|Year : 2012 | Volume
| Issue : 4 | Page : 578-585
Pre-cancerous (DNA and chromosomal) lesions in professional sports
Radhika Sharma, Shailey, Gursatej Gandhi
Department of Human Genetics, Guru Nanak Dev University, Amritsar, Punjab, India
|Date of Web Publication||29-Jan-2013|
Department of Human Genetics, Guru Nanak Dev University, Amritsar - 143 005, Punjab
Source of Support: None, Conflict of Interest: None
Context: Exhaustive exercises may become detrimental, causing disturbance of intracellular oxidant-antioxidant balance and damage to macromolecules, leading to genomic instability when DNA/chromosomes get damaged. As these are precancerous lesions, cancer occurrence is probable. Because professional sports requires high-intensity training and increasing physical demand, there may occur cellular genomic instability.
Aim: To evaluate genetic damage at DNA and chromosomal levels in hockey and baseball-soft ball players and compare with levels in age- and sex-matched sedentary controls.
Setting and Design: Players professionally active in their sport from 3 to 11 years on a daily training session of 4h/day were contacted during their camps, and the study was approved by the Institutional Ethics Committee. All participants were healthy individuals, not on any medication and were not taking any supplements.
Materials and Methods: Genetic damage using the single cell gel electrophoresis assay and buccal micronucleus cytome assay of 56 individuals (36 players and 20 controls) was evaluated.
Statistical Analysis Used: Student's t -test, ANOVA, Pearson correlation and linear regression and Chi-square analysis were performed.
Results: Players had significantly elevated levels of genetic damage. There were no gender differences and also no significant difference in the genetic damage incurred in both sports types. However, the extent of DNA migration in hockey players was higher.
Conclusion: Significantly increased genomic instability in players of both sports was observed. Both repaired and repairable genetic damage cells were observed in different tissues of the same subject. The presence of such genetic damage implies that these players are at an individual risk from cancer- and age-related diseases.
Keywords: Exercise, cancer, genetic damage, genomic instability
|How to cite this article:|
Sharma R, Shailey, Gandhi G. Pre-cancerous (DNA and chromosomal) lesions in professional sports. J Can Res Ther 2012;8:578-85
| > Introduction|| |
Although the beneficiary effects of regular physical activity in the primary and secondary prevention of chronic diseases (including cancer) and premature death and in the quality of life during and after therapy in cancer patients have been well documented, , yet, there are studies showing increased risk of cancer in athletes  and moderate-to-heavy leisure/occupational activities.  Among the studies performed on physically active individuals with moderate to strenuous activity, Srivasatva and Kreiger  reported increased risk of testicular cancer (n = 94, 45.2%) in individuals who participated in moderate to strenuous recreational activity in their mid-teens as compared with those with less activity. Mink et al.  reported a 1.5-fold increased risk of ovarian cancer in women with regular leisure physical activity than in sedentary women, and the risk was increased twice in moderate-to-heavy leisure/occupational activities. Wannamethee and Walker  also showed increased risk of bladder cancer (n = 92) in vigorously active men as compared with other cancers. The latter gains significance as professional outdoor sports requires regular specific training programmes and optimal physical fitness before competitions. Cyclic schedules of training, taper, competition and recovery periods are typically followed.  Depending on the sport, exercise training may be continuous and/or intermittent, of short and long duration, accompanied by various strengthening and conditioning practices, prolonged and high-intensity exercises, endurance training and acute and strenuous exercises. The intensive physical activities contribute toward the physical fitness and specific demands of the sport activities.
