells or cells treated with PARP1 inhibitor, PJ34. This allow us to speculate that DSB clusters
interfere with c-NHEJ and HRR and are processed by the highly error-prone alt-EJ pathway.
Work supported by grants from the „Bundesministerium für Bildung und Forschung“ (BMBF: (02NUK043B
–COLLAR)) and the DFG (GRK1739).
European Radiation Research 2018, August 21-25, Pécs, Hungary
128
Insights in the repair of clustered double-strand breaks in human cells
Veronika Mladenova1, Emil Mladenov
1 and George Iliakis
1
1 Institute of Medical Radiation Biology, University Duisburg-Essen, Medical School, Essen,
Germany
From the plethora of DNA lesions generated by ionizing radiation, double-strand breaks (DSBs) are
considered the most consequential. DSBs elicit the majority of documented detrimental radiation effects,
including genomic rearrangements, chromosome aberrations, cell death, genetic mutations, and cancer. High-
LET radiation modalities may lead to the formation of clustered DSBs (or DSB-clusters), comprising two or
multiple DSBs that destabilize the chromatin structure, which in turn compromises overall DSB processing. It
is generally assumed that DSB-cluster-formation is linked to the increased relative biological effectiveness
(RBE) of high-LET radiation modalities, such as alpha-particles produced during nuclear decay reactions, ions
present in space, or carbon ions used in cancer therapy. In order to confirm this long-known effect we utilize a
sophisticated biological model, based on the generation of clonal human cell lines, which allows direct
analysis of assumptions regarding the biological effects of single DSBs and DSB-clusters.
In this study, derivatives of human retinal pigment epithelial (RPE-1) cells harboring multiple genomic
integrations of constructs carrying I-SceI recognition sequence(s) were generated by “Sleeping beauty”
transposon technology. The cleavage of I-SceI sites by transient expression of I-SceI endonuclease results in
generation of single DSBs or DSB-clusters with increasing complexity as monitored by the accumulation of
-H2AX and 53BP1 at the sites of DSBs. Our data indicate that the classical non-homologous end joining is
the repair pathway that processes the majority of simple and clustered DSBs, as the chemical inhibition of
DNA-PKcs elicits an elevated level of persisting unrepaired breaks. Our results also point towards increased
contribution of error-free HRR in the repair of DSB-clusters, as Rad51 recombinase is recruited with lower
affinity to simple DSBs than to DSB-clusters, possibly due to competition by c-NHEJ.
Our model system allows to elucidate the biological consequences of DSB-clustering at multiple genomic
loci, its impact on chromatin integrity, its influence on the activation and efficiency of DSB-signaling and
DNA repair, as well as the interplay and interdependence of DSB-repair pathways in the restoration of DNA
integrity. Finally, it allows the analysis of inter-species differences in pathway choice and will help in the
elucidation of the underlying molecular mechanisms.
Work supported by grants from the „Bundesministerium für Bildung und Forschung“ (BMBF: (02NUK043B
–COLLAR)) and the DFG (GRK1739).
European Radiation Research 2018, August 21-25, Pécs, Hungary
129
Regulated systems of ISce-I expression for in-depth studies of the biological effects of DSBs and
DSB-clusters
Sharif Mortoga, Veronika Mladenova, Emil Mladenov and George Iliakis
Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School Hufelandstr.55, 45122
Essen, Germany
Cells exposed to radiation accumulate DNA double strand break which are the most critical DNA lesion
generating adverse biological consequences and if left unrepaired, could results in chromosomal aberration
and cell death. It has been suggested that DSBs could be classified in distinct types, from simple DSBs with
ligatable DNA end to DSBs clusters, which could destabilize locally the chromatin structure. While the repair
of simple DSBs is well characterized the responses initiated by the DSBs clusters remain obscure. To date
models based on mathematical or computational approaches have been used to assess the effect of simple
DSBs. However, a precise biological model system mimicking DSBs clustering is still missing. In order to
study such DSB-cluster associated adverse biological consequences, a model system based on the generation
of DSBs by restriction endonuclease (ISce-I) has been established. Rodent CHO cell line harboring ISce-I
sites engineered at different configurations with increasing DSB-cluster complexity was characterized for
DNA damage responses after induction of DSBs at ISce-I sites. Obtained results show significant correlation
between cell killings together with chromosomal aberrations with formation of DSBs clusters and equal
activation of DNA Damage Response (DDR) signaling measured by γ-H2AX and 53BP1 foci formation.
Surprisingly, live-cell imaging shows that with increasing complexity there is more retention of 53BP1 to the
DSBs. Recruitment of 53BP1 at DSBs follows a multitude of upstream events. Evidence suggests that RNF8
and RNF168 – two known E3 ligases – play significant roles in this upstream regulatory signaling cascade via
chromatin ubiquitylations and regulate 53BP1 assembly at DSB-flanking chromatin. Therefore, we seek to
investigate the plausible roles of these two known regulators of 53BP1 accrual at ISce-I inflicted DSB
chromatin. Some preliminary results based on involvement of two novel chromatin-associated ubiquitin
ligases (RNF8 and RNF168) in 53BP1 accrual to simple DSBs and DSB clusters as well as in chromosomal
translocation formations will be presented.
European Radiation Research 2018, August 21-25, Pécs, Hungary
130
Regulation of tumors development by intact and irradiated
mesenchymal stem cells from different mouse tissues
ElizavetaYu. Moskaleva, Yulia P. Semochkina, Valentina.G. Shuvatova, Alla V. Rodina, Anastasia P.
Kuvyrchenkova
National Research Centre "Kurchatov Institute", Department of System biology and Biomedicine, Moscow,
Russia
Objective. Mesenchymal stem cells (MSCs) are long-living cells that are present in almost all organs and
tissues and play an important role in the regulation of regeneration after various injuries. During radiotherapy,
MSCs can be exposed to ionizing radiation in the field of irradiation, accumulate mutations and undergo
malignant transformation, or alter the profile and level of cytokine secretion and thereby influence the growth
of tumours. Previously, we showed that MSCs from bone marrow (BM), in contrast to MSCs from the brain
and adipose tissue (AT), undergo malignant transformation after irradiation at doses of 1 and 6 Gy [1, 2]. The
purpose of this study was to investigate the effect of intact and irradiated MSCs from mice BM and AT on the
time of onset and on the growth rate of transplanted melanoma and mammary gland adenocarcinoma when
MSCs and tumor cells were co-administered into syngeneic mice.
Methods. MSCs were isolated from BM and AT of C57Black/6 mice, cultured in DMEM/F12 medium with
10% fetal bovine serum, 2 mmol/L L-glutamine and 100 U/ml penicillin/streptomycin. The B16 mice
melanoma and the Ca755 mammary adenocarcinoma cells were cultured in DMEM medium under the same
conditions. MSCs at passage 29 were exposed to 0.1; 1 and 6 Gy at the facility "GUT-200M" (60
Co) and
cultured for 10 passages. Before the transplantation, the cells were removed from the template and the
suspensions of MSCs and of tumor cells were prepared. The MSCs and tumor cells were mixed in a ratio of
1:1, incubated for 30 minutes at 37 oC. The cell suspension of 1x10
6 cells in 100 μl of DMEM culture medium
was injected subcutaneously into syngeneic C57Black/6 mice. The statistical processing of the results was
carried out using the Student's method.
Results. In the study of the effect of control MSC BM and MSC BM on the 10th passage after irradiation at
doses of 0.1; 1 and 6 Gy on the growth of the melanoma B16 grafted subcutaneously, there was no change in
the time of appearance of tumors and in the rate of their growth. At the same time, when using MSC AT under
the same conditions, the earlier development of B16 tumors and their faster growth were detected. When
studying the effect of control and irradiated MSC BM and MSC AT on the growth of transplanted mammary
gland adenocarcinoma Ca755, an earlier appearance and faster growth of tumors were detected both with the
use of MSC BM, and with the use of MSC AT. Irradiated MSCs stimulated the growth of B16 and Ca755
tumors more intensively than intact ones.
Conclusion. It is shown that mouse MSCs from BM and AT can stimulate the growth of tumors. The ability of
MSCs to stimulate the growth of melanoma and of the mammary adenocarcinoma increases after MSCs
irradiation at doses of 1 and 6 Gy. The stimulation of tumors growth by MSCs can be associated with the
effect of such cytokines as VEGF, HGF, IL6 and TGFβ, which are secreted by MSCs from different mouse
tissues. As we have shown earlier, the highest level of these cytokines secretion was found in MSC AT [2, 3].
1. Moskaleva E. Yu. et al. Cell and Tissue Biology. 2017. 11(5): 381.
2. Moskaleva E. Yu. et al. Abstract book 43rd Annual Meeting ERRS 2017. P.380.
3. Posypanova G.A. et al. Genes and cells. 2017. XII (3): 204.
This work was supported by RFBR grant №15-29-01234.
European Radiation Research 2018, August 21-25, Pécs, Hungary
131
Genomic Instability and non-targeted effects; are they important for environmental radiation
protection?
Carmel Mothersill and Colin Seymour
Department of Biology, McMaster University, Hamilton, ONTARIO CANADA
Radiation-induced genomic instability (RIGI) and bystander effects (RIBE) are now well established non-
targeted effects (NTE) in radiobiology but their importance for radiation protection is seldom considered. This
appears to be because the effects are so variable and the mechanisms, in particular the shape of the dose
response curve are still uncertain. RIBE and RIGI also both mean that system level effects in space (effects in
distant cells) and time (effects in progeny) occur and need consideration at multiple levels of organisation.
The implications are then that an apparently harmful effect at one level of organisation; e.g. death of a cell,
may be beneficial at a higher level of organisation as it could mean a potentially carcinogenic cell has been
eliminated. While NTE tend not to be considered in human radiation protection, recent studies in
environmental radiation protection have lead to discussion of whether so-called “memory effects” in impacted
populations in Chernobyl and Fukushima may be related to persistent NTE in populations directly exposed
and in progeny of affected species. To address these questions we performed dose reconstructions to estimate
the historic dose to populations in Chernobyl, for which good datasets were available in the literature or
directly from authors such as Otaki, Moller and Mousseau, Goronchova and others.. The reconstructed doses
were plotted against data for current mutation frequencies to determine a “historic dose response” Cellular
data from our group for similar radiation dose ranges were also plotted to see if the shape of the dose response
was similar to the classic NTE dose response which saturates at around 0.5Gy. The data for Chernobyl birds,
grasshoppers and bank voles, as well as for Fukushima butterflies closely resemble the dose response seen for
NTE in the range 0.01mGy -500mGy. This could suggest that NTE play a role in determining the extent of the
“memory effect” and could at least partly explain the observations of higher than predicted effects in some
field studies. Apart from shedding light on mechanisms involved in population responses, these discussions
should help reconcile contradictory and disputed data where field studies, model predictions and laboratory
experiments show large discrepancies.
European Radiation Research 2018, August 21-25, Pécs, Hungary
132
Assessment of long-term grey matter alterations in a mouse model of in
utero irradiation using anatomical and diffusion MRI
Laura Mouton1, Olivier Etienne
2, Fawzi Boumezbeur
1,
François Boussin2 and Denis LeBihan
1
1 CEA NeuroSpin, Frederic Joliot Institute, Gif-sur-Yvette, France
2 CEA UMR 967, François Jacob Institute, Fontenay-aux-Roses, France
Objective: Despite its efficacy to treat brain tumour, irradiation may result
in severe cognitive defects. New methods to monitor radiation-induced
brain alterations are required for the follow-up of patients. Magnetic
Resonance Imaging (MRI) is a non-invasive technique used to assess
anatomical and structural lesions at high spatial resolution. In particular,
semi-automatic morphometry allows for the investigation of those
alterations in various regions-of-interest (ROI). We recently introduced S-
index, a composite diffusion metric sensitive to microstructural changes1. In
this study, we applied this approach to investigate brain alterations in adult
mice irradiated in utero.
Methods: Pregnant C57BL/6N mice were irradiated with a single dose of
1Gy from Cs137
source on the 14.5th day of gestation. F1 male irradiated
offsprings (NIRR=12) and non-irradiated (Nsham=7) were scanned at the age
of 3 months old on a 11.7 T MRI scanner equipped with a CryoProbe
(Bruker). Whole-brain 3D T2*-weighted anatomical images were acquired
with a 0.1 mm isotropic resolution, followed by diffusion-weighted MRI
(20 b-values from 0-3000 s/mm² along 6 directions). Image analysis
consisted in: (1) removal of extracranial signal; (2) computation of brain
masks from 3D T2*w anatomical images using SPM8 and tissue prior maps
of grey (GM), white (WM) matter and cerebrospinal fluid (CSF)2; (3) co-
registration with FSL pipeline3 of masked anatomical and diffusion MRIs to
our homemade mouse brain atlas; and (4) individual mouse brain
parcellation and definition of our ROIs (Fig 1). Averaged S-index values
and volumes were calculated for each compartment and our ROIs.
Comparisons between irradiated and sham groups were assessed using
Student’s t-test (*** p<0.001; ** p<0.01; * p <0.05; ns p>0.05).
Results: Figure 2 illustrates the comparison of brain volumetry between
sham and irradiated mice. We observe a 12.5% brain atrophy that can be
attributed to a significant 17% GM atrophy without any significant
alterations of WM and CSF volumes. Except for OB, all of our ROIs
exhibited large and significant volume deficits in irradiated mice (Table 1).
These observations were strengthened by significant alterations of our S-
index in Hip, Str and Ctx (Table 2).
Conclusion: Our innovative non-invasive and semi-automatic MRI
approach revealed both the microcephaly and localized alterations induced
by a 1 Gy in utero irradiation in mice. Further experiments will determine
its sensitivity to detect brain alterations after irradiation at lower doses and
other developmental stages.
European Radiation Research 2018, August 21-25, Pécs, Hungary
133
Non-conventional approaches of drug targeting: some findings related to non-radioactive and
radioactive labeled therapeutics
Biswajit Mukherjee
Dept. of Pharmaceutical Technology, Jadavpur University, Kolkata-700032, India
Email: biswajit.mukherjee@jadavpuruniversity.in; biswajit55@yahoo.com
Researchers around the globe are trying to exploit active drug targeting strategies through the
nanocarrier mediated delivery systems in order to come up with potential therapeutic weapons to
combat with certain deadly diseases such as cancer, fungal infections in lungs for which no adequate
treatment strategies are available resulting higher incidence of morality day by day. Moreover, for
drug targeting aspects focus is concentrated on the increased binding of nanocarrier on the cancer
cell surface by targeting the receptors on the cancer cell surface by designing appropriate ligands.
Most of the cases findings from these studies revealed that ligands such as antibodies, are toxic or
they are non-specific. Therefore, in our laboratory we have explored certain non-conventional
targeting strategies to come up with smarter therapeutics. We have developed a phospholipid based
1,2-distearoyl–sn-glycero3-phosphatidylethanolamine (DSPE) incorporated nanoliposomal
formulations of docetaxel in order to exploit the abundance (45%) of DSPE in brain and other
tissues. Treatment of lung fungal infection is another formidable challenge faced by researchers
especially the infection of lower lobes. In our laboratory we have designed apparatus to successful
administration of both dry powder and nebulization to conscious animals without hampering normal
breathing pattern. The design of apparatus is unique as similar therapeutic dose can be applied to
multiple animals in noninvasive manner. We reported designing of such type of apparatus to deliver
drugs to multiple animals by avoiding exposure of whole body of animals. Through this device we
have successfully delivered voriconazole to rodents in noninvasive manner as evidenced by
tecnhnetium 99m
radiolabelling and fluorescence imaging of lung tissue after administration labelled
voriconazole encapsulated Nanoparticulate formulations. Similarly gene silencing technology by
antisense oligonucleotide is another process which seems to be promising in the area of direct drug
targeting instead of using ligand-based nanotherapeutics. We have successfully explored this mode
of drug targeting by designing antisense oligonucleotides against c-raf and IGF-II and some
promising results were found in chemical induced hepatocarcinogenesis in rats as evidenced from
histopathological examination, in-situ hybridization and localization fluroscent dye tagged antisense
oligonucleotides. Further the presentation will also highlight the success the various non-
conventional targeting approaches by focussing on promising data obtained from the studies
involving radioactive and non-radioactive labelled therapeutics both in vitro and in vivo.
European Radiation Research 2018, August 21-25, Pécs, Hungary
134
FTS regulates ROS-induced Cu-Zn SOD expression in cervical cancer
cells
Sridhar Muthusami1,2
, Prabakaran DS1, Sivaraman T
3, Jae Ran Yu
4, Woo-Yoon Park
1
1 Department of Radiation Oncology, Chungbuk National University College of Medicine, Cheongju, 28644,
Republic of Korea 2 Department of Biochemistry, Karpagam Academy of Higher Education, Coimbatore,641 021 Tamilnadu,
India. 3 Department of Biotechnology, Karpagam Academy of Higher Education, Coimbatore,641 021 Tamilnadu,
India 4 Department of Environmental and Tropical Medicine, Konkuk University College of Medicine, Chungju,
27478, Republic of Korea
Objective: Ionizing radiation (IR) is commonly advocated for the treatment of cervical cancer. The
therapeutic efficacy of IR is mainly attributed to reactive oxygen species (ROS)-induced cellular damage.
However, the efficacy of IR via ROS is largely reduced by the endogenous antioxidant systems. Superoxide
dismutase (SOD) is an important antioxidant enzyme which detoxifies superoxide into hydrogen peroxide
(H2O2), which is further converted to water and molecular oxygen by catalase and glutathione peroxidase. The
present study is aimed to delineate the effects of ROS and radiation-induced alterations in Cu-Zn SOD
(SOD1) and Fused toes homolog (FTS).
Methods: A human cervical carcinoma cell line (ME180) was used. FTS was silenced using siRNA based
approach. Western blotting was done to see protein expression. Immunoprecipitation and molecular
modelling, docking and structural analyses were done to see protein-protein interaction. ROS was measured
using H2DCF-DA.
Results and Conclusion: H2O2 treatment elicited no obvious change in the viability of cervical cancer
ME180 cells at lower concentrations whereas at higher concentrations H2O2 induced cell death. A significant
time dependent increase in the expression of SOD1 induction after H2O2 treatment at low concentration
demonstrates the scavenging of H2O2. A significant increase in FTS is also noted after H2O2 treatment at
lower concentrations. Immunoprecipitation analysis identified the physical interaction between SOD1 and
FTS after H2O2 treatment. Putative residues responsible for the interaction were identified using
computational analyses. Silencing of FTS significantly increased the ROS levels in cervical cancer cells
exposed to low levels of H2O2 resulting in increased cell death. Silencing of FTS also prevented radiation-
induced induction of SOD1 in these cells. These results demonstrate a novel role for FTS in the antioxidant
defense by SOD1 in ROS-induced cellular death and defense.
European Radiation Research 2018, August 21-25, Pécs, Hungary
135
Personal RF microenvironmental exposimetry in kindergardens and
nurseries
Péter Pál Necz1, Noémi Nagy
1 and György Thuróczy
1
1National Public Health Institute, Department of Non-Ionizing Radiations, Budapest, Hungary
Objective
The aim of this study was to measure the indoor microenvironmental level of RF exposure and
individual personal exposure in kindergardens and in nurseries. Our main hypothesis was that the RF
components of indoor exposure emitted by household wireless devices (e.g. DECT, WiFi, Bluetooth,
etc.) are already higher than the exposure from downlink frequency bands of mobile base stations.
We also supposed that the indoor exposure levels are much lower when the indoor sources are in
different room.
Methods
In this study we used an MVG EME Spy 121 exposimeter to measure and record the ambient RF
electric field in 12 predefined channels between 88 MHz and 2500 MHz: FM (88-108), TV3 (174-
223), TETRA (380-400), TV4&5 (470-830), GSM Tx (880-915), GSM Rx (925-960), DCS Tx
(1710-1785), DCS Rx (1805-1880), DECT (1880-1900), UMTS Tx (1920-1980), UMTS Rx (2110-
2170), WiFi (2400-2500). The exposimeter has an isotrope probe. The lower and upper detection
limits are 0,05 V/m and 10 V/m, respectively. The sample rate can be between 4s – 255s. The device
was lent to 15 different children institution for 5 days in each place. The exposimeter was left for 24
hours in the office and for 24 hours in each children’s room. The sampling rate was 60s.The
measurements were taken in Budapest, Hungary between January 2017 - May 2017.
Results
The detected data (less than 0.25 V/m on average) was plenty under the residential limits (41-60
V/m). Because the Wi-Fi routers and DECT telephones are in the offices, the exposure coming from
indoor sources in these rooms are significantly higher than the outdoor sources’. In the children’s
room the exposure from indoor RF sources is much lower. The ratio between downlink and non
downlink is significantly depends on the spot inside the building. That’s because the indoor sources
works with much less power than the base stations thus its field decrease much sooner.
Conclusion
The exposure from indoor sources (Wi-Fi, DECT, GSM uplink) is much lower in children’s room
in case of these sources are not inside the room. The exposure level coming from mobile base
stations is similar to the level of indoor sources when these sources are in another room. It is
recommended to use these devices only in places where children are not present (e.g. in offices) in
kindergardens and nurseries.
References
Péter Juhász, József Bakos, Noémi Nagy, Gábor Jánossy and György Thuróczy, RF personal exposimetry on employees
of elementary schools, kindergartens and day nurseries as a proxy for child exposures, Progress in Biopysics and
Molecular Biology 2011/1-7
György Thuróczy, Ferenc Molnár, , Gábor Jánossy, Noémi Nagy, József Bakos and Judit Szabó, Personal RF
exposimetry in urban area, Ann. Telecommunication 2008 63:89-96
European Radiation Research 2018, August 21-25, Pécs, Hungary
136
Effects of ultraviolet radiation on full thickness
human skin model in vitro (SKIN-RF project)
Zsuzsanna Németh1, Györgyi Kubinyi
1, József Bakos
1, Brahim Selmaoui
2 and György
Thuróczy1
1 National Public Health Institute, Department of Non-Ionizing Radiation, Budapest, Hungary
2 Unité de Toxicologie Expérimentale, L'Institut National de l'Environnement Industriel et des
Risques (INERIS), Verneuil en Halate, Franc
Ultraviolet (UV) radiation is responsible for a wide variety of different acute and chronic effects on the skin.
Acute responses of human skin to UV radiation include photodamage, erythema, mutation,
immunosuppression, synthesis of vitamin D and tanning. Chronic UV radiation effects include photoaging and
photocarcinogenesis.
Interleukin IL-1α is a primary cytokine in keratinocytes capable of initiating cutaneous inflammation via
secondary cytokines like IL-6 and IL-8. The UV-induced matrix metalloproteinase-1 (MMP-1) is a crucial
biomarker of photoaging. IL-1α and IL-6 from UVB-irradiated keratinocytes can regulate MMP-1 expression
in fibroblasts through paracrine effects.
The aim of this experiment was to evaluate the effects of exposure to UV radiation on reconstructed human
skin models regarding inflammation and photoaging. We used the ELISA method to determine the
concentration of the cytokines (IL-1α, IL-6 and IL-8) and the MMP-1 enzyme from the cell culture
supernatant.
This experiment was done as a preliminary study for the project named “Cellular response to co-exposure of
radiofrequency (RF) and solar ultraviolet (UV) radiation in human in vitro skin model (SKIN-RF)” funded by
ANSES.
In this experiment MatTek EpiDerm Full Thickness (EFT-300) reconstructed skin models were used. At least
two tissues were prepared for each test condition: sham exposed as negative control, 1 % SDS and 5 % SDS
(sodium dodecyl sulphate) as positive controls and 6 doses (1.3 SED, 1.5 SED, 3.0 SED, 3.9 SED, 7.8 SED,
15.6 SED) of UV exposure by a solar simulator lamp (SOL 500, Hönle, Germany). Standard Erythema Dose
(SED) is equivalent to an effective erythemal exposure of 100 J/m2. The UV doses were checked by the
International Light ILT-900 NIST calibrated spectroradiometer.
Viability of the full thickness skin tissues were evaluated 24 hours after UV exposure with the MTT
test. After the UV radiation tissues were incubated at 37 °C and 5 % CO2 for 24 hours and the cell culture
medium was collected and frozen until analysis. The concentration of IL-1α, IL-6, IL-8 and MMP-1 was
measured with ELISA method.
We found that the 7.8 SED and 15.6 SED UV exposure and 1 % and 5 % SDS decreased the viability of the
skin model tissues below 50 %. The concentration of the IL-1α significantly increased after UV exposure to
7.8 SED and 15.6 SED. The concentration of the IL-6, IL-8 and MMP-1significantly increased after UV
exposure to 3.9 SED and 7.8 SED, but after 15.6 SED the concentration of these endpoints significantly
decreased compared to negative control.
In this experiment we studied the influence of the UV exposure on Mattek’s EFT-300 full thickness skin
model. The concentration of the cytokines and the MMP-1 enzyme from the cell culture medium were
measured with the ELISA method 24 hours after UV exposure. In this preliminary study we managed to show
that the UV doses of 1.3 SED, 1.5 SED and 3 SED did not have significant effects compared to the negative
control, but the doses of 3.9 SED and above significantly affected the skin tissues.
