Alterations of the Sister Chromatid Exchange frequency in peripheral lymphocytes caused by an Ironman triathlon

Acknowledgements

First of all I would like to thank o. Univ.-Prof. Dr. Ibrahim Elmadfa for the possibility to accomplish my diploma thesis at the Department of Nutritional Sciences.

My special thanks go to a.o. Univ.-Prof. Dr. Karl-Heinz Wagner and Mag. Oliver Neubauer for their competent supervision and support throughout writing my diploma thesis. I also want to thank Mag. Stefanie Reichhold, I appreciated her taking time for helping me, her numerous occasions giving usefull advice and patience answering my questions.

This work also benefited from the help of many members of the Department of Nutritional Sciences.

However, most of all, I would like to thank my entire family, which I cherish very much!

I want to express my heartfelt gratitude to my mother DPäd. Margit Meisel, my aunt

MR Dr. Renate Krassnig and my late grandparents Dr. Herbert and RegR. Helga

Krassnig for their unconditional support and for their encouragement when everything seemed to fall apart.

Additionally I want to thank my mother for being an idol of strength to me!

To my siblings Marcus, Marion and Martin: Thank you for being there for me. I am grateful for having you!

Last but not least, I would like to thank Kathi for all her love, patience and understanding when work seemed to take over life. Thank you for your infinite support!