Although moderate exercise up regulates the antioxidant system,  the aerobic/anaerobic activities have been reported to increase oxidative stress.  The probable mechanisms responsible for exercise-induced intracellular oxidant-antioxidant balance are mitochondrial electron transport chain (with increased oxygen consumption and causing increased reactive oxygen species (ROS) production), auto-oxidation of catecholamine, activation of inflammatory cells due to muscle/tissue damage and ischemia-reperfusion/hypoxia/re-oxygenation damage. ,, There are defense mechanisms (viz. antioxidants, repair and preventive mechanisms) within the body, but when it exceeds the body's capacity to detoxify, it causes an imbalance between ROS and anti-oxidant defense, and results in the oxidative stressful environment in the body and concomitantly macromolecular damage, viz. lipids, proteins and nucleic acids, the latter manifesting as genomic damage. , DNA damage can result in either arrest (loss of p53 function) or induction of transcription, induction of signal transduction pathways, replication errors and genomic instability, all of which are associated with carcinogenesis. , The most common initiating noeplasia are in breast, colorectum, lung, ovary, etc., and are related to imbalance in the oxidant-antioxidant system.  Therefore, genomic damage has its own repercussions in terms of initiating neoplasia and triggering neurodegenerative changes, including Alzheimer's disease, amyotrophic lateral sclerosis (one-fifth of these familial Amyotrophic Lateral Sclerosis, a motor neuron disease) patients carry mutations in the Cu/Zn-SOD gene, suggesting the involvement of ROS), Parkinson disease  and age-related changes, including decline in physiological functions. ,
As professional sports require a rigorous workout schedule and the physical demands of the game, these persons may also be harboring cellular genomic instability. In this study, results on precancerous genetic damage biomarkers in two minimally invasive tissues (buccal epithelial cells and peripheral blood leukocytes) of some hockey and baseball-softball players are presented. To the best of our knowledge, this is the first report of its kind, although there are some studies on genetic damage in swimmers, , athletes, ,, body builders, , wrestlers  and in individuals who workout in the gymnasium. 
Hockey and baseball-softball have been classified as dynamic sports and as high- and moderate-intensity sports, where the oxygen uptake is more than 70% and 40-70%, respectively.  Hockey requires skill and movements with high physical demand, , whereas baseball-softball are dynamic sports that require the elite athletes to respond to any move.  The training sessions for Indian hockey include three phases: transition, preparatory and competitive. These include continuous, interval, strength, power, speed and flexibility training; the technical training includes dribbling, set-up movements, free hit, match practice, etc.,  and baseball-softball interval also includes strengthening and conditioning. 
As carcinogenic events are triggered by the underlying damage to the genome, various genetic damage endpoints can be screened as precancerous lesions. In the present study, both chromosomal (micronucleus) and DNA damage were assessed in buccal epithelial cells and peripheral blood leukocytes, respectively. The alkaline single cell gel electrophoresis (SCGE/comet) assay is a sensitive technique to assess DNA single-strand breaks, double-strand breaks and alkali-labile sites.  Micronuclei (MN) are acentric chromosome fragments or whole chromosomes left behind during mitotic cellular division and appear in the cytoplasm of interphasic cells as small, additional MN.  The buccal MN cytome assay  is reliable for detecting chromosome breakage/loss (clastogenicity) and aneugenicity (mechanisms for carcinogenic risk) as well as necrosis and apoptosis. With the SCGE assay, DNA damage at the cellular level can be ascertained  as DNA fragments after electrophoresis move towards the anode and can be visualized as a comet. The SCGE and the buccal MN cytome assays are validated techniques that have been widely used in population biomonitoring studies for assessing genomic damage at DNA and chromosomal levels.  As there is circulation of blood throughout the body, stress induction at any site can affect the cellular genomic integrity; therefore, peripheral leukocytes can be assessed for genetic damage as they are available in a minimally invasive manner. The buccal epithelial MN assesses genetic damage that occurred 1-3 weeks earlier in the basal cells.  The ease and acceptability of providing buccal samples and the validation of the buccal MN cytome assay has made it popular for assessing genetic damage. ,
| > Materials and Methods|| |
Healthy players (22 hockey, 14 baseball-softballs) and no exercising sedentary healthy controls (20) comprised the study group. The players were contacted from the university playground during their annual training/camping time. The baseball players also played softball as it required similar training. Informed consent was taken from each participant and the study was approved by the Institutional Ethics Committee. General and sport-specific information was recorded for each participant on a prepared proforma. Anthropometric variables (height and weight) were taken as per Weiner and Lourie  for calculating body mass index (BMI) as it can also induce genetic damage. All participants were healthy, nonsmokers, not taking alcohol or any drugs with no acute/chronic disease and not on any medication. The training sessions of hockey and baseball-softball players comprised a warm-up period of 15-30 min (entailing brisk running, body rotations, stretching and jumping) and an exercise period of 4-6 h/day.