European Radiation Research 2018, August 21-25, Pécs, Hungary
137
Effect of internal contamination with tritiated water on the
neoplastic colonies in the lungs, innate anti-tumour reactions, cytokine
profile, and haematopoietic system in radioresistant and radiosensitive
mice
Ewa M. Nowosielska1, Aneta Cheda
1, Robert Zdanowski
2, Sławomir Lewicki
2, Marek K. Janiak
1
1Military Institute of Hygiene and Epidemiology, Department of Radiobiology and Radiation
Protection, Warsaw, Poland 2 Military Institute of Hygiene and Epidemiology, Department of Regenerative Medicine and Cell
Biology, Warsaw, Poland Objective: Tritium is a potentially significant source of internal radiation exposure which, at high levels, can be carcinogenic. We evaluated whether single intraperitoneal injection of BALB/c and C57BL/6 mice with tritiated water (HTO) leading to exposure to low (0.01 or 0.1 Gy) and intermediate (1.0 Gy) cumulative whole-body doses of β radiation is immunosuppressive, as judged by enhancement of artificial tumour metastases, functioning of NK lymphocytes and macrophages, circulating cytokine’s levels, and numbers of bone marrow, spleen, and peripheral blood cells. Methods: The study was conducted on radio-sensitive BALB/c and radio-resistant C57BL/6 mice. The animals were ip. injected with HTO so that the total absorbed doses of β-radiation were 0.01 Gy, 0.1, or 1.0 Gy per mouse. From day 7 post-injection of HTO the following paramters were estimated: cytotoxic activity of NK lymphocytes, production of nitric oxide (NO) by macrophages (a marker of the cytotoxic function of these cells against susceptible tumour), production of selected cytokines, and selected haematological parameters. Results: We demonstrated that internal contamination of radiosensitive BALB/c and radioresistant C57BL/6 mice with HTO at all the absorbed doses tested did not affect the development of neoplastic colonies in the lungs caused by intravenous injection of syngeneic cancer cells. However, internal exposure of BALB/c and C57BL/6 mice to 0.1 and 0.01 Gy of β radiation, respectively, up-regulated cytotoxic activity of and IFN-γ synthesis in NK lymphocytes and boosted macrophage secretion of nitric oxide. Internal contamination with HTO did not affect the serum levels of pro- (IL-1β, IL-2, IL-6, TNF-α,) and anti-inflammatory (IL-1Ra, IL-4, IL-10) cytokines. In addition, exposure of mice of both strains to low and intermediate doses from the tritium-emitted β-particles did not result in any significant changes in the numbers of bone marrow, spleen, and peripheral blood cells. Conclusion: Overall, our data indicate that internal tritium contamination of both radiosensitive and radioresistant mice leading to low and intermediate absorbed β-radiation doses is not immunosuppressive but may enhance some but not all components of anticancer immunity. The study was funded by the grant No. DEC-2011/01/D/NZ7/05389 of the Polish National Science Centre.
European Radiation Research 2018, August 21-25, Pécs, Hungary
138
Tracking Sfpi1/PU.1 and Kras mutations in murine acute myeloid
leukaemia: role in radiation leukaemogenesis
Grainne O’Brien
1, Lourdes Cruz Garcia
1, Natalie Brown
1, Rosemary Finnon
1, Joanna Zyla
2, Joanna
Polanska2, Christophe Badie
1
1Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Oxfordshire,
U.K. 2Data Mining Group, Institute of Automatic Control, Silesian University of Technology, Gliwice,
Poland
Objective
Therapy-related acute myeloid leukaemia (t-AML) is a well-recognised potential complication of
cytotoxic therapy for the treatment of a primary cancer. Radiation induces leukaemia in mice as well
as in humans and the CBA mouse model is used in radiation leukemogenesis studies where
Sfpi1/PU.1 interstitial chromosome 2 deletion and point mutation have been identified as
mechanisms of leukaemogenesis. It represents an ideal model system to improve understanding of
the biological mechanisms underlying radiation-induced leukaemia. Kras is one of the most frequent
AML mutations in human but its role in radiation-induced AML (rAML) is unclear. Our objective
was to analyse the genetic and epigenetic changes occurring in rAML cases.
Methods
In this study we screened 134 mouse rAML spleen samples for Sfpi1 R235 and Kras G12 mutations.
The samples were analysed by Sanger sequencing and mutations confirmed by pyrosequencing. We
also examined the transcriptional expression of Sfpi1 in cases with and without a R235 mutation.
Results
We identified 3 cases with either a Kras G12D together with a Sfpi1 R235C mutation (2) or Kras
G12R mutation with no Sfpi1 R235 mutation (1). Both PolyPhen-2 and PredictSNP algorithms
predict a deleterious effect of 87% for Kras G12 mutations, a previously identified hot spot.
Additionally a significant decrease of Sfpi1 gene expression was found specifically in rAML samples
without a R235 mutation.
Conclusion
In this study we identified Kras mutations for the first time in mouse rAML cases. This allowed us to
propose a model of clonal evolution in rAML, where the sequence of mutational events in rAML
was reconstructed, confirming the co-operative effect role of Kras mutation. The lower level of Sfpi1
expression was clear in cases without R235 mutations, suggesting repression through an alternate
process could therefore be the driver of AML development in these cases. Promoter methylation in
these leukaemia cases are currently under investigation.
European Radiation Research 2018, August 21-25, Pécs, Hungary
139
Beta and Gamma ray Dose Estimates of Alpha Emitters for
Brachytherapy Uses
Kaylyn Olshanoski and Chary Rangacharyulu
Department of Physics and Engineering Physics, University of Saskatchewan, Saskatoon,
SK Canada S7N 5E2
Brachytherapy as a therapeutic tool has found a renaissance of sorts in recent decades [1] after
being forgotten during the 1970s and 1990s. The attractive feature is that the short-range alpha
particles from a radioactive nucleus deposit large amounts of energy in the surrounding cancerous
tissue, without harming the healthy tissues which are out of reach. This will be an ideal situation if
alphas are the only radiations from the source. At the very least, the decays are accompanied by
atomic x-rays of the daughter nuclides. In addition, the decay chain of candidate isotopes consists of
beta emissions and gamma ray emissions. One cannot make a sweeping assumption that the doses
delivered by secondary beta and gamma radiations to surrounding tissues can be ignored.
Below, we list the primary alpha-emitting brachytherapy isotopes of interest, along with the final
decay product and indicate the number of alpha and beta particles of each chain. In addition, several
of these decays involve gamma ray emissions of various energies and intensities. Brachytherapy
isotopes of interest are:
; ; ;
; ; ;
;
In this work, we follow the decay sequence of candidate nuclides to estimate the dose
contributions of beta and gamma radiations from the nuclear emissions to biological tissues. To this
end, we make use of MIRD values [2] and the energy and medium dependent attenuation coefficients
of biological tissues from NIST [3]. The decay chain of each brachytherapy isotope will be
considered until the radioactive half-life of the daughter isotope is on the order of 10 years.
Combined with the biological half-life, the activity isotopes of longer half-lives are negligible. We
present the quantitative dose estimates of the electron, gamma and x-ray emissions of these nuclides.
REFERENCES:
[1] See for example, Orio PF, et al. 2017 American Brachytherapy Society’s Annual Meeting Report.
Translational Andrology and Urology. 2017;6(5):1005-1013. doi:10.21037/tau.2017.09.05.
[2] National Nuclear Data Center (NNDC). “Nuclear Decay Data in the MIRD Format.”
https://www.nndc.bnl.gov/mird/.
[3] National Institute of Standards and Technology (NIST). “X-Ray Mass Attenuation Coefficients.”
https://www.nist.gov/pml/x-ray-mass-attenuation-coefficients.
European Radiation Research 2018, August 21-25, Pécs, Hungary
140
Ecological effects of ionizing radiation on earthworms
Deborah Oughtonab
, Emmanuel Lapeidab
, Andrej Rusind, Ilya Velegzhaninovac
, Cristian
Fernandezad
, Carmel Mothersillad
, Hallvard Haanesae
.
aCentre for Environmental Radioactivity (CERAD CoE), Norway;
bNorwegian University of Life
Sciences, Department of Environmental Sciences, Campus Ås, Norway; cThe Institute of Biology,
Komi Scientific Center, Russia; dMcMaster University, Canada;
eNorwegian Radiation Protection
Authority, Norway
Earthworms are key organisms for soil functioning and fertility. They participate in the decomposition of soil
organic matter, are important as engineers of the soil in transporting nutrients and creating drainage and
structure. As soil dwellers, earthworms can be exposed to high levels of ionizing radiation, either after nuclear
accidents or in areas naturally enriched with radionuclides. However, while earthworms are central to
ecotoxicology research, relatively little is known about the impacts of ionizing radiation. This largely reflects
the fact that, until about 15 years ago, studies of the biological impacts of radiation were almost entirely
focused on humans.
This paper will report a series of laboratory and field studies carried out at NMBU on the ecological impacts
of ionizing radiation on earthworms. Controlled experiments at the NMBU Co-60 irradiation facility
demonstrated that chronic radiation exposure has a negative effect on reproduction, and at much lower doses
(4-10 mGy/h) than those causing mortality (>60 Gy). Two week exposures at 10 mGy/h led to increases in
apoptosis, DNA damage and in bystander effects after 24 hour exposures, with significantly different
responses seen between tissues and worm species. Field studies at a site in Komi exposed for more than 50 yr
to waste from radium mining, showed reduction in earthworm diversity between contaminated and controlled
sites, as well as differences in DNA repair in earthworms subject to a challenging radiation dose. Differences
in bystander response to a challenging dose were also seen between control worms and worms collected from
a naturally contaminated site at Fen, Norway. The talk will conclude with some preliminary results from
studies at Chernobyl and Fukushima, and discuss implications of the results to understanding radiosensitivity
and possible adaptation in earthworm species.
European Radiation Research 2018, August 21-25, Pécs, Hungary
141
Mechanistic insights into radiation induced complex chromosomal
rearrangements, genomic instability and chromothripsis
Antonio Pantelias, Georgia Terzoudi, Ioanna Karachristou, Gabriel Pantelias
Laboratory of Health Physics, Radiobiology & Cytogenetics
Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety
National Centre for Scientific Research "Demokritos", Athens, Greece
Radiation-induced chromosomal aberrations, genomic instability and formation of complex chromosomal
rearrangements, which are the hallmark of cancer and radiation-induced cell killing, are thought to reflect
stepwise accumulation of misrepaired DNA damage over time. However, recent studies have shown that such
rearrangements can arise from a one-off catastrophic event causing complex chromosomal rearrangements.
This phenomenon has been termed chromothripsis, on the basis of its distinctive feature, which points to an
underlying process involving chromosome (chromo) shattering (thripsis), and, typically, only one or a few
chromosomes being affected per event. While the characterization of the affected chromosomes has provided
new insights into the processes by which cancer genomes can evolve, the underlying signaling events and
molecular mechanisms remain unknown. A number of hypotheses have been proposed, involving ionizing
radiation, chromothriptic rearrangements by aberrant DNA replication and chromosome shattering or
rearrangements caused by telomere crisis, a process that involves formation of dicentric chromosomes and
chromatin bridges. Cumulating evidence, however, suggests that chromothripsis may result from lagging
chromosomes encapsulated in micronuclei. In the present work, we provide experimental evidence on the
mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single
catastrophic event. Specifically, we have developed a model for the experimental study of chromothripsis by
generating micronuclei using ionizing radiation and modulating their cell cycle kinetics in relation to the
kinetics of the main nuclei, in an attempt to follow the fate of chromosomes in micronuclei under the different
experimental conditions. Following an aberrant mitosis, radiation induced chromosome fragments can find
themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and
rearrangement in a single catastrophic event. The results obtained support the hypothesis that when the main
nuclei enter mitosis, they induce premature chromosome condensation that triggers shattering and
chromothripsis in chromosomes or chromosome fragments still undergoing DNA replication in micronuclei or
asynchronous multinucleated cells.
European Radiation Research 2018, August 21-25, Pécs, Hungary
142
Laser-based radiation sources for biomedical applications
Daniel Papp, Christos Kamperidis
ELI-ALPS, ELI-HU Non-profit Ltd., Szeged, Hungary
The emergence of ultrafast high-power laser systems opened the research for new types of laser-driven
radiation sources, the application of which has already been demonstrated if fields such as ultra-high
resolution imaging and high density matter radiography. This talk will present the basic principles of the most
common laser-based sources of particle beams and hard x-rays, and introduce secondary radiation sources
under development at ELI-ALPS.
Ion acceleration by lasers is generally through the interaction of a strongly focused laser beam and solid-
density foils of ~um thickness, where ions from the foil or protons from surface contaminants are accelerated
to an energy up to 10s of MeV(/U). There are several acceleration mechanisms present, the most prominent of
them being Target Normal Sheath Acceleration (TNSA).
Laser-driven electron acceleration is primarily performed via the laser wakefield acceleration (LWFA)
mechanism, proposed as early as in 1979, where a loosely focused laser beam creates a nonlinear plasma wake
in gas targets, where the very strong (TV/m) quasi-electrostatic fields can accelerate electrons to GeVs of
energy. Most of the research efforts focus on GeV electron beams for proposed novel multi-stage accelerators.
Moderate electron energies in the order of 100 MeV has also been used for radiobiological irradiation. While
the GeV electron acceleration experiments are usually single-shot, continuous operation at 10 Hz repetition-
rate for moderate energies has been demonstrated, as well high, 1 kHz repetition-rate for very low (few MeV)
energies.
The LWFA process is also accompanied by the emission of a strongly collimated hard x-ray beam (“betatron”
radiation) with x-ray peak energies of 10s of keV, depending on the peak energy of the LWFA electron
beams. The small, ~um source size of these beams offers high-resolution radiography capabilities, and, more
prominently, the capability for high-resolution phase contrast tomography. The produced electron beams can
also produce even higher (MeVs) photon energies, e.g. by inverse Compton scattering, or in high-Z secondary
targets by Bremsstrahlung radiation.
The ELI-ALPS research institute in Szeged, one of the three pillars of the ELI project, will operate laser-
driven secondary particle sources, such as an ion acceleration beamline and a 1 kHz electron acceleration
beamline, which are currently under development and will be made available for the scientific community
upon commissioning.
European Radiation Research 2018, August 21-25, Pécs, Hungary
143
In vivo irradiation effects on activation of dendritic cells in mice
Eszter Persa1, Tünde Szatmári
1, Géza Sáfrány
1, Katalin Lumniczky
1
1National Public Health Institute, Budapest, Hungary
It is becoming clear that ionizing radiation positively influences certain immune parameters, which opens
the possibility for combining radio and immunotherapies in cancer treatment. Dendritic cells (DCs) have
active role in initiating and promoting cellular immune response via T cell - DC interaction and cytokine
secretion. DCs can also stimulate anti-tumor immunity by capturing and presenting tumor antigens. While it
has been shown that dendritic cells are relatively radioresistant, few and contradictory data are available
regarding the impact of ionizing radiation on the functional integrity of these cells.
The aim of our studies was to determine the effect of ionizing radiation on phenotypical and funcional
parameters of DCs relevant in stimulating anti-tumor immune response.
Mice were total-body irradiated with 0 (control), 0.1, 0.25 and 2 Gy X-rays. Spleen cells were isolated 24h
after irradiation and DC cell surface markers were analysed by flow cytometry. Expression of costimulatory
(CD40, CD80, and CD86), coinhibitory (B7-H1 (PD-L1)), and antigen capturing (DEC205) molecules was
determined. For testing antigen capture and antigen presentation, isolated DCs and FITC-labeled OVA peptide
were used. The amount of OVA captured by DCs was followed by flow cytometry, as well. DC cytokine
expression was measured by real-time qPCR and by intracellular cytokine staining upon LPS stimulation.
Interaction of DCs with effector and regulatory T cells was investigated in a mixed lymphocyte reaction.
Our results showed a slight raise in the level of costimulatory molecules CD80 after low dose irradiation
and significant increase of all examined surface protein after 2 Gy. While 2Gy did not influence antigen
uptake or presentation, low doses stimulated antigen uptake and reduced the level of antigen presentation.
Both low and high dose radiation induced increased IL-1, IL-1, IL-6, IL-10 and IL-12 gene expression, as
measured by qRT-PCR. Additionally, 2 Gy irradiated DCs secreted higher level of IL1α upon LPS treatment
than control DCs indicating a synergic effect between irradiation and LPS stimulation. While DCs from
control animals induced stronger proliferation in regulatory T cells, DCs from animals irradiated with 2 Gy
mainly stimulated effector T cell proliferation.
In conclusion, we showed that 2 Gy irradiation had a massive effect on activation status of DCs appeared
in higher expression of costimulatory molecules, enhanced production of pro-inflammatory and immune
activating cytokines and increase in their T-cell stimulating abilities. Pronounced activation status can reflect
in more effective role of DCs in anti-tumor immune response. In addition, low dose irradiation, by increasing
antigen uptake and lowering antigen presentation shifted splenic DCs towards a less mature phenotype.
European Radiation Research 2018, August 21-25, Pécs, Hungary
144
The Proton Boron Capture Therapy: a new proton therapy
enhancement technique
Giada Petringa1,2
, Lorenzo Manti3,4
, Francesco Cammarata1,5
, Giacomo Cuttone1, Daniele
Margarone6, Antonio Picciotto
7, Pietro Pisciotta
1,2, Lorenzo Giuffrida
6, Giorgio Russo
1,5 Valentina
Scuderi1,6
and G. A. Pablo Cirrone1,6
1Istituto Nazionale di Fisica Nucleare - Laboratori Nazionali del Sud (INFN-LNS), Catania, Italy 2Dipartimento di Fisica e Astronomia, Università degli Studi di Catania, Catania, Italy
3Dipartimento di Fisica “E. Pancini”, Università degli Studi Federico II di Napoli, Napoli, Italy 4Istituto Nazionale di Fisica Nucleare – Sezione di Napoli, Napoli, Italy,
5Institute of Molecular Bio-imaging
and Physiology - National Research Council - Cefalù, Italy, 6ELI-Beamline Project, Inst. Physics, ASCR,
PALS Center, Prague, Czech Republic, 7Fondazione Bruno Kessler (FBK), Trento, Italy
Objective: In the last 40 years, clinical protontherapy has been growing rapidly counting nowadays more than
80 facilities worldwide [1]. The interest in protontherapy stems from the physical properties of protons
allowing for a much-improved dose painting around the target and greater sparing of healthy tissue as
compared to the conventional radiotherapy with photons. Recently, research efforts in the field of photon-
based radiotherapy have reduced the dosimetric gap between photons and protons in terms of tumour
conformation. However, one of the shortcomings of protontherapy resides in the fact that it is not suitable to
treat radioresistant cancers. From a radiobiological point of view protons are, in fact, almost as effective as
photons. This is because the biological outcome of cellular irradiation strongly depends on the physical
pattern of energy deposition and specifically of the Linear Energy Transfer (LET) at the nanoscale level (e.g.
DNA). Protons, ultimately, lack distinct radiobiological advantages over photons or electrons. Higher LET
12C-ions can overcome cancer radioresistance: DNA lesion complexity increases with LET, resulting in
efficient cell killing. However, economic and radiobiological issues hamper 12C-ion clinical amenability.
Enhancing proton Relative Biological Effectiveness (RBE) is hence desirable. To this end, we exploited the
p + 11B → 3α reaction to generate high-LET alpha particles with a clinical proton beam [2]. The rationale of
this novel approach, named PBCT (Proton-Boron Capture Therapy), is two-fold: the alpha-particle emitted
shows high LET and short range, allowing a highly local damaging action; the maximum cross section of the
p-B reaction occurs at low proton energies (i.e. less than 1 MeV), corresponding to the tumour region in a
typical proton therapy scenario.
Methods: In order to demonstrate the potentiality of this technique, we performed four experimental
campaigns at CATANA (Centro di AdroTerapia ed Applicazioni Nucleari Avanzate) of INFN. We irradiate
the DU145 prostate cancer cells at the three positions along the SOBP (Spread Out Bragg Peak). Treated cells
with BSH were irradiated and assayed for clonogenic survival and DNA damage induction. A set of Monte
Carlo calculations coupling the Geant4 toolkit with a semi-empirical method are also performed in order to
quantify the effect due to the production of high-LET alpha particle at nano-metric scale.
Results: We experimentally recorded a significant increased cellular lethality and occurrence of chromosome
aberrations. Specifically, we proved that, if human cells are irradiated with a given amount of 11B the
interaction with protons results in an increase of almost a factor 2 in cell killing compared to boron-free
irradiated controls.
Conclusion: In this work, we will show the potentialities of PBCT discussing a potential modelling approach
that we are developing in the attempt to study and explain the observed effects.
References
[1] Particle Therapy Co-operative Group (PTCOG), https://www.ptcog.ch
[2] H.W. Becker et al, “Low-Energy Cross Sections for 11B(p, 3a)*”, Z. Phys. A - Atomic Nuclei
327, 341-355 (1987)
[3] G.A.P. Cirrone et al, “First experimental proof of Proton Boron Capture Therapy (PBCT) to
enhance protontherapy effectiveness”, Scientific Reports, 1141-8 (2018)
European Radiation Research 2018, August 21-25, Pécs, Hungary
145
Cell and molecular response to proton radiation treatments in breast
cancer: in vitro models and in vivo applications
Pietro Pisciotta1,2,3
, Francesco P Cammarata2,3
, Luigi Minafra2,3
, Valentina Bravatà3, Giusi I Forte
2,3,
Valentina Marchese1, Rosaria Acquaviva
4, Roberta Tringali
4, Pablo Cirrone
2, Giada Petringa
2, Giacomo
Cuttone2, Lorenzo Manti
5, Giorgio Russo
2,3
1 Department of Physics and Astronomy, University of Catania, Italy. 2 National Institute of Nuclear Physics (INFN), LNS, Catania, Italy
3 Institute of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR)
Cefalù (PA), Italy; 4 Department of Drug Science - Section of Biochemistry, University of Catania, Italy.
5 Physics Department, University of Naples Federico II, Naples, Italy
Recently, the construction of new particle therapy centres is started worldwide and some other are planned.
The strength of the use of particle to treat cancer lies in their ballistic precision that permit to deposit the vast
majority of dose on the target sparing health tissue and organ at risk as well as possible.
The use of protons in breast cancer (BC) treatment is mainly due to reduce recurrences and toxicity but
nowadays is limited to few preliminary studies and the clinical activity is very restricted. As a consequence,
the molecular response to this kind of treatment is almost unknown. This work funded by an INFN project
called ETHICS foresees to study charged particle-induced damage to healthy tissues and impact on tumour
microenvironment, evaluating the biological effects of different doses of protons (0.5, 2, 4, 6, 9 Gy) on BC
cells in terms of cell survival by clonogenic assay, gene expression profiling using cDNA microarray and
inflammatory response by Luminex assay with the final aim to discover new potential biomarkers of
radiosensitivity/radioresistance. Data on cell viability indicate different percentages of surviving fractions
according to the cell type (tumorigenic MCF7, metastatic MDA-MB-231 and nontumorigenic MCF10A) and
dose delivered.
The scope was also the study of the preliminary steps to perform particle treatment of cancer cells inoculated
in small animals. At this scope, a well-defined dosimetric protocol was developed to perform the steps needed
in order to perform a precise proton irradiation in small animals and achieve highly conformal dose.
Homemade positioning system for small animals was developed at INFN-LNS (Italy) and an accurate Monte
Carlo simulation was developed. The application, developed using Geant4.10.03 version, simulates the
CATANA proton beam line geometry and includes the capability to implement DICOM-TC images as target.
The application will be used to carry out dosimetric and LET studies using the real target composition. This
application was validated comparing its results with experimental measurements.
Finally, “OMICS” approach represents the best way to analyze biological effects induced by IR, direct or
indirect damage to principal biological molecules, allowing also to find new prognostic and predictive
biomarkers of the cell sensibility to IR. Experimental data from proteomics, genomics and transcriptomics,
termed all together “Proteogenomics”, are emerging as a fundamental step to analyze globally and
simultaneously DNA, RNA, protein expression, and epigenetic modifications in order to understand molecular
mechanisms underlying cellular processes and biological events induced by several type of stress stimuli such
as IR. The proteogenomic ways: an integrated “OMICS” approach allows to detect the molecular steps
driving cellular functions and to identify new biomarkers. It is now well recognized by the entire scientific
community that to evaluate the biological effects of IR is essential an OMIC approach to take into account
both the complexity of the different cell types involved and several types of particles and doses delivered.
European Radiation Research 2018, August 21-25, Pécs, Hungary
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Electron beams accelerated by the proposed SYLOS laser system at
ELI-ALPS – A Monte Carlo dosimetry characterization
Róbert Polanek1, Dániel Papp
1, Christos Kamperidis
1 and Katalin Hideghéty
1
1 ELI-ALPS, Szeged, Hungary
Objective In the near future, the SYLOS laser system, a 1 kHz repetition-rate “single-cycle” laser will be available at
ELI-ALPS. One use of this system will be electron acceleration via the LWFA (Laser Wakefield
Acceleration) technique, producing electrons with projected 30-60 MeV mean electron energies and above
and bunch charges of a few 10s of pC at 1 kHz repetition rate. In this work we present a Monte Carlo
calculation to demonstrate the feasibility of a SYLOS laser wakefield accelerated electron beam source for
radio-biological applications. The study focuses on the dosimetric characteristics and makes predictions
regarding the achievable dose rate, depth dose properties, and dose distribution.
Methods The feasibility of the SYLOS laser driven electron beam was assessed from dosimetry point of view using
Monte Carlo simulations that considered the proposed beam properties. The Geant4 Monte Carlo development
framework was used to build the simulation application with a semi-realistic geometry. The electron beam
characteristics were obtained from previously performed 2-D particle-in-cell (PIC) simulations using the
EPOCH code.
Results The SYLOS laser system will produce electron beams with high energies and acceptable dose rates (up to
55Gy/min) with very high precision delivery, due to the high repetition rate of the system. The electron beams
can be considered as pencil-beams with a source size of few μm and beam diameter of few mm at the phantom
surface. Simulated dose value with radiochromic films placed in the water phantom at depth of 2.8 cm are
between 3.7 – 55MeV/min which varying in function of beam energy, energy spread and beam divergence.