I

Contents

LIST OF FIGURES ................................................................................. IV

LIST OF TABLES ..................................................................................... V

  ABBREVIATIONS  VI

1.  INTRODUCTION 1  INTRODUCTION...................................................................................1

2.  BACKGROUND 3  

2.1.  Human lymphocytes  3

2.2.  Cell cycle  3

2.2.1.  Interphase  3

2.2.1.1.  Checkpoints  4

2.2.2.  Mitosis  5

2.3.  Sister Chromatid Exchange  6

2.3.1.  Mechanism of SCE  6

2.3.2.  Scientific significance of the SCE assay  8

2.3.3.  SCE inducing agents  9

2.3.4.  Persistence of SCE  9

2.3.5.  Historical background  9

2.3.6.  BrdU incorporation and visualization of SCEs  10

2.3.7.  The role of cell culture components  11

2.3.8.  Factors potentially influencing SCE frequency  12

2.3.8.1.  Culture factors  12

2.3.8.2.  Biological and physiological factors  13

2.4.  The Correlation between strenuous endurance exercise and genotoxicity  15

2.4.1.  Reactive oxygen species (ROS) and physical exercise  15

2.4.2.  Exercise-induced oxidative stress  17

2.4.3.  Exercise-induced DNA damage  18

2.4.3.1.  Relation to oxygen consumption  18

II

2.4.3.2.  Relation to a single bout of exercise  19

2.4.4.  Exercise-induced adaptation  21

2.4.5.  Regular physical exercise  24

3.  MATERIALS AND METHODS 25  

3.1.  Project description  25

3.2.  Subjects  25

3.2.1.  Inclusion criteria  26

3.2.2.  Exclusion criteria  26

3.2.2.1.  Supplementation guidelines  27

3.3  Equipment for the SCE assay  28

3.4.  Reagents of the SCE assay  29

3.4.1.  Manufacturing processes and storage of reagents for SCE assay  30

3.5.  Basic assay approach  31

3.6.  Assay description  31

3.7.  Blood collection  31

3.8.  Sister Chromatid Exchange assay  32

3.9.  Statistical analysis  35

3.10.  Guidelines for microscopic assessment  36

3.11.  Top five HFCs  36

4.  RESULTS AND DISCUSSION 37  

4.1.  Study design  37

4.2.  Subjects characteristics  37

4.3.  Preliminary testing  38

4.4.  Assay criteria  39

4.5.  Distribution of evaluated SCEs per cell  40

4.6.  Abs SCEs  40

4.6.1.  Descriptive statistics  40

III

4.6.2.  Single means of abs SCEs  41

4.6.3.Total  mean abs SCEs  42

4.7.  Top 5 HFCs  44

4.7.1.  Descriptive statistics  44

4.7.2.  Single means of Top 5 HFCs  44

4.7.3.  Total mean Top 5 HFCs  45

4.8.  Correlations  47

4.8.1.Abs.  SCEs  47

4.8.2.  Top 5 HFCs  49

5.  CONCLUSION 51  

6.  SUMMARY 53  

7.  ZUSAMMENFASSUNG 54  

8.  REFERENCES 55  

9.  APPENDIX 67  

9.1.  Single values of participant 36  67

9.2.  Single values of participant 37  68

9.3.  Single values of participant 39  69

9.4.  Single values of participant 41  71

9.5.  Single values of participant 42  72

9.6.  Single values of participant 43  73

9.7.  Single values of participant 46  75

9.8.  Single values of participant 47  76

9.9.  Single values of participant 48  77

IV

LIST OF FIGURES

  Figure 1: The events of the eukaryotic cell cycle MORGAN 2007  4

  Figure 2: Metaphase chromosome SUMMER 2003  6

  Figure 3: SCEs in human lymphocytes Cell of a male healthy athlete of the SCE cohort  

  shows 5 SCEs 46 chromosomes (arrows label SCEs)  7

  Figure 4: Generation of reactive oxygen species GARRIDO et al 2004  15

  Figure 5: Oxidative stress - imbalance between oxidants and antioxidants (GSH:  

  glutathione) GARRIDO et al 2004  17

  Figure 6: Culture flasks are incubated at 37 C (5 CO 2 ) for 70 h  32

  Figure 7: Hypotonic KCl solution is added drop by drop (slowly)  33

  Figure 8: The supernatant is discarded with an exhaustion pump inside the LF  34

  Figure 9: Single mean abs SCEs cell of each participant of the SCE-cohort (n 9) 48 h  

  pre- and 24 h postrace ( p 0 05 p 0 01 compared to 48 h prerace values)  42

  Figure 10: Total mean abs SCEs cell of the SCE-cohort (n 9) ( p 0 05 compared to  

48  h prerace values)  43

  Figure 11: Relative mean abs SCE alteration ( ) 48 h before and 24 h after the  

  triathlon race  43

  Figure 12: Single mean Top 5 HFCs cell of each participant of the SCE-cohort 48 h  

  pre- and 24 h postrace ( p 0 05 p 0 01 compared to 48 h prerace values)  45

  Figure 13: Total mean Top 5 HFC frequency of SCE-cohort (n 9) 48 h pre- and 24 h  

  postrace ( p 0 05 compared to 48 h prerace values)  46

  Figure 14: Relative mean Top 5 HFC alteration ( ) 48 h pre- to 24 h postrace  46

  Figure 15: Regression analysis of relative SCE changes 48 h pre and 24 h postrace on  

  weekly net endurance exercise training time (h) in 6 subjects of the SCE-cohort  48

  Figure 16: Regression analysis of relative SCE changes 48 h before and 24 h after the  

  triathlon on cycling training per week (km) in 8 subjects of the SCE-cohort  48

  Figure 17: Regression analysis of relative SCE changes 48 h before and 24 h after the  

  triathlon on running training per week (km) in 5 subjects of the SCE-cohort  49

  Figure 18: Regression analysis of relative Top 5 HFC changes 48 h before and 24 h  

  after the triathlon on cycling training per week (km) in 8 subjects of the SCE-cohort  50

V

LIST OF TABLES

Table 1:  RDA-based supplementation guidelines during the research project  27

Table 2:  Equipment for SCE assay  28

Table 3:  Reagents for SCE assay  29

Table 4:  Physical characteristics of the SCE-cohort (n 9)  37

Table 5:  Training parameters of the SCE-cohort and their performance in the  IM

  triathlon race  38

Table 6:  Descriptive data interpretation of mean abs SCEs cell 48 pre- and 24 h  

  postrace total cell number (n 446) of the SCE-cohort (n 9) ( p 0 05 compared to  

48  h prerace values)  40

Table 7:  Descriptive data interpretation of mean Top 5 HFCs cell 48 pre- and 24 h  

