In vitro and in vivo aspects of
intrinsic radiosensitivity
Karl Brehwens
Doctoral thesis in Molecular Bioscience
Centre for Radiation Protection Research
Department of Molecular Biosciences, the Wenner-Gren Institute
Stockholm University, Sweden, 2014
© Karl Brehwens (pages i-58)
ISBN 978-91-7447-821-1
Printed in Sweden by Universitetsservice US-AB, Stockholm 2013
Distributor: Department of Molecular Biosciences, the Wenner-Gren Institute
ii
To my family
“Some people drink from the fountain of knowledge. Others just gargle”
Robert Anthony
iii
Abstract
This thesis focuses on how physical and biological factors influence the outcome of an
exposure to low LET radiation. The first part of the thesis investigates physical factors and
their role in determining the biological effects of photon irradiation as investigated in the
human lymphoblastoid cell line TK6. That the dose rate changes during real life exposure
scenarios is undisputable, but radiobiological data regarding potential differences between
exposures at increasing, decreasing or constant dose rates is absent. In paper I, it was found
that an exposure where the dose rate decreases exponentially induces significantly higher
levels of micronuclei than exposures at an increasing or constant dose rate. Paper II describes
the construction and validation of novel exposure equipment used to further study this
phenomenon, which is described in paper III. Taken together, our studies are the first to
describe this novel radiobiological effect, which could be both dose and dose rate dependent.
In paper I we also observed a radioprotective effect when cells were exposed on ice. This
“temperature effect” (TE) has been known for decades but it is still not fully understood how
hypothermia acts in a radioprotective manner. This was investigated in paper IV, where the
DNA DSB sensing, chromatin conformation, γH2AX foci formation kinetics and cellular
survival were investigated in order to find a mechanistic explanation. The results suggest that
in TK6 cells, hypothermia does not modify the radiosensitivity per se, as the radioprotective
effect was not seen on the level of clonogenic survival or γH2AX foci formation kinetics. We
instead suggest that a transient cell cycle delay is induced in hypothermic cells, as revealed by
the micronuclei frequencies scored in sequentially harvested cells.
The last paper in this thesis is directed towards the highly important question of the role of
individual radiosensitivity in the risk of developing adverse effects to radiotherapy (RT). It
has been speculated that a substantial part of the variation seen in the patient response to RT is
caused by the inherent radiosensitivity of the individual. In the clinic, there is currently no
reliable way of predicting patient radiosensitivity prior to RT and consequently no way to
tailor treatment or aftercare accordingly. In paper V the aim was to investigate the role of
biomarkers and clinical parameters as possible risk factors of late adverse effects in a cohort
of head-and-neck cancer patients. The study was performed on a rare patient cohort of highly
radiosensitive individuals that developed osteoradionecrosis (ORN) of the mandible as a
consequence of RT. Biomarkers and clinical factors were then subjected to multivariate
analysis in order to identify ORN risk factors. The results suggest that the patient’s oxidative
stress response is a key factor in ORN pathogenesis, and support the current view that patientrelated factors constitute the largest source for the variation seen in the severity of adverse
effects to RT.
In summary, this thesis provides new and important insights to the role of biological and
physical factors in determining the consequences of low LET exposures.
iv
List of original publications
This doctoral thesis is based on the following publications/manuscripts, referred to by their
roman numerals:
I
Brehwens K, Staaf E, Haghdoost S, Gonzalez A.J, and Wojcik A.
Cytogenetic damage in cells exposed to ionizing radiation under conditions of a
changing dose rate. Radiation Research 173 (3): 283-289 (2010).
II
Brehwens K, Bajinskis A, Staaf E, Haghdoost S, Cederwall B and Wojcik A.
A new device to expose cells to changing dose-rates of ionising radiation.
Radiation Protection Dosimetry 148 (3): 366-371 (2012).
III
Brehwens K, Bajinskis A, Haghdoost S and Wojcik A.
Micronucleus frequencies and clonogenic cell survival in TK6 cells exposed to
changing dose rates under controlled temperature conditions.
International Journal of Radiation Biology (2013), in press.
DOI:10.3109/09553002.2014.873831
IV
Dang L, Lisowska H, Shakeri Manesh S, Sollazzo A, Deperas-Kaminska M,
Staaf E, Haghdoost S, Brehwens K and Wojcik A.
Radioprotective effect of hypothermia on cells - a multiparametric approach to
delineate the mechanisms. International Journal of Radiation Biology 88 (7):
507-514 (2012).
V
Danielsson D*, Brehwens K*, Halle M, Marczyk M, Polanska J, MunckWikland E, Wojcik A and Haghdoost S.
Reduced oxidative stress response as a risk factor for normal tissue damage
after radiotherapy: a study on mandibular osteoradionecrosis. International
Journal of Radiation Oncology•Biology•Physics, submitted.
*=authors contributed equally to the work
Papers I-IV are reproduced with permission from the publishers
v
Publications not included in the thesis
VI
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. The yield of
radiation-induced micronuclei in early and late-arising binucleated cells
depends on radiation quality. Mutation Research. Aug 14;701(1):80-5 (2010).
VII
Staaf E, Brehwens K, Haghdoost S, Nievaart S, Czub J, Braziewicz J, and
Wojcik A. Micronuclei in human peripheral blood lymphocytes exposed to
mixed beams of X-rays and alpha particles. Radiation and Environmental
Biophysics 51 (3): 283-93 (2012).
VIII
Staaf E, Brehwens K, Haghdoost S, Pachnerova-Brabcova K, Czub J,
Braziewicz J and Wojcik A. Characterization of a setup for mixed beam
exposure of cells to 241Am particles and X-rays. Radiation Protection Dosimetry
151 (3): 570-79 (2012).
IX
Staaf E, Brehwens K, Haghdoost S, Czub J and Wojcik A.
Gamma-H2AX foci in cells exposed to a mixed beam of X-rays and alpha
particles. Genome Integrity. 3 (1) 8-9414-3-8 (2012).
X
Staaf E, Deperas-Kaminska M, Brehwens K, Haghdoost S, Czub J, Braziewicz
J and Wojcik A. Complex aberrations in lymphocytes exposed to mixed beams
of 241Am alpha particles and X-rays. Mutation Research Aug 30;756(1-2):95100 (2013).
XI
Skiöld S, Näslund I, Brehwens K, Andersson A, Wersäll P, Lidbrink E, HarmsRingdahl M, Wojcik A and Haghdoost S. Radiation-induced stress response in
peripheral blood of breast cancer patients differs between patients with severe
acute skin reactions and patients with no side effects to radiotherapy
Mutation research 2013 Aug 30;756(1-2):152-7 (2013).
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vii
Table of contents
Abstract ..................................................................................................................................... iv
List of original publications ....................................................................................................... v
Publications not included in the thesis ...................................................................................... vi
Abbreviations ............................................................................................................................ ix
Introduction ................................................................................................................................ 1
Introduction to the thesis ........................................................................................................ 1
Ionizing radiation: a short historical perspective ................................................................... 2
Ionizing radiation: A closer look ............................................................................................ 3
Factors influencing the cellular response to low LET radiation ................................................ 5
Physicochemical factors ......................................................................................................... 5
Dose rate ............................................................................................................................. 5
Changing dose rates ........................................................................................................... 6
Temperature at irradiation .................................................................................................. 9
The oxygen effect ............................................................................................................. 10
Oxidative stress ................................................................................................................ 11
Biological factors ................................................................................................................. 13
DNA damage and repair ................................................................................................... 13
Cell cycle phases and radiosensitivity .............................................................................. 15
Non-DNA targets of ionizing radiation ............................................................................ 17
Radiotherapy ............................................................................................................................ 19
Adverse effects of radiotherapy ........................................................................................... 20
Mandibular osteoradionecrosis ........................................................................................ 20
Individual radiosensitivity ........................................................................................................ 24
The present investigation ......................................................................................................... 26
Aims of this thesis ................................................................................................................ 26
Results and discussion .......................................................................................................... 27
Paper I .............................................................................................................................. 27
Paper II ............................................................................................................................. 28
Paper III ............................................................................................................................ 29
Paper IV............................................................................................................................ 30
Paper V ............................................................................................................................. 32
Concluding remarks and future perspectives ....................................................................... 34
Populärvetenskaplig sammanfattning ...................................................................................... 36
Acknowledgements .................................................................................................................. 39
References ................................................................................................................................ 41
viii
Abbreviations
γH2AX
8-oxo-dG
8-oxo-dGMP
8-oxo-dGTP
ADR
ANOVA
ASMase
AT
ATM
BAX
BER
BNC
BR
CRP
DDR
DDRE
DMSO
DSB
EGF
ELISA
Gy
HBO
HDRP
HNC
HPLC
HR
H-RAS
ICRP
IDR
IR
LET
MFROM
MN
MTH1 (NUDT1)
MTO
MUTYH
NAD(P)H
NER
NSMase
NHEJ
H2A histone family, member X, phosphorylated on serine 139
8-oxo-7,8-dihydro-2´-deoxyguanosine
8-oxo-7,8-dihydro-2´-deoxyguanosine monophosphate
8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate
Average dose rate
Analysis of variance
Acidic sphingomyelinase
Ataxia telangiectasia
Ataxia telangiectasia mutated
BCL2-associated X protein
Base excision repair
Binucleated cell
Bystander response
Chemical radioprotectants
Decreasing dose rate
Decreasing dose rate effect
Dimethylsulfoxide
Double-strand break
Epidermal growth factor
Enzyme-linked immunosorbent assay
Gray; Joule/kg
Hyperbaric oxygen
High dose rate point
Head and neck cancer
High-performance liquid chromatography
Homologous recombination
V-Ha-ras Harvey rat sarcoma viral oncogene homolog
International commission on radiological protection
Increasing dose rate
Ionizing radiation
Linear energy transfer
Moving away from the source
Micronuclei
Nudix (nucleoside diphosphate linked moiety X)-type motif 1
Moving towards the source
MutY homolog
Nicotinamide adenine dinucleotide phosphate-oxidase
Nucleotide excision repair
Neutral sphingomyelinase
Non-homologous end joining
ix
OGG1
ORN
PBL
RIF
ROS
RNS
RT
SSB
Sv
TE
TGF
SNP
8-oxoguanine DNA glycosylase
Osteoradionecrosis
Peripheral blood lymphocytes
Radiation-induced fibroatrophy
Reactive oxygen species
Reactive nitrogen species
Radiotherapy
Single-strand break
Sievert; dose equivalent (Joule/kg)
Temperature effect
Transforming growth factor
Single nucleotide polymorphism
x
Introduction
Introduction to the thesis
This thesis focuses on radiosensitivity from two angles; either the biological component (the
cell system) is well-defined and the physical factors are modulated, or the biological
component is variable (unique) with respect to the individual, but the physical aspect (the
radiotherapy [RT]) is highly controlled. The first part deals with radiosensitivity on the level
of the individual cell from a well-characterized cell line. The use of a cell line minimizes the
biological variation and allows physical factors to be the important variables in the
experiments. In papers I-III a novel radiobiological phenomenon was discovered and further
studied by constructing new exposure devices. In paper IV, exposure temperature (a wellknown factor influencing the cellular response to ionizing radiation [IR]) was investigated in
further detail as the underlying mechanism behind the radioprotective effect of hypothermia is
largely unknown.
In the second part of this thesis (paper V) the approach was the opposite compared to the first
part, in the sense that it is the biological variation (on the level of the individual cancer patient
treated with RT) that is in focus. The physical aspect (RT) is instead highly controlled in this
study. The biological variation or “individual radiosensitivity” in cancer patients treated with
RT is increasingly implicated in the occurrence of adverse effects. As of now the individual
radiosensitivity cannot be reliably assessed prior to RT, and there is consequently no
possibility to account for the individual radiosensitivity when planning the treatment or
aftercare. In paper V a rare cohort of head and neck cancer (HNC) patients that developed the
late adverse effect osteoradionecrosis (ORN) as a consequence of RT was compared to a
control group. The aim was to evaluate the in vitro capacity to handle oxidative stress, and the
influence of single nucleotide polymorphisms (SNP) in oxidative stress pathways together
with clinical parameters, as factors used in modeling to identify ORN risk factors.
