Biomarkers of oxidative stress and their application for ...198402/FULLTEXT01.pdf · individual radiosensitivity would allow for individually designed radiotherapy and • increase

Biomarkers of oxidative stress and their application for assessment of

individual radiosensitivity

Siamak Haghdoost

Department of Genetics, Microbiology and Toxicology

Stockholm 2005 ISBN 91-7155-150-6

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Abstract Radiotherapy is one of the most common therapeutic methods for

treatment of many types of cancer. Despite many decades of development and experience there is much to improve, both in efficacy of treatment and to decrease the incidences of adverse healthy tissue reactions. Around 20 % of the radiotherapy patients show a broad range in the severity of normal tissue reactions to radiotherapy, and dose limits are governed by severe reactions in the most radiosensitive patients (< 5 %). Identification of patients with low, moderate or high clinical radiosensitivity before commencing of radiotherapy would allow individual adaptation of the maximum dose with an overall increase in the cure rate. Characterization of factors that may modify the biological effects of ionizing radiation has been a subject of intense research efforts. Still, there is no assay currently available that can reliably predict the clinical radiosensitivity. The aim of this work has been to investigate the role of oxidative stress in individual radiosensitivity and evaluate novel markers of radiation response, which could be adapted for clinical use.

8-Oxo-7,8-dihydro-2´'-deoxyguanosine (8-oxo-dG), a general marker of oxidative stress, is one of the major products of interaction of ionizing radiation with DNA and the nucleotide pool of the cell. As 8-oxo-dG is highly mutagenic due to incorrect base pairing with deoxyadenosine, various repair mechanisms recognize and remove 8-oxo-dG. The repaired lesions are released from cells to the extracellular milieu (serum, urine and cell culture medium) where they can be detected as markers for free radical reactions with the nucleic acids.

Significant variations in background levels as well as in radiation induced levels of 8-oxo-dG in urine have been demonstrated in breast cancer patients (paper 1). Two major patterns were observed: high background and no therapy-related increase vs. low background and significant increase during radiotherapy for the radiosensitive and non radiosensitive patients respectively.

Studies in paper 2 indicated major contribution of the nucleotide pool to the extracellular 8-oxo-dG levels. The results also implicated induction of prolonged endogenous oxidative stress in the irradiated cells. RNA “knock-down” experiments on the nucleotide pool sanitization enzyme hMTH1 in paper 3 lend further experimental evidence to this assumption.

The applicability of 8-oxo-dG as a diagnostic marker of oxidative stress was demonstrated in paper 4. Studies on dialysis patients revealed a good correlation between inflammatory responses (known to be associated with persistent oxidative stress) and extracellular 8-oxo-dG.

In summary, our results confirm that extracellular 8-oxo-dG is a sensitive in vivo biomarker of oxidative stress, primarily formed by oxidative damage of dGTP in the nucleotide pool with a potential to become a clinical tool for prediction of individual responses to radiotherapy.

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List of original publications

1. Haghdoost S, Svoboda P, Näslund I, Harms-Ringdahl M, Tilikides A,

Skog S. Can 8-oxo-dG be used as a predictor for individual

radiosensitivity? Int J Radiat Oncol Biol Phys. 2001 Jun 1;50(2):405-10.

2. Haghdoost S, Czene S, Näslund I, Skog S, Harms-Ringdahl M.

Extracellular 8-oxo-dG as a sensitive parameter for oxidative stress in

vivo and in vitro, Free Radic Res. 2005 Feb 39(2):153-62

3. Haghdoost S, Sjölander L Czene S, Harms-Ringdahl M.

The nucleotide pool is a significant target for oxidative stress. Submitted

4. Haghdoost S, Maruyama Y, Pecoits-Filho R, Heimburger O,

Seeberger A, Anderstam B, Suliman ME, Czene S, Lindholm B,

Stenvinkel P, Harms-Ringdahl M. Systemic inflammation in

hemodialysis patients is associated with increased oxidation of the

nucleotide pool as revealed by serum levels of 8oxo-dG. Submitted

Additional publication not included in this thesis:

5. Kämpfer P, Lindh J.M, Terenius O, Haghdoost S, Falsen E, Busse H.-J,

Faye I. Thorsellia anophelis gen. nov., sp. nov., a new genus of the

gammaproteobacteria, IJSEM Paper in Press.

Reprints were made with permission from the publisher.

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Abbreviations

BER: base excision repair

CRP: C-reactive protein

CVD: cardio vascular disease

dNTP: deoxynucleotide triphosphate

DSB: double strand break

ELISA: enzyme-linked immunosorbent assay

ESRD: end-stage renal disease

GSH: glutathione peroxidase

Gy: gray

HD: hemodialysis

HPLC-EC: high performance liquid chromatography with electrochemical

detection

LET: linear energy transfer

MDS: multiply damaged site

NER: nucleotide excision repair

NIR: nucleotide incision repair

8-Oxo-G: 8-oxo-guanine

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

8-Oxo-Guo: 8-oxo-guanosine

ROS: reactive oxygen species

siRNA: short interfering RNA

SLE: systemic lupus erythematosus

SOD: superoxide dismutase

SSB: single strand break

Sv: sievert

UV: ultraviolet

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TABLE OF CONTENTS

AIM.......................................................................................................................... 6 INTRODUCTION.................................................................................................. 7

Ionizing and non-ionizing radiation ............................................................................. 7 Effects of ionizing radiation on biological materials ................................................... 9 Background radiation and cancer incidence (stochastic effects) ................................. 9 Cellular targets of ionizing radiation ......................................................................... 10 Radiation and radiotherapy........................................................................................ 12 Side effects of radiotherapy (deterministic effects) and dose limiting factors............ 14 In vitro assays used for determination of radiosensitivity .......................................... 15 DNA repair and radiosensitivity ................................................................................. 16 Oxidative stress and cellular damage......................................................................... 18 8-Oxo-2´-deoxyguanosine – formation, repair and mutagenesis ............................... 20 Sources of extracellular 8-oxo-dG.............................................................................. 21 Correlation between radiosensitivity and oxidative stress ......................................... 24

RESULTS AND DISCUSSION .......................................................................... 25 Quantitative measurement of extracellular 8-hydroxy-2'-deoxyguanosine ................ 25 Individual radiosensitivity and urinary 8-oxo-dG (paper 1) ...................................... 27 Extracellular 8-oxo-dG as a marker of in vitro oxidative stress (paper 2) ................ 29 The nucleotide pool is a major source of extracellular 8-oxo-dG (paper 3).............. 30 8-oxo-dG as diagnostic marker of oxidative stress in dialysis patients (paper 4)...... 31

FUTURE PERSPECTIVES................................................................................ 32 ACKNOWLEDGEMENTS................................................................................. 35 REFERENCES..................................................................................................... 38

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Aim The main purpose of the research studies included in this thesis was to

develop and evaluate diagnostic tools for reliable diagnosis of radiosensitive

individuals and make these tools available for the needs of individually

designed radiation therapy. Clinical availability of predictive assays for

individual radiosensitivity would allow for individually designed radiotherapy

and

• increase the probability of a better tumor control in the group of

patients with mild and moderate reactions

• decrease adverse tissue reaction in the highly radiosensitive

group of patients

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Introduction During our whole lives we are exposed to various kinds of radiation,

ultraviolet and ionizing radiation being the dominant types with known

biological adverse effects. While the sun is the main source of UV radiation,

the major sources of ionizing radiation include medical uses, cosmic radiation,

radiation from the ground, internal radiation from 40K and radon. Human

exposures to high doses of ionizing radiation are rare. A special case of

exposure to high doses of ionizing radiation is its use for therapeutic purposes.

