The aim of the present study was to investigate the effect of a triathlon (3.8 km swim, 180 km cycle, 42,2 km run) on the genomic stability of nine highly trained non-
professional athletes.
Therefore, the SCE assay, a relevant biological response marker for genotoxicity in
human biomonitoring studies [PENDZICH et al., 1997] was performed using peripheral
lymphocytes, on account of their effortless accessibility [WILKOSCY and RYNARD,
1990].
Duplicate lymphocyte cell cultures, of each participant, were incubated for 72 h (37°C, 5% CO2) according to a short-term human lymphocyte cell culture. For each participant at least 50 metaphases, containing 43-46 chromosomes were scored, to evaluate the mean SCE frequency. The number of SCEs per cell was calculated to a chromosome set of a normal diploid human cell, containing 46 chromosomes.
In the present study the alteration of SCE frequency, 48 h pre- and 24 h postrace was evaluated. As an additional endpoint Top 5 HFCs (highest five absolute SCE means) were assayed. It could be demonstrated that both the total mean SCE frequency and the mean Top 5 HFC frequency (n=9) 24 h postrace were significantly decreased (*p< 0.05) compared to 48 h prerace values. Considering the training status, a significant negative correlation between the relative SCE changes before and after the triathlon was observed for the cycling training per week (km) (r=-0.86; **p< 0.01), the running training per week (km) (r=-0.90; *p< 0.05)
as well as for the weekly net exercise training time (h) (r=-0.89; *p< 0.05). The relative changes of Top 5 HFCs before and after the race correlated significantly with the cycling training per week (km) (r=-0.79; *p< 0.05) and with the body mass index
(kg/m2) (r=-0.69; *p< 0.05). These findings suggest the existence of endogenous repair mechanisms which seem to prevent DNA damage.
Contents
LIST OF FIGURES
LIST OF TABLES
ABBREVIATIONS
1. INTRODUCTION
2. BACKGROUND
2.1. Human lymphocytes
2.2. Cell cycle
2.2.1. Interphase
2.2.1.1. Checkpoints
2.2.2. Mitosis
2.3. Sister Chromatid Exchange
2.3.1. Mechanism of SCE
2.3.2. Scientific significance of the SCE assay
2.3.3. SCE inducing agents
2.3.4. Persistence of SCE
2.3.5. Historical background
2.3.6. BrdU incorporation and visualization of SCEs
2.3.7. The role of cell culture components
2.3.8. Factors potentially influencing SCE frequency
2.3.8.1. Culture factors
2.3.8.2. Biological and physiological factors
2.4. The Correlation between strenuous endurance exercise and genotoxicity
2.4.1. Reactive oxygen species (ROS) and physical exercise
2.4.2. Exercise-induced oxidative stress
2.4.3. Exercise-induced DNA damage
2.4.3.1. Relation to oxygen consumption
2.4.3.2. Relation to a single bout of exercise
2.4.4. Exercise-induced adaptation
2.4.5. Regular physical exercise
3. MATERIALS AND METHODS
3.1. Project description
3.2. Subjects
3.2.1. Inclusion criteria
3.2.2. Exclusion criteria
3.2.2.1. Supplementation guidelines
3.3 Equipment for the SCE assay
3.4. Reagents of the SCE assay
3.4.1. Manufacturing processes and storage of reagents for SCE assay
3.5. Basic assay approach
3.6. Assay description
3.7. Blood collection
3.8. Sister Chromatid Exchange assay
3.9. Statistical analysis
3.10. Guidelines for microscopic assessment
3.11. Top five HFCs
4. RESULTS AND DISCUSSION
4.1. Study design
4.2. Subjects characteristics
4.3. Preliminary testing
4.4. Assay criteria
4.5. Distribution of evaluated SCEs per cell
4.6. Abs. SCEs
4.6.1. Descriptive statistics
4.6.2. Single means of abs. SCEs
4.6.3.Total mean abs. SCEs
4.7. Top 5 HFCs
4.7.1. Descriptive statistics
4.7.2. Single means of Top 5 HFCs
4.7.3. Total mean Top 5 HFCs
4.8. Correlations
4.8.1.Abs. SCEs
4.8.2. Top 5 HFCs
5. CONCLUSION
6. SUMMARY
7. ZUSAMMENFASSUNG
8. REFERENCES
9. APPENDIX
9.1. Single values of participant 36
9.2. Single values of participant 37
9.3. Single values of participant 39
9.4. Single values of participant 41
9.5. Single values of participant 42
9.6. Single values of participant 43
9.7. Single values of participant 46
9.8. Single values of participant 47
9.9. Single values of participant 48
LIST OF FIGURES
Figure 1: The events of the eukaryotic cell cycle [MORGAN, 2007]
Figure 2: Metaphase chromosome [SUMMER, 2003]
Figure 3: SCEs in human lymphocytes. Cell of a male healthy athlete of the SCE cohort shows 5 SCEs/ 46 chromosomes (arrows label SCEs)