The aim of the following introductory part of this thesis is not to give a comprehensive “crash
course” in radiobiology, but to provide the reader with a context for the publications included
herein.
1
Ionizing radiation: a short historical perspective
In 1895, Wilhelm Conrad Röntgen was performing experiments with a cathode ray tube that,
when filled with a certain gas, would produce a fluorescent glow if a high current was passed
through it. Röntgen discovered that if he covered his fluorescing tube with light-proof
material, a fluorescent glow could still be seen on a barium platinocyanide-painted screen a
short distance away. Röntgen continued to investigate this “invisible light”, the “X-rays”, and
found that it to various extents could penetrate different objects, as seen on exposed film
pieces. It could also penetrate the human body, making visible what previously only surgery
could reveal. After thoroughly validating his findings, Röntgen wrote a paper about the new
X-rays (Röntgen 1895). When his scientific breakthrough was published, other laboratories
(for example the laboratory of Thomas A. Edison) quickly began to reproduce and further
investigate the new X-rays, since cathode ray tubes were fairly common in physics
laboratories at this time. It did not take long for the X-rays to find use in a vast range of
applications in society, and a new field in science was born, earning Röntgen the first Nobel
Prize in physics in 1901 for his discovery of this new type of light that we now call IR.
The subsequent discovery of radioactive elements by Henri Becquerel and Marie and Pierre
Curie, which resulted in them sharing the 1903 Nobel Prize in physics, further expanded this
scientific field. It was discovered that there were various types of invisible radiation
emanating from certain elements. Some could, like the X-ray, penetrate various materials.
Others could not stain a photographic film through a piece of paper. But what effect could this
“invisible light” have on the human body?
Probably owing to its widespread use in medicine, it was not long after Röntgen’s discovery
that there were reports of detrimental effects of exposure to the X-rays. Deep “burns” and
dryness of the exposed skin, ulcerations and loss of hair were some of the symptoms.
Scientists working extensively with X-rays were often diagnosed with carcinoma, and many
had to amputate fingers, hands and arms. Thomas A. Edison’s chief assistant Clarence Dally
had worked extensively with X-rays and eventually died in 1904 from X-ray related injuries,
which made Edison stop conducting X-ray related research (Goodman 1995). Many scientists
working with X-rays had to pay a high price for their scientific advances, as described by
Percy Brown in the 12-article series “American martyrs to radiology” published in American
Journal of Roentgenology in 1995 (see (Brown 1995) for the first part in this series).
The realization of the detrimental effects of X-rays (and of course, also from radioactive
substances) initiated work regarding radiation protection, today one of the most important
aspects of radiation research. X-rays (and eventually other forms of IR) also found use not
only in diagnostic applications, but also in the direct treatment of various forms of cancer.
Importantly, it was soon realized that the response to IR was heterogeneous among the
exposed individuals, a fact that forms the foundation for the second part of this thesis. As IR
steadily became something encountered by the general public at home, at work and in the
hospital, a demand arose to investigate the biological consequences of these exposures. By
irradiating biological material with IR the aim is, and has been, to increase the understanding
2
of how cells handle the inflicted damage, and eventually try to put this in a greater perspective
that may involve mechanistic understanding, risk assessment or therapeutic applications.
Ionizing radiation: A closer look
As years have passed, the knowledge regarding IR has increased tremendously. What we
today call IR is in fact further subdivided into electromagnetic radiation (highly energetic
photons, such as X-rays and γ-rays) and particle radiation (electrons, neutrons, protons, and
heavy ions). Particle radiation will not be further discussed in this thesis, but it is worth
mentioning that the complexity and distribution of DNA damage from particulate radiation is
very different compared to that of photon radiation (resulting in its higher relative biological
effectiveness). When high energy photons are absorbed by matter, they interact with the
electrons of an atom. The energy of the photon is partially or completely converted into
kinetic energy of an electron, resulting in the release of a fast electron from the now ionized
atom. These fast electrons further ionize other molecules, releasing new electrons until their
energy falls below a critical level. Figure 1A illustrates this, but also shows that the
ionizations and excitations are not uniformly distributed in the target. The formation of local
ionization clusters of varying size at the end of the electron track has implications for the
potential level of complexity of the damage inflicted to the target (Pouget, Mather 2001, Van
der Kogel, Joiner 2009, Hall, Giaccia 2012). However, the density of ionizations (energy
deposited per unit track length) along the track of a fast electron is low compared to that of a
charged particle such as an α-particle. For this reason, photon radiation is classified as low
linear energy transfer (LET) radiation.
It is now widely acknowledged that DNA is the most critical target in the cell. It is not,
however, the most abundant target; it is more probable that a water molecule is the target for
the incident photon. Highly energetic photons can ionize water molecules, resulting in the
ejection of a fast electron that in turn damages the DNA (direct action, figure 1B). However, it
is more probable that this ejected electron interacts with water molecules close to the DNA,
resulting in radical formation, which then exert the damaging effect on the DNA (indirect
action, figure 1B). This indirect action is responsible for 60-70% of the DNA damage
following X- and γ-ray exposure (Hall, Giaccia 2012).
3
Figure 1. A: An illustration of low-LET track structure in the target volume. Fast electrons (and possibly also
scattered photons, γ’) are ejected and further interact with other atoms. Ionizations and excitations are not
uniformly distributed but tend to be localized in clusters along the particle track. B: The direct and indirect
action, in which γ/X-rays cause damage to DNA. Water molecules represent an abundant non-DNA target in the
cell. 60-70% of the damage occurs through the indirect action in the case of photon radiation. Modified from
(Pouget, Mather 2001).
With radicals as the major effector molecules of low LET radiation, the biological outcome of
an absorbed dose will also depend on the extent of radical scavenging (Hall, Giaccia 2012),
either through the cell’s own defense systems or through chemical compounds added to the
cell’s environment (Limoli et al. 2001). There are several physical and biological parameters
that influence how cells react and respond to ionizing radiation, of which some of the more
prominent ones are described in the following section.
4
Factors influencing the cellular response to low LET
radiation
Physicochemical factors
Dose rate
To say anything about the expected biological outcome of an exposure to IR, the dose
absorbed and the radiation type (low or high LET, or a mix of the two) must be known. But
given that the dose and radiation type are known it is still necessary to know the dose rate (the
dose delivered per unit time) as the dose rate is a key factor in determining the effect of a dose
of X- or γ-rays. As will be discussed below, the dose rate is important for the outcome of an
experiment: just at the author’s department, a total of four 137Cs γ-sources are available
making it possible to irradiate cells with γ-rays at a dose rate between ≈ 1.3 mGy/h to ≈ 7
Gy/min, a difference of five orders of magnitude.
If a delivered dose is split in two equal fractions separated by ≈30 minutes, the effect of the
dose will generally be reduced compared to the effect of the whole dose given in one fraction
(Hall, Giaccia 2012). The general consensus is that in between fractions, DNA damage (the
so-called potentially lethal damage) is repaired and this reduces possible interactions (leading
to lethal chromosomal aberrations) with lesions occurring during the second fraction. During
protracted exposures, there is by definition no fractionation, but the same thought can be
applied. Already in 1939 Karl Sax studied chromosomal aberrations in plants following
exposure to X-rays at various intensities (dose rates). He found that for a given dose of Xrays, lowering the intensity reduced the number of chromosomal aberrations, which he
attributed to the lower probability of two chromatid breaks interacting when the irradiation
time was extended (Sax 1939). A sparing effect of lowering the dose rate has since then been
found for several endpoints such as clonogenic survival (Hall, Bedford 1964, Holmes et al.
1990), micronuclei (MN) induction (Bhat, Rao 2003), mutation induction (Russell et al. 1958,
Russell et al. 1959, Elmore et al. 2006, Kumar et al. 2006, Okudaira et al. 2010) and
chromosomal aberrations (Tanaka et al. 2009).
If the dose rate is reduced in the range of ≈1 Gy/min to ≈10 mGy/min, more DNA repair can
take place during the irradiation, reducing the possibility of interactions between lesions in the
DNA. Above ≈1 Gy/min, the time to deliver a dose is too short for DNA repair to play a
significant role, and there is generally no dose rate effect seen above this dose rate. For
example, with MN induction in peripheral blood lymphocytes (PBL) as the endpoint studied,
a clear dose rate effect for γ-rays was seen in the range of 3 Gy/min to 2.1 mGy/min (Bhat,
Rao 2003) but no dose rate effect was seen when the dose rate of an electron beam was
lowered from 352.5 Gy/min to 35 Gy/min (Acharya et al. 2010).
5
However, the dose rate effect has also been observed outside the range of dose rates where it
usually occurs. In the 1960’s Hornsey and Alper reported an unexpected increase in percent
surviving mice four days after exposure to an electron beam if the dose rate was lowered from
60 Gy/min to 1 Gy/min (Hornsey, Alper 1966). There are also reports of an inverse dose rate
effect, where lowering the dose rate within a certain dose rate range increases the biological
effect of irradiation (Mitchell et al. 1979). A possible explanation is that at a certain dose rate,
cells progress in the cell cycle but are blocked in G2, a radiosensitive phase. During protracted
irradiations, this will lead to the exposure of more and more cells accumulated in a more
radiosensitive phase of the cell cycle. It has also been suggested that in a certain dose rate
range (≈0.3-10 mGy/min), lesions in the DNA are produced at a similar rate to the
endogenous production, and that such lesions therefore are detected (and repaired) with
optimal efficiency as compared to both lower and higher dose rates (Vilenchik, Knudson
2006).
Changing dose rates
If we exclude most medical applications almost all IR exposures take place at a changing dose
rate. In radiation accidents, it is not uncommon for the source or the exposed person to be in
motion with respect to each other. In an environmental radiological accident, such as the
Chernobyl/Fukushima disaster, radionuclides were dispersed in the atmosphere and then
gradually accumulated in the soil and water as fallout. Also, by virtue of radioactive decay the
activity (and consequently the dose rate) of any radionuclide source will decrease
exponentially with time (with half-lives ranging from fractions of a second to millions of
years). Both temporal variation of dose rate and also isotope composition has been
demonstrated following the Fukushima disaster (Hosoda et al. 2011), demonstrating that the
dose rate indeed is a dynamic factor following radionuclear accidents. One of the more
frequently encountered scenarios involving changing dose rates is during aircraft flight.
During take-off and landing the dose rate of cosmic radiation can change 16-fold (Zeeb et al.
2002, Zeeb et al. 2003). Although the dose rates and doses involved are very low, no one
knows how the effects of such an exposure scenario compares to equivalent exposures at
constant dose rates, from which current risk models are derived. Even with sophisticated
computational modeling techniques available, biological data is still necessary to provide a
starting point for the modeling.
Virtually all radiobiological experiments performed today are performed at a constant dose
rate. These exposures are relatively simple to perform, and equipment for this is readily
available. But these experiments are not representative of the vast majority of occurring IR
exposures, and it is surprising that no one until now has investigated the effects of changing
dose rates of IR. There are to the author’s knowledge no studies investigating changing dose
rates of IR, making a literature review pointless. Instead, the preliminary studies conducted so
far in Poland by Andrzej Wojcik et al, and later during the author’s master thesis will be
presented.
6
Several years ago Abel J. Gonzalez of the Argentine Nuclear Regulatory Authority
hypothesized that a changing dose rate might influence the outcome of an absorbed dose of IR
(Gonzalez 2004). A few years later, this hypothesis was tested in Poland by Andrzej Wojcik
and co-workers. The setup used was very similar to the setup described in paper I (based on
the movement of cells towards/away from the source), but using a 60Co RT source to expose
whole blood samples to 3 Gy γ-rays at room temperature either with the dose rate increasing
(moving towards the source, MTO) or with it decreasing (moving away from the source,
MFROM). As in paper I of this thesis, MN induction was the chosen endpoint. The results
indicate (figure 2, Wojcik et. al., unpublished data) that samples exposed to a decreasing dose
rate (MFROM) of IR on average suffers more damage than samples exposed to an increasing
dose rate (MTO) of IR. These interesting preliminary results were the basis for the author’s
master thesis project performed in the fall of 2008 in Andrzej Wojcik’s group.