Electron energies up to 60 MeV are realistic with the currently proposed SYLOS laser and the higher electron
energies were considered for the future upgrades (100 mJ pulse energy with sub 5 fs pulse duration). With the
expected pulse charge the dose rate, at with the same electron energy, is similar what was reached with the
JETI laser system at Helmholtz-Institute Jena and is in range of 3-12 Gy/min. These study showed also, that in
general, the shot-to-shot variation of the beams parameters is properly compensated with low dose per shot
(few µGy) and the high repetition rate of the system.
Conclusion Electron beams produced and accelerated in plasmas by the SYLOS laser system at ELI-ALPS may provide a
promising initial alternative to conventional accelerators, especially for higher electron energies which
combined with intensity and energy modulation may open new opportunities in clinical radiotherapy. This
laser system can produce electron beam with high energies and acceptable dose rates and dose delivered with
very high precision, due to the high repetition rate of the system.
Acknowledgements: This work was supported in part by the European Union through the ELI-ALPS Project
under Grant GOP-1.1.1-12/B-2012-0001 and in part by the European Union’s Horizon 2020 research and
innovation programme under grant agreement no 654148 Laserlab- Europe.
European Radiation Research 2018, August 21-25, Pécs, Hungary
147
Low dose radiotherapy enhances iron oxide nanoparticles internalizing
and toxicity for MG-63 osteosarcoma cells
Roxana Cristina Popescu1,2
, Ecaterina Andronescu2, Mihai Straticiuc
3, Cosmin Mustaciosu
1,
Mihaela Temelie1, Laurentiu Mogoanta
4, George Dan Mogosanu
5, Bogdan Vasile
2, Adina Boldeiu
4,
Alexandru Mihai Grumezescu2, Mihai Radu
1, Marlon R. Veldwijk
5, Diana Savu
1.
1“Horia Hulubei” National Insitute of Physics and Nuclear Engineering, Department of Life and
Environmental Physics, Magurele, Romania; 2 “Politehnica” University of Bucharest, Department of Science and Engineering of Oxide Materials
and Nanomaterials, Bucharest, Romania; 3“Horia Hulubei” National Insitute of Physics and Nuclear Engineering, Department of Applied
Nuclear Physics, Magurele, Romania; 4University of Medicine and Pharmacy of Craiova, Research Center for Microscopic Morphology
and Immunology, Craiova, Romania; 5 University of Medicine and Pharmacy of Craiova, Department of Pharmacognosy & Phytotherapy,
Faculty of Pharmacy, Craiova, Romania; 6National Institute for Research and Development in Microtechnologies, Laboratory of
Nanobiotechnology, Bucharest, Romania; 7Heidelberg University, Medical Faculty Mannheim, Universitätsmedizin Mannheim, Department of
Radiation Oncology, Mannheim, Germany;
Objective: In this study, we have obtained iron oxide nanoparticles (IONPs) directly conjugated
with the chemotherapeutic doxorubicin (DOX) for enhanced internalizing and targeted delivery into
human osteosarcoma cells after low dose radiation therapy.
Methods: For this, we have designed and synthesized DOX-IONPs, using the co-precipitation
method. The NPs were characterized by means of morphology, composition and crystallinity.
Human osteosarcoma MG-63 was used as tumor cell model to study the cytotoxic effects and
mechanisms of DOX-IONPs following radiation. Short-term cytotoxicty was measured using MTT
and the comet assay was used to determine the genotoxic effects. The efficiency of the DOX-IONPs
cellular uptake was evaluated through fluorescence microscopy and quantitative measurements were
done using PIXE. In vitro tests were done in comparison for both unexposed and cells exposed to 1
Gy X-Rays (40 keV). In vivo biologic evaluation was done using Balb/c mice by intraperitoneal
injection of 1 mg/mL DOX-IONPs suspension.
Results: Highly stable (ZetaP = -36.79 mV) and crystalline IONPs conjugated with DOX had a
diameter of 108 nm. DOX-IONPs were internalized by MG-63 and located in the perinuclear area.
DOX-IONPs reduced viability of MG-63 cells with 25.6% at 72h (500 μg/mL). Conjugated IONPs
resulted in an increase in DNA damage up to 2.5 fold at 48h (500 μg/mL). Exposure to a single 1 Gy
dose followed by (DOX-)IONPs showed a reduction in metabolic activity with 24.2% at 48h, while
DNA damage was 1.3 folds higher. DOX-IONPs internalizing in MG-63 cells was 1.8 fold higher in
cells exposed to 1 Gy compared to 0 Gy at 48h. In vivo biodistribution showed no systemic
cytotoxicity or histopathological alteration of the main organs, but accumulation of DOX-IONPs in
spleen (at 7 and 14 days).
Conclusions: The exposure of the tumor cells to 1 Gy radiation, followed by DOX-IONPs treatment,
led to higher cytotoxic effects due to an increased internalization of the nanoparticles into the cells
and increased genotoxicity, while metabolic activity was reduced. The system showed no in vivo
systemic toxicity for the exposed animals, proving to be safe and promising for cancer therapy
applications. Further experiments considering the effect on cell cycle and DNA damage and repair
are currently undergoing.
European Radiation Research 2018, August 21-25, Pécs, Hungary
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From Radiation Tracks to Immune Signalling: a Bystander Perspective
Kevin M. Prise
Centre for Cancer Research & Cell Biology, Queen’s University Belfast, 97 Lisburn Road, Belfast, BT9 7AE
Our understanding of the fundamental mechanisms of action of ionizing radiations for over 70 years has been
built on target theory linked to a DNA centric model, where the production of DNA double strand breaks,
repair and survival are inextricably linked. The advent of microbeam technologies has allowed the testing of
these fundamental assumptions using subcellular targeting approaches even at the level of delivering single
radiation tracks. These approaches have provided unequivocal evidence for biological responses to
cytoplasmic irradiation and the production of bystander responses where cells respond, even if they have not
been directly irradiated. Much of the signalling mechanisms underpinning bystander responses are still being
elucidated but pro- and anti-inflammatory pathways are known to play a key role.
In parallel, the delivery of radiotherapy has also rapidly advanced into an extremely precise and effective
treatment in around 50% of all cancers. Much of this is a technological evolution from large field conformal
treatments to voxel-based painting of dose linked to image guidance including the use of particle therapies.
Alongside this more precise delivery of radiotherapy the potential role of abscopal (out-of-field) effects has
been increasingly acknowledged with good evidence that immune signalling may play a role. Understanding
the fundamentals of radiation-mediated immune modulation is now a key area of research and it is clear that
there may be close synergies between bystander signalling and longer range abscopal effects. Overall, this
suggests that our understanding of the fundamental role of spatial and temporal radiation responses in
biological systems needs continued, focussed research and the development of new radiobiological models.
European Radiation Research 2018, August 21-25, Pécs, Hungary
149
CONTEMPORARY RADIATION PROTECTION TRENDS IN IR
AND IC - NEW ELECTRONIC DOSIMETRY DEVELOPMENTS -
Do we need a new type of digital personal dosemeters?
Ivica Prlić1, Marija Surić Mihić
1, Mihaela Justić
1,Luka Pavelić
1, Tomislav Meštrović
1, Mladen
Hajdinjak2, Zdravko Cerovac
3
1 Unit for Radiation Dosimetry and Radiobiolgy, Institute for Medical Research and
Occupational Health, 10000 Zagreb, Republic of Croatia 2 Haj-Kom d.o.o, Zagreb, Republic of Croatia
3 ALARA ltd. Zagreb, Rapublic of Croatia
Legal personal dosimetry is based on the use of passive dosemeters such as film, thermoluminiscent (TLD) or
optically simulated (OSL) dosemeters. Modern technology, extensive use of radiation imaging in medicine
and industry imposes the use of upgraded dosimetric devices which could provide additional information on
radiation exposure for health pysics and epidemiological purposessuch as : information on exposure dose
rates, data on the exact time moment of the exposure, duration of the exposure, etc.
Majority of available active electronic personal dosemeters (AEPD), due to a type of detector used and
electronical platform used, are not suitable for measurements in pseudo-pulsed or pulsed radiation fields used
in interventional radiology (IR) and cardiology (IC). An AEPD type ALARA OD, based on GM tube with a
proper electronical platform, was used to measure the levels and structure of the occupational exposure of
workers that are predominantly exposed to scattered X-ray radiation of the continuous and pulsed radiation
fields in IR.
The 3D H*´(10) isodose patterns representing the exposure situation in a frozen point of time were
constructed giving a good base starting point for a construction of time dependent exposure pattern (animated)
of a given point in space in operating room (to an operator) during the given IR procedure.
E-mail address of main author: iprlic@imi.hr
European Radiation Research 2018, August 21-25, Pécs, Hungary
150
EARLY AND DELAYED EFFECTS OF LOW-DOSE X-RAY EXPOSURE IN
HUMAN MESENCHYMAL STEM CELLS: DNA DOUBLE-STRAND
BREAKS, PROLIFERATION, SENESCENCE
Margarita Pustovalova1,2
, Anna Grekhova1,3
, Natalia Vorobyeva1,2
, Andreyan N. Osipov1,2
1State Research Center ‐ Burnasyan Federal Medical Biophysical Center of Federal Medical
Biological Agency,Moscow 123098, Russia 2Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow 119991,
Russia 3Emanuel Institute for Biochemical Physics, Russian Academy of Sciences, Moscow 119991,
Russia
Mechanisms underlying the effects of low‐dose ionizing radiation (IR) exposure (10‐100
mGy) still remain controversial. At the same time, rapidly advancing methods of
regenerative medicine and the use of human stem cells, including mesenchymal stem cells
(MSCs), in clinical practices are becoming a routine and often require concurrent diagnostic
imaging that delivers low doses to patients and cells. High proliferation capacity of stem cells
and a potential transmission of the accumulated DNA damage and mutations to the
differentiated progeny of exposed cells require thorough understanding of DNA damage and
repair responses in human stem cells. This is particularly important for low-dose diagnostic
X-ray procedures. Repair of low-dose induced DSBs has been hypothesized to be inefficient,
potentially causing various malfunctions in the progeny of the irradiated stem cells, such as
accelerated cellular senescence or malignant transformation. In this study we show that after
exposure to intermediate doses of X‐ray (160 and 250 mGy) the number of γH2AX foci in
cultured human mesenchymal stem cells (MSCs) significantly decreased (40–60%) between
60 and 240 min post-irradiation, indicating rejoining of DNA double-strand breaks. In
contrast, γH2AX foci produced by low doses (20–80 mGy) did not change after 60 min.
Comparative analysis of early (up to 24h) and delayed (up to 11 post‐irradiation passages)
radiation effects caused by low (80 mGy) vs intermediate (1000 mGy) dose X‐ray exposure
showed that γН2АХ foci induced by an intermediate dose decreased to the control level by
24 h post‐irradiation. In contrast, low‐dose irradiation resulted in residual γН2АХ foci still
present at 24 h. Notably, these low dose induced residual γН2АХ foci were not co‐localized
with рАТМ foci and were observed predominantly in the proliferating Кi67 positive (Кi67+)
cells. The number of γН2АХ foci and the fraction of nonproliferating (Кi67‐) and senescent
(SA‐β‐gal+) cells measured at passage 11 were increased in cultures exposed to an
intermediate dose compared to unirradiated controls. These delayed effects were not seen in
the progeny of cells that were irradiated with low‐dose X‐rays, although such exposure
resulted in residual γН2АХ foci in directly irradiated cells. Taken together, our results
support the hypothesis that the low‐dose IR induced residual γH2AХ foci do not play a role
in delayed irradiation consequences, associated with cellular senescence in cultured MSCs.
European Radiation Research 2018, August 21-25, Pécs, Hungary
151
New radiobiology setup for proton irradiation adapted at the TR19
cyclotron of IFIN-HH
Mihai Radu1, Liviu Craciun
1, Mihaela Temelie
1, Mihaela Bacalum
1, Mihai Straticiuc
1, Ana
Chiriacescu1,2
, Tiberiu Esanu1, Radu Vasilache
3 and Diana Savu
1
1 Horia Hulubei National Institute for Physics and Nuclear Engineering (IFIN-HH), Department
of Life and Environmental Sciences Magurele / Applied Nuclear Physics Department, Romania 2 University of Bucharest, Faculty of Physics, Bucharest, Romania
3 SC Canberra Packard SRL, Bucharest, Romanaia
Objective.
In recent years, considerable work on proton radiobiology was done using accelerators with dedicated beam
lines and a fully equipped and instrumented platform for a wide range of studies. Here we report an irradiation
setup dedicated for proton radiobiology. The setup is designed around the external beam line of the TR-19
cyclotron which is the core of IFIN-HH Radiopharmaceuticals Research Centre. It is a negative machine that
y in the range 14-19 MeV. For proton radiobiology studies we
need currents less than picoamps to produce dose rate around 1 Gy/min. The main application of the
cyclotron, namely production of radiopharmaceuticals uses a high-power beam, while for radiobiology a very
low power beam is required. Here we present a solution, based on a very solid management, that can govern
both of power regimes allowing proton radiobiology experiments.
Methods
To manage the cyclotron in a low power regimen the arc current in the ion source was reduced still
maintaining the general stability conditions. The quadrupoles triplet of the external beam line was unlike
electrically powered to obtain a defocus of the air extracted beam. The proton beam crossing two scattering
tungsten foils (25 m thickness) passes through a vacuum tube (1 m long) to avoid interactions with air and is
extracted in air through a thin aluminium window. Picoamps instruments, plane-parallel ionisation Markus
chamber, GAFChromic films and TLD detectors were used for dosimetry. The cell cultures were done in 12
well culture plate that can be precisely positioned and irradiated using a computer-controlled positioning
samples holder and a shutter.
Results
Monte Carlo simulations have been made using the FLUKA simulation code to estimate the effects generated
by the scattering of the beam, as well as the interaction of the protons with the environment. In good stability
conditions the proton current was reduced to 0.33 pA that correspond to a dose rate less than 600 mGy/min on
the sample position. Preliminary irradiation on dosimetric film shows a good uniformity (around 93%) on a 30
mm diameter circular spot. V79 cell cultures at subconfluency were exposed in the range of 0.5 – 5 Gy and
the surviving rate, evaluated by clonogenic test, showed a typical reduction with the exposure dose.
Conclusion
The experiments performed in this first phase demonstrate the possibility of using radiopharmaceutical
isotopes production-oriented cyclotron to produce stable currents in the range of a few picoamps or less
allowing protons radiobiology experimental conditions in the low and therapeutical dose range.
European Radiation Research 2018, August 21-25, Pécs, Hungary
152
Blocking hemichannels protects against radiation-induced endothelial cell
damage
Raghda Ramadan1,2
, Els Vromans3, Dornatien Chuo Anang
4,Elke Decrock
2, Sarah Baatout
1,5,
Luc Leybaert2, An Aerts
1
1 Radiobiology Unit, Belgian Nuclear Research Centre (SCK•CEN), Mol, Belgium;
2 Department of Basic Medical Sciences, Physiology group, Ghent University, Ghent, Belgium;
3 Centre for Environmental Health Sciences, Hasselt University, Hasselt, Belgium;
4Biomedical Research Institute, Hasselt university, Hasselt, Belgium;
5 Department of Molecular Biotechnology, Ghent University, Ghent, Belgium,
Introduction: Medical applications of ionizing radiation (IR) have become widely used for diagnostic as well
as therapeutic purposes. Emerging evidence indicates an excess risk of late occurring cardiovascular diseases,
especially atherosclerosis, after IR exposure. IR induces cellular effects which may induce endothelial cell
dysfunction, an early marker for atherosclerosis. In addition, intercellular communication through channels
composed of transmembrane connexin (Cx) proteins, i.e. gap junctions (GJs; direct cell-cell coupling) and
hemichannels (paracrine release/uptake pathway) can modulate IR-induced responses and therefore the
atherosclerotic process. However, the role of intercellular endothelial communication, particularly the role of
Cx channels, in IR-induced atherosclerosis has never been described before.
Materials & methods: Telomerase immortalized human Coronary Artery/Microvascular Endothelial cells
(TICAE/TIME) were exposed to X-rays (0.1, 0.5 and 5 Gy). Several biological endpoints were investigated:
Cx gene expression, Cx protein levels, GJ and hemichannel function. In addition, production of reactive
oxygen species, senescence, cell death and inflammatory responses were assessed with or without applying a
hemichannel blocker (TAT-Gap19).
Results: Exposure to IR induced acute and persistent upregulation of the pro-atherosclerotic Cx43 and
downregulation of anti-atherosclerotic Cx37 and Cx40 gene and protein levels in a dose-dependent manner. In
addition, IR exposure increased GJ communication and induced hemichannel opening. Moreover, IR induced
a dose-dependent increase in cell death, senescence, inflammatory responses (Il-6, MCP-1 and PECAM-1)
and ROS production. These effects were significantly reduced in the presence of the Cx43 hemichannel-
targeting peptide TAT-Gap19.
Discussion & conclusions: An increase in intercellular communication after IR exposure may alter the
transfer of IR damaging signals (ROS, senescence, cell death, inflammation) between the cells, resulting in an
increase in endothelial cell damage, which could be protected by blocking the hemichannels. In addition,
similar alterations in Cx expression levels have been reported in the literature in endothelial cells covering
atherosclerotic plaques. Therefore, these results suggest that IR may contribute to atherosclerosis progression,
and blocking hemichannels might have a potential radioprotective application.
Acknowledgements: We would like to thank Dr. Ken Raj for TICAE cell line donation. Raghda Ramadan is
supported by a SCK•CEN/Ghent University doctoral grant.b
European Radiation Research 2018, August 21-25, Pécs, Hungary
153
Radiation Hormesis - A Universal Phenomenon or an Uncommon
Antidote?
Chary Rangacharyulu and Kaylyn Olshanoski
Department of Physics and Engineering Physics, University of Saskatchewan,
Saskatoon, Canada, S7N 5E2
It is well known that there are two schools of thought for the radiation effects on living organisms. The first school of thought, a conservative approach, is the Linear No Threshold model (LNT), which calls for ALARA principle. This model can be easily dismissed since the main composition of the living beings is carbon, calcium and potassium, which contain long lived activities and that ALARA, a subjective reasoning cannot be a scientific principle. Also, we are surrounded by natural radioactivity both in the ground, environment and atmosphere. The second is the threshold model, which suggests that there is a finite minimum below which the radiation is not harmful. A recent detailed statistical analysis of Dobrzyński et al [1] shows that the relative risk is constant (zero effect) below a threshold value. One may not be sure where the threshold lies but one feels confident that low doses and low dose rates will allow the repair mechanisms of the living organisms to counter the negative effects of radiation. The less well known and hotly debated issue in some circles is radiation hormesis [2], which says that low dose is not just harmless and that it may even be beneficial to prevent and cure cancers. Despite some claims to beneficial hormesis, the presently available data is, at best, consistent with but not conclusive evidence of hermetic effects. The main complexity is that these data concern with integrated dose as Grays and not the dose rates and also they are limited to effects of low energy gamma and beta radiations. As present-day radiation therapy resorts to high energy particle beams ( >100 MeV per nucleon) and photon/electron beams of up to about 20 MeV, the physical effects may include nuclear phenomena which are irreversible. In that scenario, one has to be more careful in the quantification of the physico-chemical processes and the dose distributions before one can assess the biological effects and their consequences. We will present our analyses with a few MeV photons (<20 MeV), proton beams of up to 250 MeV with a purpose to identify the physical phenomena, secondary and tertiary radiations and alert the medical physics community. It is essential that one is aware of interactions and resulting products which vary with radiation species and their energies to determine the radiation effects on living organisms and that harmful thresholds of diverse species of radiations do not correspond to one single dose value deduced from energy deposits. Thus, we reason that hormesis, if true, cannot be assessed with dose as the sole variable. REFERENCES: [1] Ludwik Dobrzyński, Krzysztof W. Fornalski and Joanna Reszczyńska (2017), Meta-analysis of thirty-two case–control and two ecological radon studies of lung cancer, Journal of Radiation Research, pp. 1–15 doi: 10.1093/jrr/rrx061
[2] E.J. Calabrese et al (2007), Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning stress within a hermetic dose–response framework, Toxicology and Applied Pharmacology 222, pp. 122–128 [3] Mohan Doss (2013), Linear No-Threshold Model vs. Radiation Hormesis, Dose Response. 11(4): 495–
512. doi: 10.2203/dose-response.13-005.Doss
European Radiation Research 2018, August 21-25, Pécs, Hungary
154
Radon exposure and its impact on the immune system and genetic risks
Felicitas Rapp1, Udo S. Gaipl
2, Benjamin Frey
2, Anna Donaubauer
2, Ina Becker
2, Lisa Deloch
2,
Franz Rödel3, Stephanie Hehlgans
3, Sylvia Ritter
1, Kateryna Shreder
1, Andreas Maier
1, Carola
Hartel, Gerhard Kraft1 and Claudia Fournier
1
1 GSI Helmholtz Center for Heavy Ion Research, Biophysics Department, Darmstadt, Germany
2 Department of Radiation Oncology, Universitätsklinikum Erlangen, Friedrich-Alexander-
Universität Erlangen-Nürnberg, Erlangen, Germany, 3Department of Radiation Therapy and
Oncology, Goethe-University, Frankfurt am Main, Germany
Objective: Radon plays a role in environmental exposure and poses a health risk for humans. On the other
hand, radon baths or galleries are used since decades to treat patients suffering from chronic inflammatory and
degenerative musculoskeletal diseases for pain relief. In the frame of a joint project of 7 partners from 4
institutions (GREWIS and GREWIS-α), we investigate beneficial, but also potentially harmful aspects of low
dose radiation exposure.
Methods: In a first study, we used blood samples from 100 patients who underwent radon spa treatment for
detailed biomedical monitoring before and after therapy (RAD-ON01, ethics#: 12131). In parallel, a study
with patients treated with LDRT (low dose X ray radiotherapy) is ongoing to investigate differences between
radon- and photon- specific effects (clinicaltrials: NCT02653079). In parallel, in vitro experiments with
human cell types involved in radiation responses (endothelial cells, osteoclasts, osteoblasts, fibroblast-like
synoviocytes, adipocytes, blood lymphocytes) and genetic analyses from peripheral blood lymphocytes (PBL)
are performed to better understand the molecular mechanisms.
Results: Analyzing genetic damage in PBL from patients in the RAD-ON01 study via the mFISH technique
showed no effect of radon exposure on the yield of chromosomal aberrations. To reach a higher sensitivity, we
established in the frame of the ongoing LDRT study a high throughput analysis of dicentric chromosomes,
allowing the detection of low dose exposures (< 25 mGy).
Further results revealed changes which could be related to the clinical effects, i.e. a temporary increase of
regulatory T cells and a long-lasting changed activation status of immune cells after therapy. Serum markers
of bone resorption, i.e. collagen fragments (CTX-I) were decreased after therapy and remained at lower levels
until the end of observation (30 weeks). Disease-related inflammatory marker, such as visfatin, related to the
severity of autoimmune disease, were decreased, which correlated with pain relief.
Our in vitro data endorses an involvement of the immune system, and bone and joint cells in low dose
radiation response. We found that bone degrading osteoclasts were reduced in their differentiation to mature,
functional cells. An anti-inflammatory effect was indicated by a decreased lymphocyte adhesion to endothelial
cells under physiological shear stress and a reduced ROS defense in line with a non-linear regulation of
antioxidative enzymes. Based on dosimetry experiments, fat tissue, which mainly consists of adipocytes,
turned out to be a potential target of radon exposure. However, the differentiation of adipocytes and the
release of the inflammatory marker visfatin were unchanged after exposure.
Conclusion: So far, we have no indication for typical α-particle induced genetic damage for technical reasons
However, one important physiological target of radon is the immune system, which is most likely involved in
the beneficial clinical effects. Reduced inflammation and changes in activation of immune cells, as well as
bone cells, were detected. A possible involvement of adipose tissue is under investigation.
Funding by BMBF grant No. 02NUK017/ 050
European Radiation Research 2018, August 21-25, Pécs, Hungary
155
Understanding the enhanced radiobiological effectiveness of high-LET
particles utilizing ion mircoirradiation and super resolution
microscopy
Judith Reindl1, Katarina Ilicic
2, Werner Friedland
3, Thomas Friedrich
4, Stefanie Girst
1, Christoph
Greubel1, Matthias Sammer
1, Benjamin Schwarz
1, Christian Siebenwirth
5, Dietrich W.M. Walsh
1,
Anna A. Friedl6, Thomas E. Schmid
2,3 , Michael Scholz
4 and Günther Dollinger
1
1Universität der Bundeswehr München, Germany;
2Klinikum Rechts der Isar, TU Munich, Germany;
3Helmholtz Zentrum München, Germany;
4GSI, Darmstadt, Germany;
5RARAF Laboratory,
Columbia University, New York, USA; 6Klinikum der Universität München, LMU, Germany;
Objective: Radiotherapy is besides surgery, chemo- and immunotherapy one of the four pillars of
tumor therapy. Recent improvements make radiotherapy more effective and more tolerable for the
patient regarding side effects. The major improvement arises from the use of particles rather than x-
rays due to their unique depth dose distribution in tissue. Additionally high-LET (linear energy
transfer) particles exhibit an enhanced radiobiological effectiveness, e.g. cell killing is significantly
higher at the same dose. It is speculated that the underlying mechanism is the local DNA damage
density, which is much higher for high-LET particles.
