Cellular effects after exposure to mixed beams of ionizing radiation Elina Staaf

Cellular effects after exposure to
mixed beams of ionizing radiation
Elina Staaf
Doctoral thesis in Molecular Genetics at Stockholm University, Sweden 2012
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©Elina Staaf, Stockholm 2012
ISBN 978-91-7447-588-3 (pages 1-58)
Printed in Sweden by Universitetsservice US-AB, Stockholm 2012
Distributor: Department of Genetics, Microbiology and Toxicology
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Abstract
Mixed beams of ionizing radiation in our environment originate from space, the
bedrock and our own houses. Radiotherapy patients treated with boron neutron capture
therapy or with high energy photons are also exposed to mixed beams of gamma
radiation and neutrons. Earlier investigations have reported additivity as well as
synergism (a greater than additive response) when combining radiations of different
linear energy transfer. However, the outcome seemed to be dependent on the
experimental setup, especially the order of irradiation and the temperature at exposure.
A unique facility allowing simultaneously exposure of cells to X-rays and
241
Am
alpha particles at 37 ºC was constructed and characterized at the Stockholm University
(Paper I). To investigate the cytogenetic response to mixed beam irradiation (graded
doses of alpha particles, X-rays or a mixture of both) several different cell types were
utilized. AA8 Chinese Hamster Ovary cells were analyzed for clonogenic survival
(Paper I), human peripheral blood lymphocytes were analyzed for micronuclei and
chromosomal aberrations (Paper II and Paper III respectively) and VH10 normal
human fibroblasts were scored for gamma-H2AX foci (Paper IV).
For clonogenic survival, mixed beam results were additive, while a significant
synergistic effect was observed for micronuclei and chromosomal aberrations. The
micronuclei dose responses were linear, and a significant synergistic effect was present at
all investigated doses. From the analysis of micronuclei distributions we speculated that
the synergistic effect was due to an impaired repair of X-ray induced DNA damage, a
conclusion that was supported by chromosomal aberration results. Gamma-H2AX foci
dose responses were additive 1 h after exposure, but the kinetics indicated that the
presence of low LET-induced damage engages the DNA repair machinery, leading to a
delayed repair of the more complex DNA damage induced by alpha particles. These
conclusions are not necessary contradictory since fast repair does not necessarily equal
correct repair. Taken together, the observed synergistic effects indicate that the risks of
stochastic effects from mixed beam exposure may be higher than expected from adding
the individual dose components.
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Populärvetenskaplig sammanfattning
Cellulära effekter från blandad strålning.
Joniserande strålning är en naturlig del av våra liv, vår omgivning och även våra
kroppar. Vi lever med bakgrundsstrålning från berggrunden och i maten vi äter, vid
flygresor är vi närmare rymden och den kosmiska strålningen. Strålning är även ett
vanligt verktyg inom medicin (främst diagnostik och cancerbehandling) samt inom
industrin. Det finns två huvudtyper av joniserande strålning, elektromagnetisk strålning
och partikelstrålning. Den elektromagnetiska strålningen består av fotoner, energikvanta
som levererar sin energi jämt fördelad och utspridd. Partikelstrålningen inducerar istället
många och täta skador längst partikelns spår. Båda dessa stråltyper kan skada DNA-baser
samt bryta ena eller båda DNA-strängarna samtidigt, vilket kan leda till mutationer och i
värsta fall cancer. Dock har vi effektiva DNA-reparationssystem i våra celler för att
hantera detta och de krävs många skador (dvs hög dos), eller en ökad skadekomplexitet
för att reparationen inte ska fungera korrekt.
Blandad strålning inträffar när båda dessa stråltyper finns närvarande samtidigt,
t.ex. bakgrundsstrålning som inkluderar radon, den kosmiska strålningen under flygresor
och vid vissa sorters cancerbehandling. Den stora frågan är ifall effekten av blandad
strålning är additiv (1 + 1 = 2) eller ifall de två typerna interagerar och därmed inducerar
en synergistisk effekt (1 + 1 = 3). Ett flertal forskargrupper har studerat denna fråga men
eftersom både synergism och additivitet har observerats har ingen konsensus gällande
risker från blandad strålning nåtts. De svagheter som finns för tidigare studier är att
bestrålning med en stråltyp före den andra (med en paus emellan) har klassificerats som
blandad, att temperaturkontroll har saknats samt att resultaten bara har undersökts med en
enda analysmetod. Vi har därför konstruerat ett system för blandad strålning där celler i
en 37°C-inkubator utsätts för röntgenstrålning och alfapartiklar samtidigt. Denna
avhandling är baserad på resultaten från detta unika bestrålningssystem.
Den första artikeln fokuserar på att beskriva och karaktärisera detta
bestrålningssystem. Alfa-källans dos per tid (så kallad dos-rat) beräknades teoretiskt och
mättes sedan med hjälp av en speciell plastfilm. Dos-raten var 0.265 Gy per minut.
Biologisk reproducerbarhet mättes med cellöverlevnad i hamsterceller och observerades
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vara god, och effekten av blandad strålning var additiv. Dock ökade skillnaden mellan
observerad och beräknad effekt av blandad strålning med ökande dos och procent
alfapartiklar i blandningen, så en synergistisk effekt kunde ha observerats om högre doser
och högre procent alfapartiklar (> 50 %) använts.
Den andra artikeln fokuserar på dos-responsen för mikrokärnor i vita
blodkroppar från människoblod utsatt för blandad strålning. Genom att tillsätta en
speciell kemikalie stoppas celldelning men inte kärndelning, och hela kromosomer och
kromosombitar som hamnade utanför vid kärndelningen kan ses som mindre kärnor
bredvid två större kärnor i en cell. För mikrokärnor observerades en signifikant
synergistisk effekt efter blandad strålning (35 % alfa-partiklar). Denna effekt var synlig
vid alla undersökta doser.
Den tredje artikeln undersöker kromosomala aberrationer i mänskligt blod efter
blandad strålning. Aberrationer inträffar när cellen inte har lyckats laga ett
dubbelsträngsbrott, eller har lagat det fel (t.ex. klistrat ihop två kromosomer som inte hör
ihop, eller gjort en skadad kromosom till en ring), och syns vid metafas när cellen har
kondenserat DNAt till kromosomer. Aberrationer klassificeras som komplexa ifall de
skapades från minst tre dubbelsträngsbrott från två olika kromosomer, och simpla ifall
två eller färre brott var inblandade. En synergistisk effekt observerades för mittendosen
(25 % alfapartiklar) och den högsta dosen (40 % alfapartiklar) för komplexa aberrationer,
men bara för den högsta dosen för simpla aberrationer. Dos-responskurvan för komplexa
aberrationer var linjär-kvadratisk, vilket också indikerar en synergistisk effekt.
Den fjärde och sista artikeln undersöker dos- och tids-respons för gammaH2AX foci i en mänsklig hudcellinje. DNAt är tätt packat runt proteiner (bl.a H2AX),
och när DNA ändrar form (t.e.x vid ett dubbelsträngsbrott) fosforyleras dessa proteiner.
De kan sedan ses som lysande prickar – foci - under mikroskop med hjälp av färgade
antikroppar. H2AX-foci uppkommer snabbt efter DNA-skada och försvinner när skadan
har reparerats. Dos-respons för blandad strålning var additiv 1 timme efter bestrålning,
men tids-responsen indikerade att närvaron av skador från elektromagnetisk strålning
störde reparationen av partikel-inducerade skador.
Sammanlagt indikerar dessa resultat att biologiska effekten från blandad strålning
kan vara större än vad som kan beräknas från de individuella stråltyperna i bladningen.
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Publications
List of original publications
This thesis is based on data presented in the following publications:
I
Staaf E, Brehwens K, Haghdoost S, Pachnerova-Brabcova K, Czub J, Braziewicz
J and Wojcik A. (2012) “Characterization of a setup for mixed beam exposure of
cells to 241Am alpha particles and X-rays.” Radiat Prot Dosimetry, 151(3):570-79
II
Staaf E, Brehwens K, Haghdoost S, Nievaart S, Pachnerova-Brabcova K, Czub J,
Braziewicz J and Wojcik A. (2012) “Micronuclei in human peripheral blood
lymphocytes exposed to mixed beams of X-rays and alpha particles.” Radiat Env
Biophys, 51(3):283-93
III
Staaf E, Deperas-Kaminska M, Brehwens K, Haghdoost S, Czub J and Wojcik A.
(2012) “Higher than expected frequencies of complex aberrations in lymphocytes
exposed to mixed beams of 241Am alpha particles and X-rays.” Manuscript,
submitted to Acta Oncololgica
IV
Staaf E, Brehwens K, Haghdoost S, Czub J and Wojcik A. (2012) “GammaH2AX foci in cells exposed to a mixed beam of X-rays and alpha particles.”
