Cellular effects after exposure to mixed beams of ionizing radiation

Elina Staaf

Doctoral thesis in Molecular Genetics at Stockholm University, Sweden 2012

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©Elina Staaf, Stockholm 2012

ISBN 978-91-7447-588-3 (pages 1-58)

Printed in Sweden by Universitetsservice US-AB, Stockholm 2012

Distributor: Department of Genetics, Microbiology and Toxicology

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Abstract Mixed beams of ionizing radiation in our environment originate from space, the

bedrock and our own houses. Radiotherapy patients treated with boron neutron capture

therapy or with high energy photons are also exposed to mixed beams of gamma

radiation and neutrons. Earlier investigations have reported additivity as well as

synergism (a greater than additive response) when combining radiations of different

linear energy transfer. However, the outcome seemed to be dependent on the

experimental setup, especially the order of irradiation and the temperature at exposure.

A unique facility allowing simultaneously exposure of cells to X-rays and 241Am

alpha particles at 37 ºC was constructed and characterized at the Stockholm University

(Paper I). To investigate the cytogenetic response to mixed beam irradiation (graded

doses of alpha particles, X-rays or a mixture of both) several different cell types were

utilized. AA8 Chinese Hamster Ovary cells were analyzed for clonogenic survival

(Paper I), human peripheral blood lymphocytes were analyzed for micronuclei and

chromosomal aberrations (Paper II and Paper III respectively) and VH10 normal

human fibroblasts were scored for gamma-H2AX foci (Paper IV).

For clonogenic survival, mixed beam results were additive, while a significant

synergistic effect was observed for micronuclei and chromosomal aberrations. The

micronuclei dose responses were linear, and a significant synergistic effect was present at

all investigated doses. From the analysis of micronuclei distributions we speculated that

the synergistic effect was due to an impaired repair of X-ray induced DNA damage, a

conclusion that was supported by chromosomal aberration results. Gamma-H2AX foci

dose responses were additive 1 h after exposure, but the kinetics indicated that the

presence of low LET-induced damage engages the DNA repair machinery, leading to a

delayed repair of the more complex DNA damage induced by alpha particles. These

conclusions are not necessary contradictory since fast repair does not necessarily equal

correct repair. Taken together, the observed synergistic effects indicate that the risks of

stochastic effects from mixed beam exposure may be higher than expected from adding

the individual dose components.

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Populärvetenskaplig sammanfattning Cellulära effekter från blandad strålning.

Joniserande strålning är en naturlig del av våra liv, vår omgivning och även våra

kroppar. Vi lever med bakgrundsstrålning från berggrunden och i maten vi äter, vid

flygresor är vi närmare rymden och den kosmiska strålningen. Strålning är även ett

vanligt verktyg inom medicin (främst diagnostik och cancerbehandling) samt inom

industrin. Det finns två huvudtyper av joniserande strålning, elektromagnetisk strålning

och partikelstrålning. Den elektromagnetiska strålningen består av fotoner, energikvanta

som levererar sin energi jämt fördelad och utspridd. Partikelstrålningen inducerar istället

många och täta skador längst partikelns spår. Båda dessa stråltyper kan skada DNA-baser

samt bryta ena eller båda DNA-strängarna samtidigt, vilket kan leda till mutationer och i

värsta fall cancer. Dock har vi effektiva DNA-reparationssystem i våra celler för att

hantera detta och de krävs många skador (dvs hög dos), eller en ökad skadekomplexitet

för att reparationen inte ska fungera korrekt.

Blandad strålning inträffar när båda dessa stråltyper finns närvarande samtidigt,

t.ex. bakgrundsstrålning som inkluderar radon, den kosmiska strålningen under flygresor

och vid vissa sorters cancerbehandling. Den stora frågan är ifall effekten av blandad

strålning är additiv (1 + 1 = 2) eller ifall de två typerna interagerar och därmed inducerar

en synergistisk effekt (1 + 1 = 3). Ett flertal forskargrupper har studerat denna fråga men

eftersom både synergism och additivitet har observerats har ingen konsensus gällande

risker från blandad strålning nåtts. De svagheter som finns för tidigare studier är att

bestrålning med en stråltyp före den andra (med en paus emellan) har klassificerats som

blandad, att temperaturkontroll har saknats samt att resultaten bara har undersökts med en

enda analysmetod. Vi har därför konstruerat ett system för blandad strålning där celler i

en 37°C-inkubator utsätts för röntgenstrålning och alfapartiklar samtidigt. Denna

avhandling är baserad på resultaten från detta unika bestrålningssystem.

Den första artikeln fokuserar på att beskriva och karaktärisera detta

bestrålningssystem. Alfa-källans dos per tid (så kallad dos-rat) beräknades teoretiskt och

mättes sedan med hjälp av en speciell plastfilm. Dos-raten var 0.265 Gy per minut.

Biologisk reproducerbarhet mättes med cellöverlevnad i hamsterceller och observerades

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vara god, och effekten av blandad strålning var additiv. Dock ökade skillnaden mellan

observerad och beräknad effekt av blandad strålning med ökande dos och procent

alfapartiklar i blandningen, så en synergistisk effekt kunde ha observerats om högre doser

och högre procent alfapartiklar (> 50 %) använts.

Den andra artikeln fokuserar på dos-responsen för mikrokärnor i vita

blodkroppar från människoblod utsatt för blandad strålning. Genom att tillsätta en

speciell kemikalie stoppas celldelning men inte kärndelning, och hela kromosomer och

kromosombitar som hamnade utanför vid kärndelningen kan ses som mindre kärnor

bredvid två större kärnor i en cell. För mikrokärnor observerades en signifikant

synergistisk effekt efter blandad strålning (35 % alfa-partiklar). Denna effekt var synlig

vid alla undersökta doser.

Den tredje artikeln undersöker kromosomala aberrationer i mänskligt blod efter

blandad strålning. Aberrationer inträffar när cellen inte har lyckats laga ett

dubbelsträngsbrott, eller har lagat det fel (t.ex. klistrat ihop två kromosomer som inte hör

ihop, eller gjort en skadad kromosom till en ring), och syns vid metafas när cellen har

kondenserat DNAt till kromosomer. Aberrationer klassificeras som komplexa ifall de

skapades från minst tre dubbelsträngsbrott från två olika kromosomer, och simpla ifall

två eller färre brott var inblandade. En synergistisk effekt observerades för mittendosen

(25 % alfapartiklar) och den högsta dosen (40 % alfapartiklar) för komplexa aberrationer,

men bara för den högsta dosen för simpla aberrationer. Dos-responskurvan för komplexa

aberrationer var linjär-kvadratisk, vilket också indikerar en synergistisk effekt.

Den fjärde och sista artikeln undersöker dos- och tids-respons för gamma-

H2AX foci i en mänsklig hudcellinje. DNAt är tätt packat runt proteiner (bl.a H2AX),

och när DNA ändrar form (t.e.x vid ett dubbelsträngsbrott) fosforyleras dessa proteiner.

De kan sedan ses som lysande prickar – foci - under mikroskop med hjälp av färgade

antikroppar. H2AX-foci uppkommer snabbt efter DNA-skada och försvinner när skadan

har reparerats. Dos-respons för blandad strålning var additiv 1 timme efter bestrålning,

men tids-responsen indikerade att närvaron av skador från elektromagnetisk strålning

störde reparationen av partikel-inducerade skador.

Sammanlagt indikerar dessa resultat att biologiska effekten från blandad strålning

kan vara större än vad som kan beräknas från de individuella stråltyperna i bladningen.

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Publications List of original publications This thesis is based on data presented in the following publications:

I Staaf E, Brehwens K, Haghdoost S, Pachnerova-Brabcova K, Czub J, Braziewicz

J and Wojcik A. (2012) “Characterization of a setup for mixed beam exposure of

cells to 241Am alpha particles and X-rays.” Radiat Prot Dosimetry, 151(3):570-79

II Staaf E, Brehwens K, Haghdoost S, Nievaart S, Pachnerova-Brabcova K, Czub J,

Braziewicz J and Wojcik A. (2012) “Micronuclei in human peripheral blood

lymphocytes exposed to mixed beams of X-rays and alpha particles.” Radiat Env

Biophys, 51(3):283-93

III Staaf E, Deperas-Kaminska M, Brehwens K, Haghdoost S, Czub J and Wojcik A.

(2012) “Higher than expected frequencies of complex aberrations in lymphocytes

exposed to mixed beams of 241Am alpha particles and X-rays.” Manuscript,

submitted to Acta Oncololgica

IV Staaf E, Brehwens K, Haghdoost S, Czub J and Wojcik A. (2012) “Gamma-

H2AX foci in cells exposed to a mixed beam of X-rays and alpha particles.”