Sampling was done early in the day before warm-ups and the daily exercise schedule. For the micronucleus (MN) test,  buccal epithelial smears were made, and for the DNA damage assessment, finger-prick peripheral blood samples (50μL) were collected in heparinised vials. The samples were placed on ice and transferred to the laboratory and assessed within 2-3h for DNA and cytogenetic damage assessment.
DNA damage assessment
Blood samples were tested for cell viability using the standard Trypan blue exclusion test.  Samples with cell viability greater than 75% were processed for the SCGE assay. DNA damage was assessed using the alkaline SCGE/comet assay,  with slight modifications in respect of frosted slides (agarose-coated), silver staining  and use of local chemicals. In brief, on dried 1% mormal melting agarose (SRL, Mumbai, )-coated slides, a mixture of 30 μL blood and 100 μL of low melting point agarose (LMPA; 0.5%; SRL) was sandwiched after slide-setting. A coverslip was then placed over the slide and it was kept for refrigeration for 15 min. The preparations were then lysed (2.5 M NaCl, 100mM Na 2 EDTA, 1% Triton-X, 10% DMSO [Qualigens, Mumbai, ] and 10 mM Tris [SRL] pH 10.0) for 2-3 h at 4°C, incubated in alkaline electrophoresis buffer (300mM NaOH, 1mM Na 2 EDTA, pH >13) at 4°C for 25 min for DNA unwinding. Electrophoresis was performed (25 V, 300 mA) for 30 min. The preparations were then neutralized for 5 min with neutralizing buffer (0.4M Tris base [SRL Mumbai,], pH 7.5) and dried overnight at room temperature followed by staining with silver nitrate. All slides were coded and scored blind at 40X under a binocular light microscope (Magnus, MLX, Olympus Private Limited, New Delhi, India). For each sample, two slides were prepared and each was manually scored for 50 nucleoids (100/sample). For DNA migration measurements, a calibrated ocular micrometer was used. DNA migration length was taken as the difference between the length of the comet and the radius of the nucleoid; the cells were also visually grouped into 0-IV categories.  Damage frequency (DF; percent cells with tails), damage index (DI)  and DNA migration length were expressed as mean ± SEM. DI was calculated as the number of cells in zero degree damage×0+number of cells in I degree damage×1+number of cells in II degree damage×2+number of cells in III degree damage×3.
Cytogenetic damage assessment
Cytogenetic damage was assessed by the buccal micronucleus (MN) test  and scoring for the buccal cytome parameters (MN, nuclear buds, cell death, cytokinetic effects and cell proliferative parameters) was carried out as per Thomas et al. 
Buccal epithelial scrapes were taken using clean glass slides from the left and right cheeks after the participant had thoroughly rinsed his/her mouth and their separate slide preparations were made. These were fixed in Carnoy's fixative (3 methanol:1 glacial acetic acid; Ranbaxy, Chandigarh, ) for 20-25 min followed by hydrolysis (1N HCl; Qualigens Mumbai) for 8-10 min at 60°C followed by washing, air-drying and staining with 2% aceto-orecin (SRL, Mumbai) and counter staining with 1% Fast Green (CDH, New Delhi). Mounted preparations were coded and scored blind under a binocular microscope (Magnus, MLX) initially at 40X. MN was confirmed under oil immersion (100X). For each sample, 2000 cells (1000/slide) were scored for MN preceded by scoring of 500/slide (1000/sample) for nuclear buds (DNA amplification), cell proliferation (basal cells), cytokinetic effects (binucleated cells) and cell death (apoptotic and necrotic cell) indices. A separate record for each participant was maintained. The repair index (RI) was calculated as RI=(KL+KR)/(MN+BE) ([karyolysis + karyorrhexis] / [micronucleated + broken egg])  as the production of KL and KR anomalies represents the repair process before cytogenetic damage due to intercellular selection. This index reflects the dynamics of carcinogenesis as MN decreased with age in cancer patients. Values were recorded as mean ± SEM. Criterion of scoring MN and micronucleated cells as given by Thomas et al.,  was followed.