Figure 4: Generation of reactive oxygen species [GARRIDO et al., 2004]
Figure 5: Oxidative stress - imbalance between oxidants and antioxidants. (GSH: glutathione) [GARRIDO et al., 2004]
Figure 6: Culture flasks are incubated at 37°C (5% CO2) for 70 h
Figure 7: Hypotonic KCl solution is added drop by drop (slowly)
Figure 8: The supernatant is discarded with an exhaustion pump inside the LF
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)
Figure 10: Total mean abs. SCEs/cell of the SCE-cohort (n=9); (*p< 0.05 compared to 48 h prerace values)
Figure 11: Relative mean abs. SCE alteration (%) 48 h before and 24 h after the triathlon race
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)
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)
Figure 14: Relative mean Top 5 HFC alteration (%) 48 h pre- to 24 h postrace
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
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
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
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
LIST OF TABLES
Table 1: RDA-based supplementation guidelines during the research project
Table 2: Equipment for SCE assay
Table 3: Reagents for SCE assay
Table 4: Physical characteristics of the SCE-cohort (n=9)
Table 5: Training parameters of the SCE-cohort and their performance in the IM triathlon race
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)
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)
ABBREVIATIONS
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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.
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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 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.
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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].
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 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].
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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]. 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].
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 3H-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 introduced in 1974 by Perry and Wolff and provided permanent, non-fading preparations of differentially stained chromosomes, which made it possible to observe SCEs with great precision and clarity [WOLFF and PERRY, 1974; PERRY and EVANS, 1975]. SCEs are known to occur as a normal feature of cell division in mammalian tissues [ZHANG and YANG, 1992].
2.3.6. BrdU incorporation and visualization of SCEs
If cells are grown in culture under assay conditions, BrdU is efficiently incorporated into the newly evolved daughter strand of each double-stranded polynucleotide molecule during semi-conservative replication (S-phase) [WOLFF and AFZAL, 1996; WILSON and THOMPSON, 2007; WILCOSKY and RYNARD, 1990]. To obtain differential staining of sister chromatids, BrdU has to be present for two cell cycles [HAAF and SCHMID, 1991].
During the first metaphase in culture each sister chromatid has one unaltered parent strand and one newly formed BrdU-substituted strand. During second S-phase BrdU is again introduced into the newly formed DNA strand. Then, when cells enter second metaphase, one of the chromatids contains a parent and a BrdU-substituted DNA strand (unifilarly) and the other contains two strands of BrdU-substituted DNA (bifilarly).
The asymmetrical distribution of BrdU-substituted DNA in the second metaphase allows visualization of SCEs. When stained according the FPG-technique or any other approved Giemsa technique the unifilarly substituted chromatid stains more intense with Giemsa than the bifilarly substituted chromatid, due to the bleaching effect of BrdU. SCEs appear as a discontinuity in the stain intensity along the chromatid and can be observed clearly with great resolution [WILCOSKY and RYNARD, 1990; WOLFF and AFZAL, 1996; WOLFF and PERRY, 1974]. The exchange of stain has to be reciprocal, i.e., a darkly stained segment on one chromatid must be accompanied by a lightly stained region in the reciprocal part of the sister chromatid. Exchanges of stain occurring at the centromere should be included as an SCE unless there is an obvious twist of the chromatids [CARRANO and NATARAJAN, 1988].