Figure 2. PBL were exposed at room temperature to 3 Gy of γ-rays from a 60Co source, during either an
increasing (moving towards the source, MTO) or a decreasing (moving away from the source, MFROM) dose
rate. Micronuclei were then scored in binucleated cells. Blood was drawn from the same donor except in
experiment 5 and 6 where two donors (A and B) were used. * = significant difference with p<0.05, χ2 test for
Poisson-distributed events. Wojcik et. al., unpublished data.
Building on the findings of this preliminary study, the author’s master thesis aimed at further
investigating this phenomenon. Two devices similar to that described in paper I were
constructed, exposing cells to X-rays at room temperature. To reduce the interexperimental
variation seen in the Polish pilot study where PBL were used, the human lymphoblastoid cell
line TK6 was used. A third sample was also included in the exposure (average dose rate,
ADR) resulting in three samples receiving the same total dose in the same total time (which
became the standard sample lineup used in papers I and III). The initial endpoints were the
MN assay and DNA damage assessed by the alkaline comet assay. The results indicate that
there was a significant effect on the level of MN induction (as seen in the Polish study)
between MFROM and MTO/ADR at the higher dose of 4.3 Gy (figure 3), with a clear
7
difference seen also after 3.4 Gy but here the low number of replicates (2) makes statistical
testing difficult. When comparing DNA repair kinetics between MTO and MFROM, there
was nothing to suggest a difference (figure 4). Due to sample number limitations in the comet
assay, all three samples could not be included in the same experimental run. This is important
as the comet assay is a method that can exhibit large interexperimental variation. However, it
was possible to include one ADR sample (0 min repair) and nothing suggested that initial
levels of DNA damage following exposure differed among the three samples (data not
shown).
Figure 3. MN induction in TK6 cells following exposure to 3.4 (left panel) or 4.3 Gy (right panel) of X-rays
under conditions of a changing dose rate. MN were scored in binucleated cells after 27 h incubation with
cytochalasin B. Figure shows the mean MN frequency from two (left panel) and three (right panel) experiments,
respectively. Error bars represent the standard deviation. *=p<0.05 and **=p<0.01, 1-way repeated measures
ANOVA followed by Tukey’s post test.
Figure 4. DNA repair kinetics in TK6 cells following exposure to 3.4 (left panel) or 4.3 Gy (right panel) of Xrays under conditions of a changing dose rate. DNA repair was studied using the alkaline comet assay. Error
bars represent the standard deviation of the mean from three experiments.
These results and those from the Polish study were the first to indicate that the directionality
of dose rate change could influence the outcome of an absorbed dose of IR. Although
preliminary, these studies and the total lack of any other experimental data regarding
changing dose rates encouraged the investigations described in papers I, II and III.
8
Temperature at irradiation
The incubation of samples on ice is a common procedure in most molecular biology
laboratories. The reason for doing so is often to inhibit or strongly reduce DNA repair, protein
synthesis or other cellular processes during manipulation or transportation of the samples.
Importantly, the cellular response to IR is not uniform for the temperatures (0-37 °C) most
often employed in experiments, with the terms “on ice” and “room temperature” being
inaccurate definitions which ultimately can affect cellular behavior and experimental
reproducibility. In itself, lowering the temperature of cells can have profound effects on many
cellular processes such as cell cycle progression, transcription, translation, metabolism and
lead to the induction of cold shock proteins (Fujita 1999, Al-Fageeh, Smales 2006).
It has long been known that the temperature at exposure can affect the level of damage in
exposed cells, as observed by Karl Sax already in the 1930’s and 1940’s. He noticed increased
levels of chromosomal aberrations when exposing cells at a lower temperature, but also
concluded that the results from other studies at the time were inconclusive (Sax, Enzmann
1939, Sax 1947). In recent years more and more data support the view of a lower irradiation
temperature actually acting in a radioprotective manner (Bajerska, Liniecki 1969, Elmroth et
al. 1999a, Elmroth et al. 2003, Brzozowska et al. 2009, Brehwens et al. 2010). This
radioprotective effect has since been termed the “temperature effect” (TE) and has also been
observed in other biological systems and endpoints such as enzyme preparations (Kempner,
Haigler 1982), virus inactivation (DiGioia et al. 1970), survival in mice (Levan et al. 1970),
frequency of chromosomal aberrations (Bajerska, Liniecki 1969, Gumrich et al. 1986),
frequency of MN (Brzozowska et al. 2009, Brehwens et al. 2010, Dang et al. 2012) and DNA
supercoil rewinding (Elmroth et al. 1999b). Still, the TE remains somewhat elusive as it is not
always detected for different endpoints in the same cell system. This has been observed in
MCF-7 breast cancer cells where the TE was visible on the level of DNA supercoil rewinding
(Elmroth et al. 1999a) but not on the level of MN (Larsson et al. 2007). In human PBL the TE
was observed on the level of MN but not on the level of DNA damage as measured by the
comet assay (Brzozowska et al. 2009).
Despite the last decade’s efforts, a mechanistic explanation behind the TE is lacking. The TE
appears to be more pronounced for low LET than for high LET radiation, suggesting that the
indirect action of IR plays an important role (Elmroth et al. 2003). Treatment with the radical
scavenger dimethylsulfoxide (DMSO) abolishes the TE further supporting the importance of
the indirect action (Elmroth et al. 2000, Brzozowska et al. 2009). This further implies that the
chromatin conformation, meaning it’s susceptibility to radical attack could be an important
parameter. This is also supported by the finding that the TE was less pronounced in intact or
permeabilized Hs27 cells, as compared to nucleoids (Elmroth et al. 2003). It has also been
shown that chromosomal regions with a low level of gene expression (more condensed
chromatin) is less sensitive to γ-radiation than regions with higher expression levels (more
open chromatin) (Falk et al. 2008). The TE is not a mere experimental artifact in the
radiobiology laboratory, but also a potential source of much unwanted variability in biological
dosimetry, where the dicentric assay long has been the “gold standard” (International Atomic
9
Energy Agency 2001). Here, the aim is to estimate the dose in an accidently exposed
individual by analyzing the frequency of dicentric chromosomes in PBL. The result is then
compared to a standard curve generated by in vitro exposure of PBL. As the TE can result in a
20-50% reduction in observed cytogenetic damage (Gumrich et al. 1986, Brzozowska et al.
2009, Brehwens et al. 2010, Dang et al. 2012) it is evident that calibration curves must be
generated with the utmost consideration taken to irradiation temperature to allow for high
reliability and valid interlaboratory comparisons of the results.
The oxygen effect
Another well-known factor of importance in radiobiology is oxygen concentration, as oxygen
acts as a chemical radiosensitizer (Wardman 2007, Wardman 2009). The cell’s status as either
hypoxic, normoxic or hyperoxic at the time (or milliseconds after, (Michael et al. 1973) ) of
exposure influences the amount of DNA damage sustained following (primarily) low LET
radiation. This is explained by the so-called “oxygen fixation hypothesis” (figure 5) where
oxygen and chemical radioprotectants (CRP) (for example cysteamine) compete for the DNA
radical (DNA•) formed from reactions with radiolysis products.
Figure 5. The “oxygen fixation hypothesis” in which oxygen competes with chemical radioprotectants (CRP) for
the DNA radical (DNA•) formed by water radiolysis products. If oxygen outcompetes the CRP, the result will be
a chemically modified DNA molecule, and the damage is then considered to be “fixed” in the DNA. Modified
from (Bertout et al. 2008).
In this context it is also important to recognize that standard cell culture (5% CO2, 95%
humified air containing 21% O2) conditions are not to be considered normoxic, but in fact
quite hyperoxic. As extensively reviewed elsewhere (Ivanovic 2009), oxygen concentration
can exhibit great variation in human cells, from around 14% down to 0%. Importantly,
although cells are most radioresistant at 0% oxygen, increasing the oxygen concentration to
0.5% or 5% results in half or almost complete radiosensitization, respectively. Increasing the
oxygen concentration above 5% appears to have little effect on radiosensitization (Van der
Kogel, Joiner 2009, Hall, Giaccia 2012). This suggests that most cell-based radiobiological
experiments are being performed at oxygen concentrations where the cells are maximally
sensitized with respect to the oxygen effect. Therefore “normoxic” experiments in vitro do not
necessarily reflect the oxygen concentration (and consequently not the response) of these cells
in situ. The oxygen fixation hypothesis implies that if the oxygen concentration of the cell can
10
be increased, the cell will also become more radiosensitive. This is an important aspect of
fractionated RT, as discussed further below, where hypoxic (and more radioresistant) tumor
cells can be made more radiosensitive by allowing time for reoxygenation before the next
fraction is given.
Oxidative stress
Cells are under the condition of oxidative stress when the level of oxidizing molecules
exceeds the reducing capability of the cell’s defense systems. The ROS molecules of the
largest biological relevance are hydrogen peroxide (H2O2), superoxide ([O2]•−), singlet oxygen
(1O2), hypochlorous acid (HOCl), hydroxyl radical ([OH]•), ozone (O3) and lipid peroxides
(ROOH) (Dickinson, Chang 2011), and also reactive nitrogen species (RNS). ROS/RNS are
not xenobiotic to the cell; superoxide (and subsequently the formation of H2O2) is formed in
mitochondria as a consequence of the “leaky” electron transport chain in mammalian
metabolism (Zhao et al. 2007), with ≈ 2 % of O2 consumption leading to H2O2 production
(Chance et al. 1979). ROS is also used by immune cells in unspecific pathogen killing, such
as the NAD(P)H-mediated production of superoxide by activated neutrophils in their
“respiratory burst” (Robinson 2009, Martin-Ventura et al. 2012). Other endogenous sources
are peroxisomes and the cytochrome P450 enzymes. Not only can ROS damage cellular
components, but they also have important biological functions in cellular signaling. It has
been proposed that ROS are the initiator but RNS the effector molecules in ROS/RNS
mediated signaling. Most ROS are too reactive and unspecific compared to the RNS that have
lower reactivity and higher reaction specificity, which are important properties of biological
signaling molecules (Mikkelsen, Wardman 2003).
It is estimated that the cell suffers 50000 DNA lesions on a daily basis as a result of the
endogenous ROS production from the respiratory chain (Swenberg et al. 2011). In view of
this, it is interesting to ask why a 2 Gy dose of low LET radiation, causing only around 3000
DNA lesions per cell exposed (Lomax et al. 2013) leads to significant cell killing. This is
partly explained by the difference in the distribution and type (IR also causes DNA doublestrand breaks [DSB] which are potentially lethal) of the lesions induced. While endogenous
lesions are produced randomly in the DNA, a considerable proportion of IR-induced lesions
occur in clusters, which are a characteristic of IR exposure (Goodhead 1994, O'Neill,
Wardman 2009) and increase the difficulty of repair. Although radiolysis-derived ROS can
result in complicated clustered damage, it is still evident that the effects of IR extend further
than simply damaging the DNA directly/indirectly. Non-DNA targets are also highly
important in the effects of IR such as the nucleotide pool (Rai 2010). One example of this is
the finding that the serum level of 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxo-dG) 1 h
following a 1 Gy exposure of whole blood was around 35 times higher than what could be
expected to form in the nuclear DNA alone. This difference was attributed to oxidation of
deoxyguanosine triphosphate (dGTP) in the nucleotide pool to form 8-oxo-dGTP, which is
subsequently excreted from the cell as 8-oxo-dG (Haghdoost et al. 2005) (described in detail
below). It has also been demonstrated that irradiated mitochondria can release Ca2+ that
diffuses to nearby mitochondria which propagate (and thereby amplify) this Ca2+-mediated
11
signal, ultimately resulting in an increased cellular ROS/RNS production (Leach et al. 2001).