Methods: At the ion microprobe SNAKE at the 14 MV tandem accelerator in Garching near
Munich, cells can be irradiated with a wide range of particles from low-LET 20 MeV protons
(LET=2.6 keV/µm) up to high-LET 33 MeV lithium (85 keV/µm) and 55 MeV carbon
(360 keV/µm) ions. These ions can be applied either randomly over the cell sample or focused to a
spot size of <1 µm. This allows to simulate the irradiation of high-LET particles by focusing the
corresponding amount of low-LET particles, in order to study the mechanistic effects of damage
induction. Additionally, cells can be irradiated under a small angle using a broad beam at the same
setup. This allows to investigate the nanoscopic structural differences in damage induction using a
super resolution STED microscope, which is also available in our group.
Results: The irradiation with randomly distributed protons has the same effectiveness as x-ray
irradiation related to cell killing or genetic damage induction. In contrast, the effect can be
significantly enhanced by focusing the same number of protons to 1µm sized spots. It is attributed to
the interaction of double strand breaks (DSB) leading to enhanced damage structures. However, the
effectiveness of carbon ions cannot be reached due to enhanced DSB production in the inner core of
the high LET particles.
Despite the different damage distribution in the cell nucleus between high and low LET particles,
super resolution microscopy interestingly showed no differences in nanostructural clustering of DNA
double-strand break repair factors. Counting of DSB, visualized by 170 nm protein clusters is used to
compare with the number of predicted DSB from simulations.
Conclusion: Spatial focusing of low-LET ions can be, to a certain extend be used to simulate
damage induction of high-LET particles. However detailed microscopic analysis of DNA repair
showed no difference between high- and low-LET induced damage. But for the first time it was
possible to proof that the number of induced DSB is different for radiation of different LET. The
performed measurements will help to understand and to accurately predict the effects of high-LET
particles to tissue and to further improve radiation therapy of tumors.
European Radiation Research 2018, August 21-25, Pécs, Hungary
156
Treatment of Radiation Lesions with Secretome of Stem Cells
Mohi Rezvani
Swiss Biosana GmbH, Wagistrasse 27 A , 8952 Schlieren, Zurich ,Switzerland
Faculty of clinical Medicine, University of Oxford, Oxford, UK (Reader Emeritus)
Objective:
Most authors reporting the results of stem cell transplantation for tissue regeneration or repair of damaged
tissues cannot trace the transplanted stem cells in the damaged tissues or report a very low level of
engraftment that could not justify the functional improvements observed after stem cell transplantation.
Alternatively, therapeutic potential of stem cells can be attributed to their paracrine effect rather than their
direct integration in regeneration of damaged tissues. This means that the transplanted stem cells, rather than
integrating in the structure of damaged tissues, secrete biologically active substances, in the form of
extracellular vesicles or microvesicles, that stimulate and mobilise the endogenous stem cells to repair the
damage. The objective of this study is to investigate the effectiveness of stem cell secretions in the treatment
of radiation induce normal tissue lesions.
Methods: Mode of action of transplanted mesenchymal stromal cells was investigated through experimental
studies on irradiated brain, intestine and skin and demonstrated that transplanted stem cells act in a paracrine
fashion and exert their effect through nanoparticles released from transplanted stem cells. As a proof of
concept conditioned media from autologous adipose tissue derived mesenchymal cells were tested in different
tissues.
Results: Cell free extracts derived from mesenchymal stem cells injected in irradiated animals was as
effective as transplanted cells in modification of gut and skin lesions in rats.
Conclusion: It was concluded that secretome derived from adipose tissue-derived stem cells can be used as
potent therapeutics, perhaps as an alternative, or in combination with stem cell transplantation, in the
treatment of a number of damaged tissues.
European Radiation Research 2018, August 21-25, Pécs, Hungary
157
Molecular cytogenetics : from biological dosimetry to deciphering mechanistic effects
Michelle Ricoul, Tamizh Gnana-Sekaran, , Patricia Brochard, and Laure Sabatier
PROCyTOX Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA), Fontenay-
aux-Roses, France. laure.sabatier@cea.fr
After an overexposure to genotoxic agents such as ionizing radiations an estimation of the absorbed
dose is required in order to predict the health consequences and to implement countermeasures.
Cytogenetic biomarkers are considered to be the most sensitive and reliable ones (Hall et al 2017,
Mut Res). The dicentric assay is the international gold-standard method for biological dosimetry and
classification of genotoxic agents. The introduction of telomere and centromere staining renders the
scoring of dicentric chromosomes more reliable and robust not only in stimulated metaphases
(MKacher et al, 2014 MutRes, Kaddour et al, SciRep 2017) but also in non-stimulated lymphocytes
following premature chromosome condensation in which it permits their identification in
lymphocytes following cell fusion mediated premature chromosome condensation (PCC) which is
not possible by conventional staining (MKacher et al, 2015 IJROBP). TC Staining and image
analysis in PCC-fusions can be improved by using an alternate to CHO-fusion that showed huge
interstitial telomeric signals. TC Staining improves also MN scoring and discreminates between
aneugenic and clastogenic agents.
Combining protein (H2AX) immunostaining, DNA (telomeres, chromosomes) painting are useful
tools for studying the kinetic of chromosome aberration formation and deciphering some process as
clastogenic effect versus loss of telomere maintenance (Pottier et al, Plos One 2013).
These approaches can be used both for new insights in toxicity mechanisms and for biological
dosimetry in radiation emergency medicine or molecular epidemiology.
European Radiation Research 2018, August 21-25, Pécs, Hungary
158
Application of CLI (Cherenkov Luminescence Imaging) and other imaging
modalities in the preclinical research
1Ritter Zsombor,
3Balogh Péter,
2Szöllősi Dávid,
1Schmidt Erzsébet,
4Michael Irvine,
4David Tuch,
5Kunal Vyas,
2Horváth Ildikó,
2Máthé Domokos,
1Zámbó Katalin
1PTE Department of Nuclear Medicine, Pécs,
2Semmelweis University Department of Biophysics and
Radiation Biology, Budapest 3PTE Department of Immunology and Biotechnology, Pécs,
4Lightpoint Medical Ltd, Rickmansworth, United Kingdom,
5Sagentia Ltd, Cambridge, United
Kingdom
Objective: The baseline aim of the study was to investigate the spreading of Bc-DLFL.1 lymphoma
in vivo and ex vivo with high resolution CLI (Cherenkov Luminescence Imaging) and other imaging
modalities.
Bc-DLFL.1 is a novel spontaneous high grade lymphoma from BALB/c mice which shows
preferentially peritoneal distribution and propagate via the lymphatic vessels towards the mesenteric
lymph nodes and the spleen.
Methods: First we compared the FDG uptake of the lymphoma cells and normal limphocytes in vitro.
The lymphoma cells were adjusted to 106/recipiens, and were injected intraperitoneally. After several
day (1,2,4,5,6,9,11,13) of the injection we administered intravenous and in early stage (24-48 hour
after lymphoma inoculation) also intraperitoneal FDG, or Ga-67 citrate to the mice. After the
radiotracer injection we performed in vivo PET and SPECT/MRI and ex vivo CLI and
autoradiography. For the anti-FITC immunhistochemistry detection CFSE-labeled lymphoma cells
were adjusted to the recipiens. The abnormal structures were verified histologically with
hematoxilin-eozin (HE) staining and immunofluorescence.
Results: We found that FDG uptake of lymphoma cells are 20-25 times higher than in normal
lymphocytes.
Most effective imaging method has varied among the different stages of the disase in our lymphoma-
modell. In early stages with autoradiography (after the intraperitoneal injection of FDG) and CFSE-
based immunohistochemical labeling we found the same: delineation of microscopic lymphoma
clusters within the mesentery and along the gut wall. Despite that ex vivo CLI was not suitable for
early detection, at more advanced stages CLI allowed accurate detection of FDG accumulation in
tumors. We observed lymphoma foci accumulating FDG in the expected regions of adipose tissue
along mesenteric veins, within enlarged mesenteric lymph nodes, in the omentum and also in the
splenic hilus. In the late stage tumor-bearing mice (11-13 days after LI) with the combination of
SPECT/MRI and ex vivo CLI we could clearly reveal lymphoma accumulation in the parathymic
lymph nodes, which were unnoticeable with MR imaging only.
Conclusion: The CLI was capable of identifying 200-300 micrometer sized structures at the late
stage of disease both with FDG and also with Ga67-citrate, which reveals the importance of CLI
both in clinical and preclinical practice, to imaging radionuclides and threrefore detect and
emphasize tumorous tissues and to guide the surgical resection. Beside CLI with other imaging
modalities we can detect the exact intraperitoneal spreading ways which contributes the
understanding of tumor cell migration via the lymphatics and peritoneal cavity.
European Radiation Research 2018, August 21-25, Pécs, Hungary
159
Late radiobiological effects in mice following combined low-dose
extended gamma and mixed gamma, neutron irradiation
Alla V. Rodina, Semen S. Arzumanov, Victor V. Safronov, Yulya P. Semochkina, Marina Yu.
Kopaeva, Elizaveta Yu. Moskaleva
NRC "Kurchatov Institute", Department of System Biology and Biomedicine, Moscow, Russia
Objective. Radiation environment in extended duration space flights is scrutinized in the context of the
probability of undesirable late effects of irradiation. The risk for the development of learning and memory
deficits might be significantly increased due to exposure to secondary neutrons. The pathogenesis of cognitive
injury under these conditions is unknown but may involve neuroinflammation due to increase of the number
of microglia cells and their activation. Recent studies have shown that microglia affects the fate of neural stem
cells in response to ionizing radiation, which suggests a role of microglia in radiation-induced degenerative
outcomes. The aim of this study was to determine the late effects of the combined low-dose whole-body γ-
irradiation and cranial γ,n-irradiation on microglia and cognitive functions of animals.
Methods. 2-months-old male C57BL/6 mice were exposed to prolonged whole-body gamma irradiation at
0.1, 1, 2, 3 Gy alone or cranial mixed γ,n-irradiation at 1 and 2 Gy alone or combined prolonged whole-body
γ-irradiation at 0.1, 1, 2 Gy and cranial γ, n-irradiation at 1 and 2 Gy in 7 days after γ-irradiation. Mice were
exposed to γ-quanta 60
Со at the facility "GUT-200M" at the dose rate of 0.8 mGy/min. The mixed γ,n-
irradiation was carried out in a collimated beam of neutrons and gamma quanta at a special station of a nuclear
reactor IR-8. Estimated (using the Geant4 software package) absorbed dose rate in the sample was 0.6 Gy/h.
Of these, 0.2 Gy/h is due to neutron irradiation, and 0.4 Gy/h due to γ-quanta. On the day 7, 14, 21 and 60 the
mice were tested in the Open Field. Movement and exploration were tracked and analyzed with video
software from Noldus Information Technologies (Ethovision XT 8.5, The Netherlands). At the 60th day after
exposure mice tested in Morris water maze. Three months post irradiation murine microglia cells were
isolated from the mouse brain for cell surface staining and flow cytometry analysis.
Results.The study of irradiated animals in the Open Field test showed that the changes of cognitive functions
occurred 7 days after prolonged whole-body γ-irradiation at a dose of 3 Gy and cranial γ, n-irradiation at a
dose of 1 Gy were normalized in 14 days. The combined effect of whole-body γ-irradiation at a dose of 1 Gy
and cranial γ,n-irradiation at a dose of 1 Gy, and also whole-body γ-irradiation at a dose of 2 Gy and cranial
γ,n-irradiation at a dose of 1 Gy resulted in decrease in motor activity and was accompanied by an increased
level of anxiety 3 weeks after irradiation, which persisted for 2 months. The results of the study of spatial
learning and memory assessed by Morris water maze test showed a violation of spatial orientation and
memory in animals 60 days after the combined effect of prolonged whole-body γ-irradiation at a dose of 0.1
Gy and cranial γ,n-irradiation at a dose of 1 Gy. The number of activated microglia cells increased in 60 days
after combined prolonged whole-body γ-irradiation at a dose of 0.1 Gy and cranial γ,n-irradiation at a dose of
1 Gy compared with control mice and another tested groups.
Conclusion. Disturbance of spatial orientation and memory in animals 60 days after the combined effect of
prolonged whole-body γ-radiation at a dose of 0.1 Gy and cranial γ,n-radiation at a dose of 1 Gy is
characterized by an increase in the population of microglia and its activation. The microglia activation may
indicate the development of neuroinflammation in the late period that leads to cognitive impairment. Thus,
combined prolonged whole-body low-dose γ-irradiation and cranial γ,n-irradiation, induces late
radiobiological effects.
This work was supported by RFBR grant № 17-29-01033.
European Radiation Research 2018, August 21-25, Pécs, Hungary
160
Long term imaging of cells after targeted irradiation of mitochondria
Sarah Rudigkeit1,2
, Judith Reindl2, Nicole Matejka
1,2, Matthias Sammer
2, Benjamin Schwarz
2,
Günther Dollinger2, Dietrich W.M. Walsh
2
1 Technical University of Munich, Department of Physics, Garching bei München, Germany 2 Universität der Bundeswehr München, Institut für Angewandte Physik und Messtechnik,
Neubiberg, Germany
Objective: For many years the central dogma of radiation biology stated, that the main radiotoxic effects of
ionizing radiation originate from damage to the nucleus. So only little research has investigated the role of
cytoplasm. Mitochondria are the “power plants” of the cell and cover 30-40% of the cytoplasmic volume in
HeLa cells. Additionally they are the main organelles for the survival of the cell and play an important role in
signalling pathways such as apoptosis. Furthermore they build large highly dynamic networks throughout the
whole cell, which react to the energy demand of the cell. So the morphology of the network is an indicator for
the cells stress level and healthiness. All this makes them a substantial component of the cytoplasm and
therefore a target for irradiation in the cytoplasm.
Methods: To investigate the response of mitochondria to ionizing radiation we performed targeted irradiation
with carbon ions at the ion microbeam SNAKE (Super conducting Nanoprobe for Applied Nuclear (Kern)
physics Experiments) at the 14 MV tandem accelerator in Garching near Munich, with a beam spot size of ~1
µm. To enable monitoring of the mitochondrial networks a method was established to selectively plate out
only few cells (ca. 80 cells) at four points with an area of 0.0012 cm² on a live cell imaging system (LCI).
With this method it is possible to target mitochondria in these areas with a defined number of 55 MeV Carbon
ions and afterwards perform long-term observation. In the experiments presented the cells were tracked for 3.5
days every 15 minutes with a phase contrast microscope (20x objective, PH2) and additionally once a day
with epifluorescence (63x objective). The LCI was placed in a live cell imaging incubator (Tokai-hit, Japan).
For the targeting of the mitochondria a low concentration TMRE staining was implemented. TMRE is a
cationic dye, which accumulates only on functional mitochondria due to a membrane potential at the
mitochondrial inner membrane. For irradiation of the mitochondria in the cells three different rasters (8x8
irradiation points with 80 ipp (ions per point), 6x6 irradiation points with 142 ipp and 4x4 irradiation points
with 320 ipp) with a point distance of 750 nm were used. So each cell cytoplasm undergoes a dose of 142
carbon ions/µm2 (~100 Gy/µm²).
Results: An irradiation with minimum 80 ipp is enough to depolarize the mitochondria at this point. The
irradiated cells show the targeted area as decrease in TMRE signal or a “dark spot” in the TMRE staining. The
cells in all 4 areas keep on growing during the 3.5 days. While the cell number of the unirradiated control
group showed an exponential growth curve, the cell numbers in the three irradiated groups stay relatively
constant. The three groups irradiated with the three different areas but the same doses yield the same results
within the variances. In the unirradiated group the cells divide themselves ca. twice as often as in the
irradiated groups, the rate of cell death is 2-2.5 times more frequent in the irradiated groups. A CR39 solid-
state track detector shows 3 % parasitic ions up to 500 µm next to the targets, what approximately yields a
dose up to 3.5 Gy per nucleus.
Conclusion: We have shown that mitochondrial irradiation does not kill the cells although a high dose was
used. The decreased propagation of the cells and the increased cell death might come from the parasitic ions,
so we have to optimize the ion microbeam for the next experiments. The irradiation depolarizes the
mitochondria only locally and the unirradiated mitochondria remain functional during the observation time.
We assume that the dynamic mitochondrial networks can compensate the few depolarized, non-functional
mitochondria.
European Radiation Research 2018, August 21-25, Pécs, Hungary
161
Effect of different radiation qualities on functional behavior of cancer cells using a bio-printed
3D comprehensive model of chondrosarcoma embedded in healthy cartilage.
Yannick Saintigny, Vidhula Ahire, Marie Brocquehaye
LARIA – CIMAP. CEA. Campus Jules Horowitz, Bd Henri Becquerel, Caen, France
Although recent advances in the understanding and treatment of human cancer have resulted in
markedly improved overall patient survival, some cancers remain hard-to-treat, with no established
or satisfactory therapy. Chondrosarcomas are usually treated with surgery, due to poor responses to
conventional chemo- and radiation therapy. There is an increasing realization that tumor
heterogeneity and the tumor microenvironment (TME) may play a crucial role in treatment
resistance. Cancer stem cells (CSCs) were proposed to be responsible for tumor development and
heterogeneity, and have been linked with treatment failure, relapse and metastasis. Recent evidence
indicates that CSCs interact with the surrounding TME and that such interplay should be taken into
account for developing new therapeutic strategies. For example, mesenchymal stem cells (MSCs)
have the ability to migrate into tumor sites and exert effects on cancer cells and CSCs through direct
and/or indirect interaction. We and others have shown that the maintenance of CSC phenotype and
properties (chemo- and radio-resistance, tumor-initiating and metastasis-promoting abilities) requires
specific signals and molecular regulations that are controlled by the TME and that may serve as
potential therapeutic targets for CSC-targeting treatments.
While conventional X-ray radiation therapy is relatively ineffective in treating radioresistant tumors,
new radiation therapy modalities using accelerated particles (protons or heavy ions) have shown
promising results in those hard-to-treat cancers. There is strong agreement that approaches
combining particle therapy and targeted therapy (for example, anti-CSC molecular therapy) may
provide some breakthrough in the treatment of tumors with bad prognosis.
2D cell cultures models are obviously not suitable for studies aiming at studying tumor heterogeneity
and microenvironment. Conventional 3D cultures, based on hydrogels (like spheroids/organoids) or
porous scaffolds, have been developed, but are still limited by oversimplification and size
limitations. The recent rise of 3D bioprinting technologies provides broad flexibility in designing
such models. Specific spatial patterns of heterogeneous living cells can be deposited, using various
“bioink” formulations. It is now possible to develop models with complexity approaching that of in
vivo TME.
We develop here a cartilage tumour implanted in a healthy tissue within an extracellular matrix
secreted by chondrocytes or differentiated MSCs. Interactions between cancer cells and non-
differentiated MSCs may be directly observed. Similarly, bio-printing may allow understanding the
role of the stromal environment in radiation quality differential response.
European Radiation Research 2018, August 21-25, Pécs, Hungary
162
Selective heart irradiation induces cardiac overexpression of the pro-
hypertrophic miR-212
Márta Sárközy1, Renáta Gáspár
1, Ágnes Zvara
2, Laura Kiscsatári
3, Zoltán Varga
3, Bence Kővári
4,
Mónika G. Kovács1, Gergő Szűcs
1, Gabriella Fábián
3, Gábor Cserni
4, László G. Puskás
2, Thomas
Thum5, Zsuzsanna Kahán
3, Tamás Csont
1, Sándor Bátkai
5
1Metabolic Diseases and Cell Signaling Group, Department of Biochemistry, Faculty of Medicine,
University of Szeged, Szeged, Hungary 2Department of Functional Genomics, Biological Research Center, Szeged, Hungary
3Department of Oncotherapy, Faculty of Medicine, University of Szeged, Szeged, Hungary
4Department of Pathology, University of Szeged, Szeged, Hungary
5Hannover Medical School, IMTTS, Hannover, Germany
Objective A deleterious, late-onset side effect of thoracic radiotherapy is the development of
radiation-induced heart disease (RIHD). It is often manifests as heart failure with preserved ejection
fraction (HFpEF) characterized by left ventricular hypertrophy and diastolic dysfunction. The miR-
212 is a crucial regulator of pathologic cardiac hypertrophy and has been proposed as a therapeutic
target for heart failure. Therefore, our aim was to investigate whether the miR-212 and its
hypertrophy associated targets play a role in the development of RIHD.
Methods RIHD was induced in a clinically relevant chronic rat model. A single dose of 50 Gy was
delivered to the whole heart of the animals and 19 weeks later, cardiac morphology and function was
assessed by transthoracic echocardiography and tissue samples were collected for histology and
molecular analysis.
Results Echocardiography and histology revealed left ventricular hypertrophy with preserved
ejection fraction, diastolic dysfunction and interstitial fibrosis in the irradiated group. MiR-212 was
overexpressed and FOXO3 mRNA – which is a validated direct anti-hypertrophic target of miR-212
- was repressed in the irradiated hearts. In contrast, total FOXO3 protein level failed to decrease in
response to heart irradiation. However, cardiac phospho-FOXO3 level and phospho-FOXO3/total
FOXO3 ratio showed a non-significant increase in irradiated hearts. Another regulator of the FOXO3
protein, the phospho-AKT/total AKT ratio failed to change in the irradiated hearts as compared to
controls.
Conclusions Cardiac overexpression of miR-212 might play a role in the development of HFpEF in
RIHD. The development of cardiac hypertrophy seems to be independent of the AKT/FOXO3
mediated pathways in RIHD.
European Radiation Research 2018, August 21-25, Pécs, Hungary
163
Proton minibeam radiotherapy (pMBRT) as an innovative
radiotherapy approach
T.E. Schmid1,2
, A. Hunger1, M. Sammer
3, E. Zahnbrecher
2, J. Reindl
3, K. Ilicic
2, D. Walsh
3, C. Greubel
3, B.
Schwarz3, J.J. Wilkens
2, G. Dollinger
3, S.E. Combs
1,2.
1Klinikum rechts der Isar, Technische Universität München, Munich, German
2Institute of Innovative Radiotherapy, Helmholtz Zentrum München, Neuherberg, Germany 3Institut of Applied Physics and Metrology, Universität der Bundeswehr München, Germany
Objectives
Proton minibeam radiotherapy (pMBRT) using submillimeter beam dimensions allows to enhance normal
tissue sparing in the entrance channel by spatial fractionation additionally to advantageous proton depth dose
distribution. In this study, acute side effects of proton minibeam irradiation were examined in an in-vivo
mouse model to account for immune system, vasculature and higher complexity. In this study, partially
widened proton minibeams were applied as they occur on their way to the tumor within the normal tissue in an
in vivo mouse model.
Methods
A total of six different minibeam sizes were applied to the ear of Balb/c mice using 20 MeV protons. The
average dose of 60 Gy was distributed in 4x4 minibeams with beam sizes of σ = 0.09, 0.2, 0.31, 0.45, 0.56 and
0.9 mm and a beam-to-beam distance of 1.8 mm. Inflammatory response, i.e. ear swelling and skin reactions,
were monitored for 90 days following irradiation.
Results
The results show a correlation between the applied beam sizes and the dimension of acute side effects after
irradiation. The largest beam sizes lead to significant ear swelling (up to 3-fold), erythema and desquamation
3-4 weeks after irradiation. With decreasing beam sizes, the maximum skin reactions were reduced until
almost no ear swelling or other visible skin reactions to the irradiation could be detected. However, all sizes of
minibeams were superior to homogeneous irradiation
Conclusion
Our results show that the tissue sparing effect of proton minibeams is highest in the superficial parts of the
beam channel whereas it is possible to create almost homogenous irradiation in the tumor area, which could
be used to obtain tumor control while sparing the surrounding healthy tissue. However, as even larger
minibeams have significantly reduced acute side effects than a broad beam, proton minibeam radiotherapy
may offer various possibilities for new approaches in clinical proton and/or heavy ion radiotherapy. Skin
fibrosis as a radiation induced late effect is currently investigated 90 days after irradiation.
Supported by the DFG Cluster of Excellence: Munich-Centre for Advanced Photonics.