  postrace total cell number (n 45) of the SCE-cohort (n 9) ( p 0 05 compared to  

48  h prerace values)  44

VI

ABBREVIATIONS

abbr. abbreviation

abs. absolute

appr. approximately

bidist.H 2 O bidistilled water

cm cetimeter

°C degree centigrade

CO 2 carbon dioxide

d day

DNA desoxyribonucleic acid

dept. department

g gram

h hour

3 H tritium

HFC high frequency cell

k kilo

km kilometer

kg kilogram

l liter

m meter or milli

M mol

max maximal

min minute or minimal

µ micro

pos. control positive control

s second

SCE sister chromatid exchange

SD standard deviation

p probability of error

VO 2max maximal oxygen acceptance

vs. versus

1

1. INTRODUCTION

Physical exercise is regarded to promote health and well-being in general. Nevertheless, it has been claimed that prolonged exhaustive exercise, such as a long-distance triathlon race, could be detrimental to health because of an accelerated formation of reactive oxygen species (ROS) [MOLLER et al., 2000]. These highly reactive molecules are able to facilitate deleterious oxidation reactions with cellular proteins, lipids and DNA [POWERS et al., 2004; NIESS et al. 1999], thus forcing the generation of oxidative-, muscular- and systemic- stress, and eventually genomic instability [PITTALUGA et al., 2006; MOLLER et al., 2000]. Therefore, ultra-endurance athletes may be particularly vulnerable to oxidative cytogenetic damage [KNEZ et al., 2007].

The available data suggest that long-duration and intense exercise increases DNA damage of peripheral lymphocytes [RADAK et al., 1999; PEAKE and SUZUKI, 2004;

RADAK et al., 2000], yet on the contrary, investigators proved, by negative results of

SCE assays that ultra-endurance exercise apparently does not result in cytogenetic

damage [MOLLER et al., 2000] implicating an adequate repair of DNA lesions [TSAI et al., 2001].

Regular exercise, which is obviously performed in the current study population, contingently induces adaptive responses in antioxidant- and DNA damage- repair systems, resulting in a decreased buildup of oxidative damage, which may contribute to a limitation of exercise-induced DNA damage [TSAI et al., 2001; MASTALOUDIS et al., 2004; NIESS et al., 1999].

In this context, Niess et al. demonstrated a reduction in DNA damage levels in endurance trained individuals, due to adaptation to the regular aerobic resistance training [NIESS et al., 1996].

However, exercise-induced DNA damage and subsequent deficient DNA repair may have influence on the genesis of cancer, diabetes, atherosclerosis [GIDRON et al.,

2006] and premature ageing [POULSEN et al., 1996].

The Austrian Science Fund-project “Risk assessment of Ironman triathlon participants” was therefore designed to gain further insight into the magnitude of a single bout of ultra-endurance exercise to induce sustained oxidative tissue-damage or adverse health responses in highly trained athletes.

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The FWF-project, which is coordinated by Prof. Karl-Heinz Wagner at the Dept. of Nutritional Sciences of the University of Vienna, is scheduled from January 2006 to January 2008. The cooperative Departments, which evaluated several additional parameters, are the Dept. of Rehabilitative and Preventive Sportmedicine/ Medical University-Policlinic Freiburg (Germany), the Institute for Cancer Research, the Dept. for Internal Medicine I and IV/ Medical University Vienna, the Dept. for Pulmology and the Alpentherme Bad Hofgastein.

Within the scope of this project, at the Dept. for Nutritional Sciences, Mag. Oliver Neubauer analyzed several oxidative stress parameters, Mag. Stefanie Reichhold investigated DNA effects, Lucas Nics and Norbert Kern determined the status of enzymatic and non-enzymatic antioxidants and Anna Chalopek assessed the nutritional and training status of the particapants.

In this work, the sister chromatid exchange (SCE) assay, as a relevant biological indicator of DNA damage in human epidemiology studies [PENDZICH et al., 1997], was chosen to investigate the effects of a single bout of strenuous exercise on the genomic stability of highly trained athletes. Peripheral blood lymphocytes were used to investigate SCE frequency, on account of their effortless accessibility [WILCOSKY and RYNARD, 1990].