The data were parametric and, therefore, the Student's t-test was used. Chi (χ2 ) square analysis was performed on demographic variables of players and controls and on categories of cells. Student's t-test was performed for evaluating differences between and within the groups. For determining whether there was any relationship of DNA damage and chromosomal damage with sport-specific and confounding variables, correlation and multiple regression analyses were performed; for finding their association with damage parameters, the analysis of variance (ANOVA) was performed. All statistical analyses were performed using SPSS version 16.0 for windows (except χ2 , which was carried out manually). P-value at P<0.05 were taken as significant.
| > Results|| |
Genomic damage in hockey and baseball-softball players was assessed and compared with controls. The demographic- and sports-related information of the study group (n = 56) is presented in [Table 1]. Hockey (n = 22; 19.13 ± 0.23 years) and baseball-softball players (n = 14; 19.42 ± 0.36 years) matched healthy controls (n = 20; 19.40 ± 0.26 years) for age, sex, BMI, dietary pattern and lifestyle on Chi-square analysis. The physical attributes of the study group are presented in [Table 2]. The hockey players had been training/participating in matches over the past 3-11 years and baseball-softball players for 4-9 years. The players were contacted during their participation in training camps where training and competition (national and state level) were being conducted over a period of 10 days. The daily training session comprised a warm-up period in which running, body rotations and stretching were done from 15 to 30 min followed by the match practice.
The Student's t-test analysis [Table 3] revealed highly significant elevated difference (P = 0.000) in players of both games for all the scored SCGE assay parameters (DF, DI, mean DNA migration length) and for the buccal MN cytome assay parameters (micronucleated cells, binucleated cells, karyorrhectic cells, condensed chromatin cells, karyolitic and pyknotic cells) as compared with the respective values in control subjects. However, the RI of the epithelial cells was significantly decreased (P = 0.000) in both hockey and baseball-softball players. There were no significant genetic differences between genders in each sport; also, players of both sports did not exhibit significant differences for genetic damage from each other.
A significant association (multivariate ANOVA) of diet (P = 0.009) and of mobile phone usage (P = 0.003) with mean DNA migration length, of age (P = 0.032) with basal cells, of BMI (P = 0.011) with pyknotic cells and of duration in sports activity in hockey players with karyolytic cells (P = 0.033) was found [Table 4]. In baseball-softball players, diet (P = 0.037), age (P = 0.052) and BMI (P = 0.008) were found to be significantly contributing to increased frequencies of micronucleus, binucleated cells and karyolitic cells, respectively. The same factors were also found to be linearly correlated (Pearson correlation, multiple linear regression).
|Table 4: Multivariate ANOVA, pearson correlation and multiple linear regression analysis of comet parameters in players|
Click here to view
The cells with varying tail lengths as observed in the SCGE assay are presented in [Table 5]. The Chi-square analysis revealed almost equal number of cells without tails (category 0) in both hockey and baseball-softball players (P = 0.4836), although these cells were decreased from those in the control group. For categories I (P = 0.0001), II (P = 0.0001) and III (P = 0.0054), more cells with varying tail lengths were observed in hockey players in comparison withthose in baseball-softball players. The cells in these three categories observed (both in hockey and baseball-softball) were significantly elevated from those in the control group.
|Table 5: Cells with different grades of DNA damage of players and control individuals|
Click here to view
| > Discussion|| |
In the present study, DNA and chromosomal damage in hockey and baseball-softball players was observed to be significantly increased (P = 0.000) in peripheral blood leukocytes and buccal epithelial cells of both hockey and baseball players in comparison with baseline damage in matched healthy, sedentary controls. Comparison of the respective genetic damage between players of both sport categories was however nonsignificant. In hockey players, the variables that showed an association in the buccal MN cytome assay parameters included age, BMI and years of activity in the game; in baseball-softball players, BMI and age were associated. In the SCGE assay indices, diet and mobile usage showed association in hockey players, while no variable was confounder in baseball players. Correlation and linear regression analysis also related the same variables in affecting DNA and chromosome damage.
The increased genomic damage in players may have arisen via a number of pathways. As intensive training sessions and competitive events for dynamic sport include both aerobic and anaerobic exercises, , the increased (10-15-fold) oxygen consumption can initiate a stress response if the quenching of the produced ROS does not occur by the endogenous/exogenous antioxidants.