Ionizing and non-ionizing radiation

The various types of radiation fall into two broad categories, ionizing

and non-ionizing. When the energy of radiation is high enough it will produce

ionizations and excitations in the target. Both particle radiation (electrons,

protons, α-particles, or neutrons) and electromagnetic radiation (high energetic

photons such as X- and γ-rays) belong to the category of ionizing radiation.

Direct ionizations are produced when photons or charged particles interact

directly with the target. Indirect ionizations are produced by transfer of energy

from photons to cellular water molecules (radiolysis) forming highly reactive

intermediates e.g. hydroxyl radicals which then interact with the target.

Radiation with lower energies, on the other hand, does not produce ionizations

when interacting with matter. Examples of non-ionizing radiation are UV and

visible light, infrared radiation (IR) and micro- and radio waves.

Important definitions, terms and units in radiation biology include

Dose - radiation energy deposited in tissue or other material divided by the mass of the tissue or material. The SI unit for absorbed dose is gray (Gy), equal one joule of energy deposited in one kg of tissue or other material. Dose rate - absorbed dose divided by the time of its delivery. Due to limitations of the repair capacity in a living cell per unit of time, high dose rates are usually more damaging.

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Linear energy transfer (LET) - is expressed in terms of the mean energy released in keV per micrometer (µm) of the tissue traversed (keV/µm). Examples of low LET and high LET radiation are medical X-rays and alpha particles respectively. Equivalent dose - relates absorbed dose (Gy) in tissue to the biological effects of a specific radiation quality. The unit for equivalent dose is sievert (Sv). To determine equivalent dose (Sv), an absorbed dose (Gy) is multiplied by a radiation quality factor (Q). For example Q for gamma is 1, for alpha 20. In other words, one Gy equals 1 Sv for gamma radiation, but in case of alpha particles 1 Gy will equal 20 Sv.

A schematic illustration of radiation qualities with different LET is shown in

figure 1. Considering DNA as target, it is apparent that the quality of damage

induced by one Gy of high LET radiation will considerably differ from that

observed after the same dose of low LET radiation.

Low LET radiation

High LET radiation

• •

•• •

••

•• •• •• ••

• ••• • • •

•••• •••• ••

• ••

Gamma rays

Alfa particles

Target

••• • ••• •• • ••• •• • •• ••• • •• ••• •• •• ••• ••• ••• ••• • • •• ••

Figure 1. Schematic picture of ionization events (black dots) caused by high

and low LET radiation.

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Effects of ionizing radiation on biological materials

The biological effects of radiation arise from interactions with various

components of the cell, such as proteins or nucleic acids. Radiation-induced

modifications of cellular components may destroy their proper functions and

lead to loss of cell viability, decreased enzyme activity, mutations, initiation of

cancer, and hereditary diseases. The immediate effects of high acute radiation

exposures are caused by cell membrane rupture resulting in loss of cellular

content and cell death. Acute whole body irradiation [1] can lead to organ

failure as exemplified (in rodent) by

• loss of coordination (including breathing problems) after

exposure of the central nervous system to doses above 100 Gy,

with death occurring within few minutes up to 2 days.

• nausea, vomiting, and diarrhea caused by damage to the

gastrointestinal tract from doses 9 to 100 Gy. Progressive

dehydration will result in death within 3-5 days.

• loss of appetite and hair, hemorrhaging, inflammation, and

secondary infections such as pneumonia from doses 3 to 9 Gy

due to damage to the bone marrow and other haematopoietic

tissues. This can result in death within 10-30 days. These effects

are also found in patients undergoing radiation therapy.

• loss of appetite and hair, hemorrhaging, and diarrhea observed at

doses of around 2 Gy, the consequences are rarely lethal.

Background radiation and cancer incidence (stochastic effects)

Sources of ionizing radiation include those naturally occurring and man-

made. The principal types and sources of natural background radiation are:

• Cosmic radiation from the outer space

• Terrestrial sources of naturally disintegrating radioactive

materials e.g. radium, thorium, uranium

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• Internal sources of radioactive isotopes that are normally present

within living cells (e.g. potassium-40, carbon-14).

The background levels of radiation from natural sources show large variations

in different parts of the world (1.5 up to 100 mSv/year). Exposure levels from

radiation for diagnostic purposes (ex. nuclear medicine and diagnostic

radiology) contribute on the average with doses around 2 mSv/per year to the

Swedish population.

Increased rates of cancer and mutations are the major long-term risks of

concern associated with radiation exposure. Risk estimates for both these

effects, especially in low-dose rate chronic exposure situations, are however

difficult to perform due to confounding life-style factors (smoking, diet,

sunlight exposure) involved in the etiology of cancer. The average background

radiation dose in Sweden (including radiation from medical investigation and

radon) is 5 to 6 mSv per year which cause a few hits per cell per year. The total

cancer incidence in Sweden is around 40 000 cases per year. Genetic factors

have been estimated to cause about 20 % of the cancer incidence [2] while

contribution of life style factors may be around 70 % [3]. Based on linear

extrapolation of the relation between dose and cancer incidence, estimated from

epidemiological investigations for doses > 50 mSv, down to the low doses the

estimated number of cancers caused by background radiation in Sweden will be

1000-2000 cancers per year.

Cellular targets of ionizing radiation

Since living cells consist of ~70 % water, the majority of ionizations

(produced by radiation) take place through interactions with water molecules.

Radiolysis of cellular water will produce reactive oxygen species such as the

hydroxyl radical OH• that may interact with various cellular components. The

remaining 30 % of radiation effects in cells are mediated by direct interactions

of photons with a particular cellular component such as DNA, proteins and

membranes leading to ionizations and excitations. Of note, non-ionizing

radiation may interact with biological systems in other ways represented by e.g.

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the photochemical reactions in the human eye, the photosynthesis of plants, and

the UV radiation-induced damage in skin.

Among the various cellular modifications induced by ionizing radiation,

persistent alterations of DNA (mutations) are of particular biological

importance, as they can be passed over to the progeny and result in hereditary

disorders. Elevated mutation frequencies and gross chromosomal changes also

play an essential role in the pathogenesis of many human disease states

including cancer, as well as in the processes of aging.

