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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


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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


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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

http://www.pxinc.com/

http://www.accela.eu/

http://aok.pte.hu/en

https://www.oki.hu/

http://www.mbft.hu/index-en.php

http://www.softflowbio.com/

http://www.frank-diagn.hu/


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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.


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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,


1221 Budapest,

Anna u. 5.

Hungary

Tel. +36-1-4822010

E-mail: [email protected]

mailto:[email protected]


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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.

http://aok.pte.hu/about-the-school


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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


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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.


Lecture Room IV.

13:00-14:00 Lunch


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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


10:00-11:20 Radiation and exosomes Lecture Room III.

10.00-11.20 Radioecology meets radiobiology Lecture Room IV.



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



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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


10:00-11:20 Individual radiosensitivity and radiosusceptibility

Lecture Room III.

10.00-11.20 Radiation hormesis Lecture Room IV.



Lecture Room III.

11:50-13:00 Non-targeted effects of ionising radiation

Lecture Room IV.




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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


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.



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


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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


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Wednesday, August 22, 2018


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.


Lecture Room IV.

Chairpersons: S. Tapio and B. Madas

10:00-10:10 S. Tapio: Introduction to Multidisciplinary European Low Dose

Initiative (MELODI)


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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


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


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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


15:30-18:00 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?


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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


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Thursday, August 23, 2018


Chairperson: M. Kadhim

9:00-9:45 V. Jendrossek: Linking radiation-induced DNA-damage to systemic

anti-tumor responses and normal tissue toxicity


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.



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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



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Chairperson: B. Madas

15:00-15:45 G. Kraft: Radiation Research and Society


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


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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


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Friday, August 24, 2018


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


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!


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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?



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?


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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.




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


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.


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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?


25

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.


26

Saturday, August 25, 2018


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


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


27

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.



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


28

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


29

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


30

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


31

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.


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


33

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


34

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


35

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

https://www.bioem.org/user/2993




36

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?


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.


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.


39

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.


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.


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.


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.


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)


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


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.


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


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


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).


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.


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.


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.


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.


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.


54

Long-term cerebrovascular and cognitive impairments following childhood exposure

to radiation

Rafi Benotmane

[email protected]

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.



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.


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.


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.


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.


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.


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.


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.


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.

[email protected]



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.


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.


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.


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.


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.


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: [email protected] (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.



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: [email protected] (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.



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.


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.


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.


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.


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.


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: [email protected]

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.


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.


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.


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.


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.


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.


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: [email protected]

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.


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 radiotherapy. 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.


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.


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


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.


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.


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.


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.


89

Biomedical potential of laser-driven particle acceleration

Katalin Hideghéty1,2

, Róbert Polanek1, Rita Emilia Szabó

1, Szilvia Brunner

1, Tünde Tőkés

1,3


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.


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.


91

RADIATION LEVELS IN HUNGARIAN BUILDINGS

Zsolt Homoki


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.


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.


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.


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.


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: [email protected]

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


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.


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).


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]).


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


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


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.


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.


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.


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.


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.


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.


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: [email protected] (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.



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.


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.


110

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.


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.


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.


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.


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.


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).


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

https://arxiv.org/abs/1803.02630


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).


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.


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.


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)


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.


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.


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

https://mailtrack.io/trace/link/547c2b147fdd4cfbb53fb49e75549f2d6044e2bb?url=http%3A%2F%2Fwww.pxinc.com%2F&userId=2762999&signature=b5cb291382918c6c


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.


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.


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.


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).


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).


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.


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.


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.


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.


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: [email protected]; [email protected]

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.




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.


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





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.


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.


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.


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.

https://www.nist.gov/pml/x-ray-mass-attenuation-coefficients


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.


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.


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.


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.


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)


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.


146

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.


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.


148

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.


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: [email protected]


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.


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.

http://dfvm.nipne.ro/

http://dfvm.nipne.ro/

http://www.nipne.ro/research/departments/dfna.php


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


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

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3834742/

https://dx.doi.org/10.2203%2Fdose-response.13-005.Doss


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


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.


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.


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. [email protected]

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.



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.


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.


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.


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.


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.


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.


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.

http://www.storedb.org/


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


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/

https://cos.io/our-services/top-guidelines/


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.


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?


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.


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.


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


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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: [email protected]

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.



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.


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.


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.


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.


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.


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


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.


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.


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


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.


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.


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.


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.


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.


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.


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.


199

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)


200

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.


201

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