2.3.7. The role of cell culture components
- Heparin: Thus considering EDTA as a known inducer of SCEs [TUCKER et al., 1993], the anticoagulant heparin is added to the culture in order to prevent clotting of human whole blood [PARK, 1999].
- PHA: To stimulate cells in culture to undergo mitosis, phytohemagglutinine (PHA) is added which especially stimulates T-cells to divide [WILKOSCY and RYNARD, 1990].
- BrdU: See chapter 2.3.6. for a detailed description of BrdU incorporation.
- Penicillin-streptomycin: Antibiotics are added to restrict bacterial infections of the cell culture [PARK, 1999].
- Incubation time: A culture length of 72 h is recommended [LAMBERT et al., 1982], at which the highest percentage of second division cells is yielded [PARK, 1999].
- Colchicine: The plant alkaloid colchicine acts as a mitotic arrestant, which inhibits the construction of the mitotic spindle apparatus, causing a cell-arrest at metaphase [HSU and ELDER, 1991], and therefore allows accumulation of metaphase chromosomes [SUMMER, 2003].
- Hypotonic KCl solution: Treatment with hypotonic solution causes on the one hand haemolysis and on the other hand a swelling of lymphocytes from the influx of water. This allows the chromosomes to disperse freely within the cell membrane, separating the paired chromatids but leaving them still connected at the centromere.
- Fixative: To harden the chromatin and enhance the morphology, the cells are fixed.
- Giemsa solution: Due to the Giemsa staining the differential incorporation of BrdU between sister chromatids can be evidenced [PARK, 1999].
2.3.8. Factors potentially influencing SCE frequency
The baseline SCE frequency in human peripheral lymphocytes averages about 7-10 per cell [ALBERTINI et al., 1985]. There are many potential sources influencing SCE frequency. The baseline value even varies within the same laboratory, by a factor of two or more. In order to minimize variation and maximize sensitivity, the use of standardized protocol is necessary [TAWN and HOLDSWORTH, 1992].
2.3.8.1. Culture factors
- Blood storage
Heparinized whole blood can be stored for up to one week, between 4 and 37°C, without consistent alterations in SCE frequencies. However, prolonged storage may have an adverse effect upon the mitotic index or proliferation kinetics, or could lead to an overall decrease in lymphocyte numbers and modification of lymphocyte subclasses. Thus, the samples should be placed in culture as quickly as possible [TUCKER et al., 1993; CARRANO and NATARAJAN, 1988].
- BrdU concentration
BrdU itself causes SCEs in a dose-dependent manner, therefore its concentration should be kept to a minimum to allow differential labeling of the chromatids [TAWN and HOLDSWORTH, 1992; LAMBERT et al., 1982]. The frequency of SCE has been shown to increase by as much as 50%, whether the BrdU concentration is raised 10-fold [ALBERTINI et al., 1985].
As the actual spontaneous SCE rate was investigated, it was demonstrated that the SCE value depends on the incorporated nucleoside, necessary to allow their detection. If SCEs were determined autoradiographically, the SCE rate was significantly elevated when compared to the BrdU application method.
By interpolating the results obtained with low concentrations of BrdU to those expected at zero concentration, the true spontaneous level of SCE formation was determined [WOLFF and PERRY, 1974].
- Incubation time
The influence of incubation time reflects the presence of different subpopulations of lymphocytes with different cell advancement and SCE values [TAWN and HOLDSWORTH, 1992]. Santesson et al. found that B-lymphocytes had a significantly lower SCE frequency than T-lymphocytes [SANTESSON et al., 1979].
- Other factors
The utilized serum in the culture medium has been identified to influence the baseline SCE frequency [ABDEL-FADIL et al., 1982], whereas higher SCE values were found in cultures that used autologous serum compared to cultures that used fetal calf serum [LAMBERT et al., 1982]. Different culture media and culture temperature also influence SCE frequencies [ABDEL-FADIL et al., 1982].