Both the direct and indirect effects of radiation can initiate damage to

DNA. Direct effects involve the formation of radicals and excited intermediates

as a result of the deposition of energy within the DNA structure. Indirect

effects involve the interaction of the DNA with radiolysis products of water

such as OH radicals, H-atoms or hydrated electrons. Low LET ionizing

radiation causes many different types of damages on DNA such as double

strand breaks (DSB, 20-40 breaks/cell per Gy), single strand breaks (SSB, 1000

breaks/cell per Gy) and base modifications (1000 modifications/cell per Gy) [4-

7]. A particular feature of radiation induced chemical alterations is the

production of unique types of damages in a small section of the DNA. These

so-called multiply damaged sites (MDS), consisting of clusters of strand breaks

and base damages within one or two turns of the DNA (10 to 20 base pairs) [8],

are suggested to be a specific signature of ionizing radiation.

Among the DNA lesions the DNA double strand breaks are of principal

interest. The genotoxic properties of many physical and chemical agents are

often closely associated with their ability to induce DSBs. Because of their high

cytotoxicity (unrepaired or misrepaired DNA double-strand breaks are often

lethal) and ability to induce chromosomal aberrations (that may ultimately lead

to carcinogenesis), cell survival and maintenance of genome integrity are

critically dependent on efficient repair of DNA DSBs. Cells have developed

different DNA repair mechanisms to deal with harmful effects of radiation on

DNA such as: homologous recombination and nonhomologous end joining

(both involved in repair of DSBs); base excision repair (BER) acting on base

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damages and single strand breaks; nucleotide excision repair (NER) mainly

involved in removal of bulky DNA adducts (induced by chemicals) and

thymidine dimers (induced by UV light); and the recently described pathway of

nucleotide incision repair (NIR) that is involved in repair of base damages [9]

Besides activation of DNA repair mechanisms the induction of primary

DNA damages in the genome may lead, depending on the biological context, to

activation of other cellular responses such as cell cycle arrest, stress response

and apoptosis. There is an efficient “cross talk” between these mechanisms,

ultimately deciding the fate of the irradiated cell. During cell cycle arrest cells

have time to efficiently repair damages before re-entering the cell cycle.

Depending on the severity and quantity of DNA damage, apoptosis signaling

pathways may take precedence over the DNA repair and the cell cycle arrest

mechanisms. Elimination of potentially dangerous cells that could e.g. lead to a

progeny with high probability of genomic instability is especially vital for

multicellular organisms.

Radiation and radiotherapy

In 1898 and 1899, just a few years after the discovery of X-rays in 1895

by Wilhelm Roentgen, two Swedish scientists, Thor Stenbeck and Tage

Sjögren, treated three patients with skin tumor by X-rays. One of the patients

was treated once a day under three months and became the first cancer patient

who was cured by radiotherapy. After world war II new radiation equipments

with higher energies became available allowing the treatment of deep-seated

tumors. The new 60Co sources allowed shorter treatment times and almost all

radiotherapy clinics in the world used these sources from 1950 until 1960. At

the end of 1950-ties radiation machines with energy outputs in the MeV ranges

(linear accelerators) were introduced and are still the most common radiation

equipments in clinical praxis.

The availability of new radiotherapeutic modalities was paralleled by

simultaneous improvement of diagnostic techniques. The use of computer

tomography (CT), positron emission tomography (PET), magnet resonance

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imaging (MRI) and ultra sound increased the possibilities for the oncologists to

distinguish a tumor from a normal tissue and thus optimize the dose planning

and treatment regimes.

Examples of novel approaches that have led to more effective radiation

therapy of cancer include:

• Intraoperative irradiation: a large dose (8-15 Gy) of external

radiation (usually electrons) is delivered to the tumor and

surrounding tissue during surgery (e.g. in colon cancer)

• Brachytherapy, interstitial irradiation, and intracavitary

irradiation: radioactive implants are placed directly in a tumor or

body cavity (in prostate cancer, uterus cancer)

• Radiosurgery and Gamma knife: high doses of gamma radiation

(3-5 fractions of 7-15 Gy) are delivered to small solid tumors

(liver and lung metastasis, small brain metastasis)

• Radiolabeled antibodies: radiolabeled tumor-specific antibodies

are used to deliver doses of radiation directly to the cancer cells

(radioimmunotherapy).

At present, radiotherapy is used to treat about 45 % of all cancer patients in

Sweden. Radiotherapy can be used alone as a curative, adjuvant (in

combination with surgery and chemotherapy) or palliative (pain relieving,)

treatment. The aim of modern radiotherapy is to cure patients from cancer with

limited adverse effects to the normal tissue surrounding the cancer tumor. This

can be achieved by carefully planning of the dose delivery to the tumor (dose

planning system) and more detailed understanding of the tumor and healthy

tissue responses to radiation.

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Side effects of radiotherapy (deterministic effects) and dose limiting

factors

For many years it has been recognized by clinicians that some patients

show hypersensitivity to the radiotherapy. Early and late adverse effects in

exposed healthy tissues are the major factors limiting dose escalation in

radiotherapy of cancer patients. During the radiotherapy both cancer and

normal cells are being exposed to the radiation. Radiation-induced damage in

tissues may lead to the cell death, and toxic components from dead cells may

affect the whole body and cause symptoms such as nausea.

Deterministic side effects of radiation therapy can be divided into acute

and late reactions. The dose responses for these reactions are characterized by a

threshold dose over which the effects appear. Acute reactions occur during the

ongoing treatment or within a few weeks after the treatment and are usually

reversible (ex: erythema). For example, skin reaction and pain in the irradiated

breast and chest area are the most common side effects during radiotherapy of

breast cancer patients. Acute radiosensitivity is most often manifested in tissues

with high proliferation rate such as skin, small intestine and rectum. Late

reactions occur months or years after radiation, mainly in slowly proliferating

tissues such as fatty tissues and muscles and are of permanent character

(fibrosis, telangiectasia). In addition to the radiation dose and the dose per

fraction, side effects of radiation are also dependent on the area and volume of

treatment as well as on the individual radiosensitivity. There are also

indications of the stochastic effects of radiation therapy such as increased

cancer incidence in normal tissues receiving comparatively low doses of

radiation during irradiation of the neighboring organ with the primary tumor.

Examples of stochastic effects include increased lung and esophagus cancer

incidence in breast cancer patients receiving radiotherapy [10-12].

The most commonly used system for evaluation of a patients’

radiosensitivity is the Radiation Therapy Oncology Group (RTOG) that is

based on to the intensity of the side effects (acute as well as late effects). These

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acute and late radiation morbidity scoring criteria were developed in 1985 and

an example of such a protocol used for evaluation of the acute radiosensitivity

in breast cancer [13] is as follows:

Grade 0: no change over baseline

Grade 1: follicular or dull erythema

Grade 2: tender or bright erythema, moderate edema

Grade 3: confluent moist desquamation, skin folds, pitting edema

Grade 4: ulceration, necrosis.

About 20 % of the patients who undergo radiotherapy of breast cancer

will develop adverse reactions to the therapy [14, 15], as many as 5 % of the

patients will develop severe reactions during the course of the treatment

(RTOG 3-4). Notably, patients with adverse reactions often have better cure

rates comparing to non-reacting patients. Positive correlations between

radiation side effects and local tumor control in breast, head & neck and colon

cancer patients have been previously reported [15-17]. Local tumor recurrence

after radiation therapy is due primarily to failure to eradicate all of the cancer

cells within the irradiated fields. Theoretically, all cancers could be controlled

locally if a sufficiently high radiation dose could be delivered to a target that

encompasses all of the cancer cells. However, at the present time, doses used in

radiotherapy are adapted to what can be tolerate by the most radiosensitive

patients, keeping the risk of severe persistent normal tissue damage below 5-10

%, despite the fact that many patients could tolerate a larger dose without

severe tissue reactions. Hence, radiotherapy would greatly benefit from a

diagnostic tool providing information on individual radiosensitivity.