Manuscript, revised submitted to Genome Integrity
Articles I and II were reprinted with the kind permissions of the publishers: Oxford
Journals and Springer.
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Additional publications
Not included in this thesis:
V
Johannes C, Dixius A, Pust M, Hentschel R, Buraczewska I, Staaf E, Brehwens
K, Haghdoost S, Nievaart S, Czub J, Braziewicz J and Wojcik A. (2010) “The
yield of radiation-induced micronuclei in early and late-arising binucleated cells
depends on radiation quality.” Mutat Res 701(1):80-5
VI
Brehwens K, Staaf E, Haghdoost S, González A.J, and Wojcik A. (2010)
“Cytogenetic damage in cells exposed to ionizing radiation under conditions of a
changing dose rate.“ Radiat Res 173(3):293-9
VII
Brehwens K, Bajinskis A, Staaf E, Haghdoost S, Cederwall B and Wojcik A
(2012) “A new device to expose cells to changing dose rates of ionising
radiation.” Radiat Prot Dosimetry 148(3):366-71
VIII
Dang L, Lisowska H, Manesh S.S, Sollazzo A, Deperas-Kaminska M, Staaf E,
Haghdoost S, Brehwens K and Wojcik A. (2012) “Radioprotective effect of
hypothermia on cells – a multiparametric approach to delineate the mechanisms.”
Int J Radiat Biol 88(7):507-14
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Table of contents
Abstract ............................................................................................................................... 3
Populärvetenskaplig sammanfattning ................................................................................. 4
Introduction....................................................................................................................... 11
Introduction to ionizing radiation ................................................................................. 11
A part of our lives ..................................................................................................... 11
Direct and indirect effect .......................................................................................... 11
Linear energy transfer ............................................................................................... 12
Relative biological effectiveness .............................................................................. 13
Complexity of radiation-induced DNA damage ....................................................... 14
Repair of radiation-induced DNA damage ................................................................... 15
Overview of DNA double-strand break repair systems............................................ 15
Homologous recombination...................................................................................... 16
Non-homologous end-joining ................................................................................... 17
Outcomes of DNA damage repair ............................................................................ 17
The concept of risk........................................................................................................ 18
Risk considerations ................................................................................................... 18
Synergism and additivity .......................................................................................... 20
Mixed beam studies – reviewing the literature ............................................................. 21
The cell system and endpoint.................................................................................... 21
The LET combination, doses and dose regimes ....................................................... 24
Irradiation characteristics – temperature, time and order of exposure...................... 25
Summary of findings ................................................................................................ 27
The present investigation .................................................................................................. 28
Aims of this thesis......................................................................................................... 28
Novel approaches of the experiments ....................................................................... 28
Materials and methods .................................................................................................. 29
Cell types .................................................................................................................. 29
The irradiation setup ................................................................................................. 29
Cell exposure ............................................................................................................ 30
Clonogenic cell survival ........................................................................................... 31
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Chromosomal aberrations ......................................................................................... 31
The micronucleus assay ............................................................................................ 32
The gamma-H2AX assay.......................................................................................... 32
Results and discussion .................................................................................................. 34
Paper I ....................................................................................................................... 34
Paper II...................................................................................................................... 36
Paper III .................................................................................................................... 38
Paper IV .................................................................................................................... 40
Concluding remarks and future perspectives................................................................ 42
Concluding remarks .................................................................................................. 42
Future perspectives ................................................................................................... 43
Acknowledgements........................................................................................................... 45
References......................................................................................................................... 46
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Abbreviations
Artemis
ATM
bp
CHO
DNA-PKcs
DSB
FISH
γ-H2AX
Gy
HR
IMRT
IR
Ku70/80
LET
LF
MN
MRE11
MRN
NBS1
NHEJ
PCC
PBL
Rad50/51/52
RBE
RT
SF
SSB
Sv
XLF
endonuclease active in NHEJ and V(D)J recombination
ataxia telangiectasia mutated
base pair (of DNA)
Chinese hamster ovary
DNA-dependent protein kinase catalytic subunit
double strand break (in DNA)
fluorescence in situ hybridization
phosphorylated form of histone 2AX protein
Gray = Joule per Kg. Unit in radiation biology.
homologous recombination repair
intensity modulated radiotherapy
ionizing radiation
heterodimer of the proteins Ku70 and Ku80
linear energy transfer
“large” gamma-H2AX foci
micronuclei (or micronucleus, depending on context)
meiotic recombination 11 homolog
complex of MRE11-Rad51 and NBS1, active in HR
Nijmegen breakage syndrome 1
non-homologous end-joining
premature chromosome condensation
peripheral blood lymphocytes
radiation sensitive protein 50/51/52
relative biological effectiveness
room temperature
“small” gamma-H2AX foci
single strand break (in DNA)
Sievert. The absorbed dose is modified by correction factors.
XRCC4-like factor
XRCC3/4
X-ray repair cross-complimenting group 3 / 4 protein
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Introduction
Introduction to ionizing radiation
A part of our lives
Ionizing radiation (IR) is a continuous part of our lives, our environment and our
bodies. The natural exposure from IR is around 3 mSv per year in Sweden, with 2.1 mSv
from natural and 0.9 mSv from artificial sources (1). Natural radiation consists of cosmic
radiation, radioactive substances in the earth’s crust (and their decay products) as well as
radioactive isotopes within our own bodies. The cosmic radiation originates from outer
space and the sun and though it mostly affects astronauts in space it can also penetrate the
atmosphere, reaching us during airplane flight and at the ground level (2, 3). Radon gas
released from the bedrock and building materials in our houses adds alpha particles to the
natural exposure, an effect contributing to the risk for lung cancer (4). Our bodies contain
natural isotopes such as
14
C,
210
Pb and
40
K (3, 5) and through our food and drink
additional radioactive particles can be ingested. Artificial sources originate from human
inventions and activities. Medical diagnostic procedures (6) along with radiation therapy
of cancer (7) are becoming more common worldwide, and 137Cs from Chernobyl fall-out
and other nuclides from nuclear tests are present in our food (in Sweden, mainly in
mushrooms and wild animals) (8, 9). Most individuals will not experience any
measurable negative stochastic effects (such as cancer) from this background radiation.
However, exposure to IR has the potential to cause harm.
Direct and indirect effect
IR induces damage by two processes. When IR is absorbed by cells there is a
possibility that it will directly interact with DNA, the critical target. This occurs through
direct ionization and/or excitation in the DNA from interaction with the radiation.
11
Damage to DNA can also be induced indirectly. The indirect effect is a two-step process
where the first step is IR-induced ionization of liquid molecules. This produces radicals,
which in turn can diffuse to and react with the target molecule (10, 11).
Linear energy transfer
An important property of IR is the linear energy transfer, LETΔ, defined as energy
transferred per unit path length traversed by an ionizing particle, excluding deltaelectrons with energy above Δ (12). LETΔ, hereafter referred to as LET, is an average
quality, describing the whole exposure and not each individual particle. IR can be
classified as low or high LET.
Low LET radiation generally consists of electrons and photons, which originate
from natural decay of radioactive isotopes (for example
137
Cs or
60
Co) or man-made X-
rays where electrons are accelerated into a target (usually 79Au or 74W), thereby emitting
photons as bremsstrahlung (11). Photons are electromagnetic radiation/waves that have
short wavelengths at higher energies. One ionizing photon generates about 30 ionizations
within a cell nucleus and about 70 % of the induced damage is from the indirect effect
(11). The LET of photons is usually below 1 keV/μm.
High LET radiation consists of particles: protons, neutrons, alpha particles and
heavy ions and can be generated by decay of radioactive isotopes or in purpose-built
devices (accelerators and reactors). Upon interaction with the target, secondary particles
(for alpha-particles mainly delta-electrons) are produced and in turn cause ionizations
along the path of the original particle. For alpha particles, about 10 000 ionizations are
induced per track (13), giving rise to clusters of damage, the majority from direct
interaction. High LET values vary from 10-100 keV/μm for neutrons to 100-200 keV/μm
for alpha particles and more than 1000 keV/μm for heavy ions (11, 14). The differences
between high and low LET IR is exemplified in Figure 1.
The uncharged state of photons means that it can penetrate far into matter before
encountering a target with which to react. This means that within an irradiated cell
population low LET IR will induce an even dose distribution, since the cells will on
average have received the same number of photon interactions and thus the same dose.
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For high LET, the massive amount of energy delivered per traversed length result in a
very short reach (alpha particles: a few nm in tissue), and a characteristic dose delivery
called the Bragg peak (11). An alpha particle-irradiated cell population will show an
uneven dose distribution, since each track traversing a nucleus will by definition induce
heavy damage and an irradiated cell population will always contain cells that received
different numbers of hits.
Figure 1: Track structure for IR of different LET. The circle represents a cell nucleus. To the left: Low
LET (photons). To the right: High LET (α-particle). Figure adapted from (10).