European Radiation Research 2018, August 21-25, Pécs, Hungary
164
The STORE database; a platform for data and resource sharing in
radiation biology, radioecology and epidemiology
Paul Schofield1, Michael Gruenberger
1, Mandy Birschwilks
2, Shin Saigusa
3, Bernd Grosche
2,
Gayle Woloschak4, Soile Tapio
5, Nick Beresford
6, and Ulrike Kulka
2
1 University of Cambridge, Department of Physiology, Development and Neuroscience, Downing Street, UK. 2 Bundesamt fuer Strahlenschutz (BfS) Ingolstaedter Landstrasse 1 85764 Oberschleissheim Germany
3 National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology,
4-9-1 Anagawa, Inage-ku, Chiba-shi, 263-8555 Japan 4
Departments of Radiation Oncology, Radiology, and Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University 303 E. Chicago Ave., Ward 13-002, Chicago, IL 60611
5 Helmholtz Zentrum München, Ingolstädter Landstr. 1 , 85764 Neuherberg , Germany
6 Centre for Ecology & Hydrology, CEH Lancaster,
Lancaster Environment Centre, Library Av., Bailrigg, Lancaster, LA1 4AP, UK
Motivation and Objectives There is major concern about the availability and reuse of primary scientific data, in relation to accountability, reproducibility and value of publicly funded science. Sharing of legacy data especially from large-scale experiments on radiation exposure as well as current studies in radiobiology, radioecology and epidemiology provides a huge benefit in terms of data reuse, aggregation and validation. In response to these needs in radiobiology, epidemiology and radiation safety research we have developed the STORE data-sharing platform, funded by the European Commission’s Euratom programme. Results Development of the STORE database was initiated in 2009 following the successful completion of the European Radiobiology Archive (ERA) database containing legacy datasets from the large-scale experiments on animal radiation exposure carried out in Europe, the USA and Japan between the 1950s and the 1990s. Extensive consultation with the user community has lead to the creation of an open, on-line infrastructure for effective resource sharing, now hosted by the Bundesamt fuer Strahlenschutz in Germany. STORE permits users to upload and share experimental, observational or epidemiological data from legacy or current studies. Users can archive primary or derivative data without charge, and maintain control over its dissemination through Creative Commons licensing and user-defined security and privileges. The data structure in STORE is centred on the Study which is a top level directory for data within a particular project or programme. Within a Study there are Datasets and within those individual Files or data elements. So a study might contain many datasets and each dataset many pieces of data, which can be of any type or size and annotated with standard metadata terms for retrieval and searching. Each Dataset and data item are assigned a Digital Object Identifier (DOI) and a persistent STORE ID using the STOREDB: namespace. The IDs thus assigned can be used for reference, for example when data are used in support of publications as an alternative or an adjunct to journal supplementary information sites. STORE currently contains 98 studies and more than 3000 individual data elements, which range from epidemiology data, though images to proteomics and raw mass spectroscopy data. STORE is compliant with the FAIR data principles which have been adopted by funding agencies such as the US National Institutes of Health and the European Commission and is registered with r3Data (https://www.re3data.org), the ELIXIR/EBI-based Identifiers.org registry (https://identifiers.org) of persistent identifiers, and the FAIR sharing initiative (https://fairsharing.org). Conclusions STORE is open and its use free to investigators and to institutions or funding agencies as a central repository for data sharing. It is available on http://www.storedb.org.
European Radiation Research 2018, August 21-25, Pécs, Hungary
165
A role for epigenetic mechanisms in the effects of low dose radiation
on ecosystems
Paul N. Schofield
University of Cambridge, Department of Physiology, Development and Neuroscience, Downing Street, UK.
Models of the impact of ionising radiation on the environment are currently based on a traditional mutagenic processes occurring in plants and animals in an environment otherwise considered devoid of interacting metabolic and behavioural stressors. For animals the radiation sensitivity of humans to neoplasia is used as an endpoint for damage assessment. We have recently assessed how current criteria for radiation-induced environmental damage might be reconsidered in the light of non-traditional mechanisms of action of ionising radiation and effects on the structure and dynamics of an ecosystem as opposed to individual organisms (Mothersill et al., 2018). One such mechanism with potential impacts on an exposed ecosystem is that of epigenetic damage, or epimutation. It is now well established that exposure to low doses of ionising radiation can result in heritable epigenetic change which affects the phenotype of both plants and animals (Schofield and Kondratowicz, 2017). The epigenetic mechanisms by which these changes occur are not well understood. Evidence suggests that however radiation is sensed and its effects transduced or transmitted, whether through a DNA mediated or more likely Non-targeted effect (NTE), there are persistent changes in the modification of genomic DNA, for example through alterations in CpG methylation, and chromatin. These changes are associated with various endpoints including accelerated cellular senescence, genomic instability and cell death. Introducing epigenetics into the range of potential mechanisms raises unanswered questions about the short term individual response (ie within one generation) and long term population response of organisms to acute or chronic low dose irradiation. Impacts on behaviour, reproduction and lifespan are only just beginning to be identified in contaminated ecosystems, but as yet links between these and epigenetic mechanisms are circumstantial. The speed of phenotypic changes recognised in plant and animal species to low dose contamination raise the question of whether an epigenetic mechanism may play a part in adaptive or maladaptive change at the population level over and above mechanisms familiar as part of the neo-Darwinian evolutionary synthesis. This critical review will consider the evidence for direct damage to cell physiology or genomic stability by epigenetic modifications, and evidence for epigenetic effects in the wild. I will also consider radiation-induced epigenetic effects on population fitness, indirect damage through paramutation and the possibility that a process of epimutation might interact with classical DNA mutation or preexisting genetic variance to influence changes in the genetic structure of populations through the process of genetic assimilation (Nishikawa and Kinjo, 2018).
References
Mothersill, C., et al., 2018. When a duck is not a duck; a new interdisciplinary synthesis for environmental radiation protection. Environ Res. 162, 318-324.
Nishikawa, K., Kinjo, A. R., 2018. Mechanism of evolution by genetic assimilation : Equivalence and independence of genetic mutation and epigenetic modulation in phenotypic expression. Biophys Rev. 10, 667-676.
Schofield, P. N., Kondratowicz, M., 2017. Evolving paradigms for the biological response to low dose ionizing radiation; the role of epigenetics. Int J Radiat Biol. 1-13
European Radiation Research 2018, August 21-25, Pécs, Hungary
166
FAIRing and TOPing in radiation science
Paul Schofield1, Ulrike Kulka
2, Michael Gruenberger
1, Bernd Grosche
2, Gayle
Woloschak3, and Balazs Madas
4
1 University of Cambridge, Department of Physiology, Development and Neuroscience, Downing Street, UK. 2 Bundesamt fuer Strahlenschutz (BfS) Ingolstaedter Landstrasse 1 85764 Oberschleissheim Germany
3 Departments of Radiation Oncology, Radiology, and Cell and Molecular Biology, Feinberg School of
Medicine, Northwestern University 303 E. Chicago Ave., Ward 13-002, Chicago, IL 60611 4 Environmental Physics Department, MTA Centre for Energy Research, H-1121 Budapest, Konkoly-Thege
Miklós út 29-33, Hungary.
Motivation and Objectives
The primary data produced in the course of publicly-funded science represents a common asset for
society as much as the analysed and interpreted results. Recent years have seen widespread agreement
that such data and discoveries should be as accessible as possible by other scientists and the public in
order to extract the maximum value from that investment. Open access publication has been the focus
of much attention and most investigators are now familiar with mandated open access publication
mechanisms and requirements. However the public availability of primary data particularly requires
the establishment of governance and a sharing infrastructure. International guidelines have been
established for open data (FAIR1) and transparent publication (TOP
2). We report an assessment of
needs, adherence and compliance of individual PIs and journals to these guidelines and an assessment
of the barriers to data sharing in radiation science.
Results and Conclusions
The requirement of funding agencies for PIs to comply with FAIR guidelines for the data resulting
from grant funding is very variable, with compliance often recommended but in most cases we
examined not mandated. Increasingly, however, funding agencies require a data management plan. UK
funding agencies were found to provide the most complete guidelines on what was expected from data
management plans and data sharing, with the BBSRC, EPSRC, NERC and Wellcome Trust providing
different but very extensive guidance. NIH and European Commission guidance is more general and
principle-driven. The EC Horizon2020 programme is currently running an extended pilot scheme for
Open data. We can find no evidence for specific assessment of data management plans outside normal
peer review, or monitoring of compliance with the plan and Open data requirements during or after
completion of grants. Similarly there is no evidence as yet for the operation of sanctions for non-
compliance. This is in contrast to Open access publication where more agencies require compliance,
and which is actively monitored.
The role of journals in encouraging open data access, transparency of the review process and
mandatory disclosure of the data on which publications are based, using the TOP framework as a
model, varies extensively from journal to journal, with the PLoS stable of journals arguably having the
best articulated and most enforced guidelines. In contrast the journals in which radiobiological
research is mainly published, show the least developed access and transparency policies of any
journals examined. A survey of radiobiologists and epidemiologists carried out recently by us suggests
that amongst the issues discouraging investigators from making their data open were fear of
competition and lack of time or funding to prepare data for publication. Few respondents knew
whether their Institution or funding agency had open data policies, though more knew about open
access publication policies. We conclude that the field requires more training, especially amongst
young scientists, in data management and data sharing and that reproducibility and data reuse would
be significantly enhanced if journals would articulate and enforce strong data transparency policies.
1 Wilkinson, et al. (2016).. Sci Data 3, 160018.
2 Nosek, B. A., et al. (2015). Science 348, 1422-5 and https://cos.io/our-services/top-guidelines/
European Radiation Research 2018, August 21-25, Pécs, Hungary
167
The direct and bystander effects of low-dose radiation on skin-
infiltrating model
Kazumasa Sekihara1,2,5
, Kaori Saitoh1, Haeun Yang
1,2, Takashi Miida
1, Keisuke Sasai
4
and Yoko Tabe1,3
1 Juntendo University, Department of Laboratory Medicine, Tokyo, Japan
2 Juntendo University, Leading Center for the Development and Research of Cancer
Medicine, Tokyo, Japan 3 Juntendo University, Department of Next Generation Hematology Laboratory Medicine, Tokyo,
Japan 4 Juntendo University, Department of Radiation Oncology, Tokyo, Japan
5 Kagoshima University, Department of Molecular Oncology, Kagoshima, Japan
Object: The inflammatory response of high-dose ionizing radiation used in radiotherapy have been thoroughly
demonstrated in vitro and in vivo. However, the effects of low-dose ionizing radiation (LDIR), such as for
computed tomography-guided biopsies and X-ray fluoroscopy, remain we investigated the molecular effects of
LDIR using skin-infiltrating model to assess the contribution of monocytes to the release of inflammatory
cytokines.
Methods: Cells were exposed to 0.1 Gy X-ray as LDIR. The modulation of transcription was assessed using a
cDNA array and the protein expression after LDIR exposure was investigated using isobaric tags for relative and
absolute quantification (iTRAQ) proteomic analysis at 24 hours and confirmed by immunoblotting analysis. The
direct effects of LDIR on U937 cells and human primary keratinocytes (HPKs) and the bystander effects of
irradiated HPKs on U937 cells were also investigated.
Results: LDIR induced the downregulation of c-Myc in both U937 cells and HPKs and the upregulation of
p21WAF1/CIP1protein expression in U937 cells, along with the activation of TGFβ and protein phosphatase 2A
(PP2A). In HPKs, LDIR downregulated mTOR signaling with repression of S6 and 4EBP1 activation. Similar
molecular changes were observed as bystander effects of LDIR.
Conclusion: Our findings suggest that LDIR inhibits protein synthesis and activates the cytokines associated
with inflammation in direct and bystander effects on skin-infiltrating model.
European Radiation Research 2018, August 21-25, Pécs, Hungary
168
Hormesis, chronic doses and bioaccumulation – some heretical
thoughts!
Colin Seymour and Carmel Mothersill
Department of Biology, McMaster University, Hamilton, Ontario, Canada
Hormesis was championed by Paracelsus, who thought that the dose determined the outcome.
All things were beneficial until they became toxic. Any substance even water becomes toxic
at large doses. So if small doses are beneficial do multiple small doses remain beneficial? We
are all familiar with the folk saying “an apple a day keeps the doctor away” which suggests
that repetition of a small dose can be beneficial. However a concept in radiobiology is that of
bioaccumulation - that is that small insignificant doses accumulate to give the effect of a large
toxic dose. This is embodied in the Linear-no-threshold relationship which as a dose and dose
rate effectiveness factor (DDREF) of 2 to convert between chronic and acute exposure. In
some circles a DDREF value of 1 is preferred i.e. there is no sparing from giving a dose over
a long period. We pose the question - could these small insignificant (in terms of risk) doses
give continual beneficial effects? If bioaccumulation is measurable and increasing, is it per se
above the point of any beneficial effects? Or is bioaccumulation over time more equivalent to
a fractionated radiotherapy dose i.e. producing a sparing effect or should bioaccumulation be
reserved as a term to mean concentration of small doses in organisms in the food chain to
give a toxic dose to the top predator? A number of case studies based on work from our
laboratory and others will be discussed. These will include consideration of chronic radium
studies where high, accumulated doses actually reversed the effects seen after lower amounts
of accumulation and became beneficial. The “banana dose” will be considered – Is this a “red
herring”? In terms of a biological organism, what is an acute dose? How much does it vary
with the organism’s lifespan, and metabolic rate? How does radiation affect hibernating
animals? Is lifespan accounted for correctly in radiation protection? What do hormetic tipping
points look like if dose rate rather than dose is plotted against effect?
European Radiation Research 2018, August 21-25, Pécs, Hungary
169
Determination of cluster DNA damage in testicular tissue after
chronic irradiation with EMR of 1800 MHz
A.S. Shafarost1
1 Gomel state medical university, Science and Investigation Laboratory, Gomel, Belarus
Objective. The aim of this work is to determine the number of oxidatively induced
clustered DNA lesions (OCDL) in cells of testicular tissue of rats at chronic exposure of
electromagnetic radiation (EMR) range of mobile communications (1800 MHz).
Methods. Studies were performed on male-rats F2 Wistar line of gregarious breeding
derived from exposed parents (F1). Parents and their descendants were subjected of EMR for
8 h/day during embryogenesis and postnatal development until the age of 6 months.
EMR source was the experimental installation of mobile communication allowed to
simulate the cell phone signal (1800 MHz) talk time (flux density of electromagnetic energy
in cells amounted from 2,0 to 20,0 mW/cm2). Samples were collected on the 1st day after a
cessation of the electromagnetic exposure at animals in aged 2, 4 and 6 months. Samples of
testicular tissue after the extraction were frozen in liquid nitrogen.
Isolation of DNA was carried out using High Pure PCR Template Kit (Roche)
according to [1]. To identify sites of DNA with lost oxidized or modified nitrogenous base
used the hAPE1 endonuclease according to [2], the calculation of the number of the clustered
lesions were performed according to the method of NALA [3].
Results. In chronic irradiation of EMR at 2 month old animals F2 observed an
increase of frequency АРЕ1 clusters in 2,12 times (7,27 APE1 clusters/Mbp) in compared
with the intact control. Analysis of clustered lesions at males in the age of 4 months shows an
increase of the analyzed parameter in 0,17 times at animals exposed of EMR range mobile
communications. The most notable changes of the number of clustered lesions, which may be
identified using hAPE1 endonuclease, observed at 6 month old animals. In the experimental
group showed an increase of frequency АРЕ1 clusters in 9,41 times (18,29 APE1
clusters/Mbp).
As noted in [4], the effect of electromagnetic radiation 1800 MHz leads to increased
peroxidation processes in the tissues of brain, liver, kidney and blood at rats. Peroxidation
processes are the result of formation of free radicals and indicate about development of
oxidative stress in the cells. Its leads to appear of lesions area DNA, including abasic sites.
The obtained data about the number АРЕ1 clusters in testicular tissue at irradiated rats,
indicate that the DNA reparation system is not doing its function.
Conclusion. Embryonic-postnatal exposure of rats Wistar line of EMR range of
mobile communications 1800 MHz leads to an increase of the number of abasic sites in the
DNA of cells of testicular tissue during all periods of the experiment. These lesions can be
inherited and lead to accumulation of genetic load, if the cells, which are their bearers, will
not be eliminated in the process of spermatogenesis.
References
1. Detection of oxidative clustered DNA lesions in X-irradiated mouse skin tissues and human MCF-7 breast
cancer cells / E. Gollapalle [et. al.] // Radiat. Res. – 2007. – Vol. 167. – P. 207–216.
2. Accumulation of oxidatively induced clustered DNA lesions in human tumor tissues / S. Nowsheen [et. al.] //
Mutat. Res. – 2009. – Vol. 674. – P. 131–136.
3. Quantifying clustered DNA damage induction and repair by gel electrophoresis,electronic imaging and
number average length analysis B.M. Sutherland [et. al.] // Mutat. Res. – 2003. – Vol. 531. – P. 93–107.
4. Influence of electromagnetic field (1800 MHz) on lipid peroxidation in brain, blood, liver and kidney in rats /
P. Bodera [et. al.] // Int J Occup Med Environ Health. – 2015. Vol. 28, № 4. – P. 751-759.
European Radiation Research 2018, August 21-25, Pécs, Hungary
170
Age dependence of cancer risks links to tissue stem/progenitor cell
response to ionizing radiation
Yoshiya Shimada1, Mayumi Nishimura
1, Tatsuhiko Imaoka
1, Yi Shang
1, Kokubo
Toshiaki1, Kentaro Ariyoshi
2, Masaaki Sunaoshi
1, Rena Takei
1 and Shizuko Kakinuma
1
1 QST, NIRS, Department of Radiation Effect Research, Chiba, Japan
2 Hirosaki University, Institute of Radiation Emergency Medicine (IREM), Hirosaki, Japan
Age at exposure is a critical factor that significantly influences the risk of cancer after exposure to
ionizing radiation. Although children are generally believed to be more susceptible to radiation,
susceptible age to radiogenic tumor induction varies among cancer types (UNSCEAR 2013). In this
study, we first constructed the panel of critical age at exposure for cancer risk of each organ in
experimental animal models. Secondly, since stem/progenitor cells are potential carcinogenic
candidates, from which cancer cell arises, we elucidated radiological characteristics of stem/progenitor
cells of several organs in association with age of susceptibility. Characteristics included not only the
total number of cells at risk but also radiation-induced apoptosis, cell cycle arrest, genetic instability,
proliferation following irradiation through regeneration and/or activation of microenvironment. We
show here that stem cell responses to radiation change as a function of age in tissue dependent
manners, and that susceptible age to radiation carcinogenesis corresponds to the stage when
stem/progenitor cells are either highly proliferative or resistant to radiation-induced cell death.
1. Shimada Y, Yasukawa-Barnes J, Kim R, Gould MN, Clifton KH. (1994) Age and
radiation sensitivity of rat mammary clonogenic cells. Radiat Res. 137, 118-123.
2. Imaoka T., Okamoto M, Nishimura M, Nishimura Y, Ootawara M, Kakinuma S,
Tokairin Y, Shimada Y. (2006) Mammary tumorigenesis in ApcMin/+ mice is enhanced
by X irradiation with a characteristic age dependence, Radiat Res, 165, 165-173.
3. Miyoshi-Imamura T, Kakinuma S, Kaminishi M, Okamoto M, Takabatake T, Nishimura
Y, Imaoka T, Nishimura M, Murakami-Murofushi K, Shimada Y. (2010) Unique
characteristics of radiation-induced apoptosis in the postnatally developing small
intestine and colon. Radiat Res. 173, 310-318.
4. Kokubo T, Kakinuma S, Kobayashi T, Watanabe F, Iritani R, Tateno K, Nishimura M,
Nishikawa T, Hino O, Shimada Y. (2010) Age dependence of radiation-induced renal
cell carcinomas in Eker rat model. Cancer Sci. 101, 616-623.
5. Shimada Y, Nishimura M, Amasaki Y, Shang Y, Sawai T, Hirano S, Imaoka T, Kokubo
T, Ishida Y, Yamada Y, Takabatake T, Okamoto M, Kakinuma S. (2011) Interactions of
low dose radiation with other factors in carcinogenesis in vivo. Health Phys. 100, 278-
279.
6. Ariyoshi K, Takabatake T, Shinagawa M, Kadono K, Daino K, Imaoka T, Kakinuma S,
Nishimura M, Shimada Y. (2014) Age dependence of hematopoietic progenitor survival
and chemokine family gene induction after gamma-irradiation in bone marrow tissue in
C3H/He mice. Radiat Res, 181, 302-13.
7. Shang Y, Sawa Y, Blyth BJ, Tsuruoka C, Nogawa H, Shimada Y, Kakinuma S. (2017)
Radiation exposure enhances hepatocyte proliferation in neonatal mice but not in adult
mice. Radiat Res. 188, 235-241.
European Radiation Research 2018, August 21-25, Pécs, Hungary
171
Ultraviolet B degrades ciliary zonules in vitro
Y. Shiroto1)
, Y. Kitayama1)
, R. Saga1)
, H. Yoshino1)
, S. Terashima1)
, Y. Hosokawa1)
,
and E. Tsuruga1)
1 Hirosaki University, Department of Radiation, Hirosaki, Japan
Objective
Elastic system fibres as well as collagen fibres are main extracellular matrices. The elastic system
fibres include three types, i.e., elastic, elaunin, and oxytalan fibres, which differ ultrastructurally in
their relative proportions of microfibrils and elastin. The ciliary zonules consist of oxytalan fibers,
whose main molecules are fibrillin-1 and fibrillin-2. The ciliary zonules are transparent fibres which
connect the lens to the ciliary body in the eye. The function of the ciliary zonule is to adjust the focus
by controlling the thickness of the crystalline lens. Clinically, dislocation of the lens occurs due to a
rupture of ciliary zonules exposed to the sun's ultraviolet (UV) component. However, there is no
available evidence that UV directly affect ciliary zonules.
Methods
We cultured human non-pigmented ciliary epithelial cells and confirmed that they form an oxytalan
fibres. The cell/matrix on the culture dish was then irradiated with a UV lamp at levels of 0-150
mJ/cm2
for each of UV-A and UV-B, and culture was continued for another 24 hours. We then
investigated the appearance of oxytalan fibres using immunohistochemical study. To examine
sensitivity of other types of elastic system fibres to UV-B, we cultured human pulmonary fibroblasts
which produce elastic fibre. Likewise, the appearance of fibrillin-positive fibres was investigated.
Additionally, the presence of metalloproteinase (MMP)-2 was analysed by Western blot using specific
antibody.
Results
After UV-A irradiation, the appearance of both fibrillin-1 and fibrillin-2-positive fibres in HNPCEC
was unaffected. In the case of UV-B irradiation, the structure of fibrillin1-positive fibres became
thinner at an irradiation level of 100 mJ/cm2 and became the appearance of amorphous at 150 mJ/cm
2.
On the other hand, the structure of fibrillin-2-positive fibres disappeared by 150 mJ/cm2. MMP-2
inhibitor completely inhibited these changes of fibre’ degradations. The data of Western blot showed
the presence of MMP-2. The amounts of MMP-2 tend to increase depend on an intensity of UV. In
pulmonary fibroblasts culture, an effect of UV was also confirmed.
Conclusion
The sensitivity of extracellular fibres against UV was different by the type of elastic system fibres.
These results may suggest a structural difference between oxytalan and elastic fibre.
150mJ/cm2
Inhibitor 150mJ/cm2
Inhibitor Inhibitor
European Radiation Research 2018, August 21-25, Pécs, Hungary
172
Nanocarriers for the delivery of radiosensitizing agents and
radionuclides to brain tumours
P. Sminia1, R.M. de Kruijff
2, A.J.G.M van der Meer
2, G. Becerril Aragon
1, A. Gasol Garcia
1,
R.S. Narayan1, S.M.A. van der Pol
3, F. Bikhezar
1, H.E. de Vries
3, B.J. Slotman
1, A.G.
Denkova2
1VU University medical center & Cancer Center Amsterdam,
Radiation Oncology,
Amsterdam, The Netherlands
2Delft University of Technology, Radiation Science and Technology, Delft, The Netherlands. 3VU University medical center, Molecular Cell Biology and Immunology, The Netherlands.
Objective. Recently, we identified the MAPK inhibitor MEK162 (binimetinib) to act as
radiosensitizer in preclinical in vitro and in vivo GBM models [Narayan et al., Mol Cancer
Ther. 2018; 17:347-354]. However, a major problem in brain tumour therapy is the presence
of the blood-brain barrier (BBB), which is abrogating the delivery of most chemical
compounds. An ingenious method to facilitate the transport of therapeutic agents over the
BBB is the application of surface functionalized polymeric nanocarriers (polymersomes, PS).
PS can be loaded with mixtures of therapeutics via encapsulation of water-soluble compounds
(e.g. radionuclides; De Kruijff et al., Eur. J. Pharm. Biopharm. 2018; 127:85-91) in the core
together with water-insoluble substances (most drugs) in the hydrophobic outer bilayer.
Methods. U87 human glioma cells growing as monolayer and multicellular 3D spheroids; cell
proliferation and spheroid growth assays, western blotting, human BBB endothelial cells on a
Transwell system. Self-assembled polymeric vesicles (Ø: 80 nm) composed of amphiphilic
copolymers. Therapeutic agents: the MAPK inhibitor MEK162, ү-rays and the alpha-emitting
radionuclide 225
Ac.
Results. FITC fluorescently labeled PS were taken up in the cytoplasm of U87 cells within a
few minutes. Biodistribution experiments in U87 spheroids showed that PS have diffused
almost throughout the spheroids at day 4, with a completely homogeneous distribution at day
7. In a spheroid growth assay, fractionated radiation (5 x 2Gy) resulted in a growth delay time
of 6 days relative to controls, which was further prolonged to 10 days after combined
treatment with MEK162 PS. Western blot data demonstrated that p-ERK, the downstream
target protein of MEK162 in the MAPK pathway, was not phosphorylated at 24 h, neither
after exposure to free MEK162 nor to MEK162 delivered via PS. Following treatment with
MEK162 and 2 Gy irradiation, үH2AX expression was upregulated, indicating an increase in
radiation-induced DNA double strand breaks. Next, the therapeutic potential of PS loaded
with the alpha emitter 225
Ac was investigated. Considering the biodistribution kinetics of the
PS in spheroids as well as the decay time (T½ = 9.9 days) for 225
Ac, the entire spheroid could
be irradiated. A decrease in spheroid growth was observed upon addition of only 0.1 kBq 225
Ac. At higher activity (5 kBq), spheroids were destroyed completely after two days. First
data on BBB crossing demonstrated effective passaging of the PS through a layer of human
brain endothelial cells towards the lower compartment of the Transwell system.
Conclusion. Polymeric nanocarriers might pass the BBB and are efficiently taken up by
glioma cells and distributed over the spheroid volume. PS loaded with therapeutic agents
inhibited or even abrogated spheroid growth. Therewith, nanocarriers offer exciting
challenges ahead for local delivery of therapeutics to GBM patients.
