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The Ala16Val MnSOD gene polymorphism modulates oxidative response to exercise
Clinical Biochemistry 46 (2013) 335–340
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The Ala16Val MnSOD gene polymorphism modulates oxidative response to exercise Guilherme Bresciani a, b,⁎, Javier González-Gallego a, Ivana B. da Cruz b, Jose A. de Paz a, María J. Cuevas a a b
Institute of Biomedicine (IBIOMED), University of León, Spain Programa de Pós-Graduação em Ciências Biológicas: Bioquímica Toxicológica, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria (UFSM), RS, Brazil
a r t i c l e
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Article history: Received 4 June 2012 Received in revised form 20 November 2012 Accepted 22 November 2012 Available online 5 December 2012 Keywords: MnSOD polymorphism Gene expression Enzyme activity Redox balance Physical exercise
a b s t r a c t Objectives: In humans, the manganese-superoxide dismutase (MnSOD) gene contains a polymorphism (Ala16Val) that has been related to several metabolic dysfunctions and chronic diseases. However, the obtained results suggest that risks related to this polymorphism are directly influenced by environmental factors. Because few studies have analyzed this possible influence, we performed a controlled study to evaluate if the oxidative stress caused by exercise is differentially modulated by the Ala16Val MnSOD polymorphism. Design and methods: Fifty-seven males were previously genotyped and 10 subjects per genotype were selected to perform a bout of controlled intense exercise. MnSOD mRNA expression, protein content, enzyme activity, and total glutathione and thiol content from peripheral blood mononuclear cells were evaluated before and 1 h after a bout of intense exercise. Results: The AA genotype participants showed increased post-exercise MnSOD mRNA expression and enzyme activity compared to baseline values. Conversely, MnSOD mRNA expression did not change but protein thiol content decreased significantly after the bout of exercise in VV carriers. A comparison of the genotypes showed that the AA genotype presented a higher MnSOD protein content than VV volunteers after exercise; while a dose-effect for the A allele was found for enzyme activity. Conclusion: This study supports recent evidence that genotypes of key antioxidant enzymes may be associated with differential oxidative stress modulation and the hypothesis that the risk of disease associated with the MnSOD Ala16Val gene polymorphism may be controlled by environmental factors. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction Cells express a nucleus-encoded, mitochondrial localized manganesesuperoxide dismutase (MnSOD) to protect mitochondria from superoxide (O2−)-mediated damage. Previous studies confirmed that MnSOD is essential for life as illustrated by its neonatal lethality in knockout mice [1,2]. A single nucleotide polymorphism (SNP) has been identified in the MnSOD encoding gene, with genetic variants found to contain a structural mutation of a thiamine (T) substituted for a cytosine (C) in the exon 2. The substitution affects the 16th codon, mutating a valine (GTT) into an alanine (GCT) (Ala16Val). This protein modification produces a β-sheet secondary structure instead of the expected α-helix structure [3]. The Ala16Val polymorphism has been associated with several diseases, such as breast and prostate cancers and cardiovascular dysfunctions, and risk factors, such as hypercholesterolemia [4–8]. However, the obtained results are still contradictory, suggesting environmental influences on risks of disease related to the Ala16Val MnSOD polymorphism [9], and investigations with controlled conditions of environmental variables and this polymorphism are still incipient. Because intense or unaccustomed physical exercise increases oxygen ⁎ Corresponding author at: Laboratório de Bioquímica do Exercício, Centro de Educação Física e Desportos, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail address: [email protected] (G. Bresciani).