Mitochondria are becoming increasingly implicated as an important target for IR, and also
important in the development of long-term radiation effects (reviewed in (Azzam et al. 2012,
Kam, Banati 2013), especially since they are (by volume) the second largest target for IR in
the cell (Mikkelsen, Wardman 2003). One proposed mechanism of prolonged ROS production
following IR exposure suggest that the damage mitochondria suffer (to their DNA and/or to
their proteins) from an exposure increases the ROS “leakage” from the respiratory chain
(Spitz et al. 2004). Importantly, chronic oxidative stress has been implicated in cancer
(Wiseman, Halliwell 1996, Evans et al. 2004, Halliwell 2007, Klaunig et al. 2010, Reuter et
al. 2010, Kryston et al. 2011), non-cancer disease (Wiseman, Halliwell 1996, Evans et al.
2004, Reuter et al. 2010) and adverse effects to RT (Robbins, Zhao 2004, Zhao et al. 2007).
Measuring ROS directly is technically difficult (Mikkelsen, Wardman 2003), with an
alternative approach being the measurement of a more stable molecule formed in the reaction
with ROS. A wide spectrum of DNA base modifications can result from the action of ROS
(Evans et al. 2004, Dizdaroglu, Jaruga 2012). The measurement of oxidized forms of guanine
(especially the previously mentioned 8-oxo-dG) is one of the most common endpoints due to
guanine having the lowest reduction potential of the DNA bases (Steenken, Jovanovic 1997).
8-oxo-dG can arise from direct oxidation of guanine in the DNA, or by oxidation of its
precursor dGTP in the cytoplasmic nucleotide pool (Tajiri et al. 1995). 8-oxo-dG can base
pair with both cytosine and adenine, and if formed in/incorporated into the DNA 8-oxo-dG is
potentially mutagenic causing G:CT:A and A:TC:G transversions (see figure 6). If 8oxo-dG is formed in the DNA, it is excised by the OGG1 protein of the base excision repair
(BER) pathway as 8-oxo-guanine (Michaels et al. 1992), preferentially if opposite to cytosine
(Nakabeppu et al. 2006b). If present in DNA as a template during replication, 8-oxo-dG can
mispair with adenine. This is repaired by the MUTYH protein that excises adenine in the
newly synthesized DNA strand opposite 8-oxo-dG, followed by the correct insertion of a
cytosine by DNA polymerase λ (Nakabeppu et al. 2010). To prevent incorporation of 8-oxodGTP from the nucleotide pool, the enzyme MTH1 hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP,
which is not a substrate in DNA synthesis (Nakabeppu 2001, Nakabeppu et al. 2004,
Nakabeppu et al. 2006a). 8-oxo-dGMPase further degrades 8-oxo-dGMP to 8-oxo-dG which
can be excreted to the extracellular environment (Hayakawa et al. 1995) and eventually
measured in the cell medium, blood serum or urine using HPLC or ELISA methods. Among
other applications, 8-oxo-dG has been used as a biomarker of metabolism-increased oxidative
stress following physical exercise (Harms-Ringdahl et al. 2012) and of particular interest for
this thesis, as a biomarker of individual radiosensitivity (Haghdoost et al. 2001, Skiold et al.
2013).
12
Figure 6. Pathways of formation and excretion of a commonly oxidized form of guanine (8-oxoguanine) or its
precursor in the nucleotide pool (8-oxo-dGTP), both being potentially mutagenic. The oxidized nucleotide 8-oxodGTP can be measured in extracellular fluids as 8-oxo-dG (orange). Proteins involved in prevention of this
mutagenesis are showed in green. The newly synthesized DNA strand is shown in bold. Modified from
(Nakabeppu et al. 2006b, Nakabeppu et al. 2010).
Biological factors
DNA damage and repair
DNA damage is not exclusively linked to exposure to IR, chemicals, or any other exogenous
agent. The DNA is chemically somewhat unstable (Lindahl 1993, Hoeijmakers 2001), and the
previously mentioned cellular metabolism continuously generates ROS that can damage its
structure. In view of this, it is obvious why efficient repair systems have evolved in cells to
maintain genome integrity (Friedberg 2003, Branzei, Foiani 2008, Iyama, Wilson 2013), and
13
it also comes as no surprise that defects in these repair systems can result in severe
syndromes, some of which result in radiosensitivity and cancer predisposition (McKinnon,
Caldecott 2007). DNA repair pathways are also of considerable interest from the point of
view of cancer therapy where abnormalities in the cancer cell DNA repair machinery are
exploited to selectively kill such cells (Helleday et al. 2008, Helleday 2011, Furgason,
Bahassi el 2013).
It is well known that the DNA suffers a variety of lesions from IR. Out of these, the
potentially lethal DSB is considered to be the most dangerous for the cell (Pouget, Mather
2001, Branzei, Foiani 2008, Mahaney et al. 2009). A DSB occurs if both DNA strands are
broken opposite each other, or it may be the result of two single-strand breaks (SSB) that are
in close (a few bases) proximity. Although most of the damage (SSBs, base damage) caused
to the DNA is readily repaired, damage caused by the previously mentioned ionization
clusters can cause significant problems for the cell. If a short segment of DNA (≈20 base
pairs) is simultaneously attacked by several radicals, the result can be a complex DSB,
containing several kinds of lesions in what is called a “locally multiply damaged site”. This is
considered particularly difficult for the cell to repair (Van der Kogel, Joiner 2009, Hall,
Giaccia 2012). Figure 7 gives an overview of the most relevant IR-induced DNA damage and
pathways of repair. BER is versatile, repairing abasic sites and oxidative/alkylation damage
(Robertson et al. 2009). SSBs can arise in several ways but are repaired similarly through a
“BER-like” process (Caldecott 2008). DSBs are repaired (depending on cell cycle phase)
through the “error-free” homologous recombination (HR) or the “error-prone” nonhomologous end joining (NHEJ) (Mahaney et al. 2009). DNA-DNA/protein crosslinks are
thought to be repaired by interplay between the NER and HR pathways (Barker et al. 2005,
Deans, West 2011).
14
Figure 7. The most relevant IR-induced DNA damage and the subsequent pathways aimed at their repair.
Modified from (Hoeijmakers 2001).
Cell cycle phases and radiosensitivity
The cell cycle phase of a mammalian cell plays a key role in the response to IR. It is wellknown that the radiosensitivity of the cell varies with the cell cycle phases, with the M-phase
followed by the G2-phase as the most sensitive phases. Late S-phase and possibly also early
G1 (if this phase is longer) are the more resistant phases, as illustrated in figure 8. It is mainly
the length of the G1 phase of the cell cycle that accounts for the variations in cell cycle length
seen in mammalian cell lines (Hall, Giaccia 2012).
15
Figure 8. Radiosensitivity in HeLa cells as a function of the cell cycle phases, illustrating the coincidence of
increased survival and homologous recombination that becomes available in the S/G 2 phases. Modified from
(Hall, Giaccia 2012).
In general, cell killing correlates best with DSB (Radford 1986), and the increase in survival
as cells enter S-phase is most likely due to loosening of the chromatin and the increased
proportion DSB repair by homologous recombination (HR). This is made possible utilizing
the sister chromatid that becomes available in S-phase. In G1 there is no sister chromatid
available and the cell must then rely on the error-prone NHEJ pathway for DSB repair. This is
supported by experiments showing that HR-deficient cells lack S-phase radioresistance (Hinz
et al. 2005, Wilson et al. 2010). The fact that radiosensitivity varies with cell cycle phase also
has implications for RT, and will be described further below. From a radiobiological
perspective the PBL are of great interest, as they are naturally synchronized in the G0 phase.
Not only do these cells have a more uniform radiosensitivity with respect to cell cycle phase,
but they are also very easy to obtain by a simple venipuncture. A subset of the PBL (the Tcells) can be induced to divide by the polyclonal activator phytohemagglutinin, making it
possible to study cytogenetic effects such as chromosomal aberrations or MN.
16
Non-DNA targets of ionizing radiation
The cell membrane: an unavoidable target of IR
If the nucleus is hit by a track of IR, it means by necessity that the cellular membrane was
traversed (and possibly also hit), but the reverse is not necessarily true. The plasma membrane
serves several important functions for the cell; it is the barrier between the cell and its
environment, it is the interface for endo- and exocytosis, and it also harbors a vast diversity of
proteins used for attachment and signaling. By mere change in composition and charge, the
properties of the plasma membrane can change and promote or prevent the aggregation of
signaling molecules, effecting downstream targets. For example, cells show a higher level of
survival following irradiation if grown on fibronectin (a component of the extracellular
matrix) as compared to plastic, an effect attributed to signaling by membrane-bound integrins
(Cordes, Meineke 2003).
IR can directly induce peroxidation and fragmentation of the lipids in the cell membrane,
potentially disrupting membrane integrity (Shadyro et al. 2002, Corre et al. 2010) but even
more importantly introduce changes in membrane composition. The cell membrane is mainly
composed of cholesterol, phospholipids and sphingolipids. Sphingolipids and cholesterol
interact closely and this favors the formation of microdomains, termed “lipid rafts”, in the
“sea” of phospholipids (Gulbins, Kolesnick 2003). The lipid rafts can provide proteins with a
unique environment within the plasma membrane, facilitating certain protein-protein
interactions and inhibiting others (Simons, Toomre 2000). Many signaling proteins are found
to be associated with lipid rafts, such as the T-cell, B-cell, EGF, insulin and H-RAS receptors,
and also integrins (Simons, Toomre 2000). These lipid rafts can be fused together into larger
platforms by the sphingolipid ceramide, and this clustering into ceramide-rich macrodomains
can trigger apoptosis signaling presumably through enrichment of apoptosis-promoting
receptors and/or exclusion of survival-promoting receptors (Gulbins, Kolesnick 2003).
Ceramide can be generated either through hydrolysis of sphingomyelin by acidic or neutral
sphingomyelinase (ASMase/NSMase) or by de novo synthesis by ceramide synthase (Corre et
al. 2010). IR can trigger lysosomal ASMase to relocate to the plasma membrane where it
converts sphingomyelin into ceramide, which then promotes lipid raft aggregation as
described above (Corre et al. 2010). This process has been shown to be independent of DNA
damage, and occurs rapidly following irradiation, but the de novo synthesis appears to be
dependent on ataxia telangiectasia mutated (ATM)-mediated signaling of DNA damage
(Haimovitz-Friedman et al. 1994, Vit, Rosselli 2003). Probably, both pathways are required
for sufficient apoptotic signaling (Vit, Rosselli 2003). Interestingly, cells from Niemann-Pick
disease type A patients are resistant to radiation-induced apoptosis due to faulty ASMase, but
can again be made sensitive by introducing functional ASMase (Corre et al. 2010). Ceramide
does not only play an important role in fusing lipid rafts, but also as an intracellular signaling
molecule where it can mediate BCL2-associated X protein (BAX) incorporation into the
17
mitochondrial membrane, which releases cytochrome C initiating the caspase cascade leading
to apoptosis (Prise et al. 2005).
The bystander response
The radiation-induced bystander response (BR) has attracted attention over the past two
decades, and as the name implies means that cells other than those directly hit by radiation
also suffers damage. The BR has been investigated mainly in two ways. First, cells can be
grown in such a way as to permit signaling through gap junctions, and inhibiting this gap
junction signaling can reduce the magnitude of the BR (Prise et al. 2003). Second,
experiments can be performed where the medium from irradiated cells is transferred to
unirradiated cells, implying that excreted factors mediate the response. The BR signaling
appears to be complex, but a common outcome seems to be the induction of a persistent
production of ROS/RNS, for example by plasma membrane-associated NAD(P)H induced by
TGF-β1 excreted from irradiated cells (Hamada et al. 2007). Interestingly, NAD(P)H is
localized to lipid rafts in the membrane and the inhibition of lipid raft formation can
consequently reduce the BR (Prise et al. 2003). Microbeam irradiation allows the targeting of
individual cells or specific parts of cells, making it possible to study the spatial distribution of
BR induction in a cell culture. The BR can be substantial and has been analyzed with several
endpoints but the response saturates at low doses (≈<0.2 Gy). This implies that it is
potentially an important factor in the biological outcome of exposures in the low dose region,
where there is much debate regarding the level of risk (Prise et al. 2003). The BR also infers
that cell culturing conditions during irradiation (cell density, media volume and composition,
etc.) can influence the damage sustained by cells and that such parameters need to be
carefully controlled in any experiments investigating the effects of low to moderate doses of
IR.