Funding: STOPhersentumoren.nl (grant # 2015009) and the Zabawas foundation.
European Radiation Research 2018, August 21-25, Pécs, Hungary
173
Damped-oscillator model of hormesis and its consequences
Yehoshua Socol,1 Yair Y. Shaki
1 and Ludwik Dobrzyński
2
1 Jerusalem College of Technology, Jerusalem,, Israel
2 National Centre for Nuclear Research (NCBJ), Swierk, Poland
Objective. Ionizing radiation inflicts immediate damage to living organism by causing DNA
lesions and producing reactive oxygen species (ROS). However, the radiation also triggers
many protective mechanisms at molecular, cell, tissue and organism levels – like ROS
scavenging, apoptosis, DNA repair, cell proliferation and immune response. Overall,
numerous experimental, ecological, and epidemiological studies show that low doses of
ionizing radiation may be beneficial to human health causing adaptive response, or hormesis.
The dual effect of the radiation has been summarized by the dual-probability model, which is
a qualitative model that estimates the resulting biological effect of the radiation by taking into
account both (a) dose- and time-dependent damage and (b) dose- and time-dependent
beneficial health effects (adaptive protection).
Method. We developed the dual-probability model into a novel quantitative model by
modeling time-evolution of response to radiation as time-evolution of a damped oscillator in
the critical damping regime. Continuous irradiation with arbitrary time-dependent rate has
been modeled as a limit of acute irradiations with increasing frequency and correspondingly
decreasing dose. We have introduced several assumptions that are a priori far from obvious.
However, they enabled to construct a simple analytical model described below. Our hope is
that the model predictions will be verified by future experiments, confirming a posteriori the
made assumptions
Results. It was possible to develop a model with essentially two parameters: (1) characteristic
time and (2) slope of adaptive response after acute irradiation. Both are anticipated to be
person-dependent. Characteristic time is estimated by us as 2-4 weeks based on radiation
therapy data. Slope of adaptive response should be determined by future experiments (animal
models and clinical trials). The model predicts that organism's resistance to radiation stress
can be considerably improved by "radiation training", maybe by and order of magnitude. If
the model is verified by future experiments, it should be feasible, e.g., to improve
considerably efficacy of radiation therapy by increasing therapeutic doses.
Conclusion. A simple model of radiation hormesis with two essential parameters only has
been developed. Experiments on animal models are suggested to verify the model. If verified,
the model will contribute both to understanding of radiation effects and to development of
improved radiation therapy protocols.
European Radiation Research 2018, August 21-25, Pécs, Hungary
174
Thyroid cancer: over-radiosensitivity or overdiagnosis?
Yehoshua Socol,1 Yair Y. Shaki
1 and Alexander Vaiserman
2
1 Jerusalem College of Technology, Jerusalem, Israel
2 Institute of Gerontology, Kiev, Ukraine
Objective. It is generally assumed that for thyroid cancers, children are clearly more
radiosensitive than adults [1]. Recently, pooled analysis of 9 thyroid cancer studies [2]
claimed also to reaffirm linearity of the dose response of radiation-associated cancer for dose
below 10 cGy. However, the recently-acknowledged problem of thyroid cancer overdiagnosis
[3] demands reconsideration of the entire field of radiation carcinogenesis, as far as thyroid
cancer epidemiology is involved.
Method. We estimated possible extent of overdiagnosis by reviewing existing estimations
of the above extent in the scientific literature. Afterwards, we compared thyroid cancer
incidence and its time trend in several countries, both affected and not affected by increased
radiation: Ukraine, South Korea and USA. Finally, we performed analysis of the 9 thyroid
cancer cohorts of the pooled epidemiopogical study [2] to determine whether extensive
thyroid cancer overdiagnosis was probable in these cohorts.
Results. The extent of overdiagnosis proved to reach extremely high values. In South
Korea, for example, thyroid cancer incidence increased by factor of 15 without any increase
in thyroid cancer mortality. After the Fukushima nuclear accident, subsequent massive
screening of children for thyroid cancer yielded up to 60-fold increase in cancer incidence, in
unexposed as well as in exposed prefectures of Japan [4]. It can be therefore said that thyroid
cancer incidence is greatly affected by screening extent. The cohorts of the 9 pooled
epidemiological studies included (1) childhood cancer survivors, (2) children treated by
radiation for benign diseases, and (3) atomic bombings' survivors. It can hardly be doubted
that children in all the above cohorts were subjected to increased medical attention, and
therefore to increased rate of screening. Therefore, high overdiagnosis rate is expected. The
high thyroid cancer incidence in these cohorts is most probably a result of the overdiagnosis.
Conclusion. Thyroid cancer incidence in all the 9 cohorts of the pooled epidemiological
study was seriously affected by overdiagnosis. What was claimed as radiosensitivity is most
probably an artifact due to overdiagnosis. Radiosensitivity of the thyroid gland should be
reconsidered.
[1] United Nations Scientific Committee on the Effects of Atomic Radiation. Sources and
Effects of Ionizing Radiation, UNSCEAR 2013 Report to the General Assembly, with
Scientific Annexes. New York, NY: United Nations Publications; 2013, p. 13
[2] Lubin JH, Adams MJ, Shore R. 2017. Thyroid Cancer Following Childhood Low-
Dose Radiation Exposure: A Pooled Analysis of Nine Cohorts. J Clin Endocrinol Metab. 102:
2575-2583. doi: 10.1210/jc.2016-3529
[3] Vaccarella S, Franceschi S, Bray F, Wild CP, Plummer M, Dal Maso L. 2016.
Worldwide Thyroid-Cancer Epidemic? The Increasing Impact of Overdiagnosis. N Engl J
Med. 375: 614-7.
[4] Normile D. 2016. Epidemic of fear. Science 351: 1022-1023.
European Radiation Research 2018, August 21-25, Pécs, Hungary
175
Ionizing radiation-induced endothelial senescence and role in
normal tissue injury
Frederic Soysouvanh1, Amine Benadjaoud
2, Morgane Dos Santos
3, Michele Mondini
4,
Jeremy Lavigne1, Valérie Buard
1, Georges Tarlet
1, Serge Adnot
5, Eric Deutsch
4, Olivier
Guipaud1, Vincent Paget
1, Agnes François
1and Fabien Milliat
1
1 Institute for Radiation Protection and Nuclear Safety, PSE-SANTE/SERAMED/LRMed,
Fontenay-aux-Roses, France 2 Institute for Radiation Protection and Nuclear Safety, PSE-SANTE/SERAMED,
Fontenay-aux-Roses, France 3 Institute for Radiation Protection and Nuclear Safety, PSE-SANTE/SERAMED/LRAcc,
Fontenay-aux-Roses, France 4 National Institute of Health and Medical Research, U1030, Villejuif, France
5 National Institute of Health and Medical Research, U955, Créteil, France
Radiotherapy is the main modality in cancer treatment. However, this procedure is associated with
radiation damages on healthy tissue. The endothelial compartment (layer of cells that line the inner
surface of blood vessels) plays a key role in the evolution of radiation-induced normal tissue injuries.
Irradiated endothelial cells (ECs) acquire a senescence phenotype. Cellular senescence is a powerful
tumor suppressor mechanism but, paradoxically, long-term senescence can be deleterious for the
tissue. The presence of senescent cells within the radiation-induced lesions has been shown but their
role is not well understood. We aim to identify the role of senescent EC in radiation-induced
pathophysiological process.
mRNA expression of 44 genes involved in senescence in 6 human primary irradiated ECs reveal that
Human Umbilical Vein Endothelial Cells (HUVECs) are the most relevant in term of gene expression.
The dynamic molecular profile associated to radiation-induced senescence (RIS) of HUVECs was
analyzed after 9 doses of irradiation and 7 time points. In vivo, using luciferase knock-in mice
(INK4A/p16LUC
) to detect activation of a senescence player after radiation exposure, we aim to
identify the impact of senescent cells in the development of radiation-induced pulmonary fibrosis.
In vitro analysis by mathematical methods, we decipher the dynamical transcriptional program
involved in RIS. We then identified molecular hubs, which could potentially modulate the senescence
phenotype. By altering their expression level, we shifted the transcriptional program of irradiated
HUVECs to a non-senescent profile.
After high-dose irradiation of p16LUC
mice lung, we showed the overexpression of p16 using
bioluminescence imaging and its persistence up to 16 months after radiation exposure. By combination
of immunostaining of several lung cell lines and senescence markers, we observed the majority of
senescent cells are pulmonary epithelial cells and macrophages. Some endothelial cells exhibit
senescence markers but this remains a rare event. In parallel, with an endothelium specific knockout
for PAI-1, another player in senescence, we aim to elucidate its implication in the progression of RIS
and its contribution in lung injury.
European Radiation Research 2018, August 21-25, Pécs, Hungary
176
A role for the bystander effect in the in vitro response to
microbeam radiation
H. Steel1, C. Box
1, U. Oelfke
1, S. Bartzsch
1, 2
1. Centre For Cancer Imaging, Joint Department of Physics, The Institute of Cancer Research, London,
UK
2. Institute of innovative Radiation Therapy, Helmholtz-Centre Munich, Germany
Objective: The damage to normal tissues is the main limitation of conventional radiation therapy and
often prevents the application of curative treatment doses to the tumour. One possible alternative to
conventional radiotherapy is microbeam radiation therapy (MRT), which utilizes spatially
fractionated, 25 - 100 µm wide, parallel kilo-voltage X-ray beams, spaced at intervals of 200 µm to
400 µm. This dose delivery results in peak areas in which cells receive unconventionally high doses
and valley areas where radiation doses are very low.
Work on the use of MRT in animal models has demonstrated a high therapeutic ratio; where normal
tissue can repair microbeam damage inflicted at peak doses of up to several 100 Gy. These same doses
cause significant tumour growth delay and in some cases ablation. However, the mechanism
underlying the differential response of normal tissue and tumour remains unknown; tumour
vasculature, immune response and bystander effect have all been suggested to play a role.
In this work we investigated whether the differential effect of microbeam radiation found in vivo can
also be observed in vitro, and if bystander signalling plays a role in the response of cells to MRT.
Methods: Microbeams were produced using a conventional X-ray tube and a bespoke collimator,
developed in-house, resulting in the production of 49 50 µm wide beams each 400 µm apart with peak
to valley dose ratio of 20. The effects of both broad beam and microbeam radiation on both non-
tumour cell lines, MRC-5 lung fibroblasts and human umbilical cord endothelial cells (HUVECs), and
lung tumour cell lines (A549 and NCL-H23) in vitro were investigated.
Clonogenic assays were used to assess cell survival and immunofluorescent cell staining indicated
levels of DNA damage. Mechanisms of cell death were investigated using flow cytometry.
Clonogenic survival in response to broad bream irradiation was fitted to the linear quadratic model.
The resulting survival curves were then used to calculate predicted responses to the integrated
microbeam dose. This prediction assumed that 1/8 of cells received the peak dose whilst 7/8 received
the valley dose, and that there was no communication between cells in the peaks and the valleys.
Results: The survival of the tumour cell lines fell below that of the prediction, indicating that
communication between the cells in the peaks and the valleys enhances the effectiveness of the MRT.
However, the survival of the non-tumour cells was as predicted as or higher than predicted. In
addition, the tumour cells showed a decreased ability to repair the DNA damage sustained in the
peaks, at 24 hours post-irradiation, compared to the non-tumour cells lines. Finally, an increase in cell
death was detectable in tumour cells at 48 hours post microbeam but not broad beam irradiation.
Conclusion: Our data is amongst the first to demonstrate the differences between the response of non-
tumour and tumour cells to MRT in vitro. The observed differences between the predicted cell survival
and actual survival of the tumour cells suggest that communication between cells in the peaks and
valleys plays a substantial role in the microbeam response and supports the hypothesis of a bystander
effect.
European Radiation Research 2018, August 21-25, Pécs, Hungary
177
Towards a better understanding of dose and dose-rate
effectiveness factors
Sjors Stouten1,2
, Sjoerd Verduyn Lunel1 and Fieke Dekkers
1,2
1Utrecht University, Mathematical Institute, Utrecht, the Netherlands
2Netherlands National Institute for Public Health and the Environment, Centre for Safety,
Bilthoven, the Netherlands
Objective: development of mathematical models to contribute to the ongoing debate on
the use of dose and dose-rate effectiveness factors in radiation protection.
Methods: we developed models for dose and dose-rate dependent DNA damage induction
and repair incorporating effects of cell cycle phase and radiation quality. We based our
models on the current understanding of biological mechanisms involved in the cellular
response to ionizing radiation exposure. By including time-dependent parameters, we have
modelled the activity of repair pathways throughout the cell cycle. Additionally, cells are able
to initiate cell cycle arrest at G1/S and G2/M checkpoints.
Results: utilizing our models we can replicate experimental results for biological
endpoints such as γH2AX foci, chromosome aberrations and surviving cell fractions for dose-
rates between 1-1000 mGy/min and linear energy transfer values up to 1 MeV/µm. The model
allows for quantification of dose/dose-rate effectiveness functions for these endpoints.
Furthermore, we can characterize the conditions required for observing inverse dose-rate
effects.
Conclusion: our models allow one to study dose-rate effects in synchronous and
asynchronous cell populations dependent on radiation quality, dose and cell cycle phase.
Possible dose/dose-rate effectiveness functions were derived for γH2AX foci, chromosome
aberrations and surviving cell fractions. After coupling our current models to a long-term
population model of radiocarcinogenesis we will be able to investigate the possible effects of
(low) dose and dose-rate radiation exposure on cancer incidence.
European Radiation Research 2018, August 21-25, Pécs, Hungary
178
Normal tissue reaction following proton irradiation of the mouse brain
Theresa Suckert1,2
, Johannes Müller1,3
, Elke Beyreuther1,3
, Malte Gotz1,3
, Falk Tillner1,3,5
,
Michael Schürer1,4
, Antje Dietrich1,2
, Rebecca Bütof1,4,5
, Armin Lühr1-3
, Cläre von Neubeck1,2
,
Mechthild Krause1-5
1) OncoRay – National Center for Radiation Research in Oncology, Faculty of Medicine and
University Hospital Carl Gustav Carus, Technische Universität Dresden, Helmholtz-
Zentrum Dresden – Rossendorf, Germany
2) German Cancer Consortium (DKTK), Partner Site Dresden, and German Cancer Research
Center (DKFZ), Heidelberg, Germany
3) Helmholtz-Zentrum Dresden - Rossendorf, Institute of Radiooncology – OncoRay,
Dresden, Germany
4) National Center for Tumor Diseases (NCT), Partner Site Dresden, Germany; German
Cancer Research Center (DKFZ), Heidelberg; Faculty of Medicine and University
Hospital Carl Gustav Carus, Technische Universität Dresden, Germany; and Helmholtz
Association / Helmholtz-Zentrum Dresden – Rossendorf (HZDR), Dresden, Germany
5) Department of Radiotherapy and Radiation Oncology, Faculty of Medicine and University
Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
Objective: Radiotherapy leads to inactivation of tumor cells following radiation-induced DNA
damage. Compared to conventional photon-based radiotherapy, proton therapy offers the
potential of normal tissue sparring due to its favorable depth-dose distribution. However,
acute or long-term side effects could still occur due to clinical safety margins and
uncertainties about the relative biological effectiveness (RBE). While a variable RBE has
been demonstrated in in vitro studies, especially at the end of the proton range, in clinical
practice, a constant RBE value of 1.1 is applied. To elucidate the RBE issue based on in vivo
experiments, proton irradiation of mouse brains was realized in Dresden.
Methods: Experiments were performed at the experimental beam line [1] of the University
Proton Therapy Dresden. For beam characterization and dosimetry, a 2D scintillation
detector, ionization chambers and radiochromic films [2] were used. A multi-modality mouse
bed suitable for imaging, transportation and irradiation was developed in-house. Like clinical
applications, the workflow includes computed tomography scans for treatment planning and
X-ray images for refined positioning. By combining these images with proton radiographies
[3] of the setup, it was possible to accurately locate the animals relative to the beam. To
in the irradiated mouse brain.
Results and conclusion: Proton mouse brain irradiation was successfully performed.
Distribution of DNA DSB via gH2AX revealed that the proton beam stopped in the beam
facing brain hemisphere. The setup enables the comparison to corresponding photon
experiments with SAIGRT [4] and clinically relevant long-term experiments, such as
measuring cognitive functions and anomalies in imaging, to directly relate potential photon
and proton side effects in brain radiotherapy.
European Radiation Research 2018, August 21-25, Pécs, Hungary
179
Calibration experiment of Passive type radon-thoron
discriminative monitor using radon and thoron calibration
facilities at Hirosaki University
Takahito Suzuki1, Hu Jun
1, Yuki Morita
2, Chanis Pornnumpa
3, Yuki Tamakuma
1,
Kazuki Iwaoka4, Masahiro Hosoda
1 and Shinji Tokonami
4
1 Hirosaki University Graduate School of Health Sciences, Hirosaki, Japan
2 Hirosaki University, School of Health Sciences, Hirosaki, Japan
3 Kasetsart University, Faculty of Sciences, Department of Applied Radiation and Isotope,
Bangkok, Thailand 4 Hirosaki University, Institute of Radiation Emergency Medicine, Hirosaki, Japan
Radon (222
Rn) and thoron (220
Rn) are normally generated by alpha decay from 226
Ra and 224
Ra in soil,
rocks and building materials. Inhalation of radon and thoron is believed to increase the risk of lung
cancer and is as the second most important risk factor only after tobacco smoking. Therefore, indoor
radon and thoron surveys had been carried out using a passive detector in many countries. A passive
type radon-thoron discriminative monitor called RADUET (Radosys Ltd, Hungary) was developed by
Tokonami et al, which was recognized as a suitable detector for a large scale survey and it is used by
many researchers including authors. However, in general, it is necessary to calibrate radon and thoron
monitor beforehand in order to guarantee the reliability of measured radon and thoron concentration.
On the other hand, radon and thoron exposure systems were designed and developed for calibrating
radioactive gas monitor, based on the quality assurance (QA) and quality control (QC) standards of the
monitors at Hirosaki University. In this study, calibration experiment of passive type radon-thoron
discriminative monitor called RADUET was conducted using radon and thoron calibration facilities
developed at Hirosaki University.
In exposure of radon gas, three volumes of radon gas (1000, 3000, 5000 mL) generated by natural
uranium ore were injected into a 150 L exposure chamber after installed RADUET in the chamber.
Radon concentration depends on its initial activity and decay constant. During the operation, a
portable radiation monitor (AB-5, Pylon Electronics Inc., Canada) with a scintillation cell (300A,
Pylon Electronics Inc.) was used to measure radon concentration. In exposure of thoron gas, thoron is
generated by layered commercial lantern mantles (M-7910, Captain Stag, Japan) and was continuously
introduced into 150 L exposure chamber in which RADUET was installed. An electrostatic collection
radon/thoron monitor (RAD7, Durridge. Co. Inc., USA) was installed to continuously measure thoron
concentration. Furthermore, the grab sampling technique using the 300A scintillation cell with the
AB-5 monitor was used to correct the value of the thoron concentration obtained by RAD7. In this
study, two types of CR-39 with different sensitivity were used as a solid-state track detector. One was
produced by Japanese company (BARYOTRAK, NAGASAE LANDAUER Ltd, Japan) and another
was produced by Radosys Ltd. After exposure, the CR-39 produced by NAGASE LANDAUER Ltd
was chemically etched for 24 h in a 6 M NaOH solution at 60°C. The another one produced by
Radosys Ltd was chemically etched for 4.5 h in a 6.25 M NaOH solution at 90°C. The number of
alpha tracks were counted using microscope or an automated track counting microscope (RadoMeter
2000, Radosys Ltd, Hungary). And then, the conversion factor from track density to radon and thoron
concentration for each CR-39 was calculated.
We will present the detailed results of calibration experiment and conversion factor evaluated.
European Radiation Research 2018, August 21-25, Pécs, Hungary
180
Simple vertebrate model development for radiobiology research at ELI-ALPS on laser
driven hadron beams
Emília Rita Szabó1, Tünde Tőkés
1, Róbert Polanek
1, Szilvia Brunner
1, Szabolcs Czifrus
2, András
Fenyvesi3, Barna Biró
3, Elke Beyreuther
4,5, Jörg Pawelke
4,5, Katalin Hideghéty
1
1ELI-ALPS, ELI-HU Non-Profit Ltd., Dugonics ter 13, Szeged 6720, Hungary
2Budapest University of Technology and Economics, Budapest, Hungary
3MTA-ATOMKI- Hungarian Academy of Sciences Institute for Nuclear Research, Debrecen, Hungary
4Helmholtz-Zentrum Dresden – Rossendorf, Dresden, Germany
5OncoRay – National Center for Radiation Research in Oncology, Faculty of Medicine and University
Hospital Carl Gustav Carus, Technische Universität Dresden, Germany
Purpose/objective: High power lasers provide the basis of particle acceleration, but at the actual
status of the development, low energy, limited size beams with special properties (ultrahigh dose rate,
pulsed mode) are available under technical conditions for radiobiology experiments.
Our main aim was to introduce and validate a vertebrate system for in vivo experiments to investigate
the biological effects of novel hadron beams.
Material/methods: Series of zebrafish embryos in 24 hour post fertilization ages in different holders
like tubes and 96 well plates varying the number (n) of embryos/well were prepared. For irradiation
we used fission neutron (0, 1.25, 1.875, 2, 2.5 Gy), cyclotron-based neutron (0, 2, 4, 6.8, 8.12, 10.28
Gy) and proton (0, 5, 10, 15, 20 and 30 Gy) at two positions along the proton depth-dose curve (at the
plateau and at the middle of Spread Out Bragg Peak), furthermore, with reference linear accelerator
photon (0, 5, 10, 15, 20 Gy) beams (n=96 in each group), repeated several times (≥3). Thereafter,
survival, any type of organ developmental disturbance (pericardial edema, spine curvature, shortening
of the body length and micro-opthalmia) were detected each days up to 7 days post irradiation (dpi).
Histological evaluation (size of the eye, brain necrosis, intestinal changes, liver vacuolization, hyper
eosinophilic necrotic muscle-fibers) and molecular changes were evaluated with RT-PCR method at
certain time points post irradiation.
Results: The RBE was highly sensitive in this system to time, dose and endpoints. The most robust
result could be revealed by survival analysis with RBE of definition on the base of LD50- s at the 5th to
7th dpi: RBE between 10 and 4.8 for the <En = 1 MeV> fission and <En> = 3.5 MeV cyclotron based
neutrons and around 1.1-1.4 for protons, respectively. The morphological distortions and its severity
exhibited a good agreement to the survival derived RBE with a narrow time and dose frame for the
different type (i.e. pericardial edema: 3 dpi 20 Gy, spine curvature 4 dpi 15 and 20 Gy). The gravity of
the histopathological changes on the basis of semi-quantitative analysis corresponded well to the
macro morphological abnormalities.
Conclusion: Numerous features of the zebrafish embryo model makes it amenable for large scale of
radiobiological investigations. On the basis of our experimental series, the optimal radiation setup,
radiation dose and observation time points for assessment of the different biological endpoints could
be established. This vertebrate model proved to be highly reproducible, reliable, and seems to be well
applicable for RBE determination.
Acknowledgements: The ELI-ALPS project (GINOP-2.3.6-15-2015-00001) is supported by the
European Union and co-financed by the European Regional Development Fund. The project has
received funding from the European Union's Horizon 2020 research and innovation program under
grant agreement no 654148 Laserlab-Europe and by the German BMBF, grant no. 03Z1N511.
European Radiation Research 2018, August 21-25, Pécs, Hungary
181
Ionizing radiation induces AML related changes in the cargo of
extracellular vesicles from blood and bone marrow
Tünde Szatmári, Rita Hargitai, Nikolett Sándor, Eszter Persa, Enikő Kis, Géza Sáfrány and
Katalin Lumniczky
National Public Health Centre, National Research Directorate for Radiobiology and
Radiohygiene, Div. of Radiation Medicine, Budapest, Hungary
Objectives: There are strong evidence pointing to a possible connection between
development of leukemias and exposure to ionizing radiation. Acute myeloid leukemia
(AML) is the most common radiation-induced leukemia observed in adults. Radiation-
induced damage to the hematopoietic stem cell pool is a major driver in the disease, but
communication between the tumor and its environment also contribute to the development of
the disease. Extracellular vesicles (EVs) are small membrane coated bodies released by the
cells into extracellular medium. They have an important role in intercellular communication
by carrying proteins and nucleic acids. Using an in vivo model we have recently demonstrated
that radiation-induced bystander effects can be mediated by EVs in the hematopoietic system.
Here we investigated the effects of radiation on bone marrow and blood-derived EV miRNA
profiles in the same model, and the possibility that EVs might transmit AML-related signals
both within the BM and in the blood.
MATERIALS AND METHODS: C57Bl/6 mice were total-body irradiated with 0.1 and
2 Gy, bone marrow- and plasma-derived EVs were isolated using Exoquick reagents. Total
RNA was isolated from these EVs and miRNA profile was determined by miR profiling
(Exiqon) and verified by real-time qPCR. To examine the biological function of these
miRNAs we performed a multiple miRNA effect analysis using bioinformatical tools.
RESULTS: In the EVs from bone marrow, we identified 8 miRNAs differentially expressed
following both low- and high-dose-irradiation and 27 pathways potentially regulated by these
miRNAs. In blood, there were 7 differentially expressed miRNAs in the 0.1 Gy group and 11
miRNAs in the 2 Gy group. Although there were no miRNAs affected by both doses in the
plasma, 26 signalling pathways were affected by both low and high dose irradiations.