This work was aimed to evaluate the alterations of SCE frequency, 48 h before and 24 h after an Ironman triathlon (3.8 km swim, 180 km cycle, 42 km run), in peripheral blood lymphocytes of highly trained athletes. The correlation between relative SCE changes pre- vs. postrace and several training levels of the athletes were additionally examined.

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2. BACKGROUND

2.1. Human lymphocytes

Human lymphocytes constitute a subpopulation of leukocytes, are produced in the bone marrow and the thymus [TOBIN and DUSCHEK, 1998] and contain two eminent cell types, namely T and B cells.

The addition of a mitogen, such as phytohemagglutinine (PHA), stimulates lymphocytes, adhered in the non-proliferative-G 0 phase, to reenter the cell cycle and proliferate [CARRANO and NATARAJAN, 1988].

Human population studies, performing cytogenetic analysis, typically use peripheral blood lymphocytes to investigate sister chromatid exchange (SCE) frequency, due to their effortless accessibility, constant karyotype and steady spontaneous SCE value. Some minor disadvantages are the variability between individuals on account of their metabolism of chemicals, DNA damage repair-capacity and percentage of cells responding to a particular mitogen [WILCOSKY and RYNARD, 1990]. However, lymphocytes-SCEs still serve as a relevant biological response marker of DNA damage [WILSON and THOMPSON, 2007].

2.2. Cell cycle

The cell cycle, a periodical event that achieves cell reproduction, consists of two major stages named interphase and mitosis. The duration of a single cell cycle depends on the organism and on its circumstances [TOBIN and DUSHEK, 1998].

2.2.1. Interphase

Interphase was believed to be a resting phase because cells only appeared to be active during mitosis. On the contrary it is a process in which the cell is vigorously active in order to achieve the greatest part of cellular growth and to duplicate the genetic material for an error-free cell division.

Interphase itself consists of three subsections, G 1 (first gap), S (synthetic phase) and G 2 (second gap). G 1 phase occupies most time of the cell cycle and is regulated through

4

two control checkpoints to reassure that the cell provides the machinery needed to accomplish cell division [POLLARD and EARNSHAW, 2002].

Differentiated, metabolically and physiologically active, thus non-dividing cells are considered to be in a special compartment of G 1 , called G 0 phase. Mitogens such as

PHA are able to stimulate cells resting in G 0 stage, to reenter the cell cycle and hence to

divide [CARRANO and NATARAJAN, 1988].

In S phase the genetic material is duplicated [POLLARD and EARNSHAW, 2002], according a semi-conservative replication of the DNA double helix, triggered by certain CDKs (cyclin dependent kinases) [WATRIN and LEGAGEUX, 2003], resulting in syngeneic copies of DNA strands [AUDESIRK et al., 2002]. During G 2 phase the DNA structure is proofread and preparations for mitosis are made [POLLARD and EARNSHAW, 2002]. The events of the eukaryotic cell cycle are depicted in figure 1.

Figure 1: The events of the eukaryotic cell cycle [MORGAN, 2007]

2.2.1.1. Checkpoints

Inaccuracies in cell division are devastating, may cause abnormal distribution of chromosomes [SUMMER, 2003], chromosome breakage or aneuploidy [WATRIN and LEGAGEUX, 2003]. Thus it is essential that the cell cycle is highly regulated. Checkpoints inhibit a subsequent process until the preceding event has been completed [SUMMER, 2003]. These biochemical pathways respond to external and internal

5

signals, and are able to arrest the cell’s advancement or even coerce the cell to initiate apoptosis (programmed cell death) if an error is registered.

To pass the restriction point in late G 1 , appropriate growth stimuli from the extracellular matrix must be received. Both DNA damage checkpoints, conducted at the end of G 1 and G 2 , check for DNA damage, or unduplicated centrosomes. The metaphase-, or spindle assembly checkpoint delays the commencement of chromosome segregation in mitosis until all chromosomes have attached to the mitotic spindle apparatus [POLLARD and EARNSHAW, 2002].