Manuscript, revised submitted to Genome Integrity

Articles I and II were reprinted with the kind permissions of the publishers: Oxford

Journals and Springer.

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Additional publications Not included in this thesis:

V Johannes C, Dixius A, Pust M, Hentschel R, Buraczewska I, Staaf E, Brehwens

K, Haghdoost S, Nievaart S, Czub J, Braziewicz J and Wojcik A. (2010) “The

yield of radiation-induced micronuclei in early and late-arising binucleated cells

depends on radiation quality.” Mutat Res 701(1):80-5

VI Brehwens K, Staaf E, Haghdoost S, González A.J, and Wojcik A. (2010)

“Cytogenetic damage in cells exposed to ionizing radiation under conditions of a

changing dose rate.“ Radiat Res 173(3):293-9

VII Brehwens K, Bajinskis A, Staaf E, Haghdoost S, Cederwall B and Wojcik A

(2012) “A new device to expose cells to changing dose rates of ionising

radiation.” Radiat Prot Dosimetry 148(3):366-71

VIII Dang L, Lisowska H, Manesh S.S, Sollazzo A, Deperas-Kaminska M, Staaf E,

Haghdoost S, Brehwens K and Wojcik A. (2012) “Radioprotective effect of

hypothermia on cells – a multiparametric approach to delineate the mechanisms.”

Int J Radiat Biol 88(7):507-14

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Table of contents

Abstract ............................................................................................................................... 3

Populärvetenskaplig sammanfattning ................................................................................. 4

Introduction....................................................................................................................... 11

Introduction to ionizing radiation ................................................................................. 11

A part of our lives ..................................................................................................... 11

Direct and indirect effect .......................................................................................... 11

Linear energy transfer ............................................................................................... 12

Relative biological effectiveness .............................................................................. 13

Complexity of radiation-induced DNA damage ....................................................... 14

Repair of radiation-induced DNA damage ................................................................... 15

Overview of DNA double-strand break repair systems............................................ 15

Homologous recombination...................................................................................... 16

Non-homologous end-joining ................................................................................... 17

Outcomes of DNA damage repair ............................................................................ 17

The concept of risk........................................................................................................ 18

Risk considerations ................................................................................................... 18

Synergism and additivity .......................................................................................... 20

Mixed beam studies – reviewing the literature ............................................................. 21

The cell system and endpoint.................................................................................... 21

The LET combination, doses and dose regimes ....................................................... 24

Irradiation characteristics – temperature, time and order of exposure...................... 25

Summary of findings ................................................................................................ 27

The present investigation .................................................................................................. 28

Aims of this thesis......................................................................................................... 28

Novel approaches of the experiments ....................................................................... 28

Materials and methods .................................................................................................. 29

Cell types .................................................................................................................. 29

The irradiation setup ................................................................................................. 29

Cell exposure ............................................................................................................ 30

Clonogenic cell survival ........................................................................................... 31

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Chromosomal aberrations ......................................................................................... 31

The micronucleus assay ............................................................................................ 32

The gamma-H2AX assay.......................................................................................... 32

Results and discussion .................................................................................................. 34

Paper I ....................................................................................................................... 34

Paper II...................................................................................................................... 36

Paper III .................................................................................................................... 38

Paper IV .................................................................................................................... 40

Concluding remarks and future perspectives................................................................ 42

Concluding remarks .................................................................................................. 42

Future perspectives ................................................................................................... 43

Acknowledgements........................................................................................................... 45

References......................................................................................................................... 46

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Abbreviations Artemis endonuclease active in NHEJ and V(D)J recombination ATM ataxia telangiectasia mutated bp base pair (of DNA) CHO Chinese hamster ovary DNA-PKcs DNA-dependent protein kinase catalytic subunit DSB double strand break (in DNA) FISH fluorescence in situ hybridization γ-H2AX phosphorylated form of histone 2AX protein Gy Gray = Joule per Kg. Unit in radiation biology. HR homologous recombination repair IMRT intensity modulated radiotherapy IR ionizing radiation Ku70/80 heterodimer of the proteins Ku70 and Ku80 LET linear energy transfer LF “large” gamma-H2AX foci MN micronuclei (or micronucleus, depending on context) MRE11 meiotic recombination 11 homolog MRN complex of MRE11-Rad51 and NBS1, active in HR NBS1 Nijmegen breakage syndrome 1 NHEJ non-homologous end-joining PCC premature chromosome condensation PBL peripheral blood lymphocytes Rad50/51/52 radiation sensitive protein 50/51/52 RBE relative biological effectiveness RT room temperature SF “small” gamma-H2AX foci SSB single strand break (in DNA) Sv Sievert. The absorbed dose is modified by correction factors. XLF XRCC4-like factor

XRCC3/4 X-ray repair cross-complimenting group 3 / 4 protein

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Introduction

Introduction to ionizing radiation

A part of our lives Ionizing radiation (IR) is a continuous part of our lives, our environment and our

bodies. The natural exposure from IR is around 3 mSv per year in Sweden, with 2.1 mSv

from natural and 0.9 mSv from artificial sources (1). Natural radiation consists of cosmic

radiation, radioactive substances in the earth’s crust (and their decay products) as well as

radioactive isotopes within our own bodies. The cosmic radiation originates from outer

space and the sun and though it mostly affects astronauts in space it can also penetrate the

atmosphere, reaching us during airplane flight and at the ground level (2, 3). Radon gas

released from the bedrock and building materials in our houses adds alpha particles to the

natural exposure, an effect contributing to the risk for lung cancer (4). Our bodies contain

natural isotopes such as 14C, 210Pb and 40K (3, 5) and through our food and drink

additional radioactive particles can be ingested. Artificial sources originate from human

inventions and activities. Medical diagnostic procedures (6) along with radiation therapy

of cancer (7) are becoming more common worldwide, and 137Cs from Chernobyl fall-out

and other nuclides from nuclear tests are present in our food (in Sweden, mainly in

mushrooms and wild animals) (8, 9). Most individuals will not experience any

measurable negative stochastic effects (such as cancer) from this background radiation.

However, exposure to IR has the potential to cause harm.

Direct and indirect effect

IR induces damage by two processes. When IR is absorbed by cells there is a

possibility that it will directly interact with DNA, the critical target. This occurs through

direct ionization and/or excitation in the DNA from interaction with the radiation.

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Damage to DNA can also be induced indirectly. The indirect effect is a two-step process

where the first step is IR-induced ionization of liquid molecules. This produces radicals,

which in turn can diffuse to and react with the target molecule (10, 11).

Linear energy transfer

An important property of IR is the linear energy transfer, LETΔ, defined as energy

transferred per unit path length traversed by an ionizing particle, excluding delta-

electrons with energy above Δ (12). LETΔ, hereafter referred to as LET, is an average

quality, describing the whole exposure and not each individual particle. IR can be

classified as low or high LET.

Low LET radiation generally consists of electrons and photons, which originate

from natural decay of radioactive isotopes (for example 137Cs or 60Co) or man-made X-

rays where electrons are accelerated into a target (usually 79Au or 74W), thereby emitting

photons as bremsstrahlung (11). Photons are electromagnetic radiation/waves that have

short wavelengths at higher energies. One ionizing photon generates about 30 ionizations

within a cell nucleus and about 70 % of the induced damage is from the indirect effect

(11). The LET of photons is usually below 1 keV/μm.

High LET radiation consists of particles: protons, neutrons, alpha particles and

heavy ions and can be generated by decay of radioactive isotopes or in purpose-built

devices (accelerators and reactors). Upon interaction with the target, secondary particles

(for alpha-particles mainly delta-electrons) are produced and in turn cause ionizations

along the path of the original particle. For alpha particles, about 10 000 ionizations are

induced per track (13), giving rise to clusters of damage, the majority from direct

interaction. High LET values vary from 10-100 keV/μm for neutrons to 100-200 keV/μm

for alpha particles and more than 1000 keV/μm for heavy ions (11, 14). The differences

between high and low LET IR is exemplified in Figure 1.

The uncharged state of photons means that it can penetrate far into matter before

encountering a target with which to react. This means that within an irradiated cell

population low LET IR will induce an even dose distribution, since the cells will on

average have received the same number of photon interactions and thus the same dose.

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For high LET, the massive amount of energy delivered per traversed length result in a

very short reach (alpha particles: a few nm in tissue), and a characteristic dose delivery

called the Bragg peak (11). An alpha particle-irradiated cell population will show an

uneven dose distribution, since each track traversing a nucleus will by definition induce

heavy damage and an irradiated cell population will always contain cells that received

different numbers of hits.

Figure 1: Track structure for IR of different LET. The circle represents a cell nucleus. To the left: Low

LET (photons). To the right: High LET (α-particle). Figure adapted from (10).

