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

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© 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

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To my family

“Some people drink from the fountain of knowledge. Others just gargle”

Robert Anthony

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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 patient-

related 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.

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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, Munck-

Wikland 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

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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 241

Am 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 241

Am alpha particles and X-rays. Mutation Research Aug 30;756(1-2):95-

100 (2013).

XI Skiöld S, Näslund I, Brehwens K, Andersson A, Wersäll P, Lidbrink E, Harms-

Ringdahl 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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viii

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

ix

Abbreviations

γH2AX H2A histone family, member X, phosphorylated on serine 139

8-oxo-dG 8-oxo-7,8-dihydro-2´-deoxyguanosine

8-oxo-dGMP 8-oxo-7,8-dihydro-2´-deoxyguanosine monophosphate

8-oxo-dGTP 8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate

ADR Average dose rate

ANOVA Analysis of variance

ASMase Acidic sphingomyelinase

AT Ataxia telangiectasia

ATM Ataxia telangiectasia mutated

BAX BCL2-associated X protein

BER Base excision repair

BNC Binucleated cell

BR Bystander response

CRP Chemical radioprotectants

DDR Decreasing dose rate

DDRE Decreasing dose rate effect

DMSO Dimethylsulfoxide

DSB Double-strand break

EGF Epidermal growth factor

ELISA Enzyme-linked immunosorbent assay

Gy Gray; Joule/kg

HBO Hyperbaric oxygen

HDRP High dose rate point

HNC Head and neck cancer

HPLC High-performance liquid chromatography

HR Homologous recombination

H-RAS V-Ha-ras Harvey rat sarcoma viral oncogene homolog

ICRP International commission on radiological protection

IDR Increasing dose rate

IR Ionizing radiation

LET Linear energy transfer

MFROM Moving away from the source

MN Micronuclei

MTH1 (NUDT1) Nudix (nucleoside diphosphate linked moiety X)-type motif 1

MTO Moving towards the source

MUTYH MutY homolog

NAD(P)H Nicotinamide adenine dinucleotide phosphate-oxidase

NER Nucleotide excision repair

NSMase Neutral sphingomyelinase

NHEJ Non-homologous end joining

x

OGG1 8-oxoguanine DNA glycosylase

ORN Osteoradionecrosis

PBL Peripheral blood lymphocytes

RIF Radiation-induced fibroatrophy

ROS Reactive oxygen species

RNS Reactive nitrogen species

RT Radiotherapy

SSB Single-strand break

Sv Sievert; dose equivalent (Joule/kg)

TE Temperature effect

TGF Transforming growth factor

SNP Single nucleotide polymorphism

1

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 well-

known 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.

2

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

3

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).

4

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.

5

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 137

Cs 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 X-

rays, 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).

6

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.

7

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 60

Co 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

8

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 X-

rays 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.

9

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

10

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

11

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 double-

strand 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

12

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:CT:A and A:TC:G transversions (see figure 6). If 8-

oxo-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-oxo-

dGTP 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).

13

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-oxo-

dGTP 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

14

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” non-

homologous 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).

15

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 well-

known 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).

16

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/G2 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 T-

cells) can be induced to divide by the polyclonal activator phytohemagglutinin, making it

possible to study cytogenetic effects such as chromosomal aberrations or MN.

17

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

18

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.

19

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 RT-

induced 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

20

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.

21

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.

22

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 anti-

inflammatory 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).

23

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.

24

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, Chang-

Claude et al. 2009, Pugh et al. 2009, Pratesi et al. 2011, Langsenlehner et al. 2011, Mangoni

25

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.

26

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-oxo-

dG 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.

27

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.

28

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

29

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

30

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.

31

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.

32

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 radiation-

induced 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.

33

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.

34

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

35

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.

36

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 DNA-

dubbelsträngsbrott, samt en mer öppen kromatin-konformation påverkade temperatureffekten.

Vi undersökte också temperatureffekten med flera metoder, för att se om effekten är beroende

37

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

38

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.

39

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.

40

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.

41

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