Relative biological effectiveness
The relative biological effectiveness (RBE) is a tool for evaluating differences in
effects between IR of different LET. RBE is determined by estimating the dose that gives
rise to a defined level of biological effect and comparing it to the dose from a reference
type of radiation (normally low LET) required for the same effect (15). Low LET
radiations have a RBE of 1 and this value increases with increasing LET to reach a
maximum at about 100 keV/μm, thereafter decreasing (11). For review, see (14). The
RBE is strongly dependent on factors such as cell type and endpoint and is therefore not
an absolute value (exemplified in 16).
13
Complexity of radiation-induced DNA damage
Several types of DNA lesions can be induced by IR. Base damage, base loss, DNAprotein crosslinks, along with the single and the double strand break (SSB and DSB,
respectively) are some types of damage that occur (10). The DSB is the most detrimental
of these since it is the only lesion where both DNA strands are simultaneously broken
(17). In addition, if several lesions occur within a small volume they form clustered
lesions (also called multiple damages sites), defined as >2 damages within a 20 bp region
(18). These clustered lesions are more difficult to repair than simple DSBs without
additional flanking damage (19, 20). A clustered lesion is exemplified in Figure 2.
Figure 2: Example of a clustered lesion, a DSB flanked by a variety of other lesions.
Depending on the LET, IR induces different proportions of lesions, with low LET
photons inducing the majority of its effect indirectly, while high LET particles induces a
higher level of direct interaction (21). Due to the higher density of ionizations and
excitations along the track, high LET also generates a higher proportion of clustered
lesions (22). The proportion of clustered lesions as well as the degree of complexity of
each lesion increases with increasing LET (23). All components of a clustered lesion
must be repaired or removed for the break to be fully repaired, thus delaying the repair
process (17). Several DSBs within the same small area also result in a high probability of
incorrect rejoining of the broken DNA ends (19).
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Repair of radiation-induced DNA damage
Overview of DNA double-strand break repair systems
The survival of cells and organisms is linked to the integrity of DNA, and efficient
DNA-repair mechanisms are present in our cells (24). These repair systems can handle
endogenous damage from the cell metabolism as well as exogenous damage from agents
such as IR, UV-radiation, chemicals or changes in temperature or oxygen level.
Deficiencies in DNA repair systems often lead to disease (see e.g. 25). As previously
mentioned, a DSB is the most dangerous lesion for the cell, because both DNA strands
are broken (17). Upon DSB formation, the broken DNA ends are immediately bound by
the MRN protein complex (MRE11, Rad51 and NBS1), a signalling and end resection
factor (26, 27). ATM is recruited by MRN, and both participate in H2AX
phosphorylation (28, 29). The phosphorylation rapidly spreads up to 2 million base pairs
(circa 2000 H2AX molecules) in each direction from the DSB (30, 31), where stretches
of gamma-H2AX act as accumulation signals and docking places for DNA repair proteins
(32, 33).
Two main DSB repair systems exist; homologous recombination (HR) and nonhomologous end joining (NHEJ). A simplified visualization of the two repair systems is
shown in figure 3. HR is mainly active during replication and G2, and functions by
copying information from a non-damaged, homologous double-strand of DNA to the
damaged strand. It is therefore a precise, but slow method of repair (34). NHEJ is active
during all phases of the cell cycle and is not homology-dependent (35). In NHEJ the
DNA ends are held in place, damaged nucleotides trimmed away and the ends rejoined.
This process results in a fast repair, but mutations or cell death may occur since some
genetic material is always lost around the breakpoint (36). The presence of both repair
systems leads to biphasic DSB repair kinetics (37-40), with NHEJ being mainly
responsible for the fast and HR for the slow component (34, 41). ATM and DNA-PKcs
are involved in the pathway choice, which also depends on the cell cycle phase (42-44).
15
Figure 3: Simplified models of DNA DSB repair systems HR (to the left) and NHEJ (to the right).
Homologous recombination
In HR, nuclease end-processing removes DNA in the 5´to 3´ direction to form long
3’ single-stranded DNA “tails” on both sides of the break (45). The single stranded
sections are then covered by RPA, single-stranded DNA-binding proteins which interact
with Rad51 and Rad52. Rad51 creates a hexameric structure around the end of the singlestranded part, and forms nucleoprotein filaments (46) which search for homologous
double-stranded DNA. In this step Rad54, a nuclear translocase, is essential for creating a
D-loop when invading the homologous double-stranded DNA sequence and for
depolymerising the Rad51 filaments (47, 48). New DNA is subsequently synthesized by
DNA polymerases to fill the gaps, Holliday junctions are cleaved by a resolvase complex
(49, 50) and nicks in the DNA are sealed. XRCC3 and Rad51C, (a Rad51 paralogue)
forms the CX3 complex, necessary for holding in place and resolving of the four DNA
16
strands involved in the Holliday junctions (51, 52).
Mitotic HR has recently been
reviewed (53).
Non-homologous end-joining
In NHEJ the heterodimer Ku70/80 binds specifically to open double-stranded DNA
ends (54). Ku70/80 then recruits DNA-PKcs, together forming a complex to hold the
DNA ends in place and to create a scaffold for other proteins (55). Artemis joins the
complex and carries out end-processing of the DNA via nuclease activities (56). Ku70/80
further recruits XRCC4 and ligase IV to the processed ends (57) while DNA-PKcs
stabilizes the new complex (58, 59) and finally the XRCC4-ligase IV complex ligates the
DNA-strands (60). XLF is an alternative Ku70/80 recruited protein (61), essential for
performing the ligation of complex DSBs when strands are lacking in homology or
mismatched (62). NHEJ has recently been reviewed (63).
Outcomes of DNA damage repair
The capacity and outcome of DNA repair is influenced by the complexity of
induced damage. A clustered lesion is more difficult to repair and does therefore retain
repair proteins for a longer time than a simple DSB (19, 20). This has been observed at
the level of DNA DSB signalling and repair proteins, where foci after high LET IR are
larger and persist for a longer time than if the same dose was given with low LET IR (6466). The cellular repair of DNA lesions can in itself give rise to additional DSBs, and
contribute to the complexity of the induced damage (22). In addition to the LET of the
IR, the cell cycle phase during which the cells were irradiated also plays a role (67). The
cells are most sensitive in mitosis, while the highest resistance is observed in S-phase
(11).
DSBs that failed to repair or were misrepaired result in chromosomal aberrations,
which can be visualized in metaphase as breaks, gaps and other anomalies present in the
chromosomes. There are two classes of chromosomal aberrations; simple aberrations that
include breaks, rings, and exchanges between two chromosomes (dicentrics and
translocations) and complex aberrations, consisting of at least three breaks in two or more
17
chromosomes (68). A high LET particle will give rise to many open DNA ends along the
particle track, significantly increasing the risk for misrepair relative to the same dose of
low LET. Consequently, high LET IR induces substantial numbers of complex
aberrations already at low doses, while low LET IR mainly gives rise to simple
aberrations (21). Aberrations can also be classified as stable or unstable, where stable
aberrations such as translocations or insertions are transmissible while unstable
aberrations (e.g. dicentrics and rings) will be lost from the genome and/or result in cell
death (69). The presence of complex aberrations in the genome can be viewed as a
marker of high LET exposure (70, 71).
The concept of risk
Risk considerations
It is known that high doses of IR cause harmful effects in the human body. The
major deterministic effect is the acute radiation syndrome, while cancer induction is the
main stochastic effect. The dose responses for deterministic and stochastic effects of IR
have been established epidemiologically, mainly from atom bomb survivors (72, 73),
nuclear workers (74) and patients receiving IR for medical reasons (e.g. 75). In humans,
the acute radiation syndrome appears above a threshold dose of about one Gy/Sv or
higher, where the severity of these effects (mainly on the hematopoietic system and the
skin) increase with increasing dose (76). For doses below 100 mSv (effective dose), no
significant stochastic effects have been observed at the epidemiological level (77, 78).
Several extrapolations of the dose response curve for cancer risk after radiation
exposure from the high dose region to the low dose region exist; see Figure 4 for a
schematic representation (adapted from Brenner et al. (77)). The linear no threshold
model (extrapolation “a” in Figure 4) is currently the most accepted and applied method
for radiation protection and modeling of cancer risk (79).
18
Figure 4: The dots at the upper right corner represent the 100 mSv dose point. The effect for lower doses
could be a) linear, b) downwardly curving (the slope decreasing with decreasing dose), c) upwardly curving
(slope increasing with dose), d) threshold-dependent or e) beneficial before becoming harmful, in
accordance with the hormesis hypothesis, (80).
Many studies on the effects of IR were based upon the action of a single type of IR.