consumption, enhances reactive oxidative species (ROS) production and thus causes oxidative stress [10,11], exercise may be an environmental variable used as an in vivo model to investigate acute oxidative stress effects on humans [10,12]. The relevance in the analysis of an exercise effect is based on evidence that shows that mitochondria are particularly susceptible to oxidative damage from O2− generated by metabolic pathways activated during exercise [10,13]. Because ROS are reported to induce endogenous antioxidant enzymes [14], exercise-related ROS production is a reliable method to study the molecular pathways related to the MnSOD modulation during oxidative stress. Therefore, we analyzed the influence of the MnSOD Ala16Val gene polymorphism on gene expression, protein content, enzyme activity, and the redox status of biochemical markers after a controlled bout of intense exercise in young men. 2. Methods 2.1. Participants and experimental design Fifty-seven males (20.6 ± 1.8 yr) participated in the study, which was approved by the Ethics Committee of the University of León and carried out according to the Declaration of Helsinki. Written inform consent to enroll in the study was then obtained from participants
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.11.020
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who were not taking inflammatory, hormonal, or antioxidant supplements. After obtaining a blood sample for MnSOD Ala16Val genotyping and full medical screening, the participants were weighed, had their height measured, and had their body fat calculated according to a previously described equation [15]. A VO2max test until exhaustion was carried out on a cycle ergometer with electromagnetic brakes (Ergoline, GmHLindestrable, Bitz, Germany). Participants warmed up for 5 min with 25 W workload. After, the workload was increased by 25 W/min−1 until exhaustion. Achievement of VO2max was considered to be the attainment of at least two of the following criteria: (a) participants' volitional exhaustion, (b) a plateau in oxygen consumption (VO2), or (c) a heart rate (HR) ±10 beats min−1 of age-predicted maximal HR (Polar S810, Polar Electro OY, Kempele, Finland). Ventilatory and gas exchange variables were continuously monitored and collected breath-by-breath using an automated and open-circuit system (CPX plus, Medical Graphics, St. Paul, MN, USA). The data were averaged every 5 s, and the metabolic cart was calibrated with calibration gas mixtures of known O2 and CO2 concentrations (accuracy 0.01%) as provided by the manufacturer (Medical Graphics, St. Paul, MN, USA). The highest VO2 obtained during the incremental exercise test was taken as the VO2max whereas the intensity obtained just before exhaustion is referred to as peak power (Wmax). A bout of intense exercise was performed two weeks after the all-out test. Participants had to maintain a pedaling rate of 70 r·min−1 for 40 min at 75% of the VO2max obtained on the previous test. The Wmax at 75% was adjusted by extrapolation depending on the VO2max obtained during the all-out test. The VO2 was registered along the bout of intense exercise: 1) during the first 5 min; 2) from 10 to 15 min; 3) 20 to 25 min; and 4) 30 min until the end of the test. The HR was continuously monitored along the bout of exercise. Blood samples were obtained immediately before and 1 h after the exercise bout. The participants consumed 500 mL of commercial mineral water between blood sampling to avoid dehydration. A small amount of blood sample was used as a hematocrit measurement (Haemofuge A, Heraeus Sepatech, Germany). The VO2, HR, and W were measured using the same devices along the whole study. 2.2. MnSOD Ala16Val genotyping A peripheral blood sample (5 mL) was drawn from the antecubital vein using EDTA as anticoagulant. Genomic DNA was isolated from leukocytes using a GFX Genomic Blood DNA Purification Kit (Amersham Biosciences Inc., Uppsala, Sweden), and MnSOD genotyping was performed according to Gottlieb et al. [6]. 2.3. Dietary intake A 24 h self-administered dietary recall was used to assess food intake prior to the bout of intense exercise. The food composition present in the records was calculated using the NutriberR software, version 1.1.3 (Spain), and the values are presented as the mean ± standard error of means (SEM). 2.4. Analytical procedures Blood samples (35 mL) were obtained from the antecubital vein and immediately centrifuged at 1500 ×g for 10 min at 4 °C. The peripheral blood mononuclear cells (PBMC) were separated as previously described [17] and used since they are reliable cells to investigate exercise-related oxidative stress and inflammation [16,17]. Total RNA was isolated from PBMCs by using a RiboPure™ Blood Kit (Ambion Inc., Austin, TX, USA) and quantified by the fluorescent method Ribogreen RNA Quantification Kit (Molecular Probes, Leiden, The Netherlands) as described elsewhere [18]. TaqMan primers and probes for MnSOD (Genbank M36693.1 and Hs00167309) and 18S (housekeeping