18
Radiotherapy
A majority of all cancer patients will receive RT (mainly external beam therapy,
brachytherapy, or a combination) as a part of their treatment (Dunne-Daly 1999, Hirst 2007).
Simply speaking, any form of RT aims to deliver such a dose to the tumor that the cancer cells
die. This is referred to as tumor control. A major challenge is the balance of maximizing the
dose to the tumor while minimizing the dose to surrounding healthy tissue. Consideration to
normal tissue can be taken by forming the beam to the tumor shape and to use a radiation type
that by virtue of its physical properties deposits more energy in the tumor relative to
surrounding normal tissue (Bortfeld, Jeraj 2011). Despite this the whole therapeutic dose
cannot be given at once due to the severe reactions in normal tissue that would result. It has
been known for more than 90 years that splitting the dose into fractions spares normal tissue
and allows for a larger dose to be given to the target tissue. In fractionated RT kinetics of the
four “Rs”: (DNA) Repair, (cell cycle) Redistribution, (cellular) Repopulation and (cellular)
Reoxygenation are exploited to increase tumor cell killing and decrease normal tissue damage
(Withers 1992, Fowler 1992). Three of the “four R’s of RT” (Trott 1982, Pajonk et al. 2010)
have already been discussed in this thesis. A fifth “R”, intrinsic/individual Radiosensitivity
(Steel et al. 1989), is described in a separate paragraph below.
Time between fractions will spare normal tissue by allowing time for sublethal damage repair,
thereby preventing its interaction with additional damage to form lethal damage (Bedford
1991). Fractionation also allows time for repopulation of cells in (predominantly in early
reacting) tissues, but can also result in unwanted accelerated repopulation of tumor cells
(Trott 1990, Trott, Kummermehr 1993). At the same time, fractionation increases tumor cell
killing by allowing redistribution of cells from a radioresistant to a more radiosensitive phase
of the cell cycle (Withers 1992, Chen et al. 1995). Finally, reoxygenation of hypoxic tumor
cells can occur between fractions. Hypoxic cells are known to be radioresistant (Crabtree,
Cramer 1933, Du Sault 1969, Tinganelli et al. 2013) (see “oxygen effect”) and tumor hypoxia
is recognized as an important cause of reduced therapeutic efficacy (Chaplin et al. 1986,
Moulder, Rockwell 1987, Hoogsteen et al. 2007). The specific fractionation scheme chosen is
dictated by the characteristics of the tumor in question (Marcu 2010) but practical limitations
such as five-day work weeks also have a considerable influence.
Whether or not exposure to IR occurs accidentally or in the form of RT it still raises concern
for the risk of cancer and non-cancer effects in healthy tissue. Studies on cohorts exposed in
the Chernobyl accident in 1986 (Cardis, Hatch 2011), by atom bombs dropped on Japan in
1945 (Little 2009, Douple et al. 2011), and in the future from Fukushima (Boice 2012) are
and will be important in delineating these risks. The International Commission of
Radiological Protection (ICRP) publication 103 gives risk values for cancer and hereditable
effects following (low dose rate, whole body) IR exposures. Here, the whole population’s risk
of cancer is 5.5% and for heritable effects 0.2% per Sv, respectively (ICRP 2007). For RTinduced secondary cancer risk predictions are more difficult, and the choice of risk model can
have a big influence on the resulting risk estimate (Dasu et al. 2005). With more patients
19
surviving their cancer due to improved healthcare, late adverse effects of RT is of increasing
concern.
Adverse effects of radiotherapy
Tissues can be classified as early (for example skin, intestinal epithelium, mucosa, testis, and
also tumor tissue in general where cell turnover is high) or late (for example kidney, spinal
cord, bladder, lung) responding with respect to adverse effects of RT. Early-reacting tissues
respond within days to weeks following (and during) RT, usually with the response being
transient. Examples of such effects are erythema and dry/moist desquamation of the skin, loss
of hair, and oral mucositis (Van der Kogel, Joiner 2009). Severe early effects might lead to
termination or modification of the RT regimen, and can also lead to consequential late effects,
in which an early adverse effect increases the probability of experiencing a late adverse effect
in the same tissue (Dorr, Hendry 2001). Late reacting tissues respond to IR after months to
years, in a slowly progressing and usually irreversible manner. Late reactions include fibrosis,
necrosis, sclerosis and cataracts, to mention a few (Van der Kogel, Joiner 2009). These
adverse effects to RT will affect the patient differently depending on the tissue(s) irradiated,
and consequently require different treatment strategies. In general, tumor doses are given as to
not result in more than 5% severe late toxicity (5% incidence up to 5 years post treatment)
(Emami et al. 1991). The inflammatory response and ROS imbalance appear to be important
processes in the pathogenesis of late adverse effects (Halle et al. 2011, Dorr, Herskind 2012).
As paper V focuses on the late adverse effect ORN following RT for HNC patients as an
indicator of high individual radiosensitivity, this severe late side effect will be described in
greater detail below.
Mandibular osteoradionecrosis
Worldwide, it is estimated that around 600.000 cases of HNC arises each year (Leemans et al.
2011). Adverse effects of RT in the head/neck area can be particularly devastating for the
patient as many biological and quality of life-related functions can be affected, which can also
lead to mental trauma and withdrawal from social life. ORN is a late adverse effect following
RT of HNC defined as “irradiated bone that becomes devitalized and exposed through the
overlying skin or mucosa without signs of healing for a period of more than three months,
without recurrence of tumor” (Lyons, Ghazali 2008). In ORN the mandibular bone becomes
necrotic over months-years without subsequent healing, often associated with severe pain and
sometimes with fistulation of the adjacent tissue. ORN is in most cases progressive and very
difficult and costly to manage (Chrcanovic et al. 2010a). The reported incidence of ORN is
2.6-15% (Lambade et al. 2013), and commonly occurs within the first few years after RT
(Lyons, Ghazali 2008, Jacobson et al. 2010). It has also been suggested that the incidence rate
per year after RT remains constant (Jung et al. 2001), and a recent study has shown an
increasing incidence of surgical reconstructions for ORN over the last two decades (Zaghi et
al. 2013). Figure 9 shows representative clinical features of ORN grade IIIb (Schwartz, Kagan
2002), and figure 10 the successful reconstructive surgery performed.
20
Figure 9. A: Clinical finding with established grade III ORN showing necrotic bone through a non-healing
wound in the oral mucosa. B-E: Typical progress of ORN over time (in this case 24 months) ultimately leading
to pathologic fracture of the mandible and the need for reconstructive intervention. F: Oro-cutaneous fistula
with necrotic bone visible, often associated with grade II-III ORN.
Figure 10: Reconstructive surgery following grade III ORN. Resection of necrotic bone leading to a continuity
defect. Immediate reconstruction with free vascular composite fibula flap with skin island is used to cover the
defect. Aim is to restore local anatomy, aesthetics and enable dental reconstruction with dental implants.
A: Seven days postoperative panoramic X-ray of fibula flap attached with Synthes Matrix Mandible pre-formed
reconstruction plate. B: Six months postoperative panoramic X-ray showing healing and integration of fibula to
mandibular bone. C-D: Dental reconstruction with Nobel Biocare dental implants.
21
ORN has been known for more than 90 years and considerable effort has been spent on
understanding its pathogenesis and identifying underlying risk factors. The theory of Meyer in
the 1970’s postulated that trauma permitted bacteria to invade and infect irradiated bone, and
this theory promoted the use of antibiotics to treat ORN (Meyer 1970). This was subsequently
challenged by Marx in the 1980’s who questioned the importance of bacteria and trauma as
causes of ORN, as an appreciable fraction of ORN cases occurred spontaneously and with
bacteria only appearing to be surface contaminants of the bone. He proposed a new theory
where radiation leads to “hypoxic-hypovascular-hypocellular tissue” (the so called “3H
hypothesis”) in which the tissue homeostasis is disrupted (Marx 1983b). In this tissue cellular
turnover and collagen synthesis is reduced, and wounds can thereby occur spontaneously or
be induced by trauma. In any case, the wound healing capabilities of the tissue is greatly
reduced or non-existent. Marx developed a “definitive hyperbaric oxygen protocol” that
involved a series of stages where hyperbaric oxygen (HBO) is used alone or in combination
with surgery (Marx 1983a). HBO is a treatment form where the patient is subjected to
pressurized (2-2.5 atmospheres) pure oxygen in a series of “dives” in a pressure chamber.
HBO is thought to promote wound healing by stimulating angiogenesis, collagen formation
and by high oxygen levels being bacteriostatic (Chrcanovic et al. 2010b). The consensus
appears to be that HBO therapy should be combined with surgery to be effective (Peleg,
Lopez 2006, Jacobson et al. 2010) but still HBO therapy is debated (Spiegelberg et al. 2010,
Bennett et al. 2012). Also, HBO therapy is technically demanding, expensive, not risk free,
and not available to all patients.
The most recent ORN theory is the radiation-induced fibroatrophic (RIF) process (figure 11)
(Delanian, Lefaix 2004), where damaged endothelial cells promote the differentiation of
fibroblasts into excessively proliferating myofibroblasts with a dysregulated collagen
metabolism. This in combination with the radiation-induced death of bone cells without
subsequent repopulation leads to a fragile tissue that provides a poor environment for healing
of subsequent physiochemical trauma. Occlusion of the inferior alveolar artery is also
common in ORN (Bras et al. 1990), but the impact of microcirculation and impaired
circulation in conduit vessels is not fully elucidated (Marx 1983a, Delanian, Lefaix 2004).
ROS and inflammation appear important in ORN pathogenesis, as it has been found that the
combined use of tocopherol (vitamin E, an antioxidant) and pentoxifylline (an antiinflammatory drug) can heal ORN (Delanian et al. 2005, Kahenasa et al. 2012). Oil
containing tocopherol has also been reported to reduce oral mucositis (Ferreira et al. 2004).
The current view of ORN risk factors include primary tumor site, proximity of tumor to bone,
extent of mandible in the radiation field, state of dentition, poor oral hygiene, radiation dose
>60 Gy, brachytherapy, acute/chronic trauma, concomitant chemo-radiation and advanced
stage tumors (Jacobson et al. 2010).
22
Figure 11. The radiation-induced fibroatrophic (RIF) theory as proposed by Delanian and Lefaix (Delanian,
Lefaix 2004). Adapted and modified from (Lyons, Ghazali 2008).IR; ionizing radiation, ROS; reactive oxygen
species, ORN; osteoradionecrosis.
23
Individual radiosensitivity
It has been proposed that patient-related factors could account for as much as 80-90% of the
variation seen in the patient response to RT (Safwat et al. 2002). Consequently it has been
hypothesized that this variability is due to unique properties of the individual patient (i.e
genomic variation) and this has been termed “individual radiosensitivity”. Adverse effects of
RT often have a devastating impact on quality of life for the affected patients, and the
resulting aftercare is an added burden to the healthcare system. Consequently, it has long been
a goal to predict the patient’s radiosensitivity prior to RT and to adjust treatment or at the very
least identify patients with an elevated risk for adverse effects. Considerable effort has been
put into evaluating biological endpoints that could be used to discriminate between more
sensitive and more resistant patients. Fifteen to twenty years ago, clonogenic survival in
primary fibroblasts was a common endpoint (often after 2 Gy, [SF2] ), but the results are
largely inconclusive (Begg et al. 1993, Geara et al. 1993, Burnet et al. 1994, Johansen et al.