However, after an atom bomb detonation low and high LET radiation (photons and
neutrons) is present simultaneously. Such “mixed beam” exposure situations are now
becoming more frequent. In airplane-, and especially during spaceflight people are
exposed to particles from cosmic IR, which interact with the shielding material of the
shuttle, forming a photon background (1, 81, 82). High background radiation of mixed
type can be found at several sites in the coastal region of Brazil, Yangjian in China,
Kerala in India and Ramsar in Iran (83, 84). In external beam radiation therapy mixed
beam exposures occur, especially during long irradiation sessions of intensity modulated
radiotherapy (IMRT), when the major part of the photon beam is shielded by a multi-leaf
collimator (85). Here, the absorbed neutron doses can be up to several hundred mSv (86,
87). The same effect occurs for fast neutron therapy where photons are generated through
thermalization of neutrons (88), and for boron neutron capture therapy (89) where the
captured neutrons give rise to photons,
10
He- and Li-ions through the (n,γ) and the
B(n,α)7Li reactions (90).
19
Synergism and additivity
Synergism is defined as “cooperative interaction between two or more components
in a system, such that the combined effect is greater than the sum of each part” (91).
Conversely, additivity describes a situation where the observed combined effect is not
greater than that expected from the sum of each agent. In a cellular system synergism
between two agents can occur via two mechanisms; either by one of the agents
potentiating the level of damage from the other, or by impairing cellular damage repair
mechanisms. The former effect is observed for example for the interaction of oxygen
with IR (92-94), while the latter can be exemplified by the interaction IR with heavy
metals (95-97). The classification of a biological response as additive or synergistic is
complicated if the dose response of one or both agents is non-linear, requiring the
construction of “envelopes of additivity” (98, 99) and/or the application of mathematical
models (100-102).
Currently, there are no physical reasons to assume that interactions between IR of
different LET should result in a synergistic effect. However, synergistic effects have
previously been observed for mixed beam irradiation alongside results suggesting
additivity (see Table 1) – a clear lack of consensus. The key to the mechanisms of
synergistic effects are likely to be found at the subcellular level. It is possible that the
complex DNA damage induced by high LET IR could trap and make the DNA damage
repair proteins unavailable or less effective for repair of low LET induced damage (103).
Alternatively, the presence of low LET-induced, simple and dispersed DNA damage
could occupy the DNA repair systems to such an extent that the high LET induced
damage are not efficiently repaired. Finally, it is also possible that low and high LET IR
interact to potentiate the complexity of DNA damage.
Biological effects of mixed beams is thus one of the areas in radiation research
where the potential risks are still unclear and experimental results inconclusive. The issue
of additivity or synergism is still debated and 50 years of research within this area has not
yielded a clear conclusion. It is therefore very interesting to continue research in this area
with a wider range of methods, with the aim of contributing towards solving the problem.
20
Mixed beam studies – reviewing the literature
Several investigations regarding the DNA-damaging properties of mixed beams of
radiation have already been performed (see Table 1.). No overall conclusion has yet been
reached since synergism and additivity alike have been and are still being reported. This
section will investigate and discuss the factors that may favour a synergistic or an
additive outcome of an experiment. The discussion will focus on the following set of
parameters: 1) The cell system and endpoint, 2) The LET combination, doses and doseregimes and 3) Irradiation characteristics - Temperature, time and order of exposure.
In total, 35 published studies have investigated different aspects of the cellular
response to mixed beam irradiation (Table 1). Brooks et al. (124) and Furusawa et al.
(128) examined two different endpoints, multiple cell systems were employed by
Furusawa et al. and Hornsey et al. (119, 128), and two or more LET combinations were
employed in 8 different studies (103, 104, 116, 123, 126, 128, 130, 134). The majority of
studies however focused on a single endpoint and single combination of high and low
LET IR.
The cell system and endpoint
The majority of the studies, 18 out of 35, were performed with hamster cells, either
V79 Chinese hamster lung fibroblasts (102, 112, 114-119, 121-123, 125, 126, 128, 132,
135) or Chinese hamster ovary cells (109, 111). Out of these, only 5 observed an additive
effect (109, 122, 126, 128, 132). Human cells were also often used; fibroblasts (103, 130,
133), peripheral blood lympohcytes (PBL) (127, 128, 136), kidney cells (105, 108) as
well as salivary gland (129), epithelial (131) and HeLa cells (134) were employed. In
total, 11 studies were thus completely or partly (128) run with human cells, and six of
these concluded synergism (105, 108, 127-129, 133). Other mammalian cells were
obtained from mouse (107, 113, 120) and rat (124), and all these studies concluded
synergism. Non-mammalian systems were barley (106), bean root (104), and S.
cerevisiae yeast (110), where the first two observed additive effects and the last
synergism.
21
Table 1: Summary of published mixed beam data. a = alpha particles, g = gamma radiation, N = neutrons,
X = X-rays, n.d. = no difference in effect, Simu = simultaneous, RT = room temperature, CS = clonogenic
survival, CA = chromosomal aberrations, MN = micronuclei, FISH = fluorescence in situ hybridization A =
additivity, A** = Additivity, but slight sign of synergism for Fe-ions. S = synergism. S* = incubation time
dependent synergism, effect disappears with time.
Author
Cells used
High
LET
Gray and Read 1944
(104)
Bean Root
N or a
X-ray / g
Barendsen et al.
1960 (105)
Human
kidney cells
a
X-rays
1.5 - 6
Both, n.d.
Heddle 1965 (106)
Barley
N
X-rays
5N + 150X
Masuda 1970 (107)
Mousle L
cells
N
X-rays
Raju and Jett 1974
(108)
Human T1
kidney cells
a
Railton et al. 1974
(109)
CHO
N
Murthy et al. 1975
(110)
S.Cerevisiae
BZ34
a
60-Co
2 - 17.5
Simultaneous
Railton et al. 1975
(111)
CHO
N
60-Co
1-9
Both, n.d.
Durand and Olive
1976 (112)
V79
Hornsey et al. 1977
(113)
Mice, in
vivo, small
intestine
Ngo et al. 1977 (114)
V79
N
N
N
Endpoint
Effect
RT, 15-22
Bean survival
A
RT, < 120 min
RT
CS
A
Both, 1 diff
RT, 13-15 min
RT, cold
CA
A
5-7
Both
RT, 5-10 min
RT
CS
S
X-rays
8, 11, 15
a -> X
RT, 1 min
RT
CS
A
60-Co
2.5 - 15
Both, n.d.
RT, 1-180 min
RT
CS
A
RT
arginine
reversion
S
RT
CS
S
RT
Survival,
monolayers &
spheroids
S
S
X-rays
X-rays
X-rays
Order of
exposure
Between
exposures
(temp, time)
Exposure
temp (°C)
Low LET
Combined
dose (Gy)
Both, n.d.
4 -16
Both
6 -23.5
4 - 12.2
RT, < 45 min
RT, 4 h
Both, n.d.
BodyTemp
2-4 h
RT
Jejunum
crypts in small
intestine
N -> X-rays
Ice, or 37°C
2, 3 or 5 h
On ice
CS
S
On ice
CS
S*
Ngo et al. 1981 (115)
V79
10-Ne
X-rays
5.5 - 12.9
Both
Ice, or 37 for
0.5-24h
Bird et al. 1983 (116)
V79
2-H
3-He
X-rays
7-15 / 5-13
H/He -> Xrays
RT, < 5 min
RT
CS
S
Higgins et al. 1983
(117)
V79
N
60-Co
2 - 15
Simu + Both
37°C, 5 min
37°C
CS
S*
Higgins et al. 1984
(118)
V79
N
60-Co
1 - 15
Simu + Both
37°C, <3 min
37°C
CS
S
Hornsey et al. 1984
(119)
V79, Erlich
ascites,
mouse
jejunum
stem cells
N
X-rays
4 - 16
N -> X-rays
10°C,
2 min, 4 or 8 h
20°C
CS, stem cell
survival
S
Joiner et al. 1984
(120)
In Vivo
Mouse
fibroblasts
N
X-rays
10 - 60
Simultaneous
RT
Average skin
reaction
S
McNally et al. 1984
(121)
V79
N
X-rays
3 - 12
Both
6 min RT,
3 h 37°C
RT
CS
S*
McNally et al. 1988
(122)
V79
a
X-rays
4 - 12
a -> X
RT, 4 min
RT
CS
A
Ne/Ar ->X
4 °C
15-30 min
4°C
CS
S*
Ngo et al. 1988 (123)
V79
Ne, Ar
X-rays
3 - 12
22
Brooks et al. 1990
(124)
Rat lung
epithelial
cells
a
X-rays
1.5 - 3.5
Simultaneous
RT?
RT?
CS, MN
S
Suzuki 1993 (125)
V79
N
60-Co
1.5 - 7.5
Both, n.d.
RT, <3 min
RT
CS
S
Kanai et al. 1997
(126)
V79
12-C
3-He,4-He
1-7
He -> C?
RT; 3 – 5 min
RT?