Two miRNAs (miR-150-5p and miR-491-5p) were downregulated both in plasma and BM
EVs in mice following 2 Gy irradiation and none after 0.1Gy, but several pathways were
altered irrespective of the origin of EVs (BM or blood) or the dose. A part of these pathways
have already been associated with irradiation and bystander responses. Interestingly, AML
was one of the most affected pathways both in the peripheral blood and BM, regardless of the
applied dose. We analysed this pathway in more detail, and found that its major components,
such as Sos1, Ras GTPases, Raf kinases and Pi3K family members, as well as the major
transcription factors Runx1, and NFκB, are targets of differentially expressed miRNAs found
in EVs of both low and high dose irradiated mice.
CONCLUSION: In vivo low and high dose irradiation caused similar alterations in the
miRNA cargo of EVs and there is a substantial overlap in the pathways regulated by these
miRNAs. Our preliminary data show that the altered miRNA cargo of EVs regulates AML
associated pathways, but the results remain to be further validated by functional studies.
European Radiation Research 2018, August 21-25, Pécs, Hungary
182
Radiation-induced senescence: a possible mechanism of resistance
and a tumour-promoting pathway in NSCLC.
Antonella FS Tabasso1, Keyury Desai
1, 3, Ioannis Lamprou
1, Donald JL Jones
1,
Salvador Macip2 & George DD Jones
1
1 University of Leicester, Leicester Cancer Research Centre, Leicester, UK
2 University of Leicester, Department of Molecular & Cell Biology, Leicester, UK
3 Current Address: CRUK & UCL Cancer Trials Centre, 90 Tottenham Court Road, W1T
4TJ, UK.
Objective: Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide,
with radiotherapy (RT) being a common treatment option. However, despite continuous advances in
the delivery of RT, outcomes for the disease treated by this modality (either alone or combined with
chemotherapy) remain poor. Cellular senescence refers to the irreversible arrest of cell proliferation
that occurs when cells are exposed to damaging stress, including ionising radiation (IR). Senescence is
known to be a powerful tumour-suppressive mechanism so, initially at least, may be beneficial to
treatment outcome. However, senescent cells remain metabolically active and are able to secrete a
number of factors, collectively known as the senescence-associated secretory phenotype (SASP). A
number of these factors have tumour-promoting functions and so it is possible that an accumulation of
senescent cells post-RT will adversely affect RT efficacy. It is the aim of this study to characterise the
impact of radiation-induced senescent cells in NSCLC resistance to RT.
Results: H460 cells irradiated with 1-6 Gy showed an enlarged/flattened morphological appearance
coupled with positive SA-β-gal staining; low reproductive integrity coupled with high measures of
viability; and increased expression of p16 and CDKI-p21. All this confirmed extensive IR-induced
senescence (IRIS). Significant increases in wound healing activity and invasion noted for the CM from
IRIS cells indicates that their secretome had pro-proliferative activity on neighbouring cells.
Preliminary proteomic analysis in combination with unsupervised multivariate analysis (PCA) reveals
that IRIS cells were secreting more proteins than the non-irradiated cells and that these proteins
paralleled the exposure to radiation.
In further studies, the relative radiosensitivity of a panel of four NSCLC cell lines plus one normal
lung fibroblast cell line was tested over two weeks at several different doses of IR, using cell viability
and proliferation assays. The same panel were also investigated for the presence of IRIS under the
same conditions. We propose a potential relationship between the extent of IR-induced senescence and
the resistance of the cell lines to IR.
Conclusion: Our results demonstrate that clinically relevant doses of IR effectively induce senescence
in H460 cells and that IRIS H460 cells secrete pro-proliferative proteins. This may contribute to the
noted high level of radiotherapy failure.
European Radiation Research 2018, August 21-25, Pécs, Hungary
183
The REQUITE project: integrating biomarkers and clinical predictors of radiotherapy side
effects
CJ Talbot1, D Azria
4, T Burr
5, J Chang-Claude
2, A Dunning
14, C Herskind
2, D De Ruysscher
10, R
Elliott8, S Gutiérrez-Enríquez
9, P Lambin
10, A Müller
2, T Rancati
3, B Rosenstein
11, T Rattay
1, P
Seibold2, L Veldeman
12, A Vega
13, F Wenz
15, R Valdagni
3, A Webb
1, C West
8
1University of Leicester, Leicester;
2German Cancer Research Center (DKFZ), Heidelberg;
3Fondazione IRCCS
Istituto Nazionale dei Tumori, Milano; 4University of Montpellier, Montpellier;
5Source Bioscience, Manchester;
6The Christie NHS Foundation Trust, Manchester;
7University Hospitals Leuven/KU Leuven, Leuven ;
8University of Manchester, Manchester;
9Vall d’Hebron Institute of Oncology-VHIO, Barcelona;
10Stichting
Maastricht Radiation Oncology (Maastro), Maastricht; 11
Mount Sinai School of Medicine, New York; 12
Universiteit Gent, Gent; 13
Fundación Pública Galega Medicina Xenómica , Santiago de Compostela; 14
University of Cambridge; 15
University hospital Mannheim.
Corresponding author: cjt14@le.ac.uk
The European Union funded REQUITE consortium aims to validate predictive models of
radiotherapy-related adverse reactions. REQUITE is a multi-centre, observational study
(www.requite.eu). Enrolment was open for 2.5 years through 10 clinical centres. Follow-up is being
collected for two years ending in September 2018, with primary endpoints: change in breast
appearance (breast), rectal bleeding (prostate) and breathlessness (lung). 4442 patients have been
enrolled in REQUITE: 2071 breast, 562 lung and 1809 prostate cancer patients.
All patients who complete the study are being SNP genotyped using Infinium OncoArrays. RILA
was carried out on 1322 patients in three of the European centres using a standardised protocol. RILA
assesses the percentage of radiation-induced apoptosis in lymphocytes, detected by flow cytometry.
The levels of apoptosis range from 2.4% to 62.4%, confirming large inter-patient variability. Factors
that affect RILA have been identified, including cancer type and smoking status. Preliminary analysis
has shown that RILA predicts acute breast pain and in prostate patients urinary toxicity at one year. A
pilot RNA sequencing experiment has been carried out using 50 lung cancer cases.
18 sub-studies have been approved for use of the REQUITE data and/or samples to address a
number of important questions e.g. the role of mitochondrial DNA, circadian rhythm effects, effect of
integral dose on fatigue, modelling of the α/β ratio for prostate toxicity, exploring patient attitudes to
predictive testing.
This large scale prospective observational study will be the largest to date to assess the use of
predictive biomarkers for assessing radiotherapy related toxicity.
European Radiation Research 2018, August 21-25, Pécs, Hungary
184
Potential mechanisms contributing to radiation-induced heart
disease
Soile Tapio
Helmholtz Zentrum München, German Research Center for Environmental Health GmbH,
Institute of Radiation Biology, Neuherberg, Germany,
Epidemiological data indicate that exposure to ionizing radiation increases the risk of cardiovascular
mortality and morbidity in a moderate but significant manner. Two main radiation targets have been
identified in the heart: the vascular endothelium and the myocardium (heart muscle). Ionizing
radiation causes persistent endothelial alterations characterised by inflammation and early senescence
in vitro and in vivo. Irradiated endothelial cells communicate with surrounding non-irradiated cells by
secreting inflammatory and senescence-associated proteins that trigger activation of STAT-mediated
pathways in the bystander cells. In the myocardium, ionizing radiation induces alterations in lipid
metabolism, glycolysis, and mitochondrial function (respiration, ROS production). In these energy-
related processes a dose- and dose-rate dependent activation or inactivation of peroxisome
proliferator-activated receptor alpha (PPAR alpha) plays a central regulatory role. Countermeasures to
mitigate negative effects of ionizing radiation to the heart are discussed.
European Radiation Research 2018, August 21-25, Pécs, Hungary
185
In vitro neuronal differentiation leads to enhanced cellular
vulnerability to genotoxic stress induced by UV-irradiation
Mihaela Temelie1, Mustaciosu Cosmin
1, Nicoleta Moisoi
2and Diana Savu
1
1 “Horia Hulubei” National Insitute of Physics and Nuclear Engineering, Department of
Life and Environmental Physics, Magurele, Romania 2De Montfort University, Leicester School of Pharmacy, Faculty of Health Sciences,
Leicester, UK
Introduction: Accumulation of DNA damage is a common hallmark of brain aging and it is
associated with neurodegenerative processes. Brain cells are exposed to genotoxic factors throughout
the life span either from natural or unwanted exposure to physical and chemical agents or due to
medical irradiation or drug treatments. The DNA damage accumulation appears to be dependent on
cell types with neuronal cells being more vulnerable in the brain. This is often correlated with
enhanced neurodegenerative processes.
Objective: Our work uses a human neuroblastoma cell line known to differentiate towards
dopaminergic neurons in order to analyse the cellular vulnerability to UV-induced genotoxic stress in
relationship with neuronal differentiation, and the mechanisms involved.
Methods: The study was performed in SH-SY5Y human neuroblastoma cells commonly used as a
cellular model to investigate neurodegenerative processes related to Parkinson’s disease Cellular
differentiation was done by a well characterised method involving serum deprivation (1%) and
addition of Retinoic Acid (RA). Cell were characterised by morphological observation and
immunofluorescence using beta-III-tubulin. Genotoxic stress was induced by UV-C exposure. Cellular
and genotoxic stress was determined by several methods (MTS, caspase induction, morphological
apoptosis, ROS measurement, ATP levels, γ-H2AX/53BP1 foci formation, alkaline comet assay).
Mechanisms of cellular stress response were analysed by qRT-PCR.
Results: Cellular differentiation of SH-SY5Y neuroblastoma line using RA and serum deprivation
lead to a higher sensitivity to genotoxicity induced by UV-C exposure. Differentiated cells presented a
lower viability accompanied by increase of apoptosis markers and reactive species, together with an
increase of DNA damage (as measured by DSB associated foci and comet assay). Cellular stress
response showed different response to genotoxic stress in relation with cellular phenotype.
Conclusion: Neuronal differentiation of SH-SY5Y cells lead to a higher vulnerability to genotoxic
stress induced by UV-C.
European Radiation Research 2018, August 21-25, Pécs, Hungary
186
Intracellular and intercellular signaling following DNA damage is
modulated by the mitochondrial kinase PINK1
MihaelaTemelie1, Nicoleta Moisoi
2, Diana Iulia Savu
1
1Horia Hulubei National Institute of Physics and Nuclear Engineering, Department of Life
and Environmental Physics,Magurele-Bucharest, Romania 2 De Montfort University, Leicester School of Pharmacy, Faculty of Health Sciences,
Leicester, UK
Introduction: Impaired mitochondrial function and accumulation of DNA damage have been
recognized as hallmarks of aging and age related diseases. Along with maintenance of
mitochondria function, preserving nuclear DNA integrity has a pivotal role in determining the
fate of the cells challenged throughout the life with endogenous threats (ROS, DNA repair
errors) as well as exogenous stress comprising physical and chemical agents.
Objective: Here we have initiated a study that addresses how mitochondria nucleus
communication may occur in conditions of combined mitochondrial dysfunction and
genotoxic stress induced by ionizing radiation and radiomimetics and what are the
consequences of this interaction on the cell system.
Methods: In this work, we used cells deficient for PINK1, a mitochondrial kinase involved in
mitochondria quality control whose loss of function leads to accumulation of dysfunctional
mitochondria, challenged with inducers of DNA damage namely X-rays and bleomycin.
Results: Combined stress at the level of mitochondria and the nucleus impairs both
mitochondrial and nuclear functions. Our findings revealed exacerbated sensibility to
genotoxic stress in PINK1 deficient cells. The same cells showed an impaired induction of
bystander phenomena following stress insults. Instead, these cells respond adaptively when a
challenge dose is applied subsequently to a low dose treatment to the cells.
Conclusion: The data demonstrates that PINK1 modulates intracellular and intercellular
signalling pathways particularly adaptive responses and transmission of bystander signaling,
two facets of the cell protective mechanisms against detrimental agents.
European Radiation Research 2018, August 21-25, Pécs, Hungary
187
TransExpo: International study of childhood leukemia and
residences near electrical transformer rooms
György Thuróczy1, Nagy Noémi
1, Gábor Jánossy
1 and Zsuzsanna Jakab
2
1National Public Health Institute, Department of Non-Ionizing Radiation, Budapest,
Hungary 2Hungarian Pediatric Cancer Registry, 2nd. Department of Pediatrics, Semmelweis
University, Budapest
To reduce the scientific uncertainty surrounding the epidemiologic association between extremely low
frequency magnetic fields (ELF-MF) and childhood leukemia, new approaches in epidemiology are
required. Childhood leukemia and average exposures to ELF-MF above 0.3/0.4 μT are both quite rare.
Epidemiologic studies are needed that are designed to minimize biases from different sources and
maximize the ability to detect an association, should one exist.
The appeal of the study design is its selection of study subjects (affected or not by the disease of
interest) from similar environments and its subsequent ascertainment of exposure status based on
residential location, which does not require subject participation. The TransExpo study is meant to
avoid certain control selection challenges and participation biases plaguing previous ELF-MF studies,
while focusing on a population with higher than average exposure to ELF-MF. Pilot work results
completed in several countries demonstrate that protocol classification of ELF-MF exposure based on
apartment location is feasible with remarkable specificity and sensitivity. The full study will require
additional country participation to achieve a sufficient sample size and ensure adequate precision.
In Hungary there are many multi-level residential buildings where transformer stations are built
inside the buildings, usually on the first floor or in the basement, with an apartment typically located
directly above the transformer station. According to our estimation approximately 1500-2000
apartments are above and/or neighbouring situation to the transformer chambers. Hungary also has a
reliable childhood cancer registry and population registry which allows it to participate in such an
international study. To confirm that the location of transformers can reliably be used to categorize MF
exposure with sufficient accuracy we conducted this measurement study.
In Hungarian cities 50 Hz magnetic field exposure in several multi-level apartment buildings with
built-in step-down transformer stations was evaluated. In each building, minimum three apartments
were selected; one apartment located immediately above the transformer room (index apartment), one
located on the same floor, and one on a higher floor. The mean value of measured magnetic fields was
0.48 T in apartments above transformers, 0.10 T on the same floor, and 0.05 T in on higher floors.
We conclude that apartments in building with built-in transformers can be reliably classified into high
and low exposure categories based on their location in relation to transformers.
European Radiation Research 2018, August 21-25, Pécs, Hungary
188
Response of isogenic head and neck cancer cell sublines to
ionizing irradiation
Vesna Todorovic1, Ajda Prevc
1, Martina Niksic Zakelj
1, Primoz Strojan
2,3, Maja
Cemazar1,4
and Gregor Sersa1,5
1 Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana,
Slovenia 2 Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia
3 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
4 University of Primorska, Faculty of Health Sciences, Izola, Slovenia
5 University of Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia
Radiotherapy is one of the standard treatment modalities for head and neck cancer. However,
locoregional tumor control remains a significant problem. Management of recurrent tumors,
specifically in previously irradiated area, is limited to second course of radiotherapy (re-irradiation)
for many reasons, including radioresistance of the recurrent tumor.
Tumor radioresistance, intrinsic or acquired, is an important cause of treatment failure. Improved
understanding of the mechanisms underlying the radioresistant phenotype (i.e. intrinsic
radioresistance) can be achieved by direct comparison of isogenic cell lines with different
radiosensitivity levels in which the emergence of radioresistance can be directly attributed to specific
biological response.
Therefore, our objective was to establish a radioresistant subline from parental FaDu cells (FaDu-RR)
after exposure to repeated irradiation (total dose received 120 Gy) and to evaluate the response of the
newly established subline FaDu-RR to ionizing radiation by clonogenic assay. In addition,
chemosensitivity to cisplatin, oxaliplatin and bleomycin, cell cycle distribution, H2AX
immunofluorescence and gene expression analysis of genes involved in DNA damage signaling (RT2
ProfilerTM
PCR Array Human DNA Damage Signaling Pathway) were explored to further characterize
the response of radioresistant FaDu-RR subline in comparison to parental FaDu cells.
Newly established FaDu-RR cells were significantly more radioresistant than parental FaDu cells. The
half-maximal effective dose (ED50) for FaDu-RR was 2.6 Gy, resulting in 1.6 dose-modifying factor in
comparison to parental FaDu cells (ED50 1.6 Gy). In addition, FaDu-RR cells were also cross-resistant
to cisplatin (1.8-fold potentiation in half-maximal inhibitory concentration (IC50) value) and in lesser
extent to oxaliplatin (1.5-fold potentiation in IC50 value), but not to bleomycin (0.95-fold potentiation
in IC50 value). No difference in cell cycle distribution after 5 Gy irradiation was observed. In both,
FaDu and FaDu-RR cells, percentage of cells in G1 and G2 phase was increased, while percentage of
cells in S phase was decreased. Temporal expression of H2AX foci, markers of DNA double-strand
breaks, differed significantly. Namely, a peak of H2AX foci in FaDu-RR cells was observed 30
2 hours after irradiation. In addition, less H2AX foci/nuclei and lower percentage of H2AX-positive
cells was observed in FaDu-RR cells. Basal gene expression of DNA damage signaling-associated
genes differed between FaDu and FaDu-RR cells with 13 genes under-expressed in FaDu-RR cells. In
response to 5 Gy irradiation, 5 genes were over-expressed in FaDu, while in FaDu-RR 12 genes were
over-expressed and 2 genes were under-expressed. Majority of these genes were involved in
ATM/ATR signaling and DNA double-strand break repair.
In conclusion, the emergence of radioresistance in FaDu-RR cells after repeated exposure to ionizing
radiation was associated also with chemoresistance to cisplatin and oxaliplatin (but not to bleomycin),
reduced susceptibility to formation of H2AX foci and more efficient DNA double-strand break repair.
European Radiation Research 2018, August 21-25, Pécs, Hungary
189
Acute and late consequences of partial brain irradiation in rats
Tünde Tőkés1,5
, Imola Plangár2, Emília Rita Szabó
1, Imola Mán
3, Kitti Brinyiczki
4, Mihály
Boros5, Katalin Hideghéty
1,6
1ELI-HU Non-profit Ltd., Szeged, Hungary
2MTA-KOKI, Neuronal Signaling Group, Budapest, Hungary
3Avidin Ltd., Szeged, Hungary
4University of Szeged, Department of Pathology, Szeged, Hungary
5University of Szeged, Institute of Surgical Research, Szeged, Hungary 6University of Szeged, Department of Oncotherapy, Szeged, Hungary
Objective: Radiotherapy plays major role in the treatment of brain tumors. Our goals were to detect
the early, possible biochemical signs of peripheral inflammatory activation, and to examine the late
histological consequences of hippocampus irradiation in an in vivo rat model. As an additional aim, we
investigated the possible protective effects of the L-alpha-glycerylphosphorylcholine (GPC) in this
experimental protocol.
Methods: Anesthetized Sprague-Dawley rats were subjected to 40 Gy irradiation of the hippocampus,
with or without GPC treatment. Other groups served as saline-treated control (n = 6, each). Blood
samples were obtained 3 h after the end of irradiation in order to examine the early changes in tumor
necrosis factor-alpha (TNF-α), interleukin 1-beta, interleukin 6 (IL-6) and interleukin 10 (IL-10); and
liver tissue samples were taken to determine adenosine triphosphate (ATP) concentrations. To
determine the late effects of this hippocampus irradiation, Morris water maze (MWM) behavioural test
was used to examine the spatial orientation and learning ability of the rats, and histopathological (HP,
hematoxylin-eosin staining) evaluation was performed 4 months after irradiation.
Results: The hepatic ATP levels were significantly diminished, while plasma concentrations of
circulating TNF-α, IL-6, IL-10 were significantly increased after hippocampus irradiation. GPC
treatment significantly reduced the irradiation-induced release of cytokines, while the liver ATP level
was maintained at the control value. The 40 Gy irradiation resulted in a moderate neurological deficit
at the levels of both cognitive function and morphology 4 months after the irradiation. The site
navigation of the rats was impaired by the irradiation, but the GPC treatment markedly decreased the
cognitive impairment. HP examination revealed lesser amounts of macrophage density, reactive
gliosis, and necrosis in the GPC-treated group.
Conclusions: Targeted brain irradiation produced measurable pro- and anti-inflammatory cytokine
changes in the systemic circulation. GPC supplementation provided significant protection against
irradiation-induced peripheral pro-inflammatory activation and ATP depletion. GPC treatment led to
significant protection against the cognitive decline and cellular damage, evoked by focal brain
irradiation at 40 Gy dose level.
Supports: The ELI-ALPS project (GINOP-2.3.6-15-2015-00001) is supported by the European Union
and co-financed by the European Regional Development Fund. The project has received funding from
the European Union's Horizon 2020 research and innovation programme under grant agreement no
654148 Laserlab-Europe.
European Radiation Research 2018, August 21-25, Pécs, Hungary
190
Combined effects of irradiation in a dose of 1.0 Gy and
magnetic field of industrial frequency (50Hz) on cells death sperm
of rat
Alena Tsukanava, Natalia Chueshova, Ihar Cheshyk, Natalia Veyalkina
1 Institute of Radiobiology of National Academy of Sciences of Belarus,
Gomel, Belarus Well known that radiation and magnetic field influence first affects mitotically active cells.
Spermatogenic epithelium is one of the most radiosensitive tissues of the body.
There are various evidences showing that magnetic fields could affect on reactive oxygen
species and apoptosis/necrosis induced by ionizing radiation.
The aim of this study was to estimate the effect of isolated and combined action of magnetic
fields of industrial frequency and acute single ionizing radiation dose of 1.0 Gy on the number of
apoptotic and necrotic epididymal sperm of rats on the 3, 15 and 30th day after exposure.
Materials and methods. Male Wistar rats (4,5 - weeks-old) were irradiated of magnetic fields
of industrial frequency (MF 50 Hz, 0,5 mT, 4 h/day, 5 day/week, for a total of 30 days) and/or once at
a dose of 1 Gy to the whole body. Then rats were sacrificed 3 days, 2 and 4 weeks after irradiation and
rats, which were not exposed to irradiation were used as controls. At each time point, six rats of each
group were sacrificed and their epididymal sperm samples were harvested for analysis. Analysis using
Annexin V and propidium iodide (PI) was carried out by means of the cytofluorimeter. Mann–
Whitney U test was used to investigate the significance of difference between the groups.
Results. There was the 10% decrease in viability of epidimal spermcells after influence of MF
50 Hz, in 20 % after influence of 1 Gy and in 26% after combined influence of MF 50 Hz+1 Gy.
The number of apoptotic sperm cells increased in 3 day after influence of 1 Gy and MF 50
Hz+1 Gy only. Wheras the number of necrotic sperm cells was increased in all studied groups.
There were increase in apoptotic cells in all groups at 15th day after exposure, which reached
2,85% after 50 Hz influence, 3,68% after 1Gy influence and 2,72% after combined influence at 30th
day. In control group the proportion was 0,67% at 30th day.
The level of necrotic sperm cells at 15th day also was increased and it reached 2,75% after 50
Hz influence, 3,85% after 1Gy influence and 4,67% after combined influence at 30th day. In control
group the proportion was 1,0% at 30th day.
Conclusion. It was found that prolonged exposure MF (50 Hz) causes a significantly increases
sperm death by necrosis. After irradiation at a dose 1.0 Gy observed marked deterioration of the
studied quantitative and qualitative indicators of spermatozoa epididymal, thus affecting the viability
and cells death. The combined effect of anthropogenic factors also leads to significant deviations of
the studied parameters of the reproductive system, which is greater than the effect of each of the
studied factors separately.
European Radiation Research 2018, August 21-25, Pécs, Hungary
191
Comparative gene expression analysis after exposure to
123
I-iododeoxyuridine, γ- and α-irradiation
– a possible tool for biodosimetry?
Marcus Unverricht-Yeboah1, Ulrich Giesen
2, Ralf Kriehuber
1
1
Radiation Biology Unit, Department of Safety and Radiation Protection,
Forschungszentrum Jülich, D-52425 Jülich, Germany 2
Physikalisch-Technische Bundesanstalt (PTB), D-38116 Braunschweig, Germany
Gene expression analysis was carried out in Jurkat cells in order to identify candidate genes showing
significant gene expression alterations allowing robust discrimination of the Auger emitter 123
I,
incorporated into the DNA as 123
I-iododeoxyuridine (123
IUdR), from α- and γ-radiation.
The γ-H2AX foci assay was used to determine equi-effect doses or activity, and gene expression
analysis was carried out at similar levels of foci induction. Comparative gene expression analysis was
performed employing whole human genome DNA microarrays. Candidate genes had to show
significant expression changes and no altered gene regulation or opposite regulation after exposure to
the radiation quality to be compared. The gene expression of all candidate genes was validated by
quantitative real-time PCR.
The functional categorization of significantly deregulated genes revealed that chromatin organization
and apoptosis were generally affected. After exposure to 123
IUdR, α-particles and γ-rays, at equi-effect
doses/activity, 155, 316 and 982 genes were exclusively regulated, respectively. Applying the
stringent requirements for candidate genes, four (PPP1R14C, TNFAIP8L1, DNAJC1 and PRTFDC1),
one (KLF10) and one (TNFAIP8L1) gene(s) were identified, respectively allowing reliable
discrimination between γ- and 123
IUdR exposure, γ- and α-radiation, and α- and 123
IUdR exposure,
respectively. The Auger emitter 123
I induced specific gene expression patterns in Jurkat cells when
compared with γ- and α-irradiation, suggesting a unique cellular response after 123
IUdR exposure.