1994, Brock et al. 1995, Johansen et al. 1996, Burnet et al. 1996, Rudat et al. 1997, Kiltie et
al. 1999, Peacock et al. 2000, Oppitz et al. 2001). In the last decade, focus has instead shifted
to PBL as the cell system, with cytogenetic (MN, G0 and G2 assays) DNA damage (γH2AX
foci assay, comet assay, gel electrophoresis), apoptosis or blood biomarker assays as the
endpoints (Barber et al. 2000, Borgmann et al. 2002, Ruiz de Almodovar et al. 2002, Hoeller
et al. 2003, De Ruyck et al. 2005a, Severin et al. 2006, Perez et al. 2007, Borgmann et al.
2008, Werbrouck et al. 2011, Brzozowska et al. 2012, Finnon et al. 2012, Goutham et al.
2012, Padjas et al. 2012). As with the fibroblast investigations, none of these assays has
emerged as a reliable endpoint correlating the cellular radiosensitivity with the frequency of
adverse effects to RT.
That radiosensitivity has a genetic component is evident from the existence of syndromes
rendering the carrier radiosensitive and cancer-prone, such as Nijmegen breakage syndrome
(Digweed, Sperling 2004) and ataxia telangiectasia (AT) (McKinnon 2012). Although these
syndromes are rare and usually so severe that they are diagnosed early, AT can lack an
obvious phenotype and require extensive biochemical and cytogenetic analysis for diagnosis
(Claes et al. 2013). This further implies that it is possible that some individuals undiagnosed
for a radiosensitivity syndrome undergoes an unsuitable course of RT in the case they develop
cancer. Contrasting to these syndromes featuring high penetrance, low-frequency mutations
are the SNP, constituting low penetrance mutations of higher frequency in the population
(definition of a point mutation as a SNP is that it is present in more than 1% of the
population). In later decades the advances in DNA genotyping and associated computational
methods has led to the rapid and automatic analysis of large parts of the genome for SNP in
hundreds of samples, potentially allowing the identification of novel markers of disease. In
most cases the SNP investigated in the context of adverse effects to RT reside in genes from
pathways involved in DNA repair, oxidative stress and inflammation. Similarly to the past
decade’s investigations in fibroblasts and PBL, results are conflicting with both positive (De
Ruyck et al. 2005b, Ambrosone et al. 2006, Edvardsen et al. 2007, Azria et al. 2008, ChangClaude et al. 2009, Pugh et al. 2009, Pratesi et al. 2011, Langsenlehner et al. 2011, Mangoni
24
et al. 2011, Talbot et al. 2012, Lyons et al. 2012, Falvo et al. 2012) and negative (Andreassen
et al. 2006, Kuptsova et al. 2008, Zschenker et al. 2010, Farnebo et al. 2011, Barnett et al.
2012b, Reuther et al. 2013) correlations with radiosensitivity. In one of the largest SNP
studies to date which aimed at evaluating many of the SNP previously reported to be
correlated to adverse effects to RT, no single SNP emerged as a reliable indicator of
radiosensitivity (Barnett et al. 2012a). Still, despite the large cohort size (1613 patients) the
study contained relatively few highly radiosensitive patients. It is becoming increasingly
unlikely that any single SNP will render the carrier radiosensitive, which is in good agreement
with the theory that radiosensitivity is a multifactorial trait. Frequent points of criticism
regarding the SNP studies performed so far is that the number of patients (and consequently
the number of highly radiosensitive individuals) was small, and that no correction for multiple
testing was applied. Paper V approached these challenges by recruiting only the most
radiosensitive individuals (thereby reducing the need to recruit many patients) and by
evaluating several endpoints together through multivariate analysis.
25
The present investigation
Aims of this thesis
This thesis focuses on how physical and biological factors influence the outcome of an
exposure to low LET radiation. The first part of the thesis investigates physical factors and
aimed at:
 Designing and constructing novel irradiation equipment allowing cells to
be exposed to changing dose rates of X-rays.
 Investigating the effects on cells from exposures using such equipment.
 Addressing recent hypothesis regarding the mechanism behind the TE.
The second part of this thesis is directed towards the seemingly unavoidable adverse effects
following RT, where the problem is the current incapacity to assess patient radiosensitivity
prospectively. The aims were to, in a unique cohort of highly radiosensitive individuals and
their controls, study:



Radiation-induced oxidative stress by measuring the biomarker 8-oxodG as a potential indicator for the risk of severe late effects.
Clinical parameters as potential risk factors for late effects.
Selected SNP previously implicated in radiation sensitivity, involved in
the oxidative stress response and potentially the pathogenesis of late
effects to RT.
26
Results and discussion
Paper I
Cytogenetic damage in cells exposed to ionizing radiation under conditions of a changing
dose rate
In this study, TK6 cells were exposed to changing dose rates of X-rays. Three cell samples
were simultaneously exposed to an X-ray beam where one sample was moving towards the
source (MTO), one away from the source (MFROM), and one was stationary in the beam,
exposed to the average dose rate (ADR) of the two moving samples. The total dose to each of
the three samples was 1.2 Gy and the total exposure time was approximately 48 minutes. The
samples were exposed at 37 °C which permits all biological processes in the cells, or at 0.8 °C
in order to prevent DNA repair, protein synthesis, and other processes during the course of the
exposure. After exposure, cytogenetic damage was determined by scoring MN in binucleated
cells (BNC). The proliferative capacity of the cells was also assessed by scoring the
percentage of BNC, indicating completion of one round of nuclear division following the
exposure. The results indicate the same relationship between the MTO and MFROM samples
as was observed in the Polish pilot study and the author’s master thesis. Our results suggest
that cells exposed to a decreasing dose rate suffer more damage than cells exposed to an
increasing or a constant dose rate, a phenomenon we chose to call the “decreasing dose rate
effect”, or DDRE. Experiments were also performed at 0.8 °C with the hypothesis that the
prevention of DNA repair and protein synthesis would ameliorate any DDRE, but this proved
not to be the case. Instead, the same intersample relationship was seen as for the 37 °C
exposures, but with a general reduction of cytogenetic damage. This sparing effect of
irradiating samples at a lower temperature is a well-known phenomenon and was investigated
further in paper IV.
The initial interpretation of the results was that there might be some kind of adaptive response
taking place in the MTO sample during the exposure which makes the cells handle higher
dose rates better than the cells in the MFROM sample. The experimental conditions should
not promote an adaptive response, and it does not explain the difference seen between the
MFROM and ADR samples. Instead, it is possible that in the MFROM sample, an initial
alarm signal ceases to operate as the dose rate drops below a threshold, leading to the
accumulation of DNA damage. Ultimately, the mechanism behind the DDRE seen in this
study is not fully understood.
Main findings in paper I:
 A significantly higher level of MN were seen in the MFROM sample as compared to
the MTO and ADR sample
 Hypothermia exhibited an overall radioprotective effect (the TE was seen), but did not
change the intersample relationship.
27
Paper II
A new device to expose cells to changing dose rates of ionizing radiation
One reason for the lack of studies on changing dose rates of IR is that there is no compact
device available that could be mounted on top of a radiation source. Therefore, the aim of this
study was to design and construct a device that would fit into a standard cell incubator
positioned on top of a radiation source
The basic principle of the device is the attenuation of the X-ray beam using a near-saturated
aqueous solution of barium chloride. The device consists of three plexiglass tanks separated
by lead shielding. Two of the tanks are interconnected via silicone tubing connected to a
peristaltic pump. Consequently, the shielding medium can be transferred from one tank to the
other during exposure. When irradiating from below during shielding media transfer, the dose
rate will exponentially increase on top of the tank being emptied (increasing dose rate, IDR),
and consequently decrease on the top of the tank being filled (decreasing dose rate, DDR).
Included is also a third tank, filled with shielding media to yield the average dose rate from
the two interconnected tanks similar to the ADR sample in the setup used in paper I. The
exposure is monitored with an ionization chamber on top of the IDR tank, and terminated
when the initial dose rates on top of the IDR and DDR tanks have been reversed.
The results show that the device is well-positioned in the X-ray beam, and that it gives highly
reproducible exposure runs. We could also show that the field on top of each tank was
homogenous and through spectrometry that the X-ray beam spectrum did not change
noticeably with changing shielding media thickness.
Main findings in paper II:
 The device works as intended and yields reproducible exposure runs
 The fields on top of the tanks are homogenous
 The photon spectrum of the X-ray beam does not change noticeably as shielding
media thickness is increased/decreased
28
Paper III
Micronucleus frequencies and clonogenic survival in TK6 cells exposed to changing dose
rates under controlled temperature conditions
This study aimed at investigating the biological effects of changing dose rates of X-rays by
exposing TK6 cells at 37 °C using the MARK IV device, characterized in paper II. To this
end, the MARK IV device was also modified to allow a higher total dose (1.1 Gy) by
increasing the current to the X-ray tube and by using sample rafts floating on the liquid
shielding media in each tank. In addition to scoring MN, a second endpoint (clonogenic
survival) was added.
At the lower dose (0.48 Gy) there was no intersample difference observed at either endpoint.
At the higher dose (1.1 Gy) a significant intersample difference on the level of MN was
observed, similarly to the observation in paper I, albeit with a significant difference only
between the IDR and DDR samples. There was no difference in replication index between the
samples at either dose. It could be expected that a significant difference on the level of MN
induction would translate into a difference in clonogenic survival, but this was found not to be
the case.
Similarly to paper I, we observed the highest MN frequency in the DDR sample, an effect we
termed “decreasing dose rate effect”, DDRE. The hypothesis was that as the dose rate drops
below a threshold level, the DNA damage sensing and repair induction is reduced and damage
accumulates. The fact that this was not reflected on the level of clonogenic survival suggests
that if DNA damage sensing and repair induction is the underlying mechanism, the effect
might simply be too small to be seen on the level of survival. As radiation damage is
nonuniform in the cell population, the rate of progression through the cell cycle might exhibit
variation in a sample. Since the manifestation of MN is dependent on cells undergoing one
round of postexposure replication, an early time point of cell harvesting will contain the less
damaged cells, compared to those harvested at a later time point. This further implies that a
difference in cell cycle progression could be observed on the replication index, but this was
not the case. We also speculate that interphase death is delayed in the DDR sample, leading to
the expression of lethal damage as MN in the first postirradiation mitosis. If it is the dose or
the dose rate fold change (or a combination of the two) that causes the DDRE, we can only
speculate.
Main findings in paper III:
 The MARK IV device was successfully modified to also yield a higher dose of X-rays
 The DDRE, observed in paper I was also evident in this study, making this the third
study to date observing this intersample relationship following exposure to changing
dose rates of X-rays.
 The difference seen between the IDR and DDR samples on the level of MN induction
was not reflected on the level of replication, or clonogenic survival
29
Paper IV
Radioprotective effect of hypothermia on cells –a multiparametric approach to delineate the
mechanisms
The radioprotective effect of hypothermia (the TE) remains an elusive phenomenon in
radiobiology. Despite decades of investigations, a definitive explanation is lacking as to how
hypothermia exerts its radioprotection. This study aimed at investigating several potential
mechanisms behind the TE using multiple endpoints. TK6 cells were irradiated at 0.8 or 37
°C and clonogenic survival, MN induction and γH2AX foci formation kinetics were
investigated. The impact of chromatin conformation and DNA DSB sensing on the TE were
also investigated by inhibiting histone deacetylation (with trichostatin A, TSA) and by
inhibiting the ATM kinase (with KU55933, KU).