Relative biological effectiveness

The relative biological effectiveness (RBE) is a tool for evaluating differences in

effects between IR of different LET. RBE is determined by estimating the dose that gives

rise to a defined level of biological effect and comparing it to the dose from a reference

type of radiation (normally low LET) required for the same effect (15). Low LET

radiations have a RBE of 1 and this value increases with increasing LET to reach a

maximum at about 100 keV/μm, thereafter decreasing (11). For review, see (14). The

RBE is strongly dependent on factors such as cell type and endpoint and is therefore not

an absolute value (exemplified in 16).

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Complexity of radiation-induced DNA damage

Several types of DNA lesions can be induced by IR. Base damage, base loss, DNA-

protein crosslinks, along with the single and the double strand break (SSB and DSB,

respectively) are some types of damage that occur (10). The DSB is the most detrimental

of these since it is the only lesion where both DNA strands are simultaneously broken

(17). In addition, if several lesions occur within a small volume they form clustered

lesions (also called multiple damages sites), defined as >2 damages within a 20 bp region

(18). These clustered lesions are more difficult to repair than simple DSBs without

additional flanking damage (19, 20). A clustered lesion is exemplified in Figure 2.

Figure 2: Example of a clustered lesion, a DSB flanked by a variety of other lesions.

Depending on the LET, IR induces different proportions of lesions, with low LET

photons inducing the majority of its effect indirectly, while high LET particles induces a

higher level of direct interaction (21). Due to the higher density of ionizations and

excitations along the track, high LET also generates a higher proportion of clustered

lesions (22). The proportion of clustered lesions as well as the degree of complexity of

each lesion increases with increasing LET (23). All components of a clustered lesion

must be repaired or removed for the break to be fully repaired, thus delaying the repair

process (17). Several DSBs within the same small area also result in a high probability of

incorrect rejoining of the broken DNA ends (19).

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Repair of radiation-induced DNA damage

Overview of DNA double-strand break repair systems

The survival of cells and organisms is linked to the integrity of DNA, and efficient

DNA-repair mechanisms are present in our cells (24). These repair systems can handle

endogenous damage from the cell metabolism as well as exogenous damage from agents

such as IR, UV-radiation, chemicals or changes in temperature or oxygen level.

Deficiencies in DNA repair systems often lead to disease (see e.g. 25). As previously

mentioned, a DSB is the most dangerous lesion for the cell, because both DNA strands

are broken (17). Upon DSB formation, the broken DNA ends are immediately bound by

the MRN protein complex (MRE11, Rad51 and NBS1), a signalling and end resection

factor (26, 27). ATM is recruited by MRN, and both participate in H2AX

phosphorylation (28, 29). The phosphorylation rapidly spreads up to 2 million base pairs

(circa 2000 H2AX molecules) in each direction from the DSB (30, 31), where stretches

of gamma-H2AX act as accumulation signals and docking places for DNA repair proteins

(32, 33).

Two main DSB repair systems exist; homologous recombination (HR) and non-

homologous end joining (NHEJ). A simplified visualization of the two repair systems is

shown in figure 3. HR is mainly active during replication and G2, and functions by

copying information from a non-damaged, homologous double-strand of DNA to the

damaged strand. It is therefore a precise, but slow method of repair (34). NHEJ is active

during all phases of the cell cycle and is not homology-dependent (35). In NHEJ the

DNA ends are held in place, damaged nucleotides trimmed away and the ends rejoined.

This process results in a fast repair, but mutations or cell death may occur since some

genetic material is always lost around the breakpoint (36). The presence of both repair

systems leads to biphasic DSB repair kinetics (37-40), with NHEJ being mainly

responsible for the fast and HR for the slow component (34, 41). ATM and DNA-PKcs

are involved in the pathway choice, which also depends on the cell cycle phase (42-44).

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Figure 3: Simplified models of DNA DSB repair systems HR (to the left) and NHEJ (to the right).

Homologous recombination

In HR, nuclease end-processing removes DNA in the 5´to 3´ direction to form long

3’ single-stranded DNA “tails” on both sides of the break (45). The single stranded

sections are then covered by RPA, single-stranded DNA-binding proteins which interact

with Rad51 and Rad52. Rad51 creates a hexameric structure around the end of the single-

stranded part, and forms nucleoprotein filaments (46) which search for homologous

double-stranded DNA. In this step Rad54, a nuclear translocase, is essential for creating a

D-loop when invading the homologous double-stranded DNA sequence and for

depolymerising the Rad51 filaments (47, 48). New DNA is subsequently synthesized by

DNA polymerases to fill the gaps, Holliday junctions are cleaved by a resolvase complex

(49, 50) and nicks in the DNA are sealed. XRCC3 and Rad51C, (a Rad51 paralogue)

forms the CX3 complex, necessary for holding in place and resolving of the four DNA

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strands involved in the Holliday junctions (51, 52). Mitotic HR has recently been

reviewed (53).

Non-homologous end-joining

In NHEJ the heterodimer Ku70/80 binds specifically to open double-stranded DNA

ends (54). Ku70/80 then recruits DNA-PKcs, together forming a complex to hold the

DNA ends in place and to create a scaffold for other proteins (55). Artemis joins the

complex and carries out end-processing of the DNA via nuclease activities (56). Ku70/80

further recruits XRCC4 and ligase IV to the processed ends (57) while DNA-PKcs

stabilizes the new complex (58, 59) and finally the XRCC4-ligase IV complex ligates the

DNA-strands (60). XLF is an alternative Ku70/80 recruited protein (61), essential for

performing the ligation of complex DSBs when strands are lacking in homology or

mismatched (62). NHEJ has recently been reviewed (63).

Outcomes of DNA damage repair

The capacity and outcome of DNA repair is influenced by the complexity of

induced damage. A clustered lesion is more difficult to repair and does therefore retain

repair proteins for a longer time than a simple DSB (19, 20). This has been observed at

the level of DNA DSB signalling and repair proteins, where foci after high LET IR are

larger and persist for a longer time than if the same dose was given with low LET IR (64-

66). The cellular repair of DNA lesions can in itself give rise to additional DSBs, and

contribute to the complexity of the induced damage (22). In addition to the LET of the

IR, the cell cycle phase during which the cells were irradiated also plays a role (67). The

cells are most sensitive in mitosis, while the highest resistance is observed in S-phase

(11).

DSBs that failed to repair or were misrepaired result in chromosomal aberrations,

which can be visualized in metaphase as breaks, gaps and other anomalies present in the

chromosomes. There are two classes of chromosomal aberrations; simple aberrations that

include breaks, rings, and exchanges between two chromosomes (dicentrics and

translocations) and complex aberrations, consisting of at least three breaks in two or more

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chromosomes (68). A high LET particle will give rise to many open DNA ends along the

particle track, significantly increasing the risk for misrepair relative to the same dose of

low LET. Consequently, high LET IR induces substantial numbers of complex

aberrations already at low doses, while low LET IR mainly gives rise to simple

aberrations (21). Aberrations can also be classified as stable or unstable, where stable

aberrations such as translocations or insertions are transmissible while unstable

aberrations (e.g. dicentrics and rings) will be lost from the genome and/or result in cell

death (69). The presence of complex aberrations in the genome can be viewed as a

marker of high LET exposure (70, 71).

The concept of risk

Risk considerations

It is known that high doses of IR cause harmful effects in the human body. The

major deterministic effect is the acute radiation syndrome, while cancer induction is the

main stochastic effect. The dose responses for deterministic and stochastic effects of IR

have been established epidemiologically, mainly from atom bomb survivors (72, 73),

nuclear workers (74) and patients receiving IR for medical reasons (e.g. 75). In humans,

the acute radiation syndrome appears above a threshold dose of about one Gy/Sv or

higher, where the severity of these effects (mainly on the hematopoietic system and the

skin) increase with increasing dose (76). For doses below 100 mSv (effective dose), no

significant stochastic effects have been observed at the epidemiological level (77, 78).

Several extrapolations of the dose response curve for cancer risk after radiation

exposure from the high dose region to the low dose region exist; see Figure 4 for a

schematic representation (adapted from Brenner et al. (77)). The linear no threshold

model (extrapolation “a” in Figure 4) is currently the most accepted and applied method

for radiation protection and modeling of cancer risk (79).

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Figure 4: The dots at the upper right corner represent the 100 mSv dose point. The effect for lower doses

could be a) linear, b) downwardly curving (the slope decreasing with decreasing dose), c) upwardly curving

(slope increasing with dose), d) threshold-dependent or e) beneficial before becoming harmful, in

accordance with the hormesis hypothesis, (80).

Many studies on the effects of IR were based upon the action of a single type of IR.