Since BrdU is sensitive to light (other than red or yellow), exposing the culture to light increases SCE frequency as a function of time and intensity, due to photolysis of BrdU containing DNA. Light protection of cultures during incubation is therefore obligatory [DAS, 1988].
2.3.8.2. Biological and physiological factors
- Age and sex
Many investigators found a positive correlation between baseline SCE value and age [CARRANO and NATARAJAN, 1988]. A statistically higher mean frequency of SCEs in females than in males with significant positive regression with age was observed by Bender et al. [BENDER et al., 1988]. Pregnancy and oral contraceptive use has been associated with increased SCEs [CARRANO and NATARAJAN, 1988].
- Smoking
Although contradictory results of the effect of smoking exist [PENDZICH et al., 1997; ANDERSON et al., 1993], the majority of evidence indicates that individuals who smoke have significantly elevated SCE frequencies [BENDER et al., 1988; WILCOSKY and RYNARD, 1990; BARALE et al., 1998].
- Disease
Several studies observed elevated SCE frequencies in patients with Bloom’s syndrome, Crohn’s disease, multiple sclerosis [KANG et al., 1997], ankylosing spondylitis [SÖNMETZ et al., 1997] and ovarian cancer [BALTACI et al., 2002]. Yet, an association between SCEs and cancer risk could not be approved in a study of 1621 subjects [NORPPA et al., 2006].
- Lifestyle factors
Alcohol consumption has not been found to have an effect on SCE rate, except at the level consumed by alcoholics, when an increase in both CA and SCE has been observed [NORPPA et al., 2006; PERRY and EVANS, 1975]. Also caffeine intake was associated with an increased SCE frequency [BUKVIC et al., 1998].
- Free radicals
Free radical inducing agents are known to increase SCE frequency in human lymphocytes [MATSUOKA et al., 2004]. A dose-dependent relationship between free radicals and SCE rate was also investigated in Chinese hamster ovary cells [LEE et al., 1989].
2.4. The Correlation between strenuous endurance exercise and genotoxicity
2.4.1. Reactive oxygen species (ROS) and physical exercise
Free radicals, such as superoxide anions (O2.-), hydrogen peroxide (H2O2), hydroxyl radicals (OH.) and nitric oxide (NO.) [URSO and CLARKSON, 2003] are continuously generated in the human body (figure 4). These highly reactive molecules feature an unpaired electron in their outer orbital and can facilitate deleterious oxidation reactions with cellular proteins, lipids or desoxyribonucleic acids (DNA), giving rise to oxidative stress and impaired cellular function [POWERS et al., 2004; NIESS et al., 1999]. ROS play an eminent role in a variety of physiological processes and are implicated in the genesis of inflammatory diseases, cancer and aging [SCHNEIDER and REISCHAK DE OLIVEIRA, 2004].
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Figure 4: Generation of reactive oxygen species [GARRIDO et al., 2004]
Specific sources of ROS during exercise include leakage of electrons from the mitochondrial electron transport chain, xanthine oxidase activation, acidosis, haemoglobin oxidation [URSO and CLARKSON, 2003], neutrophil activation [TAULER et al., 2006], prostanoid metabolism and NAD(P)H-oxidase activation [URSO and CLARKSON, 2003].
The leakage in the respiratory chain, in the mitochondrial inner-membrane, is regarded as the primary source of radical formation, thus 2-5% of the oxygen flux results in the genesis of superoxide radicals [POWERS et al., 2004; DEATON and MARILIN, 2005].
Secondary sources of radical formation during exercise include autoxidation of catecholamines, radical generation by phagocytic white cells and damage to iron-containing proteins [POWERS et al., 2004].
Physical exercise provokes an increased production of ROS, depending mainly on the intensity of physical performance [APOR and RADI, 2006]. ROS are believed to be a considerable cause of oxidative DNA modification [POULSEN et al., 1996], actually the cytotoxic effects include the oxidative damage of cellular DNA [RADAK et al., 2000].