The TE was visible as a ≈50% reduction in MN frequency, which is in agreement with the
lab’s previous results using this cell line. Interestingly, this strong effect was not visible on the
level of clonogenic survival or γH2AX foci formation kinetics. The TE was still observed on
the level of MN induction following ATM inhibition using KU, but was abolished when the
chromatin was forced in a more open conformation by TSA. At a first glance this suggested
that the indirect (radical-mediated) effect of IR was responsible for the TE, a hypothesis
supported by previous studies (Elmroth et al. 2003, Brzozowska et al. 2009). However TSA
has other effects on the cell such as G1 arrest, which was visible as a lower replication index.
This further implied that TSA retarded both the 0.8 and 37 °C samples equally in the cell
cycle, and that cell cycle retardation is one possible mechanism behind the TE. If exposure to
low temperature delays cells in the cell cycle, it could explain why there is a difference
observed on the level of MN (scored after the first postexposure mitosis) but not on the level
of survival (scored as colonies after two weeks of growth). Indeed, when a sequential
harvesting regimen was applied and cells were harvested and scored also at later timepoints,
the TE disappeared supporting the idea that a low temperature induces a temporary cell cycle
delay in the samples exposed at 0.8 °C.
Ultimately, this suggests that at least in TK6 cells hypothermia does not actually modify the
radiosensitivity of the cells (as there is no difference on the level of survival). This finding
indicates the importance of a multiparametric approach in experimental design, and that some
endpoints are sensitive to physical factors that must be controlled carefully.
Main findings in paper IV:
 The TE was observed as a ≈ 50% reduction in observed MN frequency.
 The TE was not observed on the level of clonogenic survival or γH2AX foci formation
kinetics.
 Inhibition of histone deacetylase (by TSA), but not ATM kinase (by KU55933),
abolished the TE.
30


The TE disappeared if cell harvest was delayed three hours, suggesting that a
temporary cell cycle delay is induced in the cells exposed at 0.8 °C. However, any
such perturbation was not observed on the level of the replication index.
As TSA is known not only to force the chromatin into a more open conformation but
also to induce a G1 block, this could explain why the TE was abolished by the TSA
treatment; both the 0.8 and 37 °C samples were equally delayed in the cell cycle.
31
Paper V
Reduced oxidative stress response as a risk factor for normal tissue damage after
radiotherapy: a study on mandibular osteoradionecrosis
RT is commonly used to treat HNC, but despite technical advances in dose delivery it is
inevitable to expose normal tissue in the head and neck region, which is also anatomically
complex. In addition to this, improved healthcare not only means that patients live longer and
thereby experience an increased risk of both secondary cancers but importantly also of late
adverse effects to the RT. Mandibular osteoradionecrosis (ORN) is a late adverse effect
occurring in 2.6-15% of HNC patients in which the mandible over months-years becomes
necrotic and exposed through the mucosa, with possible fractures. Trismus, local infection
and severe pain are common symptoms. ORN is generally progressive and its management is
very resource demanding, sometimes requiring significant surgical procedures. Consequently,
it would be of great value to assess patient radiosensitivity before RT is started, with the
prospect of modifying the RT plan or at the very least “highlight” potentially radiosensitive
patients for increased medical supervision after treatment.
In this study, 37 patients with ORN were recruited together with a matched control group, and
PBL were collected from the patients, together with clinical data. Oxidative stress has been
implicated as a key factor in the pathogenesis of late adverse effects, and we have previously
observed that patients with pronounced side effects to RT appear to have reduced levels of the
oxidative stress-induced biomarker 8-oxo-dG. Therefore the capability to handle radiationinduced oxidative stress was analyzed using an in-house developed ELISA method measuring
8-oxo-dG concentration in serum 60 min post exposure. As radiosensitivity is strongly
suspected to have a significant genetic component eight SNP involved in the oxidative stress
response, all previously implicated in the occurrence of late effects to RT, were analyzed. The
two biomarkers together with clinical patient parameters were also subjected to multivariate
analysis with the aim of creating an ORN risk model.
Univariate analysis of the clinical parameters suggested a good matching between the two
patient cohorts, and also highlighted factors that were potentially useful for inclusion in the
modeling. The in vitro measurement of 8-oxo-dG indicated a significantly reduced serum
level of 8-oxo-dG in the ORN+ patient group 1 h following a 2 Gy dose of γ-radiation, which
could indicate that the ORN+ patients have a reduced capacity to handle the radiation-induced
oxidative stress. This hypothesis is supported by the results from the SNP analysis where it
was found that a point mutation in GSTP1, which is known to alter both the protein’s
enzymatic and regulatory properties, was significantly more frequent in the ORN+ group. A
logistic regression-derived model, initially consisting of both the two biomarkers and clinical
parameters, were subjected to manual tuning and the optimized model consisted of the factors
brachytherapy, serum level of 8-oxo-dG following 2 Gy and the SNP rs1695 in GSTP1. This
model had a sensitivity of 85% and a specificity of 47 %. Taken together the results support
the theory of oxidative stress being a key factor in the pathogenesis of late adverse effects to
RT.
32
Main findings in paper V:
 The patient groups were well-matched. Some clinical parameters emerged as
potentially useful for the multivariate analysis.
 ORN+ patients appear to have a reduced capacity to handle radiation-induced
oxidative stress, as measured by serum levels of 8-oxo-dG.
 The SNP rs1695 in GSTP1 was significantly more frequent in ORN+ patients
 The multivariate analysis resulted in a model which could identify 85% of ORN cases,
based on measurement of 8-oxo-dG levels after 2 Gy, genotyping of the SNP in
GSTP1 and whether or not brachytherapy was/is planned to be used.
33
Concluding remarks and future perspectives
The first three papers of this thesis describe the discovery and first steps towards the
understanding of what appears to be a novel radiobiological phenomenon; the DDRE. Starting
out, it was necessary to construct hardware that would allow these exposures to be performed
and to ascertain that dosimetric differences were not the cause for any observed effects. Taken
together, the DDRE has now been observed in the Polish pilot study, in the author’s master
thesis and in papers I and III, using a total of four different exposure setups. This “track
record” supports that the DDRE indeed is a biological effect. Even though the MARK IV
device (paper II) allows for precise exposures to be performed, the particular X-ray tube used
sets an upper limitation for the dose rates (and consequently the total dose) that can practically
be used. Prolonging the exposure over several hours is questionable from a technical
standpoint (this particular X-ray tube is not designed to run for such extended periods of
time). The inherent limitation in dose delivered was overcome to some extent by the addition
of the rafts floating on the shielding media surface, but this on the other hand limited the
space available for samples. For an extension of the study, it would therefore be preferable to
use a stronger IR source which would allow a greater range of dose rates, total doses, and
exposure times to be used. It would also be interesting to investigate if a linear dose rate
change (as opposed to an exponential, as used so far) in any way alters the biological outcome
of the exposure.
The relevance of the (relatively) small biological effect seen is another interesting question. It
is obvious from the example of aircraft flight that it would be absurd to think that airplanes
only should take off but avoid to land. It is however more meaningful to consider the (small)
effect in the context of collective risk. For the sake of argument, let’s assume that a risk model
for stochastic effects for aircraft flight does not differentiate between the ascent, cruise and
descent phases of the flight; the risk per unit dose is the same. Now instead imagine that the
descent part of an aircraft flight is associated with a larger (but only very marginally so) risk
of stochastic effects compared to the risk during ascent and flight at cruising altitude. The first
model would then, ever so slightly, underestimate the risk of stochastic effects. Still, the risk
at the level of the individual would in reality not differ between the two scenarios; it would be
very small. However, analogous to a lottery, given the extremely large number of aircraft
passengers (lottery ticket buyers), even an almost negligible increase in risk (chance to win)
could still translate into a group of affected individuals (ultimately, someone always wins the
lottery).
The attempts in paper IV to elucidate the underlying mechanisms behind the TE resulted in
the hypothesis that, at least in TK6 cells, hypothermia induces a temporary cell cycle delay.
This would then explain why there was no difference between the samples when the
incubation time was extended past 27 hours. Surprisingly, when this hypothesis was
investigated in a follow-up study focusing on cell cycle progression there was no difference
seen whatsoever between the 0.8 and 37 °C samples (Lisowska 2013, under review). In
addition to this, sequential harvesting did not result in the disappearance of the TE past the
34
standard incubation time of 72 h for human PBL (Wojcik group, unpublished data), a cell
system where the TE is very well documented in the literature. In conclusion, the TE remains
an elusive phenomenon which makes it even more important to control the temperature during
the exposure as the consequences can be difficult to predict.
The final part of this thesis (paper V) aimed at identifying factors that could be used to predict
the risk of late adverse effects to RT prior to therapy onset, or at the very least permit high
risk patients to be followed more closely after treatment. This study had the alternative
approach of sampling only the most radiosensitive patients (those that developed ORN) and
their controls, thereby reducing the number of total patients needed but still having samples
from the patients with the largest observed difference in radiosensitivity. This approach also
made the study more manageable as a part of a PhD thesis. However, if the strategy of
biomarkers and clinical factors being used in a mathematical model is to be implemented in
the clinic, it is of course also necessary to include the full spectrum of observed clinical
presentations following RT. Assuming a 10% ORN incidence it would for this study have
meant continuous sampling of 370 patients in order to have 37 ORN cases, which was not
feasible. It would also be interesting to investigate the possible correlation between early
effects (oral mucositis) and late effects (ORN). In our study, there was data available for
mucositis but with missing values being unevenly distributed between the two patient groups.
With this limitation in mind it is still interesting to note that the severity of oral mucositis was
found to be significantly higher in the ORN+ group, but that this finding still must be treated
with caution. From our patients, we have frozen lymphocytes suitable for cytogenetic or DNA
damage assays and also lymphocytes isolated from whole blood exposed in vitro to 0, 1 and
150 mGy. To further investigate the underlying mechanisms behind ORN pathogenesis, a
future approach could involve proteomic analysis of these samples. The role of the two
biomarkers in the risk of other adverse effects (both early and late) is also an area of potential
future studies.
35
Populärvetenskaplig sammanfattning
In vitro och in vivo-aspekter av strålkänslighet
Ända sedan joniserande strålning (JS) upptäcktes i form av röntgenstrålning 1896 (av
Wilhelm Röntgen) har det forskats intensivt på dess inverkan på biologiska system. Man vet
nu att höga doser av JS dödar celler och skadar vävnad, vilket utnyttjas i cancerterapi. I lägre
doser orsakar JS cancer och ärftliga effekter, och detta är en potentiell risk för många
människor efter exempelvis kärnkraftsolyckor. På grund av dessa egenskaper är det mycket
viktigt att studera faktorer som kan påverka den biologiska responsen vid exponering för JS,
både på cell- och individnivå.
Avhandlingens första del fokuserar på fysikaliska effekter (dosrat och temperatur under
bestrålning) och deras inverkan på cellulära effekter av JS. Trots att de flesta icke-medicinska
exponeringar för JS sker vid en stigande/sjunkande dosrat (t.ex. vid flygresor, och vid
exponering för radioaktivt nedfall) utförs strålningsbiologiska experimet vid en konstant
dosrat, mycket beroende på att det tekniskt sett är relativt okomplicerat att utföra dessa
bestrålningar. I manuskript I upptäckte vi en ny radiobiologisk effekt. Vi såg att TK6-cellers
skadenivå, mätt i frekvensen mikrokärnor, var högre efter 1.2 Gy röntgenstrålning om dosen
givits med en exponentiellt sjunkande dosrat, jämfört med en konstant eller exponentiellt
stigande dosrat. I manuskript II designade, konstruerade och validerade vi en ny teknisk
lösning för att fortsatt kunna studera effekterna av stigande/sjunkande dosrater. Manuskript
III beskriver hur vi med den nya anordningen bestrålade TK6 med en måttlig eller en hög dos
röntgenstrålar, vid en stigande, sjunkande eller konstant dosrat. Vid den högre dosen (1.1 Gy)
kunde vi återigen se att de celler som exponerats för en sjunkande dosrat var de som hade
högst frekvens mikrokärnor. Denna effekt var dock inte synlig efter bestrålning vid den lägre
dosen (0.48 Gy). Effekten var inte heller påvisbar vid någon av doserna när klonogen
cellöverlevnad studerades. Detta kan tyda på att effekten på mikrokärnenivå beror på en
förskjutning i cellcykelprogressionen (då mikrokärnor endast kommer till uttryck efter en
mitos) i provet som exponeras under en sjunkande dosrat, jämfört med de andra två proven.
