Universität Wien

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

angestrebter akademischer Grad
Magistra der Naturwissenschaften (Mag. rer.nat.)

Marlies Meisel
Studienrichtung (lt. Studienblatt): A 474 Ernährungswissenschaften

Wien, August 2007

 

 

Contents

LIST OF FIGURE ... IV

LIST OF TABLE ... V

ABBREVIATION ... VI

1. INTRODUCTION ... 1

2. BACKGROUND ... 3

2.1. Human lymphocyte ... 3

2.2. Cell cycle ... 3
2.2.1. Interphase ... 3
2.2.1.1. Checkpoint ... 4
2.2.2. Mitosi ... 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 agent ... 9
2.3.4. Persistence of SCE ... 9
2.3.5. Historical background ... 9
2.3.6. BrdU incorporation and visualization of SCE ... 10
2.3.7. The role of cell culture component ... 11
2.3.8. Factors potentially influencing SCE frequency ... 12
2.3.8.1. Culture factor ... 12
2.3.8.2. Biological and physiological factor ... 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 stre ... 17
2.4.3. Exercise-induced DNA damage ... 18
2.4.3.1. Relation to oxygen consumption ... 18
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 METHOD ... 25

3.1. Project description ... 25

3.2. Subject ... 25
3.2.1. Inclusion criteria ... 26
3.2.2. Exclusion criteria ... 26
3.2.2.1. Supplementation guideline ... 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 analysi ... 35

3.10. Guidelines for microscopic assessment ... 36

3.11. Top five HFC ... 36

4. RESULTS AND DISCUSSION ... 37

4.1. Study design ... 37

4.2. Subjects characteristic ... 37

4.3. Preliminary testing ... 38

4.4. Assay criteria ... 39

4.5. Distribution of evaluated SCEs per cell ... 40

4.6. Abs. SCE ... 40
4.6.1. Descriptive statistic ... 40
4.6.2. Single means of abs. SCE ... 41
4.6.3.Total mean abs. SCE ... 42

4.7. Top 5 HFC ... 44
4.7.1. Descriptive statistic ... 44
4.7.2. Single means of Top 5 HFC ... 44
4.7.3. Total mean Top 5 HFC ... 45

4.8. Correlation ... 47
4.8.1.Abs. SCE ... 47
4.8.2. Top 5 HFC ... 49

5. CONCLUSION ... 51

6. SUMMARY ... 53

7. ZUSAMMENFASSUNG ... 54

8. REFERENCE ... 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

 

 

 

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.

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.

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-G0 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, G1 (first gap), S (synthetic phase) and G2 (second gap). G1 phase occupies most time of the cell cycle and is regulated through 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 G1, called G0 phase. Mitogens such as PHA are able to stimulate cells resting in G0 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 G2 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.

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 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 G1, appropriate growth stimuli from the extracellular matrix must be received. Both DNA damage checkpoints, conducted at the end of G1 and G2, 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 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, whereas the kinetochores of each chromatid serve as connection to the spindle microtubules, which emanate from opposite poles of the cell [HAAF and SCHMID, 1991].

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