ROS generated postexercise, due to reparation processes of tissue damage, may be responsible for the delayed increase in DNA damage after a massive bout of anaerobic exercise [TSAI et al., 2001].
ROS are scavenged by a complex antioxidant defense system, consisting of enzymes such as catalase, superoxide dismutase, glutathione peroxidase, and numerous non-enzymatic antioxidants, including vitamins A, E and C, glutathione, ubiquinone, and flavonoids [URSO and CLARKSON, 2003].
The available data support the occurrence of exercise-induced oxidative stress but it remains unclear what causes the increase in production of ROS. Several mechanisms may act synergistically, and it is possible that different types of exercise involve different mechanisms of free radical production [MASTALOUDIS et al., 2001].
Many observations suggest that enhanced ROS genesis is a desired or even required consequence of exercise [VOLLAARD et al., 2005]. However, the exact physiological functions of ROS in exercise remain to be established [URSO and CLARKSON, 2003].
2.4.2. Exercise-induced oxidative stress
Oxidative stress is defined as the disequilibrium between pro-oxidative and anti-oxidative molecules in a complex biological system, where oxidants overwhelm defensive systems (figure 5) [GARRIDO et al., 2004; POWERS et al., 2004; TAULER et al., 2006].
illustration not visible in this excerpt
Figure 5: Oxidative stress - imbalance between oxidants and antioxidants. (GSH: glutathione) [GARRIDO et al., 2004]
Damage to biomolecules, such as lipids, proteins and DNA, may occur by the time when ROS exceed the protective capacity of the antioxidant defense system [RADAK et al., 2007]. The harmful effects caused by oxidative stress are related to decreased physical performance, muscular fatigue, muscle damage, overtraining [KÖNIG et al., 2001] and several diseases such as diabetes, arteriosclerosis and cancer [SCHNEIDER and REISCHAK DE OLIVEIRA, 2004].
There is a body of evidence suggesting that endurance exercise causes oxidative stress. [MASTALOUDIS et al., 2001; TAULER et al., 2006; TSAI et al., 2001] The most important factors in the genesis of exercise-induced oxidative stress are the intensity and the exhaustion level of the subject submitted to exercise and, thus, the exposition to a higher flux of oxygen [SCHNEIDER and REISCHAK DE OLIVEIRA, 2004]. However, it is still unknown whether or not oxidative stress and subsequent oxidative damage are associated with physical exercise [KÖNIG et al., 2001; MASTALOUDIS et al., 2001].
Despite the many known detrimental effects of exercise, physical training is suggested to ease the results of oxidative stress, mainly through an adaptation process in the activity of the antioxidant system [WOZNIAK et al., 2001], therefore it may be necessary to contemplate exercise-induced oxidative stress in a more positive perspective [VOLLAARD et al., 2005], as ROS are suggested to contingently trigger adaptive processes [URSO and CLARKSON, 2003] by acting in signal transduction pathways [POULSEN et al., 1999].
However, it is still ambiguous whether exercise-induced oxidative modifications have little impact, induce insalubrious oxidative damage, or are an integral part of redox regulation [URSO and CLARKSON, 2003].
2.4.3. Exercise-induced DNA damage
2.4.3.1. Relation to oxygen consumption
Many studies have investigated that high-intensity exercises occur with an enhanced formation of free radicals [KÖNIG et al., 2001], due to the activation of specific metabolic pathways as well as the increased requirement of oxygen [SCHNEIDER and REISCHAK DE OLIVEIRA, 2004], which increases 100- to 200 times the resting value in skeletal muscles [MASTALOUDIS et al., 2001].
It is proposed that individual oxygen consumption is a predictor of the rate of oxidative modification of macromolecules [POULSEN et al., 1996]. Actually, it has been claimed that prolonged physical exercise is detrimental to health because of the augmented ROS-genesis due to enhanced oxygen uptake [RADAK et al., 1999].
A clear relationship between individually measured oxygen consumption and urinary excretion of 8-oxodG was demonstrated [POULSEN et al., 1996], further lending support to the notion that the increased uptake of oxygen in the body accounts for the exercise-induced DNA damage [UMEGAKI et al., 2000].
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