However, after an atom bomb detonation low and high LET radiation (photons and

neutrons) is present simultaneously. Such “mixed beam” exposure situations are now

becoming more frequent. In airplane-, and especially during spaceflight people are

exposed to particles from cosmic IR, which interact with the shielding material of the

shuttle, forming a photon background (1, 81, 82). High background radiation of mixed

type can be found at several sites in the coastal region of Brazil, Yangjian in China,

Kerala in India and Ramsar in Iran (83, 84). In external beam radiation therapy mixed

beam exposures occur, especially during long irradiation sessions of intensity modulated

radiotherapy (IMRT), when the major part of the photon beam is shielded by a multi-leaf

collimator (85). Here, the absorbed neutron doses can be up to several hundred mSv (86,

87). The same effect occurs for fast neutron therapy where photons are generated through

thermalization of neutrons (88), and for boron neutron capture therapy (89) where the

captured neutrons give rise to photons, He- and Li-ions through the (n,γ) and the 10B(n,α)7Li reactions (90).

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Synergism and additivity

Synergism is defined as “cooperative interaction between two or more components

in a system, such that the combined effect is greater than the sum of each part” (91).

Conversely, additivity describes a situation where the observed combined effect is not

greater than that expected from the sum of each agent. In a cellular system synergism

between two agents can occur via two mechanisms; either by one of the agents

potentiating the level of damage from the other, or by impairing cellular damage repair

mechanisms. The former effect is observed for example for the interaction of oxygen

with IR (92-94), while the latter can be exemplified by the interaction IR with heavy

metals (95-97). The classification of a biological response as additive or synergistic is

complicated if the dose response of one or both agents is non-linear, requiring the

construction of “envelopes of additivity” (98, 99) and/or the application of mathematical

models (100-102).

Currently, there are no physical reasons to assume that interactions between IR of

different LET should result in a synergistic effect. However, synergistic effects have

previously been observed for mixed beam irradiation alongside results suggesting

additivity (see Table 1) – a clear lack of consensus. The key to the mechanisms of

synergistic effects are likely to be found at the subcellular level. It is possible that the

complex DNA damage induced by high LET IR could trap and make the DNA damage

repair proteins unavailable or less effective for repair of low LET induced damage (103).

Alternatively, the presence of low LET-induced, simple and dispersed DNA damage

could occupy the DNA repair systems to such an extent that the high LET induced

damage are not efficiently repaired. Finally, it is also possible that low and high LET IR

interact to potentiate the complexity of DNA damage.

Biological effects of mixed beams is thus one of the areas in radiation research

where the potential risks are still unclear and experimental results inconclusive. The issue

of additivity or synergism is still debated and 50 years of research within this area has not

yielded a clear conclusion. It is therefore very interesting to continue research in this area

with a wider range of methods, with the aim of contributing towards solving the problem.

20

Mixed beam studies – reviewing the literature

Several investigations regarding the DNA-damaging properties of mixed beams of

radiation have already been performed (see Table 1.). No overall conclusion has yet been

reached since synergism and additivity alike have been and are still being reported. This

section will investigate and discuss the factors that may favour a synergistic or an

additive outcome of an experiment. The discussion will focus on the following set of

parameters: 1) The cell system and endpoint, 2) The LET combination, doses and dose-

regimes and 3) Irradiation characteristics - Temperature, time and order of exposure.

In total, 35 published studies have investigated different aspects of the cellular

response to mixed beam irradiation (Table 1). Brooks et al. (124) and Furusawa et al.

(128) examined two different endpoints, multiple cell systems were employed by

Furusawa et al. and Hornsey et al. (119, 128), and two or more LET combinations were

employed in 8 different studies (103, 104, 116, 123, 126, 128, 130, 134). The majority of

studies however focused on a single endpoint and single combination of high and low

LET IR.

T

The majority of the studies, 18 ou

he cell system and endpoint

t of 35, were performed with hamster cells, either

V79 Chinese hamster lung fibroblasts (102, 112, 114-119, 121-123, 125, 126, 128, 132,

135) or Chinese hamster ovary cells (109, 111). Out of these, only 5 observed an additive

effect (109, 122, 126, 128, 132). Human cells were also often used; fibroblasts (103, 130,

133), peripheral blood lympohcytes (PBL) (127, 128, 136), kidney cells (105, 108) as

well as salivary gland (129), epithelial (131) and HeLa cells (134) were employed. In

total, 11 studies were thus completely or partly (128) run with human cells, and six of

these concluded synergism (105, 108, 127-129, 133). Other mammalian cells were

obtained from mouse (107, 113, 120) and rat (124), and all these studies concluded

synergism. Non-mammalian systems were barley (106), bean root (104), and S.

cerevisiae yeast (110), where the first two observed additive effects and the last

synergism.

21

Table 1: Summary of published mixed beam data. a = alpha particles, g = gamma radiation, N = neutrons,

X = X-rays, n.d. = no difference in effect, Simu = simultaneous, RT = room temperature, CS = clonogenic

survival, CA = chromosomal aberrations, MN = micronuclei, FISH = fluorescence in situ hybridization A =

additivity, A** = Additivity, but slight sign of synergism for Fe-ions. S = synergism. S* = incubation time

dependent synergism, effect disappears with time.

Author Cells used High LET Low LET

Combined dose (Gy)

Order of exposure

Between exposures (temp, time)

Exposure temp (°C) Endpoint Effect

Gray and Read 1944 (104) Bean Root N or a X-ray / g Both, n.d. RT, 15-22 Bean survival A Barendsen et al. 1960 (105)

Human kidney cells a X-rays 1.5 - 6 Both, n.d. RT, < 120 min RT CS A

Heddle 1965 (106) Barley N X-rays 5N + 150X Both, 1 diff RT, 13-15 min RT, cold CA A

Masuda 1970 (107) Mousle L cells N X-rays 5 - 7 Both RT, 5-10 min RT CS S

Raju and Jett 1974 (108)

Human T1 kidney cells a X-rays 8, 11, 15 a -> X RT, 1 min RT CS A

Railton et al. 1974 (109) CHO N 60-Co 2.5 - 15 Both, n.d. RT, 1-180 min RT CS A Murthy et al. 1975 (110)

S.Cerevisiae BZ34 a 60-Co 2 - 17.5 Simultaneous RT

arginine reversion S

Railton et al. 1975 (111) CHO N 60-Co 1 - 9 Both, n.d. RT, < 45 min RT CS S

Durand and Olive 1976 (112) V79 N X-rays 4 -16 Both RT, 4 h RT

Survival, monolayers & spheroids S

Hornsey et al. 1977 (113)

Mice, in vivo, small intestine N X-rays 6 -23.5 Both, n.d.

BodyTemp 2-4 h RT

Jejunum crypts in small intestine S

Ngo et al. 1977 (114) V79 N X-rays 4 - 12.2 N -> X-rays Ice, or 37°C 2, 3 or 5 h On ice CS S

Ngo et al. 1981 (115) V79 10-Ne X-rays 5.5 - 12.9 Both Ice, or 37 for 0.5-24h On ice CS S*

Bird et al. 1983 (116) V79 2-H 3-He X-rays 7-15 / 5-13

H/He -> X-rays RT, < 5 min RT CS S

Higgins et al. 1983 (117) V79 N 60-Co 2 - 15 Simu + Both 37°C, 5 min 37°C CS S* Higgins et al. 1984 (118) V79 N 60-Co 1 - 15 Simu + Both 37°C, <3 min 37°C CS S

Hornsey et al. 1984 (119)

V79, Erlich ascites, mouse jejunum stem cells N X-rays 4 - 16 N -> X-rays

10°C, 2 min, 4 or 8 h 20°C

CS, stem cell survival S

Joiner et al. 1984 (120)

In Vivo Mouse fibroblasts N X-rays 10 - 60 Simultaneous RT

Average skin reaction S

McNally et al. 1984 (121) V79 N X-rays 3 - 12 Both

6 min RT, 3 h 37°C RT CS S*

McNally et al. 1988 (122) V79 a X-rays 4 - 12 a -> X RT, 4 min RT CS A

Ngo et al. 1988 (123) V79 Ne, Ar X-rays 3 - 12 Ne/Ar ->X 4 °C 15-30 min 4°C CS S*

22

Brooks et al. 1990 (124)

Rat lung epithelial cells a X-rays 1.5 - 3.5 Simultaneous RT? RT? CS, MN S

Suzuki 1993 (125) V79 N 60-Co 1.5 - 7.5 Both, n.d. RT, <3 min RT CS S Kanai et al. 1997 (126) V79 12-C 3-He,4-He 1 - 7 He -> C? RT; 3 – 5 min RT? CS A Wuttke et al. 1998 (127) PBL N X-rays 1 - 3 N -> X-rays Ice, 30 min RT MN A

Tilly et al. 1999 (102) V79 N 60-Co 1 - 8 N -> 60-Co Ice, <10 min Ice, 4°C CS S Furusawa et al., 2002 (128) V79, PBL