gene) rRNA (Genbank X03205.1 and Hs 99999901_s1) were designed from the commercially available TaqMan® Assays-on-Demand Gene (Applied Biosystems, Foster City, CA, USA). Relative changes in gene expression levels were determined using the 2−ΔΔct method as described previously [19]. For Western blot analysis, PBMCs were homogenized with 150 μl of 0.25 mM sucrose, 1 mM EDTA, 10 mM Tris and a protease inhibitor cocktail [20]. Samples containing 50 μg of protein were separated by SDSpolyacrylamide gel electrophoresis (9% acrylamide) and transferred to PVDF membranes. Non-specific binding was blocked by preincubation of the PVDF in PBS containing 5% bovine serum albumin for 1 h. The membranes were then incubated overnight at 4 °C with appropriate antibodies. Bound primary antibody was detected using a peroxidaseconjugated secondary antibody (Amersham Inc., Piscataway, NJ, USA). The blot was stripped in 6.25 mM Tris, pH 6.7, 2% SDS and 100 mM β-mercaptoethanol at 50 °C for 15 min and probed again with antibeta-actin antibodies (Sigma-Aldrich, St. Louis, MO, USA) (42 kDa) to verify equal protein loading in each lane. MnSOD activity was determined in PBMCs by following the MnSOD inhibition of the reaction of O2− with nitroblue tetrazolium (NBT) as previously described [21] and expressed as U/mg protein. The automated glutathione recycling method described elsewhere [22] was used to assess total glutathione content (tGSH) using a microtiter plate reader (Synergy™ HT Multi-Mode Microplate Reader, Bio-Tek Instruments Inc., Winooski, VT, USA). The protein thiol content (−SH) was analyzed with a spectrophotometric method adapted for use on a 96-well plate reader [23]. Total glutathione is expressed as μmol GSH/mg protein and -SH groups are expressed as nmol SH/mg protein. 2.5. Statistical analysis Data were expressed as the mean± SEM. Allele frequencies were estimated using the gene-counting method. Chi square analysis was used to estimate the Hardy–Weinberg equilibrium. The results for RT-PCR and western blotting were presented as percentages from baseline values. Student's t-test was used to determine significant differences between the mean for the individual response to the bout of exercise. A one-way analysis of variance (ANOVA) with the factor genotype was also used. Bonferroni post hoc analysis was applied where appropriate. SPSS 15.0 (Statistical Package for Social Sciences, Chicago, IL, USA) was used, and statistical significance was set at P b 0.05. 3. Results The genotype and allelic frequencies of the initial samples were calculated, and 25% AA (n=14), 33% VV (n=19), and 42% AV (n=24) were observed. Calculated allelic frequencies of the A and V alleles were of 0.456 and 0.544, respectively. The gene frequencies were all in HardyWeinberg equilibrium for the groups investigated (P=0.273), indicating the samples were homogenous, and avoiding possible genetic variations on the measurements performed. Table 1 presents results from the anthropometric and functional parameters, and Table 2 depicts food intake data. No differences between genotypes were observed. VO2 data and respiratory quotient did not significantly differ among genotypes during exercise (Table 3). Mean and maximal HR assessed during the bout of exercise and baseline and post-exercise hematocrit levels were also similar among genotypes (Table 4). Fig. 1 depicts MnSOD mRNA levels, protein content and activity for each genotype. Heterozygous and VV homozygous participants did not show significant changes in mRNA expression from baseline to post-exercise values. The AA homozygous participants registered a significant increase in mRNA gene expression after the bout of intense exercise (P = 0.038). No significant differences were found between genotypes. The AV, AA, and VV genotypes showed no differences between baseline and post-exercise values for MnSOD protein content.
G. Bresciani et al. / Clinical Biochemistry 46 (2013) 335–340 Table 1 Anthropometric and functional assessment of participants.