2.2.2. Mitosis

In eukaryotic cells mitosis ensures that the entire karyotype, containing 46 chromosomes, separates and is equally distributed (karyokinesis) into each daughter cell after cytoplasmic division (cytokinesis), resulting in two genetically identical daughter cells [WATRIN and LEGAGEUX, 2003; HSU and ELDER, 1991]. DNA replication in eukaryotic cells is semiconservative and initiates at volatile foci [LATT, 1973]. Mitosis is subdivided into four highly coordinated stages based on the appearance and behavior of chromosomes, termed pro-, meta-, ana- and telophase [AUDESIRK et al., 2002].

• Prophase: The chromatin condenses into compact chromosomes [POLLARD and EARNSHAW, 2002], the mitotic spindle forms and the nucleolus [AUDESIRK et al., 2002] and the nuclear membrane disperse. The spindle microtubules, hollow tubes of the protein tubulin [TOBIN and DUSHEK, 1998], are connected to the kinetochores of each chromatid of a chromosome to provide their proper movement along the spindle.

• Metaphase: The chromosomes are lined up along the cell’s equator (metaphase plate) [AUDESIRK et al., 2002] according to a stage classified as aster phase. To visualize SCEs, cycling cells are arrested at second metaphase by adding the mitotic spindle poison colchicine [TAYLOR, 1958]. The chromosomes observed under a light microscope at high magnification (x100) look different in size and in the positions of the centromeres [TOBIN and DUSHEK, 1998], according to so

6

called “metaphase chromosomes”, which are maximal condensed and therefore available for microscopic assessment.

In general, a metaphase chromosome (figure 2) consists of two sister chromatids, catenated at the centromere,

Figure 2: Metaphase chromosome [SUMMER, 2003]

• Anaphase: The two sets of homologues chromatids are separated and pulled to opposite poles along the spindle microtubules.

• Telophase: The nuclear membrane forms around each set of chromatids, the nucleoli appear [AUDESIRK et al., 2002] and the spindle apparatus disperses [TOBIN and DUSHEK, 1998]. Finally a ring of microfilaments surrounding the cell’s equator contracts and constricts, dividing the cytoplasm (cytokinesis) and therefore generating two new daughter cells [AUDESIRK et al., 2002].

2.3. Sister Chromatid Exchange

2.3.1. Mechanism of SCE

A natural process, spontaneously proceeding at certain rates in all cells during the

normal DNA replication [HAAF and SCHMID, 1991; SONODA et al., 1999], involving a four-fold polynucleotide strand breakage and reunion of each sister chromatid of a chromosome at apparently homologous regions, is called sister chromatid exchange [KANG et al., 1997].

Although this event, a reciprocal interchange by homologous recombination, is considered to be accurate [WILSON and THOMPSON, 2007], not resulting in alterations of overall chromosome morphology [PERRY and EVANS, 1975], cell

7

viability, cell function, or adverse health outcomes, an elevated value of SCEs indicates that cells have been exposed to a mutagen [WILCOSKY and RYNARD, 1990].

SCE induction raises to a maximum at the onset of DNA synthesis, but declines to zero

at the end of S-phase, suggesting that SCEs emerge at the replication point [TAWN and HOLDSWORTH, 1992], resulting in an absolute exigency of DNA replication for SCE formation [CARRANO and NATARAJAN, 1988].

The exact molecular mechanisms responsible for the genesis of SCEs are still inconclusive [WILSON and THOMPSON, 2007], but hypotheses have implicated the mechanics of DNA synthesis in SCE formation [ALBERTINI et al., 1985].

Figure 3: SCEs in human lymphocytes. Cell of a male healthy athlete of the SCE cohort shows 5

SCEs/ 46 chromosomes (arrows label SCEs)

Two major theoretical hypotheses of SCE occurrence have been proposed:

• The replication model involves homologous recombination (HR) during DNA replication [WILCOSCY and RYNARD, 1990; SONODA et al., 1999], which is, among others, required if single or double-strand breaks near the replication fork occur, or the progression of the replication fork is inhibited [TUCKER et al., 1993].