Ar, Si, Fe X-rays

0-10, 1.2-3.6 Simultaneous RT CS, FISH A**

Demizu et al., 2004 (129)

Human salivary gland cells 12-C X-rays 1.4 - 6.2 Both <15 RT CS A

Zhou et al. 2006 (103)

Primary human fibroblasts Fe, Ti protons 0.04 Both 2.5 / 48 h RT

anchorage-independent growth, CS S*

Bennet et al. 2007 (130)

Primary human fibroblasts Fe, Ti H, protons 0.04

H->Ti, prot->Fe 15 min RT CS S

Hada et al. 2007 (131)

Human epithelial cells Fe protons 2.75 protons ->Fe

37°C 2, 30, 60 min RT

FISH (mBAND, PCC) S*

Phoenix et al. 2009 (132) V79 a 60-Co 4 - 10.5 Simultaneous 10°C CS A

Yang et al. 2010 (133)

Normal human skin fibroblasts Fe protons 0.02 protons ->Fe

3, 30, 180 min 24 h RT MN A

Elmore et al. 2011 (134) HeLa Fe

prot + 137-Cs 0.20 Both

5, 15 min 16-24 h RT

Neoplastic transformation A

Mason et al. 2011 (135) V79 N gamma 0-4

Simu, in phantom in reactor 4°C CS S

Wojcik et al. 2012 (136) PBL N

60-Co and N-beam g- component

0-4, different depths

Simu, in phanotm 37°C CA, dicentrics A

The most frequently used endpoint was clonogenic cell survival, analyzed in 26 out

of 34 studies (102-105, 107-109, 111, 112, 114-119, 121-126, 128-130, 132, 135), 18 of

which with hamster cells (102, 109, 111, 112, 114-119, 121-123, 125, 126, 128, 132,

135). Here, the general response was synergistic; 1/3 of the studies (9 out of 26) observed

an additive effect (104, 105, 108, 109, 122, 126, 128, 129, 132). The clonogenic survival

in human cells however was more often additive than synergistic; 3 studies concluded

additivity (105, 108, 129) while two observed synergism (128, 130). The cytogenetic

assays chromosomal aberrations (106, 128, 131, 136) and micronuclei (124, 127, 133)

revealed synergism in 2 out of 7 studies (124, 131). Here, the majority of cytogenetic

23

studies were carried out with human cells (127, 128, 131, 133, 136), and only Hada et al.

(131) observed synergism. Other assays concluded synergism for arginine reversion by S.

cerevisiae (110) as well as for in vivo tissue reaction in mouse gut (113, 119) and skin

(120), while neoplastic transformation of human HeLa cells (134) was additive.

The LET combination, doses and dose regimes

The most commonly studied LET combination was neutrons in combination with

low LET radiation (18 studies) (102, 104, 106, 107, 109, 111-114, 117, 118-121, 125,

127, 135, 136). Additivity was observed in 5 studies (104, 106, 109, 127, 136), and

synergism in the other 13. 12 of the studies used clonogenic survival (102, 107, 109, 111,

112, 114, 117-119, 121, 125, 135), and only one of these observed additivity (109). The

doses were generally within the range of 1 – 15 Gy, with higher doses for the in vivo

(113, 119, 120) and non-mammalian studies (104, 106), and lower end doses (1 - 4 Gy)

for the number on micronuclei and chromosomal aberrations in human PBL (127, 136).

When alpha particles were combined with low LET, additivity was observed in 5

studies (104, 105, 108, 122, 132), and synergism in 2 (110, 124). Interestingly, additivity

was observed for clonogenic survival in hamster V79 cells (132, 122), a combination that

gave rise to synergism when neutrons represented the high LET. The doses varied from

1.5 up to 17.5 Gy, with the smallest dose range (1.5 – 3.5 Gy) observed for clonogenic

survival and micronuclei in rat lung fibroblasts (124, concluding additivity), and the

highest dose (17.5 Gy) for arginine reversion in S. cerevisiae (110, synergism).

Combining heavy ions with low LET radiation resulted in equal numbers of

synergistic (115, 116, 123) and additive (128, 129, 134) effects. All the studies

concluding synergism, along with one concluding additivity (128) investigated

clonogenic survival in V79 cells (dose rage 1 up to 15 Gy), while the additive studies

involved human cells; clonogenic survival in human salivary gland cells (129), neoplastic

transformation in HeLa cells (134) and chromosomal aberrations in PBL (128). The

doses employed for human cells were lower, with 1.4 – 6 Gy for clonogenic survival

(129), 1.2 - 3.6 Gy for chromosomal aberrations (128) and 0.20 Gy for neoplastic

transformation (134).

24

Cells were also exposed to combinations of two different high LET radiations. Only

one out of the 6 studies employed non-human cells (126), and the conclusions were

evenly distributed between additivity (126, 133, 134) and synergism (103, 130, 131).

The doses employed were between 0.02 and 2.75 Gy for the human cells, and 1-7 Gy for

the V79 cells, since that study investigated clonogenic survival (126).

It was clear that the combination of LET was of great importance. Neutrons and

low LET generated mainly synergistic effects while alpha particles and low LET

generated additivity. Heavy ions induced equal numbers of additivity and synergism,

irrespectively of which low LET they were combined with. When different responses

were observed for higher and for lower doses (as was the case for neutrons and heavy

ions in combination with low LET) it was more likely that the dose-dependent

differences were due to the switch in endpoint rather than to the dose range itself.

Irradiation characteristics – temperature, time and order of exposure

In the majority of studies, the irradiation setups did not allow simultaneous

exposure of cells to a mixed beam. The two radiation types were generally applied in

sequence, with a pause in between when the samples were moved from one source to

another. Inherent to this scenario was also that the temperature during and between

exposures was not always well controlled. In fact, the majority of studies were carried

out at room temperature.

In total, 9 studies involved simultaneous exposure (110, 117, 118, 120, 124, 128,

132, 135, 136). Only two groups exposed cells simultaneously at 37°C (117, 118, 136).

In the studies of Higgins et al. V79 hamster cells were exposed to neutrons and gamma

radiation simultaneously and sequentially, and synergism was observed on the level of

clonogenic cell survival (117, 118). For Wojcik et al. human PBL were exposed to a

neutron beam with a photon component and chromosomal aberrations concluded

additivity (136). Simultaneous exposure at room temperature was performed with low

LET plus alpha particles (110, 124), neutrons (120) or heavy ions (128). Synergism was

observed in all cases except for one (128). For lower temperatures, clonogenic cell

survival of V79 cells after simultaneous exposure led to additivity for alpha particles plus

25

gamma radiation at 10°C (132), and to synergism for the mixture of neutrons and gamma

radiation at 4°C in two different nuclear reactors (135). In total, simultaneous exposure

resulted in 6 cases of synergism and 2 of additivity (128, 132).

Sequential exposures were performed in the remaining 26 studies. The studies were

most often carried out with both high before low as well as low before high LET IR (14

studies). In 6 of these studies no differences between the order of irradiations were

present (104, 105, 109, 111, 113, 125), while the other 8 studies observed that high

before low LET IR induced a greater response than low before high LET IR (103, 106,

107, 112, 115, 121, 129, 134). When no differences were observed, synergism was

concluded in 5 out of 8 studies (103, 107, 112, 115, 121), while synergism and additivity

was observed in equal amounts in the studies when differences were found. In 8 studies

cells were exposed only to high before low LET IR (102, 108, 114, 116, 119, 122, 123,

127), with 3 conclusions of additivity (108, 122, 127) and the in remaining 4 studies cells

were exposed only to low before high LET IR (126, 130, 131, 133), with 2 being additive

(126, 133) and 2 synergistic (130, 131). In total, sequential exposure resulted in 15 cases

of synergism, and 11 of additivity. Sequential irradiation on ice or in cold conditions led

to 4 cases of synergism (102, 114, 115, 123) and 1 of additivity (106), while room

temperature gave rise to 11 cases of synergism (103, 107, 111-113, 116, 119, 121, 125,

130, 131) and 10 of additivity (104, 105, 108, 109, 122, 126, 127, 129, 133, 134).

The effect of the order of exposure was tested in 10 studies. In 4 of the studies, no

differences were observed between the different exposure scenarios (high before low

compared to low before high LET radiation) (109, 111, 113, 125). In 5 of the studies,

more detrimental effects was observed for the cells when high LET radiation was the first

radiation type employed (103, 112, 115, 121, 129). Interestingly, for Elmore et al. (134)

the effect was larger when the lower LET (protons) was employed before the higher LET

(iron ions). Six studies investigated the effect of increasing the time between irradiations

(103, 115, 117, 121, 131, 133), which led to the disappearance of the synergistic effect, if

it was observed. Yang et al (133) concluded additivity and did therefore not observe this.