In vitro assays used for determination of radiosensitivity

Several attempts have been made to correlate radiation therapy side-

effects with the cellular responses to the radiation. The various endpoints of

these assays include transcriptional response employing cDNA microarrays

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[18], assessment of DNA repair capacity by alkaline comet assay [19],

apoptosis [20] and cell survival [21, 22] as well as formation of chromosome

aberrations [23]. In a number of studies the results correlated with the clinical

observations, however these correlations only held for large groups with little

predictive value for the individual patients [24] as there was a large overlap in

the responses of radiation-sensitive and non-sensitive patients in these assays

(for review see [25]).

One of the possible explanations for the observed large variations is that

there are several underlying factors causing individual radiosensitivity. Known

risk factors include age, concurrent chemotherapy, anatomical variation (e.g.

bust size in breast cancer treatment), body mass index (BMI), tissue

oxygenation and genetic predisposition [26]. Hence, one of the potential

drawbacks of the predictive in vitro assays would be that they most often focus

on one particular cell function and thus examine only one of the many factors

involved in individual susceptibility to radiation. For instance, there are

indications that genetically defined radiosensitivity is not the predominant

clinical cause for excessive tissue reaction [27].

DNA repair and radiosensitivity

A recent review of the International Commission on Radiological

Protection (ICRP) of clinical observations and cellular as well as molecular

studies on radiosensitivity [28] has identified several genetic syndromes that

are connected with radiation hypersensitivity (table 1).

As seen from the list below, at least two groups of genes, those involved

in cell cycle control and DNA repair are strongly associated with

radiosensitivity. Seminal studies on hypersensitivity syndromes with a clear-cut

genetic component (ATM, NBS1 and Mre11 genes) identified DNA damage

recognition and repair as a central theme of radiosensitivity [14, 29]. However,

these classical cases of radiosensitivity syndromes are very rare and account

only for about one in 10 000 observed cases.

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Table 1. Genetic syndromes associated with radiosensitivity:

Syndrome Genetic disorder

Ataxia telangiectasia DSB recognition/repair

Bloom syndrome DSB repair

Fanconi anaemia DSB repair

Li-Fraumeni syndrome Cell cycle regulation, apoptosis

Nevoid basal cell carcinoma syndrome Control of cell proliferation

Neurofibromatosis Cell cycle regulation

Nijmegen breakage syndrome DSB repair

Retinoblastoma Cell cycle regulation

As most gene products are part of multi-protein complexes and function

in complex cellular pathways, mutation of a particular gene may affect the

expression of downstream genes involved in these cellular pathways, as well as

those of genes involved in related processes trying to compensate for the

defect. Therefore, it is most likely that individuals in the relatively large group

of radiosensitive patients will carry more subtle defects in these pathways due

to presence of low-penetrance mutations, which are not limited to the two

abovementioned groups of genes.

Still, the current knowledge on genetic predisposition for individual

response to radiation damage, in particular on the involvement of low-

penetrance gene mutations, is rather sparse. It is known that cancer patients

with diabetes mellitus [30], hypertension, rheumatoid arthritis and systemic

lupus erythematosus (SLE) [31], have a higher frequency of side effects to

radiotherapy. As suggested by a recent study [18, 32], combined effects of

polymorphisms in several DNA repair genes, could also lead to manifestations

of individual radiosensitivity.

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Oxidative stress and cellular damage

Reactive oxygen species (e.g. O2●- superoxide anion, OH● hydroxyl

radical, and H2O2 hydrogen peroxide) are continuously generated in aerobic

organisms both endogenously as by-products of oxygen metabolism as well as

by exogenous factors (figure 2)

O2-º

O2

e-From mitochondria

(1-5

% o

f tot

al O

2)

SOD H2O2

H2O

Cata

lase

Glu

tath

ione

pero

xidas

e

OHºFenton Reaction:Fe2++ H2O2=Fe3++OH

_+OHº

Gamma radiation,radiolysis of H2O

LipidsProteinsDNAdNTP

Antioxidants

X

From macrophage

Modification of:

Figure 2. Schematic picture of endogenous and exogenous production of free

radicals and cellular defense mechanisms.

The endogenous production of free radical derived DNA lesions has

been estimated to be up to 10 000 per day per cell in humans [33]. For instance,

the yield of superoxide anions has been estimated to be 1-5 % of the total

consumed oxygen, equaling to 2 kg of superoxide anions per year [33]. The

majority of the endogenously produced reactive oxygen species (ROS) is

derived from the mitochondrial electron transport chain [34]. Even though the

predominant sources of ROS in aerobic cells are the mitochondria, substantial

amounts of ROS are also generated in peroxisomes. Many chemical and

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physical agents, among them ionizing radiation as discussed above, are known

to exert their mutagenic and carcinogenic properties through production of free

radicals [35].

The biological effects of ROS are intimately related to their tissue

concentration. At low concentrations, ROS have many important physiological

functions e.g. as secondary messengers or part of the immune defence. On the

other hand, moderate and high levels of ROS within cells may lead to

“oxidative stress”. The term oxidative stress, in essence, refers to a serious

imbalance between production of ROS and antioxidant defence in favour of the

oxidants, potentially leading to cellular damage [36]. Oxidative stress may thus

result from

1. Diminished levels of antioxidants, for example caused by, mutations

affecting the activities of antioxidant defence enzymes such as

superoxide dismutase (SOD), or glutathione (GSH) peroxidase, or toxins

that deplete antioxidant defences. For example, many xenobiotics are

metabolized by conjugation with GSH; thus high doses can deplete GSH

and cause oxidative stress even if the xenobiotic itself is not generating

reactive species. Deficiencies in dietary minerals (e.g., Zn2+, Mg2+, Fe2+,

Cu2+, and Se) and/or antioxidants can also cause oxidative stress.

and/or

2. Increased production of ROS, for example, by exposure of cells or

organisms to elevated levels of O2; physical (ionizing radiation) or

chemical agents (e.g. paraquat) that generate excess ROS; or excessive

activation of ‘natural’ systems producing such species (e.g.

inappropriate activation of phagocytic cells in chronic inflammatory

diseases).

Interaction of ROS with various cellular components such as lipids, proteins

and DNA has been shown to result in temporary as well as permanent

modifications [37], that may have deleterious consequences. Alone or in

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combination with primary etiological factors, ROS have been implicated in

diverse diseases such as cancer, hypertension, atherosclerosis, Alzheimer’s

disease, lung injury as well as the aging process.

The detrimental effects of oxidative stress on cellular components are

counteracted by various defence mechanisms which range from low molecular

weight antioxidants through DNA repair enzymes to ultimate disposal of the

damaged cell by induction of programmed cell death (apoptosis). One of the

first and most important defences is the array of low molecular weight cellular

antioxidants, including the reduced form of GSH, vitamin E, and ascorbate

[38].