Gene expression analysis might be an effective tool for identifying biomarkers for discriminating
different radiation qualities and, furthermore, might help to explain the varying biological
effectiveness at the mechanistic level.
Funded by Bundesministerium für Bildung und Forschung (BMBF),
Project No.: 02NUK005A and 02NUK043A
European Radiation Research 2018, August 21-25, Pécs, Hungary
192
Oxidative stress and VEGF expression post-irradiation are
modulated by iodine deficiency in breast cells
Jessica Vanderstraeten1, Jasmine Buset
2, Naziha Ben Said
1, Bjorn Baselet
2, Marie-
Christine Many1, Sarah Baatout
2, Anne-Catherine Gérard
3, Hanane Derradji
2
1Pole of Morphology, Catholic University of Louvain, Brussels;
3Radiobiology Unit,
Belgian Nuclear Research Centre, Mol; 2
Department of Endocrinology and Diabetology,
Regional Hospital Center, Mons-Hainaut, Belgium
Background and objectives: Breast is a sodium/iodide symporter (NIS)-expressing organ which is
sensitive to iodine deficiency (ID) and radiation. ID, which still affects almost 2 billion people
worldwide, induces structural and functional breast alterations, and high iodine intake was negatively
linked to breast fibrosis and cancer. Besides, breast cancer risk was shown to be increased after
radiation exposure by several retrospective studies. However, while the effects of high doses of
electromagnetic radiations on cancer progression are largely documented, low doses effects are still
debated. In addition, radiations and ID can both induce oxidative stress (OS) and VEGF (vascular
endothelial growth factor)-dependent vascular responses in breast and other NIS-expressing organs.
Therefore, the objective of this work is to compare the effects of low and high doses of radiation on
OS, antioxidant system and VEGF regulation in breast cells and to determine whether these effects
could be amplified in ID conditions.
Methods: ID was induced by medium change in MCF7 (cancerous) and MCF12A (non-cancerous)
breast cell lines. Cells were then X-irradiated with 0.05, 0.1, 1, or 3 Gy doses. Cells were harvested 2
to 24 hours after medium change. mRNA and protein expression were determined by RT-qPCR and
western blot respectively. OS was assessed through immunofluorescent 4-HNE staining. Reactive
oxygen species (ROS) content was measured by flow cytometry using the DCFDA dye.
Results: ID and radiation doses from 0.1 to 3 Gy separately increased VEGF mRNA and protein from
3 to 4 h after medium change in MCF12A cells. In MCF7 cells, however, ID alone or combined with
radiations increased VEGF mRNA and protein expression after 6 h, while radiations alone did not. An
additive effect on VEGF expression, ROS production and OS was observed when a dose of 3 Gy of X-
rays was combined with ID only in MCF12A cells. Interestingly, the treatments influenced alternative
splicing in both cell lines as the observations on total VEGF mRNA hold true for the pro-angiogenic
isoform VEGF165 mRNA, while the anti-angiogenic isoform VEGF 165b was not modulated by the
treatments. ROS inhibition by N-acetylcysteine or diphenyleneiodonium prevented VEGF mRNA up-
regulation in both cell lines. However, the specific inhibition of mitochondrial ROS by mitoTEMPO
prevented VEGF up-regulation induced by radiations alone or combined with ID in MCF12A cells,
but not the effects of ID alone in both cell lines. Moreover, low doses of X-rays induced a stronger
antioxidant defence response than higher doses. Indeed, exposure to 0.1 Gy of X-rays increased
superoxide dismutase (SOD) 1 and SOD2 mRNA expression in both cell lines in ID conditions, while
exposure to 3 Gy of X-rays only increased SOD1 mRNA in iodide-deficient MCF12A cells. Catalase
mRNA remained unchanged in both cell lines.
Conclusions: Radiations differentially regulate ROS and VEGF expression in breast cells and can have
additive effects with ID according to the dose and cell type. Moreover, radiations and ID have additive
effects on antioxidant response, which are stronger with lower doses of X-rays, and which may
potentially lead to radiation effect regulation. Because ROS and VEGF are often involved in cancer
initiation and progression, a better understanding of their regulation could help improve radiation
prevention policies and/or cancer radiotherapy. Therefore, iodine status should not be ignored before
exposure to low and high doses of radiation.
European Radiation Research 2018, August 21-25, Pécs, Hungary
193
Receptor mediated radiosensitivity and DNA repair capacity of
breast cancer cell lines
Stephanie Vermeulen1,2
, Anne Vral1,2
, Ans Baeyens1,2
1 Ghent University, Department Radiobiology, Ghent, Belgium
2 Cancer Research Institute Ghent, Ghent, Belgium
Objective
Breast cancers are considered as either hormone sensitive or hormone insensitive based on the
expression of estrogen receptors (ER) and progesterone receptors (PR). Breast cancers that are
negative for the hormone receptors and the human epidermal growth factor receptor (HER2) are
considered triple negative breast cancers (TNBC). The presence or absence of these receptors will help
determine which treatment options are available for the patient. The ER has two isoforms: estrogen
receptor alpha (ERα) and estrogen receptor beta (ERβ). Depending on the present ER (ERα or ERβ),
different biological effects in the cell occur. ERα stimulates cell growth whereas ERβ stimulates
apoptosis and acts as a tumor suppressor. Normal breast tissue displays relatively higher expression of
ERβ than ERα, which drastically changes during breast tumorigenesis. A recent in vitro study of our
research group showed that lymphocytes of ER positive breast cancer patients had higher
chromosomal radiosensitivity values while lymphocytes of TNBC patients presented with the lowest
radiosensitivity values. However, the exact role of the ER in radiosensitivity remains unclear. We
aimed to investigate the link between ERα and ERβ and the radiosensitivity in hormone sensitive
breast cancer cell lines.
Methods
Two breast cancer cell lines (MCF-7 and MDA-MB-231) and a non-tumorigenic cell line MCF10A
were irradiated with 220 kV X-rays with doses ranging from 0 Gy to 4 Gy. Subsequently the
micronucleus assay was performed to evaluate chromosomal damage. The fixed cells were stained
with acridin orange and manually counted using fluorescence microscopy. Expression of ERα and
ERβ was demonstrated by immunocytochemistry/immunofluorescence.
Results and conclusions
The hormone sensitive breast cancer cell line MCF-7 showed more radiation-induced micronuclei than
hormone insensitive cell lines MCF10A and MDA-MB-231. These results confirm that hormone
positive breast cancers are more radiosensitive than TNBC cells. The radiosensitivity could be linked
with the ER status.
European Radiation Research 2018, August 21-25, Pécs, Hungary
194
Hippocampal aging following early-life X-ray exposure
Verslegers M.1, Emma Coninx
1,2, Peter Verstraelen
3, Neefs M.
1, De Vos W.H.
3,4, Moons
L.2
, Quintens R.1, Baatout S.
1,
1Radiobiology Unit, Institute for Environment, Health and Safety, Belgian Nuclear
Research Centre (SCK•CEN), Boeretang 200, 2400, Mol, Belgium 2Neural Circuit Development and Regeneration Research Group, Department of Biology,
University of Leuven, Naamsestraat 61 box 2464, 3000, Leuven, Belgium 3Laboratory of Cell Biology & Histology, Faculty of Pharmaceutical, Biomedical and
Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610, Antwerp, Belgium 4Cell Systems and Imaging, Faculty of Bioscience Engineering, University of Ghent,
Coupure Links 653, 900 Ghent
Objective:
Cranial radiotherapy is inevitable in the treatment of pediatric brain tumors, the second most common
childhood cancer. Due to therapeutic advances, the survival rates of brain cancer patients have
increased. However, the hypersensitivity of the developing brain to ionizing radiation exposure may
increase the risk for developing long-term cognitive defects, especially in patients predisposed to
developing neurodegenerative diseases. The exact mechanisms underlying this potential hazard have
remained largely unexplored. Cognitive functions, including memory and learning, are partly ascribed
to a small part of the mammalian brain, namely the hippocampus. In neurodegenerative diseases, such
as Alzheimer’s disease (AD), the hippocampus is first and most severely affected leading to memory
decline. Hence, we are evaluating hippocampal maturation and function in triple transgenic Alzheimer
disease (3xTg-AD) mice after early-life irradiation.
Methods:
Ten-day-old female 3xTg-AD mice are exposed to 1.8 Gy X-rays. Acute (DNA damage, oxidative
stress) and chronic (disrupted neurogenesis, inflammation, Aβ/tau pathology) radiation-induced effects
in the hippocampus are examined. In vivo analyses are complemented with analyses of in vitro aged
(sham-) irradiated primary mouse hippocampal neurons.
Results:
Before assessing the radiation impact, hippocampal aging processes were characterized in vitro. Live-
cell calcium imaging revealed that synchronous bursting increased up to 18 days in vitro (DIV) and
gradually deteriorated around 48 DIV. In a second trial antioxidants will be removed from the cell
medium, potentially accelerating the degeneration of the neuronal network. In the 3xTg-AD brain
slices an increased amount of DNA damage was observed in the dentate gyrus 1h after X-ray
exposure. Furthermore, quantitative histochemical analyses revealed a radiation-induced reduction of
neuronal precursors in the dentate gyrus of 3- and 6-month old mice. Finally, the hippocampus of 6-
month old mice showed increased phosphorylated-tau levels after radiation exposure. Currently,
cognitive function is investigated in 6-month old (sham)-irradiated 3xTg-AD mice by means of a
dedicated behavioral test battery.
Conclusion:
Our results provide better insights in the aging process of the hippocampus and how early radiation
can accelerate neurodegeneration. This could represent a first step towards an improved (radio-
)protection and follow-up of children receiving cranial radiotherapy.
European Radiation Research 2018, August 21-25, Pécs, Hungary
195
Evaluation of the genotoxicity of UV radiation by the
micronucleus test on rats keratinocytes
N.N. Veyalkina
1, K.N. Shafarost
1
1 SSI "Institute of Radiobiology of the NAS of Belarus",laboratory of experimental
biological models, Gomel, Belarus
Ultraviolet radiation is a constantly acting environmental factor, which has a powerful
effect on many physiological processes occurring in living organisms [1]. Protecting the skin
from the negative effects of UV-irradiation are very relevant problem. Despite the fact that the
share of UV in solar radiation reaching the ground is about 10%, the photobiological
processes initiated by UV rays are multiple. Hyperinsolation can provoke the onset,
aggravation and complication of the course of a number of diseases [1, 2].
Objective. The aim of this research was to study UV genotoxicity on rats skin
keratinocytes in vivo by a micronucleus test.
Methods. The experiment was performed on female Wistar white rats aged 2-2.5
months. At least 24 hours before irradiation, the animals were shaved with a razor on the back
section 4x4 cm in size.
The source of UV radiation (UV-B) was 4 USHIOUV-B 8W lamps (313 nm energy
maximum). The integrated power of the light flux 200-400 nm measured at a distance of 15
cm from the light source was 1253,1 μW/cm2. UV lamps were located at a distance of 15 cm
from the back of the animal. The duration of irradiation was 15 and 30 minutes for the first
and second groups of animals, the control animals were in similar conditions and were
subjected to all manipulations as well as the experimental ones, with the exception of UV
irradiation.
Animals were removed from the experiment on the 4th day after irradiation with deep
ethereal anesthesia and skin samples were taken for further research. Preparation of cell
suspensions and cytological preparations was carried out according T. Nishikawa et al. [3]
with some modifications.
Results. After 15 and 30 minutes UV irradiation was marked moderately expressed or
expressed skin erythema and the skin fold thickness increasing. In the subcutaneous tissue
was observed the expansion and fullness of the capillary network.
In UV-B-irradiated groups occurr an increase in the number of cells with micronuclei,
in comparison with the control. In the group of animals irradiated for 15 minutes, the
percentage of cells with microkernels averaged 0,19% (P25-P75 – 0,18-0,20). When the
animals are irradiated for 30 minutes, the sharp increase in this indicator to 0,49% (P25-P75 –
0,40-0,60).
Conclusion. Thus, photoinduced increases in the level of cytogenetic disorders in
keratinocytes of Wistar rats skin have been shown, which is expressed in an increase in the
number of cells with micronuclei, depending on the time of exposure.
Literature 1. Skin DNA photodamage and its biological consequences / L. Marrot [et al.] // Spine. – 2008. – Vol. 58, №
5. – P. 139–148.
2. Potapenko, A.Y. The effect of light on humans and animals / A. Y. Potapenko // Soros Educational
Journal. – 1996. – P. 20.
3. Study of a rat skin in vivo micronucleus test: data generated by mitomycin C and methyl methanesulfonate
/ T. Nishikawa [et. al.] // Mutation Research. – 1999. – Vol.444. – Р. 159–166.
European Radiation Research 2018, August 21-25, Pécs, Hungary
196
Analysis of DNA Repair Genes and Oxidative Stress in the
Murine Brain Following Acute and Chronic Ionizing Radiation
Exposure
Charlotte Webster, Ali Pour Khavari, Paulo Godoy and Siamak Haghdoost
Department of Molecular Bioscience, The Wenner-Gren Institute, Svante Arrheniusvag 20C Stockholm University, 10691 Stockholm, Sweden
I have been investigating the effect of ionizing radiation upon the murine brain. This is being
performed by exposing mice to a Cs137 source for either a dose of 100 mGy or 1Gy (along with
respective controls). The mice were further divided up into 2 groups; mice that were exposed
chronically (1.4 mGy/hour) to the radiation and mice that were exposed acutely (6Gy/sec) to the
radiation. The mice were then split into two categories, mice that were sacrificed after 3 hours post
radiation and mice that were sacrificed 21 days post radiation, to observe for any prolonged effects.
The analysis of the expression of 7 genes (SOD1, SOD2, MTH1, DNA PKcs, Keap1, NRF2 and
GSTO1) that are related to combatting oxidative stress and DNA repair was performed via qPCR.
Following testing and the performing of t tests it was concluded that there was no significant
difference between the acute and chronically irradiated samples. Furthermore, it was seen that there
were no significant patterns or trends within the 100 mGy irradiated samples. Although, when looking
at the 100 mGy and 1Gy samples there appeared to be elevated oxidative stress of the irradiation at 21
days. This indicates that radiation lead to long-term (3 weeks) oxidative stress. From the previous
studies, we plan to continue the research to investigate more genes that are known players in the
reduction of oxidative stress and DNA repair and potentially observe if there are any continued effects
of radiation at a later time point than 21 days.
European Radiation Research 2018, August 21-25, Pécs, Hungary
197
Establishment of a Psoriatic Skin Model for α-Irradiation
Julia Wiedemann1, Valeria Grünebaum
1, Maximilian Dornhecker
1,
Sylvie Lerchl1 and Claudia Fournier
1,2
1 GSI Helmholtzzentrum für Schwerionenforschung, Darmstadt, Germany
2 University of Applied Sciences, Darmstadt, Germany
Objectives
Psoriasis is a chronic inflammatory skin disease which affects more than 25 million of people in North
America and Europe. In general the disease is due to an overshooting and persisting activation of the
immune system. Psoriasis is on the indication list for radon treatment. Positive effects are reported but
sparely documented and the mechanisms underlying the clinical benefit are unknown.
To investigate effects of radon treatment we establish several model systems for psoriasis on different
levels of complexity, e.g on cellular or tissue level, which can be used for irradiation with α-particles
or for radon-exposure. For the preclinical cellular model we present here human primary keratinocytes
are used and a psoriasiform expression pattern of several markers is induced with a cytokine
combination. Due to the short range of α-particles when penetrating tissue or a cellular monolayer this
model system must meet special requirements for irradiation and cell culture had to been adapted. As
psoriasis is often treated with UV irradiation we exposed the model system to UV irradiation for
validation.
Methods
Special rings stringed with a 2 µm boPET foil are used to minimize the space between cells and the α-
source. The boPET foil is treated with oxygen plasma in order to facilitate the attachment and
proliferation of the primary keratinocytes (NHEK; normal human epidermal keratinocytes). Cells were
cultured in rings or culture dishes for 24 hours and induced with IL-17 (200 ng/ml), IL-22 (20 ng/ml)
and TNF-α (10 ng/ml). Some samples were exposed to 20 mJ/cm² of UV-B. Supernatants and cells for
protein and mRNA extraction were collected 24 hours after induction. ELISA, qPCR and Western
Blot analysis was performed.
Results
We could show that it is possible to culture primary keratinocytes on plasma treated boPET foil. The
morphology of the cells is similar to cells cultured in standard cell culture dishes. Furthermore, the
selected cytokines are able to significantly induce psoriasis-related markers e.g. IL-19 and BDEF2 on
mRNA level and the release of the cytokine IL-6: Testing if culturing of NHEK on the treated boPET
foil alone has an inflammatory effect revealed no significant differences in the expression or release of
markers compared to cells cultured in standard cell culture dishes. After UV-B exposure the selected
psoriasis markers are reduced but do not reach the control level again.
Conclusion
We conclude that the plasma treated boPET foil offers a promising option for a setup, which enables
α-irradiation of monolayer cell cultures. The induction of a psoriasis-like phenotype with the selected
cytokines is successful and leads to an enhancement of relevant markers. Further we could show that
the model system is sensitive to UV-B exposure which is used for psoriasis therapy. First results of
ongoing alpha-irradiation-experiments will be shown and discussed as well.
European Radiation Research 2018, August 21-25, Pécs, Hungary
198
Preclinical evaluation of whole-body irradiations with low doses
of X-rays combined with inhibition of immune checkpoints and a
heat shock protein as a novel therapy for lung cancer
Antoni Więdłocha1, Ewa M. Nowosielska2, Aneta Cheda2, Marek K. Janiak2
1Norwegian Radium Hospital, Centre for Cancer Biomedicine, Institute for Cancer
Research, Dept. of Biochemistry, Olso, Norway 2Military Institute of Hygiene and Epidemiology, Department of Radiobiology and
Radiation Protection, Warsaw, Poland Objective: Globally, in both more and less developed countries including Poland, lung cancer is the leading cause of cancer death among males and second cause of cancer death among women. In contrast to a considerable progress made over the recent years in the diagnosis and treatment of malignant neoplasms the prognosis for lung cancer patients, which are usually diagnosed at an advanced stage of the disease, remains poor. No wonder therefore that the new, more effective therapies of lung carcinoma are being actively investigated. Hence, the aim of the proposed project is to comprehensively evaluate the potential therapeutic efficacy of immunopotentiating whole-body exposures of lung cancer-bearing mice to low-level ionizing radiation combined with use of the selected inhibitors of immune checkpoints CTLA-4 and PD-1 and/or the function of the heat shock protein (HSP) 90 chaperone. Methods: The experiments will be carried out using the Lewis lung carcinoma (LLC) grown in C57BL/6 mice, as the only reproducible syngeneic model for lung cancer to date. The animals will be exposed to fractionated X-rays (5 days per week for one or two weeks) at total doses of 0.1 and 1.0 Gy. Antibodies directed against the CTLA-4 and PD-1 immune checkpoint receptors and synthetic inhibitors of HSP90 protein (NVP-AUY922) will be injected intraperitoneally three times a week for one or two weeks, according to the applied irradiation scheme. After completion of the irradiation, application of the CTLA-4 and PD-1 Abs and/or the HSP90 inhibitor, the following parameters will be estimated: number of spontaneous lung metastases of the tumour cells, number and activity of the tumour-associated cells, such as macrophages (TAMs) and T lymphocytes (TILs), number and activity of the regulatory T (Treg), cytotoxic T (CTL), and NK lymphocytes in the peripheral blood and lung tissue, viability and proliferation of cells obtained from the growing tumour, concentrations of IL-2 and IL-6 in the serum/plasma or in the lungs. Also, morphometric assessment of the tumour growth will be carried out. Results: The projected study is entirely original in design and is based on the state-of-the-art research currently pursued in experimental and clinical oncology. The obtained results will provide preclinical evidence for establishment of new, more effective combinations of treatment options for patients with advanced pulmonary cancer and also, possibly, other treatment-refractory malignant neoplasms. The study is funded by the grant No. 510/2017/DA of the Polish Ministry of Defence.
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Spatial ROS distribution contributes to the differences in the
invasion/migration processes of cancer stem cells in response to
photons and carbon ions
Anne-Sophie Wozny1,2
, Guillaume Vares3,4
, Gersende Alphonse1,2
, Caterina Monini5, Nicolas
Magné1,6
, Charlotte Cuerq2, Akira Fujimori
7, Jean-Claude Monboisse
8, Michael Beuve
5, Tetsuo
Nakajima4 and Claire Rodriguez-Lafrasse
1,2.
1Univ Lyon, Université Lyon 1, UMR CNRS5822/IN2P3, IPNL, PRISME, Cellular and Molecular
Radiobiology, Lyon-Sud Medical School, Oullins Cedex, France
2Hospices Civils de Lyon, Biochemistry, Lyon-Sud Hospital, Pierre-Bénite, France
3Advanced
Medical Instrumentation Unit, Okinawa Institute of Science and Technology Graduate University,
Okinawa, Japan
4Department of Radiation Effects Research, National Institute of Radiological Sciences, Quantum and
Radiological Science and Technology, Inage-ku, Chiba, Japan
5Univ Lyon, UMR CNRS5822/IN2P3, IPNL, PRISME, PHABIO, Villeurbanne, France
6Department of radiotherapy, Oncology Intitute Lucien Neuwirth, St Priest en Jarez, France
7Department of Basic Medical Sciences for Radiation Damages, National Institute of Radiological
Sciences, Inage-ku, Chiba, Japan.
8Champagne-Ardennes University, CNRS UMR 7369, Reims Hospital, Reims, France
The epithelio-mesenchymal transition (EMT) is the mechanism that enables cells to escape from the
tumor to form metastases. While conventional radiotherapy promotes the migration/invasion of cancer
stem cells (CSCs), carbon ion irradiation decreases these processes. Moreover, CSCs are localized in
hypoxic tumor niches inside tumors where hypoxia amplifies the biological effects associated with
radioresistance. Thus, understanding the differential mechanisms involved in the response to photon
and carbon ion exposures, particularly under hypoxia, would provide a better understanding of the
tumor escape process.
Motility, migration/invasion processes, and EMT signaling pathways were studied in response to
photon and carbon ion irradiations for two HNSCC cell lines and their subpopulation of CSCs in
normoxic and hypoxic conditions.
After confirming that the migration/invasion processes were increased in response to photon
irradiation and decreased after carbon ions under normoxic conditions, we showed that they were
decreased under hypoxia in response to both types of irradiation. In order to understand this
differential response to radiations, the phosphorylation profiles of the Akt/mTor, STAT3 and
ERK/p38/MSK pathways involved in the EMT were established. In response to photons, the activation
of the three kinase cascades was important, whereas it was weaker under hypoxia, and absent in
response to carbon ion irradiation regardless of the oxygen tension. These results thus demonstrate that
the phosphorylation profile of the three pathways is dependent on the type of irradiation and the
oxygen tension.
We also demonstrated that Reactive Oxygen Species (ROS) production is essential to activate the
EMT pathways. In addition, Monte Carlo simulations enable us to show the different spatial
distribution of ROS in response to both types of irradiation. The ROS are uniformly distributed in the
cell after photon irradiation, thus triggering the activation of the three EMT signaling pathways
whereas their localization around the carbon ion tracks is not sufficient to activate them.
Consequently, we propose a new paradigm where the spatial ROS distribution determines the
activation of the EMT signaling pathways.
Supported by LabEx PRIMES (ANR11LABX0063), France Hadron (ANR11INBS0007)
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Environmental Enrichment Regulates Radiation-Induced
Apoptosis in the Mouse Intestinal Crypt
Shinya Yokomizo1, 2
, Mayumi Nishimura2, Takamitsu Morioka
2, Yukiko Nishimura
2,
Chizuru Tsuruoka2, Yi Shang
2, Kazumasa Inoue
1, Masahiro Fukushi
1, Shizuko Kakinuma
2,
Yoshiya Shimada2
1Department of Human Health Science, Tokyo Metropolitan University, Tokyo, Japan
2Department of Radiation Effects Research, NIRS, QST, Chiba, Japan
Objective
Fukushima Daiichi Nuclear disaster forced the evacuation of a large number of residents and created
changes in the lifestyle of the evacuees. These changes caused limited physical activity and stressful
environment and increased the risk of many kind of disease. Radiation induced cancer risk is
considered really small but many people worry about it, especially to children. Hence, it is important
to establish a new prevention method for children’s stress and radiation effects. Environmental
enrichment (EE) provides positive physical and psychological stress. EE has been mainly reported to
activate the central nervous, immune and endocrine systems. However, whether EE modifies the
intestinal response after radiation exposure is unknown. In this study, we analysed the modifying
effects of EE against stressful events and radiation induced apoptosis in the mice.
Methods
Male B6C3F1 mice were housed in SE or EE cage from 3 or 11 weeks of age for 8 weeks. The EE
cage was 8 times larger than SE cage and consisted of a number of toys. After housing, mice were
irradiated with 0 or 2 Gy then sacrificed under the anesthesia. Small intestine and colon were collected
and incidence of apoptosis was determined histologically by HE staining. Serum samples were also
collected and lipid components and hormones were measured.
Results
EE showed decrease of body weight, white adipose tissue and brown adipose tissue weights. In the
mice housed with EE, total cholesterol, triglycerol and LDL-cholesterol were significantly decreased
than that of SE. Other hand, HDL-cholesterol were significantly increased. EE also exhibited decrease
of leptin and insulin levels. EE significantly increased the incidence of apoptosis in the both of small
intestine and colon 6 hours after irradiation.
Conclusion
Our study highlights the enhancement of apoptosis induction by EE in intestinal epithelium after
irradiation. We will discuss the possible mechanism of EE on apoptosis induction.
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