Sequential exposures at room temperature were thus more likely to result in an

additive effect than simultaneous exposures in a temperature-controlled environment. If

considering the sequential in vivo studies as temperature-controlled, the difference is

26

even more pronounced, since both such studies concluded synergism (113, 119). The

time between irradiations is a powerful factor for the detection of a synergistic response,

and the sequence of irradiation can potentially play a role as well.

Summary of findings

In conclusion, the responses differed based on the cell system, the LET

combination, the degree of control of the exposure conditions (temperature and

simultaneous or sequential exposure) as well as the endpoint employed.

Human cells were more likely to show an additive response, for survival as well as

cytogenetic endpoints. Rodent cells favoured the synergistic outcome, especially the in

vivo studies where all 3 gave a synergistic effect. Almost half of the studies (16 out of

35) measured clonogenic survival in hamster cells, and 10 of these were performed with

neutron irradiation. This means that approximately 30 % of the total mixed beam data

originated from very similar parameter combinations. When investigating the general

mixed beam result this must be taken into consideration, as not to give these results more

importance just because the number of experiments was larger.

Regarding the LET combination, the well-investigated neutron irradiation (17 out

of 35 studies) generated synergistic effects at the level of clonogenic survival, but

additivity when cytogenetic methods were applied. Alpha particles (8 studies) mainly

induced additive responses, for clonogenic survival as well as micronuclei. Heavy ions in

combination with low LET (6 studies) induced synergistic effects for clonogenic survival,

when investigated in the 20th century, but the 21st century studies, employing a wider

variety of endpoints concluded additivity. Combining high LET with a different type of

high LET (a comparably new field, 6 studies, the earliest study from 1997) gave rise to

equal numbers of synergism and additivity, with no identifiable trend.

Cells were more likely to exhibit a synergistic response after exposures at 37 °C.

The time between and potentially the order of exposure also influenced whether or not a

synergistic effect was observed. Simultaneous exposures in a temperature-controlled

environment (preferably 37 °C) were thus more likely to induce a synergistic effect than

sequential exposures at room temperature.

27

The present investigation

Aims of this thesis

In this thesis the cellular responses to mixed beams of high and low LET radiation,

represented by alpha particles and X-rays respectively, were investigated.

The aim was to ascertain whether the response was synergistic or additive and

elucidate the possible mechanisms behind the observed responses.

Novel approaches of the experiments

The novel approaches applied in this thesis were:

• A dedicated device allowing simultaneous exposure of cells to mixed beams

under controlled temperature conditions.

• Applying the gamma-H2AX assay

• FISH (has previously been applied in two studies (128, 131), but not as

extensively as presented here)

28

Materials and methods

Cell types

The AA8 Chinese hamster ovary cell line originates from CHO-K1 cells with a

mutation in the p53 gene (137). As a consequence, the G1/S checkpoint is lacking in the

AA8 cell line since it requires a functional p53. However, since no DNA repair

deficiencies have been detected, the cell line is used as wild type (138). The cell line was

chosen for clonogenic survival due to the ability of single cells to form detectable

colonies on glass cover slips.

VH10 is a diploid primary human fibroblast cell line derived from foreskin (139). It

has no detectable DNA repair deficiencies and is therefore classified as normal (140).

The flat nucleus made the cell line an excellent choice for the gamma-H2AX assay since

it was possible to capture the majority of foci within one focal plane.

Peripheral blood was donated by one male, non-smoking donor, aged 25 to 27

during the donation period. Ethical permission was obtained from the local ethical

committee at the Karolinska University Hospital, Stockholm, Sweden (diarium number

2010/27-31/1). Human peripheral blood lymphocytes (PBL) are well suited for cell-cycle

sensitive assays since they are naturally synchronized in G0 (141). Consequently, PBL

were employed for analysis of micronuclei and chromosomal aberrations.

The irradiation setup

The facility for mixed beam exposure consists of an alpha irradiator (custom-

constructed in Poland at the Institute of Nuclear Chemistry and Technology, Warszawa),

an YXLON SMART 200 X-ray tube and a MCO-15AC 164 l cell incubator. The alpha

irradiator is positioned inside and the X-ray underneath the incubator and the whole setup

is contained in a lead-plated cupboard.

29

The alpha irradiator is a 37 cm high, 30 cm deep and 47 cm wide aluminum

construction weighing circa 8 kg. The most important part is the 241Am alpha source

(AP1 s/n 101, Eckert and Ziegler, Berlin, Germany). The activity is contained in three

parallel strips with a total activity of 50 ± 7.5 MBq, as reported by the producer. A 2 μm

pure gold film gold foil overlays the 1 μm americium oxide foil, and the downward-

facing 180 * 180 mm source is glued to a steel disc, attached to a circular turn-table. The

table can be rotated (with a choice of 2 or 3 revolutions per second) for a better dose

homogenization. Under the source is a holder for a wobbling collimator, followed in turn

by a movable shelf for positioning of cells for exposure. The distance from the shelf to

the cell layer is 0.5 mm in the top position, 45 mm in the bottom position and it takes 20

seconds to move the shelf in between. The distance for exposure of cells on polyamide

discs can be adjusted in 0.1 mm increments.

The X-ray source was set to 190 kV and 4 mA during exposure and was operated

without the optional aluminum filter. The angle of the cone-shaped beam was 40 * 55 °

(according to the manufacturer). The distance from the X-ray source to the cell layer is

44.5 cm in shelf top position, and 40.0 cm in the bottom position, corresponding to dose

rates of 0.052 and 0.068 Gy/min respectively. The reason for the comparably low dose

rates was the passage of the X-rays through the bottom of the incubator and the bottom as

well as the movable shelf of the alpha irradiator.

Cell exposure

For exposure of cells, 15 mm thick, round polyamide discs with a 30 μm deep and

145 mm in diameter milled out well, along with a 1.5 µm Mylar foil lid were employed.

For attached cells, cover slips with cells facing upwards were positioned in the well,

sprayed with a small amount of medium and covered with the lid. For blood, 250 or 500

μm per irradiation was positioned in the well and smeared out as evenly as possible under

the lid (see Figure 5). The smaller volume was used for alpha particle and mixed beam

irradiation where penetration is essential, while the larger volume was used for X-ray

irradiation.

30

Due to the configuration of the setup, the cells were exposed to alpha particles from

above and X-rays from below. The differences in dose rate were taken into account by

initiating mixed beam exposure simultaneously and then lowering the polyamide disc out

of reach of the alpha particles to finish the irradiation with X-rays alone. No collimator

was used for cell exposure, since using a collimator positions the cells too far away from

the source, outside of the Bragg peak. This would result in a significant reduction of the

alpha particle dose rate.

Figure 5: Polyamide exposure disc. To the left: with cover slips seeded for survival. To the right: blood

smeared out under the Mylar foil lid.

Clonogenic cell survival

The clonogenic cell survival assay was modified from the standard protocol.

Instead of first exposing a large number of cells and thereafter seeding out for survival,

the cells (AA8 cells) were first seeded out for colony formation (4 h prior to exposure, on

round cover slips, 32 mm in diameter). After exposure, the cover slips were returned to

medium-filled wells, incubated one week and subsequently stained with methylene blue

in methanol. Colonies were counted by eye and plating efficiency and clonogenic cell

survival was assessed.

Chromosomal aberrations

Chromosomal aberrations can be viewed by staining of metaphase chromosomes

either uniformly by Giemsa or with fluorescence in situ hybridization (FISH) where

specifically coloured probes are used for individual chromosomes (142, 143), or even

parts of chromosomes (144). Giemsa staining is useful for fast scoring but can not detect

31

all types of aberrations (translocations and insertions are for example often invisible)

(145), while FISH is more time consuming but can reveal more about the complexity

behind the aberrations (146). In this study, PBL were treated with Colcemid and

Calyculin A at the end of the incubation time to trap cells in metaphase and prematurely

condense chromosomes in G2 (and anaphase) (147). BrdU addition during the culturing

time made possible the fluorescence plus Giemsa-treatment, where second mitosis cells

are differentially stained and centromers become more visible (148).

The micronucleus assay

Giemsa-stained micronuclei represent another way of visualizing the cytogenetic

damage in single cells (149). The advantage of micronuclei is that they represent a

“running average” of the damage remaining in interphase after the first cell division

(150), while chromosomal aberrations gives an instantaneous yield of aberrations.

Micronuclei consist of acentric chromosome fragments or whole chromosomes lagging

behind when the nuclei is dividing, so that when the DNA decondenses these fragments

create individual nuclear membranes (149). The addition of Cytochalasin B stops

cytokinesis while allowing karyokinesis, leading to bi-, tri- or multinucleated cells

(depending on culturing time and dose) (151). Only binucleated cells are accepted for

scoring of micronuclei (152), while scoring the number of nuclei per cell gives the

replication index, e.g. the rate of cell division. Modified scoring criteria were applied, in

that micronuclei with a diameter >30 % of the main nuclear diameters were scored if

their DNA was less dense than that of the main nuclei.