The second line of defence mechanisms involves enzymes with anti-oxidant

properties such as various SODs and catalase. For instance, superoxide anions

produced by mitochondria are first converted by SODs into the less toxic

hydrogen peroxide that in turn is disassembled into water and oxygen by

catalase [39].

Should the extent of oxidative stress overwhelm the cellular resources of

antioxidants as outlined above, the escaped ROS may damage various cellular

components, such as the DNA and nucleotide pool. While modified proteins

and lipids are removed from the cells by a normal turn over of the molecules,

specific repair pathways act to remove oxidative DNA modifications.

8-Oxo-2´-deoxyguanosine – formation, repair and mutagenesis

Oxidative modifications of nucleic acids include a range of various base

modifications such as 8-oxo-adenine, 2-hydroxy-adenine, 8-oxo-guanosine,

FaPy-guanine, thymine glycol, sugar damage, apurinic/apyrimidinic sites (AP

site), damage of the phosphodiester backbone of DNA (strand breaks) and

DNA-protein crosslinks [40]. One of the major types of base damage formed

by ionizing radiation, as well as by endogenous ROS, is 8-oxo-7,8-dihydro-2´'-

deoxyguanosine (8-oxo-dG). 8-Oxo-dG is produced mainly by hydroxylation

of the C8 position of 2´-dG in both DNA and the free nucleotide dGTP in the

21

nucleotide pool. Charge transfer events in the DNA may also contribute to

formation of 8-oxo-dG [41].

It is generally assumed that most of the oxidative DNA damage is dealt

with by base excision repair (BER) [42]. In the process of BER, DNA

glycosylases recognize and remove damaged DNA bases. After the action of

AP-endonuclease, the resulting gap is then filled by a DNA polymerase and

sealed by DNA ligase. Several glycosylases with ability to remove oxidative

base damage have been identified in both bacteria (the MutY and MutM

proteins) and higher organisms (e.g. the OGG1 and OGG2 proteins).

8-Oxo-dG is a mutagenic lesion as it can mispair with adenine [43]. If

not removed from DNA, replication of the damaged sequence will lead G→T

transversions [44]. Another mechanism for 8-oxo-dG mutagenesis is the

incorporation of free 8-oxo-dGTP from the nucleotide pool into DNA opposite

of adenine during either replication or repair. Such mis-incorporation will lead

to A→G transversions through activation of the mismatch repair pathway that

will remove A opposite of the 8-oxo-dG and replace it with C. This base pair

will be repaired in turn by base excision repair that will remove 8-oxo-dG

opposite C and replace it with G [45].

Of note, several recent studies [46, 47] indicate that the efficacy of BER

may be strongly reduced when a oxidised DNA base is a part of MDS.

Abortive repair of oxidised bases in MDS is detrimental for the cell as

indicated by decreased survival [48], increased mutation frequency [49], and

formation of DNA double strand breaks [50].

Sources of extracellular 8-oxo-dG

Despite its extensive use as a general marker for oxidative stress the

origin of extracellular 8-oxo-dG is a matter of scientific controversy. The result

of BER activity on 8-oxo-dG in DNA is 8-oxo-Gua [40] that can be detected in

urine and blood plasma. There are indications that 8-oxo-dG may be removed

from DNA also by the nucleotide excision repair (NER) [51] as well as by the

nucleotide incision repair (NIR) [52] mechanisms. As the NER pathway is

22

specific for bulky DNA adducts, its role in recognizing and removing 8-oxo-dG

from DNA is under discussion [51]. Primary fragments (containing 24-29

nucleotides) removed from DNA by NER, may undergo further reduction to 6-

7 mers or even isolated nucleosides. However, it has been demonstrated that

human urine contains only limited amounts of oligonucleotides and

mononucleotides; moreover these nucleotides do not contain oxidative

modification in form of 8-oxo-dG [53]. Thus, under normal physiological

conditions, NER seems to be of minor importance for repair of oxidative DNA

damage [54]. NIR seems to be involved in the repair of 8-oxo-dG [52], most

likely as a backup system for BER [40]. Still, the biological significance of

these findings for the production of extracellular 8-oxo-dG is yet to be shown

by further studies. Figure 3 shows the classes of repair products formed by the

NIR, NER and BER repair pathways.

C

T

C

T

G

A

G

A

NER, up to 28 bp

BER, damaged base

NIR, damaged base with sugar and phosphate

Figure 3. Schematic presentation of DNA repair products removed by BER, NIR and

NER (in bold squares)

23

Recent studies show that mitochondrial DNA (mtDNA) is yet another

target for free radical reactions. mtDNA is highly prone to oxidative damage

because of the lack of histones and also due to its proximity to one of the major

intracellular sources of ROS. The steady-state level of oxidized bases in

mtDNA is two to three fold higher than that in nuclear DNA [55]. The

importance of mitochondrial genome maintenance is demonstrated by the role

of mitochondrial gene mutations in degenerative diseases of the nervous

system, heart, muscle and kidneys [56, 57]. Still, the only documented repair

mechanism in the mitochondria up-to-date is that of BER. Thus, oxidative

mtDNA damage is not likely to contribute to the extracellular 8-oxo-dG.

Another target for radiation-induced oxidative damage is the pool of free

nucleotide triphosphates (dNTPs) available in the cytoplasm. Free radicals can

react with the dNTPs in the nucleotide pool and produce a wide spectrum of

modified dNTPs. 8-Oxo-dGTP is produced upon free radical attack on the C8

position in dGTP. 8-Oxo-dGTP can be misincorporated into DNA and cause

mutations. Bacterial cells are protected against incorporation of 8-oxo-dGTP by

8-oxo-dGTPase [45, 58] that converts 8-oxo-dGTP to 8-oxo-dGMP. The

human homologue for 8-oxo-dGTPase is named hMTH1. The importance of

nucleotide pool sanitization is demonstrated by the observation that insufficient

8-oxo-dGTPase activity may lead to cancer [44, 45].

A recurrent suggestion is that urinary 8-oxo-dG mainly originates from

the degradation and oxidation of DNA in dead cells. There is no convincing

experimental evidence however to prove this hypothesis [59]. As results from

previous studies also show that 8-oxo-dG is not absorbed through the intestinal

tract a dietary influence on extracellular 8-oxo-dG should be minimal [60-62].

It has been also discussed that oxidation of 2´-dG in serum or in urine

may significantly contribute to urinary or serum levels of 8-oxo-dG. However,

2´-dG is not oxidized in systemic circulation to 8-oxo-dG [59]. Moreover, the

presence of hydrogen peroxide or other ROS in urine did not lead to detectable

formation of 8-oxo-dG [63].