CS
A
Wuttke et al. 1998
(127)
PBL
N
X-rays
1-3
N -> X-rays
Ice, 30 min
RT
MN
A
Tilly et al. 1999 (102)
Ice, <10 min
Ice, 4°C
CS
S
RT
CS, FISH
A**
RT
CS
A
S*
V79
N
60-Co
1-8
N -> 60-Co
Furusawa et al.,
2002 (128)
V79, PBL
Ar, Si,
Fe
X-rays
0-10, 1.23.6
Simultaneous
Demizu et al., 2004
(129)
Human
salivary
gland cells
12-C
X-rays
1.4 - 6.2
Both
Zhou et al. 2006
(103)
Primary
human
fibroblasts
Fe, Ti
protons
0.04
Both
2.5 / 48 h
RT
anchorageindependent
growth, CS
Bennet et al. 2007
(130)
Primary
human
fibroblasts
Fe, Ti
H, protons
0.04
H->Ti, prot>Fe
15 min
RT
CS
S
Hada et al. 2007
(131)
Human
epithelial
cells
Fe
protons
2.75
protons ->Fe
37°C
2, 30, 60 min
RT
FISH
(mBAND,
PCC)
S*
Phoenix et al. 2009
(132)
V79
a
60-Co
4 - 10.5
Simultaneous
10°C
CS
A
Yang et al. 2010
(133)
Normal
human skin
fibroblasts
Fe
protons
0.02
protons ->Fe
3, 30, 180 min
24 h
RT
MN
A
Elmore et al. 2011
(134)
HeLa
Fe
prot + 137Cs
0.20
Both
5, 15 min
16-24 h
RT
Neoplastic
transformation
A
4°C
CS
S
37°C
CA, dicentrics
A
Mason et al. 2011
(135)
Wojcik et al. 2012
(136)
V79
PBL
N
gamma
0-4
Simu, in
phantom in
reactor
N
60-Co and
N-beam gcomponent
0-4,
different
depths
Simu, in
phanotm
<15
The most frequently used endpoint was clonogenic cell survival, analyzed in 26 out
of 34 studies (102-105, 107-109, 111, 112, 114-119, 121-126, 128-130, 132, 135), 18 of
which with hamster cells (102, 109, 111, 112, 114-119, 121-123, 125, 126, 128, 132,
135). Here, the general response was synergistic; 1/3 of the studies (9 out of 26) observed
an additive effect (104, 105, 108, 109, 122, 126, 128, 129, 132). The clonogenic survival
in human cells however was more often additive than synergistic; 3 studies concluded
additivity (105, 108, 129) while two observed synergism (128, 130). The cytogenetic
assays chromosomal aberrations (106, 128, 131, 136) and micronuclei (124, 127, 133)
revealed synergism in 2 out of 7 studies (124, 131). Here, the majority of cytogenetic
23
studies were carried out with human cells (127, 128, 131, 133, 136), and only Hada et al.
(131) observed synergism. Other assays concluded synergism for arginine reversion by S.
cerevisiae (110) as well as for in vivo tissue reaction in mouse gut (113, 119) and skin
(120), while neoplastic transformation of human HeLa cells (134) was additive.
The LET combination, doses and dose regimes
The most commonly studied LET combination was neutrons in combination with
low LET radiation (18 studies) (102, 104, 106, 107, 109, 111-114, 117, 118-121, 125,
127, 135, 136). Additivity was observed in 5 studies (104, 106, 109, 127, 136), and
synergism in the other 13. 12 of the studies used clonogenic survival (102, 107, 109, 111,
112, 114, 117-119, 121, 125, 135), and only one of these observed additivity (109). The
doses were generally within the range of 1 – 15 Gy, with higher doses for the in vivo
(113, 119, 120) and non-mammalian studies (104, 106), and lower end doses (1 - 4 Gy)
for the number on micronuclei and chromosomal aberrations in human PBL (127, 136).
When alpha particles were combined with low LET, additivity was observed in 5
studies (104, 105, 108, 122, 132), and synergism in 2 (110, 124). Interestingly, additivity
was observed for clonogenic survival in hamster V79 cells (132, 122), a combination that
gave rise to synergism when neutrons represented the high LET. The doses varied from
1.5 up to 17.5 Gy, with the smallest dose range (1.5 – 3.5 Gy) observed for clonogenic
survival and micronuclei in rat lung fibroblasts (124, concluding additivity), and the
highest dose (17.5 Gy) for arginine reversion in S. cerevisiae (110, synergism).
Combining heavy ions with low LET radiation resulted in equal numbers of
synergistic (115, 116, 123) and additive (128, 129, 134) effects.
All the studies
concluding synergism, along with one concluding additivity (128) investigated
clonogenic survival in V79 cells (dose rage 1 up to 15 Gy), while the additive studies
involved human cells; clonogenic survival in human salivary gland cells (129), neoplastic
transformation in HeLa cells (134) and chromosomal aberrations in PBL (128). The
doses employed for human cells were lower, with 1.4 – 6 Gy for clonogenic survival
(129), 1.2 - 3.6 Gy for chromosomal aberrations (128) and 0.20 Gy for neoplastic
transformation (134).
24
Cells were also exposed to combinations of two different high LET radiations. Only
one out of the 6 studies employed non-human cells (126), and the conclusions were
evenly distributed between additivity (126, 133, 134) and synergism (103, 130, 131).
The doses employed were between 0.02 and 2.75 Gy for the human cells, and 1-7 Gy for
the V79 cells, since that study investigated clonogenic survival (126).
It was clear that the combination of LET was of great importance. Neutrons and
low LET generated mainly synergistic effects while alpha particles and low LET
generated additivity. Heavy ions induced equal numbers of additivity and synergism,
irrespectively of which low LET they were combined with. When different responses
were observed for higher and for lower doses (as was the case for neutrons and heavy
ions in combination with low LET) it was more likely that the dose-dependent
differences were due to the switch in endpoint rather than to the dose range itself.
Irradiation characteristics – temperature, time and order of exposure
In the majority of studies, the irradiation setups did not allow simultaneous
exposure of cells to a mixed beam. The two radiation types were generally applied in
sequence, with a pause in between when the samples were moved from one source to
another. Inherent to this scenario was also that the temperature during and between
exposures was not always well controlled. In fact, the majority of studies were carried
out at room temperature.
In total, 9 studies involved simultaneous exposure (110, 117, 118, 120, 124, 128,
132, 135, 136). Only two groups exposed cells simultaneously at 37°C (117, 118, 136).
In the studies of Higgins et al. V79 hamster cells were exposed to neutrons and gamma
radiation simultaneously and sequentially, and synergism was observed on the level of
clonogenic cell survival (117, 118). For Wojcik et al. human PBL were exposed to a
neutron beam with a photon component and chromosomal aberrations concluded
additivity (136). Simultaneous exposure at room temperature was performed with low
LET plus alpha particles (110, 124), neutrons (120) or heavy ions (128). Synergism was
observed in all cases except for one (128). For lower temperatures, clonogenic cell
survival of V79 cells after simultaneous exposure led to additivity for alpha particles plus
25
gamma radiation at 10°C (132), and to synergism for the mixture of neutrons and gamma
radiation at 4°C in two different nuclear reactors (135). In total, simultaneous exposure
resulted in 6 cases of synergism and 2 of additivity (128, 132).
Sequential exposures were performed in the remaining 26 studies. The studies were
most often carried out with both high before low as well as low before high LET IR (14
studies). In 6 of these studies no differences between the order of irradiations were
present (104, 105, 109, 111, 113, 125), while the other 8 studies observed that high
before low LET IR induced a greater response than low before high LET IR (103, 106,
107, 112, 115, 121, 129, 134). When no differences were observed, synergism was
concluded in 5 out of 8 studies (103, 107, 112, 115, 121), while synergism and additivity
was observed in equal amounts in the studies when differences were found. In 8 studies
cells were exposed only to high before low LET IR (102, 108, 114, 116, 119, 122, 123,
127), with 3 conclusions of additivity (108, 122, 127) and the in remaining 4 studies cells
were exposed only to low before high LET IR (126, 130, 131, 133), with 2 being additive
(126, 133) and 2 synergistic (130, 131). In total, sequential exposure resulted in 15 cases
of synergism, and 11 of additivity. Sequential irradiation on ice or in cold conditions led
to 4 cases of synergism (102, 114, 115, 123) and 1 of additivity (106), while room
temperature gave rise to 11 cases of synergism (103, 107, 111-113, 116, 119, 121, 125,
130, 131) and 10 of additivity (104, 105, 108, 109, 122, 126, 127, 129, 133, 134).
The effect of the order of exposure was tested in 10 studies. In 4 of the studies, no
differences were observed between the different exposure scenarios (high before low
compared to low before high LET radiation) (109, 111, 113, 125). In 5 of the studies,
more detrimental effects was observed for the cells when high LET radiation was the first
radiation type employed (103, 112, 115, 121, 129). Interestingly, for Elmore et al. (134)
the effect was larger when the lower LET (protons) was employed before the higher LET
(iron ions). Six studies investigated the effect of increasing the time between irradiations
(103, 115, 117, 121, 131, 133), which led to the disappearance of the synergistic effect, if
it was observed. Yang et al (133) concluded additivity and did therefore not observe this.