Det är oklart om den observerade effekten är beroende på den totala dosen eller på
dosratsskillnaden (eller båda dessa faktorer).
I manuskript IV undersökte vi några hypoteser kring mekanismen bakom den
radioprotektiva effekten av hypotermi. Det har länge varit känt att temperaturen vid
bestrålning kan påverka den biologiska effekten av JS, och man ser oftast att en lägre
temperatur vid bestrålningen leder till en lägre nivå av cellulära skador. I TK6-celler har vi
tidigare observerat denna effekt som en ≈ 50% minskning av frekvensen mikrokärnor.
Fortfarande känner man inte till mekanismerna bakom denna ”temperatureffekt” men
modulerad detektion av DNA-skador, och den indirekta effekten (skador via radikaler) av JS
har föreslagits som potentiella förklaringar. Vi undersökte om inhiberad detektion av DNAdubbelsträngsbrott, samt en mer öppen kromatin-konformation påverkade temperatureffekten.
Vi undersökte också temperatureffekten med flera metoder, för att se om effekten är beroende
36
på vad (DNA-skador, cytogenetiska skador, cellöverlevnad) man studerar i cellerna. På
mikrokärnenivå såg vi en tydlig temperatureffekt, som inte försvann när cellernas förmåga att
detektera DNA-dubbelsträngsbrott kraftigt inhiberades. Däremot försvann temperatureffekten
när cellens kromatin tvingades till en mer öppen (och därmed sårbar) konformation, vilket
tydde på att en låg temperatur vid bestrålning kondenserar kromatinet och därmed skyddar det
från skadliga radikaler som producerats vid radiolys av vattenmolekyler nära DNA. Vi såg
dock inte någon temperatureffekt när vi mätte DNA-reparationskinetik i form av γH2AX-foci.
När cellens DNA skadas, fosforyleras en del på de proteiner som DNA ligger lindat kring,
som en tidig signal på skada som måste repareras. Denna signal kan man mäta över tid, och på
så sätt få en indikation på mängden DNA-skada och dess reparationskinetik. Inte heller sågs
någon temperatureffekt på klonogen cellöverlevnad. Att temperatureffekten inte observerades
när DNA-reparation och klonogen cellöverlevnad mättes kan tyda på att det inte är den
faktiska strålkänsligheten i cellerna som moduleras av en låg temperatur vid bestrålning, utan
att hypotermi inducerar en förändring som är av betydelse specifikt när man studerar
mikrokärnor. En modulation av cellcykelprogressionen skulle kunna vara en sådan
förändring, eftersom mikrokärnor endast är synliga i celler som genomgått en mitos efter
bestrålningen. Om hypotermi tillfälligt försenar cellcykelprogressionen, skulle detta kunna
förklara varför en lägre nivå mikrokärnor observerats i proven som bestrålats vid den lägre
temperaturen. För att bekräfta detta, mätte vi mikrokärnefrekvens även vid senare tidpunkter,
och kunde då se att temperatureffekten försvann om vi väntade tre timmar längre innan
cellerna skördades. Slutsatsen av denna studie är således att hypotermi inducerar en tillfällig
retardering i cellcykeln, vilket förklarar varför ingen skillnad observerades vid mätning av
DNA-repation eller klonogen cellöverlevnad. Temperaturen vid bestrålning är således en
potentiell källa till stor variation i resultaten, vilket kan få stora konsekvenser både för
grundläggande strålningsbiologiska experiment, men även för tillämpade metoder som
biologisk dosimetri.
Manuskript V behandlar individuell strålkänslighet ur ett kliniskt perspektiv. Det är känt att
ungefär 5 % av alla som får strålterapi utvecklar allvarliga sena bieffekter som en följd av
strålningens effekter på normalvävnad. I dagsläget är det inte möjligt att bestämma den
individuella patientens känslighet innan behandlingen påbörjas, vilket leder till en suboptimal
behandling för många patienter. En allvarlig sen bieffekt hos huvud/halscancerpatienter är
mandibulär osteoradionekros (ORN), där den del av mandibeln som bestrålats under loppet av
månader-år blir nekrotisk, med möjliga frakturer som följd. ORN är smärtsamt och medför en
kraftig försämring av livskvaliteten, inte minst om omfattande rekonstruktiv kirurgi blir
nödvändig. Omvårdnaden är även mycket resurskrävande för sjukvården. Följaktligen vore
det mycket värdefullt om patientens strålkänslighet kunde bedömas innan behandlingen, som
då kunde individanpassas.
Studien omfattade 37 huvud/halscancerpatienter som utvecklat ORN (ORN+) som en sen
bieffekt av strålterapin, samt 37 matchade kontrollpatienter (ORN-). Studien utfördes på
helblod från denna kohort. Då vi tidigare sett att förmågan att hantera strålinducerad oxidativ
stress verkar skilja sig mellan känsliga/normalkänsliga individer, mättes denna i form av
biomarkören 8-oxo-dG (en oxiderad form av guanin, en byggsten i DNA) i blodserum. Det är
37
även känt att punktmutationer i nyckelgener kan påverka individens strålkänslighet. En vanlig
form av sådana mutationer är nukleotidpolymorfismer (SNP), som i sig inte resulterar i
syndromlika effekter hos individen, men som i gengäld finns hos >1 % av befolkningen,
vilket gör dem intressanta som riskfaktorer inte bara för en rad sjukdomar utan också för
strålkänslighet. Därför genotypades alla patienter i åtta gener involverade i responsen på
oxidativ stress för SNP, som tidigare misstänkts kunna påverka risken för strålningsrelaterade
bieffekter. Relevanta kliniska uppgifter såsom total stråldos, alkohol/tobaksvanor, om
patienten fått brachy/kemoterapi etc. insamlades också.
En analys av de kliniska parametrarna indikerade att de två patientkohorterna var väl
matchade. Ett antal kliniska faktorer föreföll skilja mellan patientgrupperna; brachyterapi
medförde en ökad risk för ORN vilket det finns ett brett stöd för i tidigare studier. Vi kunde
uppmäta en signifikant lägre nivå av biomarkören 8-oxo-dG 60 minuter efter en dos på 2 Gy i
ORN+ gruppen, vilket kan tyda på att dessa patienter inte svarar på oxidativ stress på samma
sätt som de normalkänsliga (ORN-). Genotypningen av polymorfismerna talade också för
detta, då vi upptäckte att frekvensen av en punktmutation i enzymet glutathion S-transferas Pi
1 (GSTP1) var signifikant högre i ORN+ gruppen. GSTP1 är involverat i cellens
metabolisering av toxiska molekyler och fungerar även som en oxidativ stress-känslig
regulator av flera processer, till exempel inflammation. Denna punktmutation är studerad
tidigare, och resultaten tyder på att det muterade enzymet både har en nedsatt katalytisk och
regulativ förmåga. Tillsammans tyder dessa resultat på att kapaciteten att hantera oxidativ
stress är en viktig riskfaktor för sena bieffekter av strålterapi.
Då det är osannolikt att en enda faktor på ett tillförlitligt sätt kan användas för att bedöma
risken för allvarliga bieffekter efter strålterapi, konstruerades en multivariat modell bestående
av både kliniska och biologiska faktorer, för att på så sätt få ett mer robust verktyg. Efter en
optimering erhölls en signifikant modell bestående av faktorerna brachyterapi, SNP i GSTP1
samt serumnivån av 8-oxo-dG 60 minuter efter en in vitro- dos på 2 Gy. Känsligheten för
modellen var 85 %, vilket innebär att 85 av 100 ORN-riskpatienter i teorin skulle kunna
identifieras genom att man i ett blodprov mäter nivåerna av biomarkören 8-oxo-dG, genotypar
polymorfismen i GSTP1 samt undersöker om brachyterapi är aktuellt som en del av
behandlingen. Sammanfattningsvis utgör denna studie ett viktigt steg mot utvecklingen av ett
robust verktyg för att uppskatta risken för strålterapirelaterade bieffekter innan behandlingen,
vilket i förlängningen även kan medföra en bättre tumörkontroll för normalkänsliga patienter.
38
Acknowledgements
I would first like to thank myself for deciding to go for a PhD. Without that decision, I would
not have met many of the people I acknowledge below and life would probably be quite
different.
I would like to express my sincere gratitude to my supervisor prof. Andrzej Wojcik, for
accepting me first as a master student, and then as a PhD student. You have taught me many
invaluable skills and also let me try my “scientific wings” in different ways. You have also
promoted a development for a general interest in radiation-related sciences, and involved me
in the many aspects of science. You have supported me through the good and the bad in life.
Thank you!
Big thanks to Dr. Siamak Haghdoost, my cosupervisor. It has been very nice to be a part of
your path towards becoming a group leader and independent scientist. I especially admire
your ability to quickly resolve practical problems in the lab.
Another key person is prof. Mats Harms-Ringdahl, who held a very interesting lecture about
ionizing radiation in 2007 as a part of the “genetic and cellular toxicology” master course.
Fortunately I attended, and the rest is as they say, history.
Dr. Ainars Bajinskis was my patient supervisor in the radiation biology course in 2008.
Apparently I did not drive you completely crazy then, as we later as PhD student colleagues
enjoyed trombone playing, Russian lessons, beer tastings, working on paper II and III etc.
I admire your amazing organizational skills!
Past and present members of the RadBio group: Alice, Asal, Eliana, Elina, Marta, Ramesh,
Sara S, Sara SM, Siv and Traimate. Thank you for creating a very nice working atmosphere
and for being such fun travel companions, and for all the RadBio dinners!
Daniel Danielsson and Martin Halle at the Karolinska University Hospital. Not only have
you been my “partners in crime” in the ORN study, but you have also helped me understand
the clinical aspects and consequences from RT in normal tissue. I wish you the best of luck in
your important future work!
The students I have supervised during their research traineeship/master thesis: It has been
very developing for me to try to guide you in the jungle of science, so thank you for trusting
your supervision to me.
Engineer Leif Bäcklin at the university workshop for your technical assistance both for parts
of my thesis work, but also for the help in building my scintillation detectors and accessory
equipment. For you, no task is too complicated and no lab equipment ever too broken.
39
The whole MBW department, especially the “old GMT” people. Thank you for providing a
nice working environment! Special thanks to Görel, IB, Eva P, Eva E and Björn: You have
greatly facilitated the life as a PhD student. Without you, everything would quickly grind to a
halt.
My very first supervisor assoc. prof. Dr. Jana Jass, for guiding a rookie through 20 weeks of
intense “scientific growing-up” in your lab in 2007, which gave me increased confidence for
the rest of my studies. I will never forget your first round of corrections of my Bachelor’s
thesis, which you started with the words “Don’t be afraid of all the red ink”. Scientific writing
came to be one of the things I have enjoyed the most, so thank you for setting the standard
early on! Thanks also to your students Mattias Karlsson and Simon Lam.
Tom Hall at iRad inc. and Steven Sesselmann at Bee Research for helping me to become
involved in the world of gamma spectrometry. You guys have showed me that science can be
a very challenging and rewarding hobby, and that amateur science can be done to very high
standards. I also very much enjoy our non-scientific discussions about life in general.
My whole family for support through the ups and downs of life, and my friends, old and new,
for just being who you are and taking my mind off things. Thanks also to all my friends
within the world of orchestral music for all the wonderful music that we have made together!
The Swedish Radiation Safety Authority (SSM) for providing financial support for my
studies, as well as “Svensk förening för radiobiologi” for travel grants.
Finally I express my deepest admiration, gratitude and love to my wife, best friend and life
companion Maria. Thank you for your endless support and love, and for being a wonderful
mother. To my children Klara, Gustav and Viktor: you have shown me what really matters
in life. Nothing compares to you.
40
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