24

Correlation between radiosensitivity and oxidative stress

As the majority of primary radiation damage is mediated by free radicals

produced during radiolysis of the cellular water, the spectrum of radiation-

induced DNA damage overlaps to a considerable extent with that caused by

endogenous oxidative stress. Interestingly, high concentrations of urinary

and/or DNA 8-oxo-dG (marker for the oxidative stress) were observed in

patients with radiosensitivity syndromes, including

• Ataxia telangiectasia - high level of 8-oxo-dG in lymphocyte

DNA [64]

• Fanconi anemia - elevated urinary 8-oxo-dG [65]

• Cockaine syndrome (defect in transcription coupled repair

pathway, a sub pathway of NER) - elevated 8-oxo-dG in cellular

DNA [66]

•

It is also known that cancer patients with inflammatory nonmalignant

systemic diseases such as diabetes mellitus and SLE tolerate radiotherapy

poorly [31]. For many of these diseases enhanced oxidative stress was

indicated by increased levels of urinary and/or DNA level of the 8-oxo-dG,

such as; for diabetes mellitus [67], SLE [68], rheumatoid arthritis [69] and for

inflammatory bowel disease.

An interesting aspect of oxidative stress during radiotherapy is the

possible role of circulating neutrophiles and phagocytes. Neutrophiles contain

significant reservoirs of enzymes in their cytoplasmatic granules that can be

transported to the extracellular milieu upon stimulation, e.g. by bacteria and

low dose irradiation [70]. The cytotoxic substances released from neutrophiles

upon stimulation include hydrogen peroxide and superoxide anions [71]. The

release of ROS by neutrophiles is a fundamental step in the protection against

pathogenic agents (such as bacteria), which cause local inflammatory reactions.

It has been shown that stimulated neutrophiles can induce DNA damage (base

25

modification) [71] and can cause malignant transformation in other cells [72].

During radiotherapy, as neutrophiles pass through the exposed tissue, they will

receive a small radiation dose (in the cGy range). This dose could be enough to

activate the neutrophiles and give rise to inflammatory reactions, which could

theoretically contribute to various (therapeutic as well as side) effects of

radiotherapy.

Results and discussion

Quantitative measurement of extracellular 8-hydroxy-2'-deoxyguanosine

Figure 4 shows an overview of the methods used in our studies for

detection of 8-oxo-dG. In paper 1 urinary 8-oxo-dG was measured with a high

performance liquid chromatograph equipped with an electrochemical detector

(HPLC-EC). The advantage of the HPLC-EC method is that it measures only

the free form of 8-oxo-dG and not other 8-oxo-dG derivates (8-oxo-G, 8-oxo-

Guo) or oligomers containing 8-oxo-dG. Although HPLC-EC based methods

for detection of 8-oxo-dG are well established and reliable, they are less

suitable for a clinical laboratory due to low throughput (typically 1-3 hours per

sample), high cost, technical involvement and limited sensitivity for detection

of extracellular 8-oxo-dG (especially in blood serum).

We have evaluated serum levels of 8-oxo-dG as a predictive marker for

individual radiosensitivity after in vitro irradiation of whole blood samples

from patients assuming that the response may correlate with the organ response

to radiation. However the average serum level of 8-oxo-dG is relatively low

(~1.4 pmol/ml) and thus below the detection limit offered by our HPLC-EC (~1

pmol) based method. An alternative way to measure 8-oxo-dG is by enzyme

linked immunosorbent assay (ELISA). Commercial ELISA kits are sensitive,

fast and do not require sophisticated equipments. Unfortunately, the

monoclonal antibodies used in commercially available ELISA kits are not

specific for 8-oxo-dG and will even bind to some of its derivates such as 8-

26

oxo-Guo (of RNA origin) [73]. The usefulness of these kits for measurement of

8-oxo-dG in complex biological mixtures such as urine or blood serum is thus

questionable.

Samples (urine, serum, cell culture medium)

Purification by solid phase extraction (CH-bond Elute column)

Purified samples

Washing, eluting

First HPLC system based on ion pair chromatography

Urine, serum

Collection of fraction containing 8-oxo-dG

Second HPLC system,detection of 8-oxo-dG

Concentration of fraction

Medium, serum

Incubation of concentrated sample and primary antibodyin 8-oxo-dG precoated 96 well ELISA plate

Overnight incubation at 4ºC

Secondary antibody

Incubation at room temprature 2 hrs

Development and detection of colour

Figure 4. An overview of the methods for detection of 8-oxo-dG by HPLC-EC

and ELISA.

We have developed an improved ELISA for 8-oxo-dG that includes an

additional sample purification step that removes the interfering 8-oxo-Guo

before analysis. The capacity of our semi-manual ELISA method is about 100

samples per week per technician, and it has the same or better sensitivity as

commercially available kits (detection limit for 8-oxo-dG is around 0.1 pmol)

and is highly cost-effective. The reproducibility of our method for serum 8-

oxo-dG is about ± 20 % (standard deviations based on seven blood serum

samples, each analyzed 3 times, n=21). 8-Oxo-dG levels in blood serum

samples were between 0.2 up to 3 ng/ml, well within the sensitivity range of

27

our ELISA (marked as a bold line in the figure 5). To decrease the influence of

confounding factors, 8-oxo-dG is added to each sample as an internal standard.

Samples with and without internal standard were analyzed in duplicate. The

quantity of 8-oxo-dG was normalized using the internal standard.

0,1

0,2

0,3

0,1 1 10 100

8-oxo-dG (ng/ml)

OD

(450

nm

)

Figure 5. A representative standard curve for 8-oxo-dG produced by our

competitive ELISA.

Individual radiosensitivity and urinary 8-oxo-dG (paper 1)

Since the first reports on the presence of 8-oxo-dG and 8-oxo-G in urine

of human, rat and mouse [61, 74] both components have been extensively used

as markers of oxidative stress elicited by agents of both endogenous and

exogenous origin [75-79].The urinary levels of 8-oxo-G are usually 5-10 fold

higher than those of 8-oxo-dG. 8-Oxo-G, however, may also originate from

RNA degradation and its analysis is associated with technical difficulties [59,

80-82]. High levels of the 8-oxo-dG in DNA and/or urine has been detected in

cancer patients [33] and also in patients with non-cancerous diseases such as

28

Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, cardiovascular

disease, chronic hepatitis, diabetes mellitus and rheumatoid arthritis [33]. It

should be noted, however, that the background levels of urinary and DNA

8-oxo-dG are also influenced by life style factors [83] and that they also are age

dependent [84, 85].

Evidence of the involvement of oxidative stress in cellular

radiosensitivity emerged from a number of in vitro and in vivo studies [86-91].

High levels of 8-oxo-dG in lymphocytes of breast cancer patients receiving

radiotherapy [79] as well as a positive correlation between urinary 8-oxo-dG

and radiation responses in lung cancer patients [92] have been reported. We

hypothesized that clinical observations of the individual radiosensitivity in

breast cancer patients undergoing radiotherapy (such as acute skin reactions)

could be related to the level of pre-existing or therapy-induced oxidative stress

response measured as urinary 8-oxo-dG. Based on clinical observations the

patients were classified as radiosensitive (major skin reactions) or non-

radiosensitive (patients with minor or no acute skin changes). The advantages

to analyze extracellular 8-oxo-dG in urine or serum are that the method is not

invasive (easy to receive patient samples), and that the 8-oxo-dG molecule is

relatively stable in extracellular milieu. The dietary effect is assumedly low.