The gamma-H2AX assay

The phosphorylation of histone H2AX, called gamma-H2AX in phosphorylated

form, is a signal for DNA damage. The phosphorylation is rapid, but persists until the

break has been repaired, leading to a slower dephosphorylation rate (140). The

visualization of gamma-H2AX “foci” can be achieved by a 2-step antibody procedure,

where the primary antibody recognizes the protein, while the secondary antibody carries

a flourophore and is targeted towards the species in which the primary antibody was

32

produced. The gamma-H2AX assay has become one of the gold standards for measuring

the early cellular response to IR-induced DNA damage (153). Gamma-H2AX foci form

within minutes and can persist for days, making it possible to score the kinetics of

phosphotylation and dephosphorylation (154). For this assay, the VH10 cell line was

applied.

33

Results and discussion

Paper I

Characterization of a setup for mixed beam exposure of cells to 241Am alpha particles and X-rays

The traditional way of performing “mixed beam” studies has been to sequentially

expose cells to high and low LET radiation. In addition, the temperature at exposure has

not always been well controlled. The aim of this study was therefore to characterize and

validate a setup where cells can be simultaneously exposed to high and low LET in a

temperature-controlled environment.

The setup consists of a 241Am alpha particle irradiator and an X-ray tube, the former

positioned inside and the latter underneath a 164 liter cell incubator set to 37 °C. The

activity of the alpha source was characterized by track-etched detector by pressing the

source for 2.75 seconds against an aluminum sheet with pre-drilled holes (collimators)

under which the detector was positioned. This procedure was repeated 10 times, the foil

was etched with acid and 3 randomly chosen irradiated points in the detector foil were

analyzed for particle tracks. It was concluded that the dose rate was 0.265 Gy/min

(including a 0.025 Gy/min beta component, and a very small gamma component: below

0.001 Gy/h).

For validation of the setup, a modified version of the clonogenic cell survival assay

was employed, (see Materials and Methods). AA8-cells were exposed to X-rays, alpha

particles and two different mixtures of 25 % and 50% alpha particles, respectively. The

results revealed a RBE of 2.56 for 37 % and 1.90 for 10 % clonogenic cell survival, and

no statistically significant differences between observed and expected mixed beam data.

Envelope of additivity revealed that the observed data points were outside the envelope at

34

80 % survival, borderline at 50 % survival and within the envelope at 20 % survival. The

response was thus classified as additive.

In conclusion, the exposure device was performing as intended, and the effect of

mixed beam irradiation on clonongenic cell survival in AA8-cells was additive. However,

the distance between observed and expected values was larger for 50 % alpha particles in

the mix (as compared to 25 %), and increased with increasing dose. It is thus possible that

synergism could have been observed if mixed beam exposures with more than 50% alpha

particles and higher doses had been performed.

Main findings in paper I:

• The exposure facility is performing as intended

• Additive effect in AA8 cells after mixed beam exposure for the clonogenic

cell survival assay

35

Paper II

Micronuclei in human peripheral blood lymphocytes exposed to mixed

beams of X-rays and alpha particles.

The aim of this study was to investigate the cytogenetic effect of exposing human

lymphocytes (PBL) to a mixed beam of alpha particles and X-rays, by employing the

setup described in Paper I. PBL were exposed to increasing doses of alpha particles, X-

rays and mixed beams (35 % alpha particles), and harvested for micronuclei 96 h after

exposure.

The RBE for alpha particles was 3.2, and for mixed beam irradiation significantly

more micronuclei were observed per dose than expected from the single irradiations. The

PBL micronuclei response was therefore classified as synergistic. The sizes of individual

micronuclei were very heterogeneous, with no significant differences between the

irradiation schemes. It was however observed that alpha particle and mixed beams

irradiated cell populations contained a slightly higher percentage of “oversized”

micronuclei compared to X-rays (significantly so for mixed beams).

The results allowed speculation about possible mechanisms behind the observed

response. Previously, Zhou et al. (103) proposed that high LET radiation induces sites of

repair-resistant DNA damage that can “trap” repair proteins by making dissociation

difficult. Error-free repair proteins trapped in complexes at those sites would then be

unavailable for repair of additional low LET-inducted damage, thus making the repair of

these lesions more error-prone. Dispersion indices support this conclusion since mixed

beam-irradiated cells displayed values intermediate to those of X-rays and alpha particles,

rather than having values similar to or higher than alpha particle values. This indicates

that the synergistic effect observed after mixed beam irradiation was not due to a small

part of the cell population receiving high levels of damage, but that the increased

frequency of micronuclei was representative for the whole cell population. This is

precisely the effect expected if the repair of the X-ray induced damages was impaired.

In conclusion, the effect of mixed beam irradiation was synergistic on the level of

micronuclei. The proposed mechanism behind this observed effect was that the presence

36

of high LET radiation-induced damage inhibits DNA repair, thus potentiating the damage

induced by low LET radiation

Main findings in paper II:

• Synergistic effect for the number of micronuclei after mixed beam exposure

of PBL.

• The effect was due to an overall increase of the number of micronuclei, not

a few highly damaged cells.

• Proposed mechanism: The presence of high LET-induced damage inhibits

DNA repair, thus potentiating the effect of low LET-induced damage

37

Paper III

Higher than expected frequencies of complex aberrations in

lymphocytes exposed to mixed beams of 241Am alpha particles and

X-rays

Previously, it was observed that exposing human PBL to mixed beams of alpha

particles of X-rays and alpha particles gave rise to a significant synergistic effect on the

level of micronuclei. The aim of this study was to investigate the mechanisms behind this

effect by employing fluorescent in situ hybridization (FISH) on chromosomal

aberrations. PBL were exposed to X-rays, alpha particles and a mixture thereof (doses

0.27, 0.43 and 0.66 Gy, 25, 25 and 40 % alpha particles, respectively) and harvested for

chromosomal aberrations 54 h after exposure. FISH analysis was performed in

chromosomes 2, 8 and 14, and observed frequencies of simple and complex aberrations

after mixed beam exposure were compared to the expected (as calculated from the dose

components).

The distribution of aberrations was overdispersed for alpha particles, Poissonian

for X-rays and intermediate for mixed beams. For simple aberrations and primary breaks,

a linear equation was best fitted to the dose response curve. The dose response for

complex aberrations was linear for alpha particle exposure but linear-quadratic for mixed

beams and X-ray exposure. At the lowest X-ray dose no complexes were observed. The

highest degree of complexity (number of break points involved per complex) was

observed for alpha particles, closely followed by mixed beams. No dose response was

present for the degree of complexity. Significant differences between observed and

expected number of aberrations was detected for the highest and the median mixed beam

dose (simple plus complex aberrations, and complex aberrations, respectively). This can

be interpreted as evidence for a synergistic action of X-rays and alpha particles on the

induction of complex aberrations.

The synergism conclusion is in agreement with previous micronucleus results.

Furthermore, the linear-quadratic dose response of complex aberrations after mixed beam

exposure is an interesting finding. A linear quadratic dose response is observed when a

38

chromosomal aberration results from the interaction of two radiation tracks, a

characteristic observed for low LET radiation (155). It therefore points towards an

interaction between alpha particles and X-rays, giving the synergism conclusion

additional support. Consequently, the risks of stochastic effects from mixed beam

exposure may be higher than expected from adding the individual dose components.

Main findings in paper III:

• Simple aberrations and primary breaks were always best fitted by a linear

equation, while complex aberrations were linear for alpha particles, but

linear-quadratic for X-rays and mixed beams.

• Synergistic effect for complex aberrations at the two highest mixed beam

doses (as calculated from envelopes of additivity) and for simple aberrations

only at the highest mixed beam dose.

• Results indicate that cancer risk for exposure to mixed beams in radiation

oncology may be higher than expected based on the additive action of

individual components.

39

Paper IV

Gamma-H2AX foci in cells exposed to a mixed beam of X-rays and

alpha particles.

Traditional methods for analyzing mixed beam exposure have been clonogenic cell

survival or cytogenetic endpoints, thus leaving the early cellular response unexplored. To

fill this knowledge gap, VH10 human fibroblasts were exposed to one dose each of alpha

particles, X-rays and mixed beams (25 % alpha particles), incubated 0.5, 1, 3 and 24 h

and analyzed for gamma-H2AX foci. Dose response curves were performed 1 h after

exposure.