Sequential exposures at room temperature were thus more likely to result in an
additive effect than simultaneous exposures in a temperature-controlled environment. If
considering the sequential in vivo studies as temperature-controlled, the difference is
26
even more pronounced, since both such studies concluded synergism (113, 119). The
time between irradiations is a powerful factor for the detection of a synergistic response,
and the sequence of irradiation can potentially play a role as well.
Summary of findings
In conclusion, the responses differed based on the cell system, the LET
combination, the degree of control of the exposure conditions (temperature and
simultaneous or sequential exposure) as well as the endpoint employed.
Human cells were more likely to show an additive response, for survival as well as
cytogenetic endpoints. Rodent cells favoured the synergistic outcome, especially the in
vivo studies where all 3 gave a synergistic effect. Almost half of the studies (16 out of
35) measured clonogenic survival in hamster cells, and 10 of these were performed with
neutron irradiation. This means that approximately 30 % of the total mixed beam data
originated from very similar parameter combinations. When investigating the general
mixed beam result this must be taken into consideration, as not to give these results more
importance just because the number of experiments was larger.
Regarding the LET combination, the well-investigated neutron irradiation (17 out
of 35 studies) generated synergistic effects at the level of clonogenic survival, but
additivity when cytogenetic methods were applied. Alpha particles (8 studies) mainly
induced additive responses, for clonogenic survival as well as micronuclei. Heavy ions in
combination with low LET (6 studies) induced synergistic effects for clonogenic survival,
when investigated in the 20th century, but the 21st century studies, employing a wider
variety of endpoints concluded additivity. Combining high LET with a different type of
high LET (a comparably new field, 6 studies, the earliest study from 1997) gave rise to
equal numbers of synergism and additivity, with no identifiable trend.
Cells were more likely to exhibit a synergistic response after exposures at 37 °C.
The time between and potentially the order of exposure also influenced whether or not a
synergistic effect was observed. Simultaneous exposures in a temperature-controlled
environment (preferably 37 °C) were thus more likely to induce a synergistic effect than
sequential exposures at room temperature.
27
The present investigation
Aims of this thesis
In this thesis the cellular responses to mixed beams of high and low LET radiation,
represented by alpha particles and X-rays respectively, were investigated.
The aim was to ascertain whether the response was synergistic or additive and
elucidate the possible mechanisms behind the observed responses.
Novel approaches of the experiments
The novel approaches applied in this thesis were:
•
A dedicated device allowing simultaneous exposure of cells to mixed beams
under controlled temperature conditions.
•
Applying the gamma-H2AX assay
•
FISH (has previously been applied in two studies (128, 131), but not as
extensively as presented here)
28
Materials and methods
Cell types
The AA8 Chinese hamster ovary cell line originates from CHO-K1 cells with a
mutation in the p53 gene (137). As a consequence, the G1/S checkpoint is lacking in the
AA8 cell line since it requires a functional p53. However, since no DNA repair
deficiencies have been detected, the cell line is used as wild type (138). The cell line was
chosen for clonogenic survival due to the ability of single cells to form detectable
colonies on glass cover slips.
VH10 is a diploid primary human fibroblast cell line derived from foreskin (139). It
has no detectable DNA repair deficiencies and is therefore classified as normal (140).
The flat nucleus made the cell line an excellent choice for the gamma-H2AX assay since
it was possible to capture the majority of foci within one focal plane.
Peripheral blood was donated by one male, non-smoking donor, aged 25 to 27
during the donation period. Ethical permission was obtained from the local ethical
committee at the Karolinska University Hospital, Stockholm, Sweden (diarium number
2010/27-31/1). Human peripheral blood lymphocytes (PBL) are well suited for cell-cycle
sensitive assays since they are naturally synchronized in G0 (141). Consequently, PBL
were employed for analysis of micronuclei and chromosomal aberrations.
The irradiation setup
The facility for mixed beam exposure consists of an alpha irradiator (customconstructed in Poland at the Institute of Nuclear Chemistry and Technology, Warszawa),
an YXLON SMART 200 X-ray tube and a MCO-15AC 164 l cell incubator. The alpha
irradiator is positioned inside and the X-ray underneath the incubator and the whole setup
is contained in a lead-plated cupboard.
29
The alpha irradiator is a 37 cm high, 30 cm deep and 47 cm wide aluminum
construction weighing circa 8 kg. The most important part is the
241
Am alpha source
(AP1 s/n 101, Eckert and Ziegler, Berlin, Germany). The activity is contained in three
parallel strips with a total activity of 50 ± 7.5 MBq, as reported by the producer. A 2 μm
pure gold film gold foil overlays the 1 μm americium oxide foil, and the downwardfacing 180 * 180 mm source is glued to a steel disc, attached to a circular turn-table. The
table can be rotated (with a choice of 2 or 3 revolutions per second) for a better dose
homogenization. Under the source is a holder for a wobbling collimator, followed in turn
by a movable shelf for positioning of cells for exposure. The distance from the shelf to
the cell layer is 0.5 mm in the top position, 45 mm in the bottom position and it takes 20
seconds to move the shelf in between. The distance for exposure of cells on polyamide
discs can be adjusted in 0.1 mm increments.
The X-ray source was set to 190 kV and 4 mA during exposure and was operated
without the optional aluminum filter. The angle of the cone-shaped beam was 40 * 55 °
(according to the manufacturer). The distance from the X-ray source to the cell layer is
44.5 cm in shelf top position, and 40.0 cm in the bottom position, corresponding to dose
rates of 0.052 and 0.068 Gy/min respectively. The reason for the comparably low dose
rates was the passage of the X-rays through the bottom of the incubator and the bottom as
well as the movable shelf of the alpha irradiator.
Cell exposure
For exposure of cells, 15 mm thick, round polyamide discs with a 30 μm deep and
145 mm in diameter milled out well, along with a 1.5 µm Mylar foil lid were employed.
For attached cells, cover slips with cells facing upwards were positioned in the well,
sprayed with a small amount of medium and covered with the lid. For blood, 250 or 500
μm per irradiation was positioned in the well and smeared out as evenly as possible under
the lid (see Figure 5). The smaller volume was used for alpha particle and mixed beam
irradiation where penetration is essential, while the larger volume was used for X-ray
irradiation.
30
Due to the configuration of the setup, the cells were exposed to alpha particles from
above and X-rays from below. The differences in dose rate were taken into account by
initiating mixed beam exposure simultaneously and then lowering the polyamide disc out
of reach of the alpha particles to finish the irradiation with X-rays alone. No collimator
was used for cell exposure, since using a collimator positions the cells too far away from
the source, outside of the Bragg peak. This would result in a significant reduction of the
alpha particle dose rate.
Figure 5: Polyamide exposure disc. To the left: with cover slips seeded for survival. To the right: blood
smeared out under the Mylar foil lid.
Clonogenic cell survival
The clonogenic cell survival assay was modified from the standard protocol.
Instead of first exposing a large number of cells and thereafter seeding out for survival,
the cells (AA8 cells) were first seeded out for colony formation (4 h prior to exposure, on
round cover slips, 32 mm in diameter). After exposure, the cover slips were returned to
medium-filled wells, incubated one week and subsequently stained with methylene blue
in methanol. Colonies were counted by eye and plating efficiency and clonogenic cell
survival was assessed.
Chromosomal aberrations
Chromosomal aberrations can be viewed by staining of metaphase chromosomes
either uniformly by Giemsa or with fluorescence in situ hybridization (FISH) where
specifically coloured probes are used for individual chromosomes (142, 143), or even
parts of chromosomes (144). Giemsa staining is useful for fast scoring but can not detect
31
all types of aberrations (translocations and insertions are for example often invisible)
(145), while FISH is more time consuming but can reveal more about the complexity
behind the aberrations (146). In this study, PBL were treated with Colcemid and
Calyculin A at the end of the incubation time to trap cells in metaphase and prematurely
condense chromosomes in G2 (and anaphase) (147). BrdU addition during the culturing
time made possible the fluorescence plus Giemsa-treatment, where second mitosis cells
are differentially stained and centromers become more visible (148).
The micronucleus assay
Giemsa-stained micronuclei represent another way of visualizing the cytogenetic
damage in single cells (149). The advantage of micronuclei is that they represent a
“running average” of the damage remaining in interphase after the first cell division
(150), while chromosomal aberrations gives an instantaneous yield of aberrations.