Urine samples from a group of 9 radiosensitive and 8 non-radiosensitive

patients were collected before (overnight samples) and during the radiotherapy

session. Urinary 8-oxo-dG was analyzed by HPLC equipped with an

electrochemical detector. The urinary 8-oxo-dG levels of the radiosensitive

group were compared to the non-radiosensitive group.

Our results showed significantly higher background levels of urinary 8-

oxo-dG in the radiosensitive group as compared to the non-radiosensitive,

indicating the presence of pre-existing oxidative stress in the radiosensitive

patients before the start of the treatment. Radiotherapy of the radiosensitive

group resulted only in slight increases of the urinary 8-oxo-dG levels in

contrast to the non-radiosensitive group where low background levels and

significantly higher radiation-induced urinary 8-oxo-dG were observed. The

29

suggested mechanism for these observations was that the pre-existing oxidative

stress in radiosensitive patients led to activation of repair mechanisms that was

close to saturation. Accumulation of DNA damage due to limited availability of

the repair proteins in combination with an additional increase of ROS induced

by radiotherapy could in turn lead to clinical manifestation of radiosensitivity.

Extracellular 8-oxo-dG as a marker of in vitro oxidative stress (paper 2)

The results of paper 1 showed a significant correlation between in vivo

radiosensitivity and urinary 8-oxo-dG. Next we asked whether variations in

individual ability to cope with radiation-induced oxidative stress response

could be observed at the cellular level too. The aim of the studies in paper 2

was to establish dose response relation for radiation-induced appearance of

extracellular 8-oxo-dG in a cellular model system (whole blood samples and

isolated lymphocytes from healthy donors). For this purpose the highly

sensitive ELISA method was developed, as detailed above, for detection of

extracellular 8-oxo-dG in human serum and was used in parallel with the

HPLC method.

We found that the yields of extracellular 8-oxo-dG were dependent on

dose, individual repair capacity and secondary stress response. The observed

high yields of extracellular 8-oxo-dG suggest a two steps mechanism. The

initial one is an immediate production of 8-oxo-dG, which is primarily

mediated by radiolysis of cellular water. However, the levels of extracellular 8-

oxo-dG observed 60 min post irradiation exceeded about 30 fold the expected

yields, suggesting that radiation triggers a stress response involving production

of ROS. These in turn could lead to additional formation of 8-oxo-dG mainly

by oxidation events in the nucleotide pool. As a possible candidate for such a

stress response mechanism we suggested a radiation-induced oxidative burst in

neutrophiles present in the whole blood samples.

30

The nucleotide pool is a major source of extracellular 8-oxo-dG (paper 3)

For the past 20 years, since extracellular 8-oxo-dG has been used as a

biomarker for an oxidative stress [33, 61, 74] its origin has been discussed [51,

53, 61, 62, 93]. The amount of 8-oxo-dG excreted from cells after irradiation

(paper 2) indicated a significant contribution from other cellular target(s) then

DNA. Interestingly, a gene expression study (micro array) on blood samples

drawn from cancer patients with different radiosensitivity indicated that the

expression of the hMTH1 gene may be used to predict radiosensitivity in

cancer patients [18]. It has previously been shown that the nucleotide pool size

has an important role in the cellular radiosensitivity [94]. In this study the

authors compared human fibroblast cells derived from AT-patients and

fibroblast cells derived from normal individuals.

We wanted to investigate the role of the nucleotide pool and its

sanitization enzyme, hMTH1, in the appearance of extracellular 8-oxo-dG. The

hMTH1 enzyme in the human fibroblast cell line VH10 was down-regulated by

transfection of the cells with short interfering RNAs (siRNAs) homologous to

hMTH1 mRNA. After irradiation, the cells were incubated in serum-free cell

culture medium (DMEM). Samples of the cell culture medium were collected

after 60 and 120 min of incubation and 8-oxo-dG was analyzed by ELISA.

The “knockdown” of the hMTH1 mRNA resulted in significantly

decreased levels (60 %) of the hMTH1 protein. Irradiation resulted in increased

expression of the hMTH1 protein in control cells but not in the siRNA-

transfected cells. In the latter cells, significantly lower levels of extracellular

cellular 8-oxo-dG were observed. In another series of experiments a positive

correlation between nucleotide pool size and concentration of extracellular 8-

oxo-dG was found.

31

8-oxo-dG as diagnostic marker of oxidative stress in dialysis patients

(paper 4)

The results of papers 1-3 indicated a significant contribution to

extracellular levels of 8-oxo-dG from oxidative damage on the nucleotide pool.

As sanitization and replenishment of free nucleotides in the cytoplasm are

ubiquitous processes, the suitability to use extracellular 8-oxo-dG as a general

marker for oxidative stress was implicated. Indeed, studies reported in paper 4

seem to confirm this assumption.

Cardiovascular disease (CVD) is the main cause of mortality (50 %) in

patients with end stage renal disease (ESRD). The risk for cardiac mortality for

hemodialysis patients aged 45 years or younger has been estimated to be more

than 100-fold greater than for the general population [95]. It has also been

suggested that chronic inflammation and oxidative stress promote

atherosclerosis in ESRD patients [96]. Chronic inflammation is also associated

with an increased incidence of malignancy [97].

The plasma level of the C-reactive protein (produced by hepatocytes) is

increased in a variety of acute and chronic inflammation conditions and is used

as marker for inflammation. It is known that polymorphonuclear granulocytes

and the monocytes could be activated in hemodialysis patients by contact with

the biocompatible membrane during dialysis, leading to increased production

of ROS [98].

Under normal conditions, various defense systems (enzymes, low

molecular antioxidants, DNA repair pathways and nucleotide pool sanitization

enzymes) constantly neutralize the deleterious effects of ROS. Imbalance

between the antioxidant systems and increased ROS production may lead to

modification of lipids, proteins and also nucleic acids [99]. Persistent oxidative

stress is associated with increased oxidative damage of various cellular

components, among them DNA, and has been linked with different diseases

such as chronic inflammation, cancer and CVD.

32

In this study we have chosen a group of 14 persistently inflamed

hemodialysis patients and 19 persistently non-inflamed patients. The

classification of patients was based on their inflammatory status as revealed by

serum levels of C-reactive protein (CRP). Serum concentrations of 8-oxo-dG

were analyzed by means of ELISA. Significant differences in serum 8-oxo-dG

levels were found between the two groups of patients (p<0.05). Moreover, a

positive non-significant correlation between serum levels of 8-oxo-dG and CRP

was found. We concluded that extracellular 8-oxo-dG is a potential marker of

inflammation in dialysis patients and could, in combination with other

biomarkers of inflammation, improve early diagnosis of therapy-related side-

effects.

Future perspectives The studies included in this thesis extend the basic knowledge of the

biological significance of extracellular 8-oxo-dG and identify the nucleotide

pool as one of its major sources. Our findings implicate the suitability to use

urinary/plasma levels of 8-oxo-dG as a general biochemical marker of

oxidative stress. In particular, our studies provide experimental support for the

involvement of oxidative stress in the phenomena of individual radiosensitivity.