Two foci classes could be observed; large foci (LF) and small foci (SF). The

number of LF in alpha-particle irradiated cells corresponded well with the number of

particle tracks, as calculated by fluence (particles per second per cm2). The alpha particle

RBE was 0.76 ± 0.52 for total number of foci and 2.54 ± 1.11 for LF (as compared to X-

rays). The total number and area of foci in mixed beam irradiated cells was intermediate

to the results from X-ray and alpha particle exposed cells for dose response as well as

kinetics, and could thus be classified as additive. At the 24 h time point, the remaining

foci levels were low, and did not differ significantly. However, mixed beam LF kinetics

differed between observed and expected. The number and area of LF was significantly

lower than expected at 0.5 h after exposure, and an increase from 0.5 to 3 h for number

and area of LF was observed where no difference between time points were expected.

The relative LF (the LF contribution in percent of the total number and area of foci)

confirmed the trends as significant. Also, LF in mixed beam-irradiated cells did not reach

their maximal area until 1 h after exposure, and thus were not initially phoshporylated to

their full extent.

The differences between observed and expected in mixed beam LF kinetics

indicated that the phosphorylation process of H2AX histones around sites of complex

DNA damage was delayed, as compared to alpha particle and X-ray-induced damage.

That LF in mixed beam irradiated cells disappeared at a slower rate compared to LF in

alpha particle irradiated cells indicate that an interaction took place between the low and

40

high LET radiations. Taking the physics of the situation into account, it appeared most

likely that mixed beam exposure of cells to low and high LET radiation resulted in the

delay of the DNA damage response. In conclusion, the proposed hypothesis is that the

presence of low LET-induced DNA damage engages the DNA repair machinery, thus

leading to a delayed repair of the more complex DNA damage induced by alpha particles.

Main findings in paper IV:

• Additive effect for the dose response for number and area of total foci and

LF per cell.

• Significant differences in the observed repair kinetics for mixed beam LF,

compared to the expected. Initial frequency and area of LF were lower than

expected during the first hour, and LF were not were not phosphorylated to

their full extent until 1h after exposure.

• Proposed mechanism: the presence of low LET-induced DNA damage

engages the DNA repair machinery, thus leading to a delayed repair of the

more complex DNA damage induced by alpha particles.

41

Concluding remarks and future perspectives

Concluding remarks

Mixed beam exposures of humans are increasing due to airplane travel and the

application of specific cancer treatments. Establishing the correct approach for

determining the risks of mixed beam exposure is therefore of great importance. In this

thesis, the focus is on investigating DNA damage and repair in cells exposed to mixed

beams in a dedicated exposure facility, with the aim of establishing if the response is

additive or synergistic.

The main observation was that the synergistic effect was dependent on endpoint

and time after irradiation. For clonogenic survival in AA8 cells (7 days incubation) the

response was additive. However, for micronuclei and its precursor step chromosomal

aberrations (96 and 54 h after irradiation, respectively), a significant synergistic effect

was seen in human PBL. A pre-study for the micronuclei experiments observed

significantly more micronuclei 96 h compared to 72 h after alpha particles exposure,

indicating that heavily damaged cells did not have time to reach the first interphase at the

standard harvesting point 72 h. For gamma-H2AX foci, the significant differences in LF

kinetics between observed and expected mixed beams in VH10 cells at the 0.5 to 3 h time

points did not remain at the 24 h time point. In conclusion, it is important to optimize the

assays used as to capture the maximum magnitude of the response. If possible, the

response should be scored at multiple times after irradiation.

Secondly, the proportion of high LET in the mixture is of great importance to the

results. Originally, 3 mixtures of mixed beams were performed for clonogenic survival:

50, 25 and 12.5 % alpha particles, respectively. The 12.5 % results were omitted in the

paper since they overlapped with the X-ray results and did not add additional value.

When calculating expected values it was observed that the difference between observed

and expected increased with increasing percentage alpha particles. It is thus possible that

42

synergistic effects would have been observed also for clonogenic survival if experiments

had been carried out with even higher percentages of alpha particles. For PBL, significant

synergistic effects were observed for micronuclei after 35 % alpha particles and for

aberrations after 40 % alpha particles and 25 % alpha particles. For the 25 % however,

the difference was only significant at a higher dose. Foci results were obtained with 25 %

alpha particles, and if it had not been possible to find particle tracks based on size, the

results would have been scored as additive (all significant effects were observed for LF).

Therefore, it would be of interest also to investigate different mixtures, where the

proportions high to low LET are varied.

Taken together, the results indicate a synergistic effect for the interaction of low

and high LET ionizing radiation during mixed beam exposures. Proposed mechanisms

behind the observed responses are: 1) The presence of high LET-induced damage inhibits

DNA repair, thus potentiating the effect of low LET-induced damage, 2) The presence of

low LET-induced DNA damage engages the DNA repair machinery, thus leading to a

delayed repair of the more complex DNA damage induced by alpha particles, 3)

Combination of low and high LET radiation increases damage complexity. These are not

necessarily mutually exclusive, delayed repair of high LET induced damage and

potentiated effect of low LET induced damage can occur simultaneously.

Furthermore, the results presented in this thesis have given rise to some interesting

suggestions for future research, which will be detailed in the following section.

Future perspectives

As stated in the concluding remarks, the presence of the synergistic effect was

dependent on the proportion of high to low LET, the endpoint, and the time after

irradiation. Further experiments should therefore strive to investigate more than one

mixture, and optimize the assay as to capture the maximal response (and if possible

investigate the response at more than one time point).

The research presented in this thesis will also be continued more long-term with a

different approach to the irradiation scheme. So far, the proportion of high LET to low

LET has remained constant throughout an experiment. For the upcoming study however,

43

the dose of alpha particles will remain constant while the X-ray dose will be varied. This

approach aims to find if and how the synergies between the actions of low and high LET

IR are dependent on the percent high LET in the mixture, and if the appearance of

synergism is linear with increasing X-ray dose or not. The synergistic effect that was

observed for several of the assays of this study may appear and/or disappear when

changing the proportions. This study will start by focusing on the mutation frequency in

human TK6 lymphoblastoid cells, as a tool investigate the risks for secondary cancers

after mixed beam cancer treatment. Further on, the Comet assay and the gamma-H2AX

assay will also be applied.

The studies in this thesis have started to shed light upon the complicated field of

mixed beams research. What yet remains is to thoroughly investigate the mechanisms

behind the observed responses, by identifying cell cycle effects and which DNA repair

system is most sensitive to mixed beam-induced damage. Such data could confirm, or

possibly contradict the theories from the investigations presented here (as well as

previously published theories). Proteomic and metabolomic studies at different time

points after mixed beam irradiation could be very helpful in explaining the differences

observed between endpoints and cell lines. But to fully understand the effect of mixed

beams and the cellular mechanisms behind the interactions of radiations of different LET

requires the collaboration between several disciplines, such as correlating microdosimetry

simulations with observed chemical and biological responses.

A DNA-repair-targeted study following up the results summarized here is currently

being performed by the author of this thesis, Elina Staaf, and collaborator Dr. Daniel

Vare from the DNA repair field. We are employing the CHO cell lines AA8 (wild type),

irs1SF (HR-deficient), V3-3 (NHEJ deficient) and EM9 (single strand break repair

deficient) and investigating their response to mixed beam irradiation. Preliminary results

indicate a significant difference between observed and expected clonogenic survival after

mixed beam exposure (50% alpha particles) for AA8 and irs1sf (wild type and HR-

deficient cell lines). The study will also include gamma-H2AX and 53BP1 foci kinetics,

dose responses as well as micronuclei dose response curves.

44

Acknowledgements First and foremost I would like to thank my supervisor Andrzej Wojcik for inspiring

guidance, fruitful discussions and entertaining moments during travels and sports. Thank

you for making me a scientist!

Also, thanks to Siamak Haghdoost, my co-supervisor who always had time for guiding

my efforts in the lab and reading my manuscripts.

I am grateful for all help Mats Harms-Ringdahl gave me with the SWE-RAYS workshop

and official emails. Thank you for being so generous with your time and experience.

My previous thesis and project supervisors Björn Cedervall, Margareta Edgren, Fredrik

Elgh, Ingrid Marklund and Johan Trygg, thank you for improving my skills and for

teaching me new ones.

All former and present members of the Radiation Biology group: Ainars, Alice, Asal,

Eliana, Karl, Marta, Paulo, Ramesh, Sara S, Sara SM, Siv, Tai, thank you for making the

everyday work enjoyable and the parties, conferences and trips even more so!

Daniel Vare, thanks for being an awesome collaborator and for keeping me in shape!

I would like to thank the whole GMT department for a very good working environment.

Especially you innebandy players, you know who you are!

I would also like to thank family and friends for tolerating my workaholic periods and

information overload from my chatting.

Johan, your encouragement, patience and understanding have been invaluable. You are

the best!

This study was supported by a grant from the Swedish Radiation Safety Authority (SSM)

45

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