Micronuclei consist of acentric chromosome fragments or whole chromosomes lagging
behind when the nuclei is dividing, so that when the DNA decondenses these fragments
create individual nuclear membranes (149). The addition of Cytochalasin B stops
cytokinesis while allowing karyokinesis, leading to bi-, tri- or multinucleated cells
(depending on culturing time and dose) (151). Only binucleated cells are accepted for
scoring of micronuclei (152), while scoring the number of nuclei per cell gives the
replication index, e.g. the rate of cell division. Modified scoring criteria were applied, in
that micronuclei with a diameter >30 % of the main nuclear diameters were scored if
their DNA was less dense than that of the main nuclei.
The gamma-H2AX assay
The phosphorylation of histone H2AX, called gamma-H2AX in phosphorylated
form, is a signal for DNA damage. The phosphorylation is rapid, but persists until the
break has been repaired, leading to a slower dephosphorylation rate (140). The
visualization of gamma-H2AX “foci” can be achieved by a 2-step antibody procedure,
where the primary antibody recognizes the protein, while the secondary antibody carries
a flourophore and is targeted towards the species in which the primary antibody was
32
produced. The gamma-H2AX assay has become one of the gold standards for measuring
the early cellular response to IR-induced DNA damage (153). Gamma-H2AX foci form
within minutes and can persist for days, making it possible to score the kinetics of
phosphotylation and dephosphorylation (154). For this assay, the VH10 cell line was
applied.
33
Results and discussion
Paper I
Characterization of a setup for mixed beam exposure of cells to
241
Am alpha particles and X-rays
The traditional way of performing “mixed beam” studies has been to sequentially
expose cells to high and low LET radiation. In addition, the temperature at exposure has
not always been well controlled. The aim of this study was therefore to characterize and
validate a setup where cells can be simultaneously exposed to high and low LET in a
temperature-controlled environment.
The setup consists of a 241Am alpha particle irradiator and an X-ray tube, the former
positioned inside and the latter underneath a 164 liter cell incubator set to 37 °C. The
activity of the alpha source was characterized by track-etched detector by pressing the
source for 2.75 seconds against an aluminum sheet with pre-drilled holes (collimators)
under which the detector was positioned. This procedure was repeated 10 times, the foil
was etched with acid and 3 randomly chosen irradiated points in the detector foil were
analyzed for particle tracks. It was concluded that the dose rate was 0.265 Gy/min
(including a 0.025 Gy/min beta component, and a very small gamma component: below
0.001 Gy/h).
For validation of the setup, a modified version of the clonogenic cell survival assay
was employed, (see Materials and Methods). AA8-cells were exposed to X-rays, alpha
particles and two different mixtures of 25 % and 50% alpha particles, respectively. The
results revealed a RBE of 2.56 for 37 % and 1.90 for 10 % clonogenic cell survival, and
no statistically significant differences between observed and expected mixed beam data.
Envelope of additivity revealed that the observed data points were outside the envelope at
34
80 % survival, borderline at 50 % survival and within the envelope at 20 % survival. The
response was thus classified as additive.
In conclusion, the exposure device was performing as intended, and the effect of
mixed beam irradiation on clonongenic cell survival in AA8-cells was additive. However,
the distance between observed and expected values was larger for 50 % alpha particles in
the mix (as compared to 25 %), and increased with increasing dose. It is thus possible that
synergism could have been observed if mixed beam exposures with more than 50% alpha
particles and higher doses had been performed.
Main findings in paper I:
•
The exposure facility is performing as intended
•
Additive effect in AA8 cells after mixed beam exposure for the clonogenic
cell survival assay
35
Paper II
Micronuclei in human peripheral blood lymphocytes exposed to mixed
beams of X-rays and alpha particles.
The aim of this study was to investigate the cytogenetic effect of exposing human
lymphocytes (PBL) to a mixed beam of alpha particles and X-rays, by employing the
setup described in Paper I. PBL were exposed to increasing doses of alpha particles, Xrays and mixed beams (35 % alpha particles), and harvested for micronuclei 96 h after
exposure.
The RBE for alpha particles was 3.2, and for mixed beam irradiation significantly
more micronuclei were observed per dose than expected from the single irradiations. The
PBL micronuclei response was therefore classified as synergistic. The sizes of individual
micronuclei were very heterogeneous, with no significant differences between the
irradiation schemes. It was however observed that alpha particle and mixed beams
irradiated cell populations contained a slightly higher percentage of “oversized”
micronuclei compared to X-rays (significantly so for mixed beams).
The results allowed speculation about possible mechanisms behind the observed
response. Previously, Zhou et al. (103) proposed that high LET radiation induces sites of
repair-resistant DNA damage that can “trap” repair proteins by making dissociation
difficult. Error-free repair proteins trapped in complexes at those sites would then be
unavailable for repair of additional low LET-inducted damage, thus making the repair of
these lesions more error-prone. Dispersion indices support this conclusion since mixed
beam-irradiated cells displayed values intermediate to those of X-rays and alpha particles,
rather than having values similar to or higher than alpha particle values. This indicates
that the synergistic effect observed after mixed beam irradiation was not due to a small
part of the cell population receiving high levels of damage, but that the increased
frequency of micronuclei was representative for the whole cell population. This is
precisely the effect expected if the repair of the X-ray induced damages was impaired.
In conclusion, the effect of mixed beam irradiation was synergistic on the level of
micronuclei. The proposed mechanism behind this observed effect was that the presence
36
of high LET radiation-induced damage inhibits DNA repair, thus potentiating the damage
induced by low LET radiation
Main findings in paper II:
•
Synergistic effect for the number of micronuclei after mixed beam exposure
of PBL.
•
The effect was due to an overall increase of the number of micronuclei, not
a few highly damaged cells.
•
Proposed mechanism: The presence of high LET-induced damage inhibits
DNA repair, thus potentiating the effect of low LET-induced damage
37
Paper III
Higher than expected frequencies of complex aberrations in
lymphocytes exposed to mixed beams of
241
Am alpha particles and
X-rays
Previously, it was observed that exposing human PBL to mixed beams of alpha
particles of X-rays and alpha particles gave rise to a significant synergistic effect on the
level of micronuclei. The aim of this study was to investigate the mechanisms behind this
effect by employing fluorescent in situ hybridization (FISH) on chromosomal
aberrations. PBL were exposed to X-rays, alpha particles and a mixture thereof (doses
0.27, 0.43 and 0.66 Gy, 25, 25 and 40 % alpha particles, respectively) and harvested for
chromosomal aberrations 54 h after exposure. FISH analysis was performed in
chromosomes 2, 8 and 14, and observed frequencies of simple and complex aberrations
after mixed beam exposure were compared to the expected (as calculated from the dose
components).
The distribution of aberrations was overdispersed for alpha particles, Poissonian
for X-rays and intermediate for mixed beams. For simple aberrations and primary breaks,
a linear equation was best fitted to the dose response curve. The dose response for
complex aberrations was linear for alpha particle exposure but linear-quadratic for mixed
beams and X-ray exposure. At the lowest X-ray dose no complexes were observed. The
highest degree of complexity (number of break points involved per complex) was
observed for alpha particles, closely followed by mixed beams. No dose response was
present for the degree of complexity. Significant differences between observed and
expected number of aberrations was detected for the highest and the median mixed beam
dose (simple plus complex aberrations, and complex aberrations, respectively). This can
be interpreted as evidence for a synergistic action of X-rays and alpha particles on the
induction of complex aberrations.
The synergism conclusion is in agreement with previous micronucleus results.
Furthermore, the linear-quadratic dose response of complex aberrations after mixed beam
exposure is an interesting finding. A linear quadratic dose response is observed when a
38
chromosomal aberration results from the interaction of two radiation tracks, a
characteristic observed for low LET radiation (155). It therefore points towards an
interaction between alpha particles and X-rays, giving the synergism conclusion
additional support. Consequently, the risks of stochastic effects from mixed beam
exposure may be higher than expected from adding the individual dose components.
Main findings in paper III:
•
Simple aberrations and primary breaks were always best fitted by a linear
equation, while complex aberrations were linear for alpha particles, but
linear-quadratic for X-rays and mixed beams.
•
Synergistic effect for complex aberrations at the two highest mixed beam
doses (as calculated from envelopes of additivity) and for simple aberrations
only at the highest mixed beam dose.
•
Results indicate that cancer risk for exposure to mixed beams in radiation
oncology may be higher than expected based on the additive action of
individual components.
39
Paper IV
Gamma-H2AX foci in cells exposed to a mixed beam of X-rays and
alpha particles.
Traditional methods for analyzing mixed beam exposure have been clonogenic cell
survival or cytogenetic endpoints, thus leaving the early cellular response unexplored. To
fill this knowledge gap, VH10 human fibroblasts were exposed to one dose each of alpha
particles, X-rays and mixed beams (25 % alpha particles), incubated 0.5, 1, 3 and 24 h
and analyzed for gamma-H2AX foci. Dose response curves were performed 1 h after
exposure.