When further verified, our means to predict individual responses to

radiotherapy in order to provide individualized dose regimes will be enhanced.

The ELISA method we have developed during this work opens up new

opportunities for studying oxidative stress in general but also in response to

exogenous stimuli as listed below.

• Analysis of 8-oxo-dG in relation to oxidative stress (induced by

various agents) in cellular (cell culture medium and the nucleotide

pool of the cells) and animal model systems (serum and urine).

Examples of practical application could be analysis of endogenous

stress responses to drugs in animal and human trials for

33

pharmaceutical industry where there is an indication that a tested

substance induces oxidative stress.

• Antioxidant research: to test effectiveness of antioxidant drugs in

human, cellular or animal model systems.

• Prediction of radiosensitivity in cancer patients

Given the availability of specific monoclonal antibodies, our experimental

approach could also be applied to analyze urinary or serum levels of various

products of DNA repair, such as thymine glycol or UV photoproducts.

Ongoing projects

Prediction of radiosensitivity in prostate cancer patients

A pilot study has been initiated to evaluate radiation-induced

extracellular 8-oxo-dG as a predictor of individual radiosensitivity in prostate

cancer patients. This is a retrospective study on patients with documented late

effects and their matched controls. Our assay could predict 4 of 7 radiosensitive

patients and for the 5 matched controls no false positive were found. This pilot

study will be expanded in order to improve the statistical evaluation. The three

radiosensitive patients who were false negative may represent a group whose

radiosensitivity is caused by other mechanisms which deserve further attention.

Such investigations could include screening for genetic alterations which could

be responsible for the observed variations e.g. by means of protein

fingerprinting (2D-protein analysis) or SNP analysis of alleles in candidate

genes associated with radiosensitivity, particularly in the hMTH1 gene.

Low dose and low dose rate radiation effects

Risk estimates for stochastic effects (cancer, mutation) of radiation at

low doses (<50mSv) are based on the Linear-Non-Threshold (LNT) hypothesis.

The core of the LNT hypothesis is that the risk associated with radiation

34

exposure is directly proportional to dose, with no threshold. Thus regulatory

radiation exposure limits are based on extrapolation of linear dose response

relations, observed at high doses, down to the low dose range. Epidemiological

studies on effects of high doses of radiation have been performed on the

survivors of the Hiroshima and Nagasaki bombings, and also on various groups

of people given radiation for therapeutic and diagnostic purposes. There are no

epidemiological methods developed that can provide risk estimates in the low

dose range (<10 mSv). As similar uncertainties apply for most cellular and

animal models, there is a need to assess more sensitive biological markers of

the effects of low dose irradiation. The use of novel markers could help to fill

the gaps in our knowledge of the cellular responses to low doses and dose rates.

Based on the results of paper 2, we hypothesized that even low doses of

radiation could result in detectable formation of 8-oxo-dG by eliciting a

secondary stress response in the exposed cells. In pilot experiments, we have

studied the kinetics and yields of extracellular 8-oxo-dG in samples of whole

blood irradiated with doses in the mGy range. The results showed that a dose of

5 mGy increased the extracellular 8-oxo-dG yield 5 fold over the background

level. These results will be followed up in further studies.

hMTH1 expression and individual radiosensitivity

As a complementary method to 8-oxo-dG measurements, analysis of

basal and induced levels of the hMTH1 protein could also be used as a marker

for oxidative stress. Analysis in leucocytes from radiosensitive and non-

radiosensitive patients of the hMTH1 protein expression and activity is

planned.

35

Acknowledgements The financial support from the following societies and funds is gratefully

acknowledged:

Swedish Cancer and Allergy Foundation

Swedish Radiation Protection Authority

Swedish Cancer Society, Stockholm

Karolinska Institute Research Fund

Goljes Society

Nilsson Cancer Foundation

Fund for Medical Development at Karolinska Hospital

The Commission of European Union (F16R-CT-2003-508842)

Sven and Lilly Lawski Foundation for Natural Science

I would like to thank my supervisor prof. Mats Harms-Ringdahl for his supervising

and giving me all the help and encouragement that I needed. Your friendly and happy

attitude has made my life easier.

Special thanks are addressed to Doc. Ingemar Näslund, Doc. Sven Skog and prof.

Ulrik Ringborg from Karolinska Institute and Radiumhemmet; for all support and

help I got from you since the very beginning of my carrier until now, to enter the

exciting world of research.

To my co-supervisor and friend, Stefan Czene for always taking the time and helping

me to get through the complexity of the “radiobiology and genetic toxicology”, for all

scientific and non-scientific discussions, and many pleasent fishing days.

I will also thanks to my colleagues: Aris T, Gunilla I, Ulla Å, Agneta , Helena ,

Haideh, Mohsen, Arja, Christina E, Cecilia, Susanne, Maria F, Lars, Anders C,

Michaela P, Eija, Mojdeh, Gail, Karin, Peter C, Britta, Ulla Stina and all other people

at the Department of Hospital Physic and Department of Radiotherapy, Karolinska

Hospital, for all the upport, feedback that I got from you when I was close to give up

this work. You are and will always be important persons in my life.

36

Siv Ostermann-Golkar, thank you for being my teacher and good discussion partner.

Klaus Erixon, thank you for sharing your bright experience with me especially in

repair (of DNA as well as cars).

Gunilla Olsson, thank you for keeping everything clean and nice at the lab. Thank you

for our nice talks during our lunches at the “Pub”.

Lena Sjölander, Thank you being my friend. I appreciate your patience to work with

me during your radiation biology course and your exam work. Good luck with your

horses, fishes and children.

Jenny L, Carolina, Clara, Ole, Petri, Katarzyna, Mona T and Joakim, thank you for

your donation and being friends.

Peter Svoboda, thank you for sarcastic comments, HPLC helps, and good

collaboration.

Peter Stenvinkel, Björn Anderstam, Bengt Lindholm, Yukio Maruyama and other co-

authors from Huddinge Hospital, thank you for the nice collaboration.

I also want to thank the colleagues at the former Department of Radiobiology and

Department of Genetics, Microbiology and Toxicology for creating a nice and calm

working place; Gunnar Ahnström, Ainars Bajinskis, Tobias Cassel, Natalia K,

Yohannes Assefaw-Redda, Igor Belyaev, Cissi, Fredik, Ingrid Faje and her group,

Jesper Torudd, Dag Jenssen and his group, Elisabeth Haggård and her group, Ulf

Rannug and his group, Andres and Björn P.

To my ambitious students, Emma Eklöf, Lena Sjölander, Lena Brunefors and Anita

Bairamzadeh. It has been a pleasure supervising you.

To my father, mother and grand mother for raising and guiding me through my life, to

get me understand the philosophy of life.

37

Special thanks go to my family Lucia and Samuel for being with me, for giving me

your love, harmony and support. Living with you means fun, great food, great music

and stability.

This work was also influenced by: Ludka and Duro Trenkler, Young Ludka, Goli,

Omar Khayyam, Marko, Bandar Abbas, Nusrat Fatah Ali Khan, Hossein, Mareza,

Martin Trenkler and F1, Nitin Sawhney, Hafez, Tekitoi from Taha Rashid,

38

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