There is irrefutable evidence that free radicals are involved in disease pathogenicity (diabetes, heart, inflammation, pulmonary disease, cancer), physiological processes (muscle contractile activity, ischemia/reperfusion, xanthine oxidase metabolism, aging) and, at the cellular level, cause increase in oxidation of proteins, lipids and nucleic acids. , DNA has high susceptibility to free radicals, causing strand breakage and base deletion on oxygen radical-DNA interaction.  DNA damage induced by ROS includes single-strand breaks, double-strand breaks and modification of purines, pyrimidines, deoxyriboses, DNA-intrastrand adducts and DNA-protein cross-links.  The consequences of DNA damage cause transcription to be arrested as well as cause replication errors and genomic instability, which can manifest in carcinogenesis.  ROS can also affect the DNA repair process, activate signal-transduction molecules and influence proteins required for cellular activities like apoptosis and DNA repair and, in this manner, participate in carcinogenesis. 
As during exercising (training and sports events), there occurs a 10-15fold increase in oxygen consumption, inflammatory reactions possibly cause tissue injuries and phagocyte activation, all of which can initiate the production of free radicals.  This could well be occurring in the buccal cells and peripheral blood leukocytes of the players in the present study, leading to the observed significant increase in genetic damage. In the buccal MN cytome assay, there was increased cell proliferation (basal cells), cytokinetic effect (binucleated cells) and cell death (karyorrhectic, condensed chromatin, karyolitic and pyknotic cells), while the RI was significantly (P = 0.000) decreased, as the production of KL and KR anomalies represents the repair process before cytogenetic damage due to intercellular selection. 
Damage at the DNA level in peripheral blood leukocytes (comet assay) was also significantly raised as observed by the DNA migration length. Significant differences in DNA migration lengths (approximately thrice) between players and controls imply more genetic damage. The comet tail result from double-strand breaks, single-strand breaks and any alkali-labile sites and, the longer the tail, higher is the damage.  DF indicates the cells with tail, i.e. the proportion of index (DI) relates to cell gradation based on DNA migration lengths.  Both DF (three-folds) and DI (four-folds) were elevated and, on comparing the prevalence of various cell categories, significance for categories 0, I, II and III in hockey, baseball players and controls were observed, indicating a higher extent of DNA damage in hockey players compared with that in baseball-softball players.
Oxidative stress (excess of oxygen/nitrogen species) has been documented in soccer and handball players , but not in hockey and baseball-softball players, although given that both are explosive sports, increased oxidative stress would also be occurring in them. Sports activities and strenuous exercises with significantly increased ROS and reactive nitrogen species include handball,  football  and soccer.  Packer et al. and Halliwell et al.  documented increased production of ROS/reactive nitrogen species when exercise is strenuous or acute.
The other modalities in which oxidative stress is elicited include the inflammatory response following muscle/tissue injury, wherein it may alter the production of neutrophils leading to increased production of free radicals, superoxide anions and cause oxidative stress;  mitochondrial leakage as oxygen uptake if increased; ischemia-reperfusion , can lead to oxidative stress.
Oxygen consumption is beneficial as it can lead to the stimulation of the endogenous antioxidant mechanism, whereby cytosolic enzymes (catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase) show increased production. 
Moderate exercise also acted as an antioxidant and upregulate the antioxidant genes.  Dietary antioxidant supplements can rectify an imbalance as reports on various supplements in rats  and in iron triathlon runners  and it reversed the stress outcome with revealed reverse stress outcome. There needs to be enhancing sport-performance and mitigate oxidative stress and, subsequently, presenting damage and its live consequences in terms of age-related diseases and cancer.
| > Conclusion|| |
It is of importance to note that there was a significant increase in genomic instability at both chromosomal and DNA levels in players of the two sports. DNA damage in peripheral blood leukocytes as observed in the SCGE assay in respect of increased DNA migration, number of cells with tails (DF) and DI (single-strand breaks) relates to repairable damage that may be repaired if the leucocytes undergo further mitotic division(s) but which is presently being sustained in the leucocytes as these are terminally differentiated. The chromosomal damage visualized as MN and nuclear buds is the genetic damage that has been repaired since MN manifest (as a consequence of chromosome loss and/or chromosome nondisjunction) only after the cells have undergone cell division.
It is interesting to note that in players of both sports, there were no significant differences with respect to chromosomal and DNA damage, as also not observed between genders, indicating that the effects of periodic training and competitive events for each sport are comparable with no sex-specific influences and that the demands of the vigorous schedule are causing competitive genomic instability, predisposing them equally to enhanced risk for age-related neurodegenerative changes, atherosclerosis, cancer and precocious ageing.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]