Two foci classes could be observed; large foci (LF) and small foci (SF). The
number of LF in alpha-particle irradiated cells corresponded well with the number of
particle tracks, as calculated by fluence (particles per second per cm2). The alpha particle
RBE was 0.76 ± 0.52 for total number of foci and 2.54 ± 1.11 for LF (as compared to Xrays). The total number and area of foci in mixed beam irradiated cells was intermediate
to the results from X-ray and alpha particle exposed cells for dose response as well as
kinetics, and could thus be classified as additive. At the 24 h time point, the remaining
foci levels were low, and did not differ significantly. However, mixed beam LF kinetics
differed between observed and expected. The number and area of LF was significantly
lower than expected at 0.5 h after exposure, and an increase from 0.5 to 3 h for number
and area of LF was observed where no difference between time points were expected.
The relative LF (the LF contribution in percent of the total number and area of foci)
confirmed the trends as significant. Also, LF in mixed beam-irradiated cells did not reach
their maximal area until 1 h after exposure, and thus were not initially phoshporylated to
their full extent.
The differences between observed and expected in mixed beam LF kinetics
indicated that the phosphorylation process of H2AX histones around sites of complex
DNA damage was delayed, as compared to alpha particle and X-ray-induced damage.
That LF in mixed beam irradiated cells disappeared at a slower rate compared to LF in
alpha particle irradiated cells indicate that an interaction took place between the low and
40
high LET radiations. Taking the physics of the situation into account, it appeared most
likely that mixed beam exposure of cells to low and high LET radiation resulted in the
delay of the DNA damage response. In conclusion, the proposed hypothesis is that the
presence of low LET-induced DNA damage engages the DNA repair machinery, thus
leading to a delayed repair of the more complex DNA damage induced by alpha particles.
Main findings in paper IV:
•
Additive effect for the dose response for number and area of total foci and
LF per cell.
•
Significant differences in the observed repair kinetics for mixed beam LF,
compared to the expected. Initial frequency and area of LF were lower than
expected during the first hour, and LF were not were not phosphorylated to
their full extent until 1h after exposure.
•
Proposed mechanism: the presence of low LET-induced DNA damage
engages the DNA repair machinery, thus leading to a delayed repair of the
more complex DNA damage induced by alpha particles.
41
Concluding remarks and future perspectives
Concluding remarks
Mixed beam exposures of humans are increasing due to airplane travel and the
application of specific cancer treatments. Establishing the correct approach for
determining the risks of mixed beam exposure is therefore of great importance. In this
thesis, the focus is on investigating DNA damage and repair in cells exposed to mixed
beams in a dedicated exposure facility, with the aim of establishing if the response is
additive or synergistic.
The main observation was that the synergistic effect was dependent on endpoint
and time after irradiation. For clonogenic survival in AA8 cells (7 days incubation) the
response was additive. However, for micronuclei and its precursor step chromosomal
aberrations (96 and 54 h after irradiation, respectively), a significant synergistic effect
was seen in human PBL. A pre-study for the micronuclei experiments observed
significantly more micronuclei 96 h compared to 72 h after alpha particles exposure,
indicating that heavily damaged cells did not have time to reach the first interphase at the
standard harvesting point 72 h. For gamma-H2AX foci, the significant differences in LF
kinetics between observed and expected mixed beams in VH10 cells at the 0.5 to 3 h time
points did not remain at the 24 h time point. In conclusion, it is important to optimize the
assays used as to capture the maximum magnitude of the response. If possible, the
response should be scored at multiple times after irradiation.
Secondly, the proportion of high LET in the mixture is of great importance to the
results. Originally, 3 mixtures of mixed beams were performed for clonogenic survival:
50, 25 and 12.5 % alpha particles, respectively. The 12.5 % results were omitted in the
paper since they overlapped with the X-ray results and did not add additional value.
When calculating expected values it was observed that the difference between observed
and expected increased with increasing percentage alpha particles. It is thus possible that
42
synergistic effects would have been observed also for clonogenic survival if experiments
had been carried out with even higher percentages of alpha particles. For PBL, significant
synergistic effects were observed for micronuclei after 35 % alpha particles and for
aberrations after 40 % alpha particles and 25 % alpha particles. For the 25 % however,
the difference was only significant at a higher dose. Foci results were obtained with 25 %
alpha particles, and if it had not been possible to find particle tracks based on size, the
results would have been scored as additive (all significant effects were observed for LF).
Therefore, it would be of interest also to investigate different mixtures, where the
proportions high to low LET are varied.
Taken together, the results indicate a synergistic effect for the interaction of low
and high LET ionizing radiation during mixed beam exposures. Proposed mechanisms
behind the observed responses are: 1) The presence of high LET-induced damage inhibits
DNA repair, thus potentiating the effect of low LET-induced damage, 2) The presence of
low LET-induced DNA damage engages the DNA repair machinery, thus leading to a
delayed repair of the more complex DNA damage induced by alpha particles, 3)
Combination of low and high LET radiation increases damage complexity. These are not
necessarily mutually exclusive, delayed repair of high LET induced damage and
potentiated effect of low LET induced damage can occur simultaneously.
Furthermore, the results presented in this thesis have given rise to some interesting
suggestions for future research, which will be detailed in the following section.
Future perspectives
As stated in the concluding remarks, the presence of the synergistic effect was
dependent on the proportion of high to low LET, the endpoint, and the time after
irradiation. Further experiments should therefore strive to investigate more than one
mixture, and optimize the assay as to capture the maximal response (and if possible
investigate the response at more than one time point).
The research presented in this thesis will also be continued more long-term with a
different approach to the irradiation scheme. So far, the proportion of high LET to low
LET has remained constant throughout an experiment. For the upcoming study however,
43
the dose of alpha particles will remain constant while the X-ray dose will be varied. This
approach aims to find if and how the synergies between the actions of low and high LET
IR are dependent on the percent high LET in the mixture, and if the appearance of
synergism is linear with increasing X-ray dose or not. The synergistic effect that was
observed for several of the assays of this study may appear and/or disappear when
changing the proportions. This study will start by focusing on the mutation frequency in
human TK6 lymphoblastoid cells, as a tool investigate the risks for secondary cancers
after mixed beam cancer treatment. Further on, the Comet assay and the gamma-H2AX
assay will also be applied.
The studies in this thesis have started to shed light upon the complicated field of
mixed beams research. What yet remains is to thoroughly investigate the mechanisms
behind the observed responses, by identifying cell cycle effects and which DNA repair
system is most sensitive to mixed beam-induced damage. Such data could confirm, or
possibly contradict the theories from the investigations presented here (as well as
previously published theories). Proteomic and metabolomic studies at different time
points after mixed beam irradiation could be very helpful in explaining the differences
observed between endpoints and cell lines. But to fully understand the effect of mixed
beams and the cellular mechanisms behind the interactions of radiations of different LET
requires the collaboration between several disciplines, such as correlating microdosimetry
simulations with observed chemical and biological responses.
A DNA-repair-targeted study following up the results summarized here is currently
being performed by the author of this thesis, Elina Staaf, and collaborator Dr. Daniel
Vare from the DNA repair field. We are employing the CHO cell lines AA8 (wild type),
irs1SF (HR-deficient), V3-3 (NHEJ deficient) and EM9 (single strand break repair
deficient) and investigating their response to mixed beam irradiation. Preliminary results
indicate a significant difference between observed and expected clonogenic survival after
mixed beam exposure (50% alpha particles) for AA8 and irs1sf (wild type and HRdeficient cell lines). The study will also include gamma-H2AX and 53BP1 foci kinetics,
dose responses as well as micronuclei dose response curves.
44
Acknowledgements
First and foremost I would like to thank my supervisor Andrzej Wojcik for inspiring
guidance, fruitful discussions and entertaining moments during travels and sports. Thank
you for making me a scientist!
Also, thanks to Siamak Haghdoost, my co-supervisor who always had time for guiding
my efforts in the lab and reading my manuscripts.
I am grateful for all help Mats Harms-Ringdahl gave me with the SWE-RAYS workshop
and official emails. Thank you for being so generous with your time and experience.
My previous thesis and project supervisors Björn Cedervall, Margareta Edgren, Fredrik
Elgh, Ingrid Marklund and Johan Trygg, thank you for improving my skills and for
teaching me new ones.
All former and present members of the Radiation Biology group: Ainars, Alice, Asal,
Eliana, Karl, Marta, Paulo, Ramesh, Sara S, Sara SM, Siv, Tai, thank you for making the
everyday work enjoyable and the parties, conferences and trips even more so!
Daniel Vare, thanks for being an awesome collaborator and for keeping me in shape!
I would like to thank the whole GMT department for a very good working environment.
Especially you innebandy players, you know who you are!
I would also like to thank family and friends for tolerating my workaholic periods and
information overload from my chatting.
Johan, your encouragement, patience and understanding have been invaluable. You are
the best!
This study was supported by a grant from the Swedish Radiation Safety Authority (SSM)
45
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