8

SONODA et al. suggested that HR between sister chromatids is principally

responsible for SCE induction in higher eukaryotic cells [SONODA et al., 1999].

• The recombination model suggests chromatid exchange as part of a post- replication repair process [WILKOSCY and RYNARD, 1990] which can be necessary, among others, if unreplicated gaps remain during DNA replication and are later rejoined incorrectly [WILSON and THOMPSON, 2007].

Both hypotheses propose an impact of topoisomerases I and II on SCE generation by affecting the process of DNA strand break-induction and -rejoining [TUCKER et al., 1993].

The SCE assay is based on the incorporation of the DNA base analogue BrdU into replicating chromosomes, arrested in second metaphase, thus allowing differential labeling of chromatids and visualization of SCEs [TAWN and HOLDSWORTH, 1992;

WILCOSKY and RYNARD, 1990] (figure 3).

2.3.2. Scientific significance of the SCE assay

The SCE assay, as a popular method in toxicology and human biomonitoring [WILSON and THOMPSON, 2007], is a highly sensitive indicator of genotoxicity in human epidemiology studies [PENDZICH et al., 1997]. The assay additionally provides a simple, rapid and sensitive method for assaying chromosome instability [PERRY and EVANS, 1975; ZHANG and YANG, 1992] by monitoring DNA damage- and repair- aspects [BALTACI et al., 2002].

A replacement of the classical cytogenetic mutagenicity tests (e.g. chromosome

aberrations (CA), micronucleus assay (MN)) by the SCE technique is, on the basis of today’s knowledge, scientifically not justified [WILSON and THOMPSON, 2007]. This conclusion is supported by the facts that the biologic consequences of SCE are partly unknown [GEBHART, 1981], the mechanism of SCE is yet not exactly verified [TUCKER et al., 1993] and that there still exists a lack of uniformity in SCE procedures [FAUST et al., 2004]. Therefore the SCE technique is considered to serve as a valuable additional method for cytogenetic mutagenicity testing, providing important supplementary information [GEBHART, 1981; HSU and ELDER, 1991].

9

2.3.3. SCE inducing agents

Agents, which efficiently induce SCEs in in vitro mammalian cell lines are alkylating agents and other DNA-binding agents, certain DNA-base analogues (e.g. BrdU) [WILCOSKY and RYNARD, 1990], crosslinking agents [WILSON and THOMPSON, 2007], and S-dependent agents, which, among others, effectively evoke chromatid type aberrations and DNA double-strand breaks [CARRANO and NATARAJAN, 1988].

2.3.4. Persistence of SCE

Lymphocytes, present for several years, are classified as short-lived- and those existing for decades as long-lived lymphocytes [CARRANO and NATARAJAN, 1988]. The persistence of SCEs depends both on the rate of DNA repair and on the normal half–life of impaired cells. Long-lived lymphocytes could give a good estimate of dose integrated over time, whereas short-lived lymphocytes could provide appraisal of more recent exposures. However, an exposure itself may increase the rate of cell turnover and shift the proportion of long- and short–lived lymphocytes [WILCOSKY and RYNARD, 1990].

2.3.5. Historical background

The existence of SCEs was first suspected by McClintock in 1938, due to the behavior of ring chromosomes in maize [TUCKER et al., 1993], and finally detected by Taylor et

al. in 1957, who grew plant cells for two rounds of replication in the presence of 3 H- labelled thymidine to differentially label the DNA in order to distinguish the chromatids from one another by autoradiograpy [TAYLOR, 1958].

In 1973 Latt discovered a non-radioisotopic method to distinguish exchanged DNA strands [LATT, 1973], in fact by BrdU application, which made it possible to quench the fluorescence of the fluorochrome Hoechst 33258, a dye, which was used to subsequently stain the preparations [PERRY and WOLFF, 1974; WILSON and THOMPSON, 2007; GEBHART, 1981].

The FPG technique, a combination of Latt’s fluorescent staining technique and Ikushima’s and Wolff’s Giemsa technique [IKUSHIMA and WOLFF, 1974], was