Age (years) Weight (kg) Height (cm) BMI Body fat (%) VO2max (mL/kg·min−1) Peak power (Wmax)
AA
AV
VV
21.0 ± 0.7 76.4 ± 3.3 176.0 ± 1.8 24.6 ± 0.8 12.0 ± 1.1 58.2 ± 2.8 302.5 ± 9.4
20.0 ± 0.5 73.3 ± 2.2 177.8 ± 1.7 23.2 ± 0.7 10.6 ± 0.6 60.0 ± 1.9 312.5 ± 5.5
20.9 ± 0.4 75.2 ± 1.7 177.3 ± 1.5 23.8 ± 0.2 10.9 ± 0.5 57.8 ± 0.9 317.5 ± 7.5
Values are the mean ± SEM. BMI: body mass index; VO2max: maximal oxygen uptake.
The AA genotype presented a higher protein content after the bout of intense exercise when compared to the VV group (P=0.044). MnSOD enzyme activity increased significantly for both AA and AV genotypes after the bout of intense exercise (P=0.013; and P=0.018, respectively). A decrease in MnSOD activity was observed for VV subjects, but it did not reach a statistical significance. Moreover, post-exercise values were significantly lower in VV homozygous compared to the AA genotype (P=0.017). The total GSH content showed no differences post-exercise and between MnSOD Ala16Val genotypes. Nevertheless, thiol levels decreased significantly in VV homozygous participants (P=0.042), as observed in Fig. 2. 4. Discussion The results presented herein show differences in MnSOD mRNA expression, protein content, and enzyme activity after a bout of intense exercise according to distinct MnSOD Ala16Val gene polymorphism genotypes. To our knowledge, this is the first study to describe a differential MnSOD total status response related to the Ala16Val gene polymorphism using an in vivo model of increased ROS generation. The research was designed following the recommendations regarding data analysis of genotype prevalence and gene-disease interactions, which can also be applied to gene-exercise interactions [24]. Anthropometric assessment and performance data obtained during the bout of intense exercise showed no significant differences among genotypes. Moreover, data from a 3-day dietary questionnaire were analyzed, and no differences in the intake of antioxidant vitamins (such as C and D) and enzyme cofactors (such as zinc and copper) were found between genotypes [25]. Finally, genotypes and allele distribution were in accordance to the Hardy–Weinberg equilibrium, confirming the homogeneity of the sample to avoid major data bias. Currently, it has become clear that the ROS produced by exercise also act as signaling molecules to enhance the expression of antioxidant enzymes. MnSOD mRNA expression has been reported to increase in skeletal muscle of rats submitted to exercise, partly due to ROS production [26]. MnSOD overexpression is associated with increased levels of H2O2 [27,28], which may reach the cytosol and activate many redox
Table 2 Dietary intake estimation prior to the bout of intense exercise. AA
AV
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Table 3 Oxygen uptake (mL/kg·min−1) and respiratory quotient (arbitrary units) during the bout of intense exercise. Sampling
1 2 3 4
AA
AV
VV
VO2
RQ
VO2
RQ
VO2
RQ
39.7±1.1 43.2±1.7 42.5±2.1 42.6±1.6
1.04±0.01 0.99±0.03 0.95±0.03 0.95±0.02
41.1±1.5 46.6±1.9 45.4±1.6 46.4±2.3
1.02±0.02 1.00±0.02 0.97±0.02 0.97±0.03
41.1±1.5 46.6±1.9 45.4±1.6 46.4±2.3
1.05±0.02 1.00±0.01 0.98±0.02 0.96±0.03
Values are the mean±SEM. 1: first 5 min; 2: from 10 to 15 min; 3: from 20 to 25 min; 4: from 30 min until the end of the test. VO2: oxygen uptake. RQ: respiratory quotient.
signaling events [29,30]. In a study that investigated the differences of H2O2 production between men and women after a bout of exercise, lymphocyte MnSOD mRNA expression and enzyme activity were found to be higher in men; this result was attributed to their higher H2O2 production [31]. H2O2 could be the key molecule implicated in the increased MnSOD mRNA expression observed in the AA participants, in which a higher O2− dismutation would result in greater H2O2 concentrations. Due to its membrane permeability, H2O2 would easily reach the cytosol, where its signaling pathways would activate transcription factors and, in turn, increase the mRNA levels of the gene encoding MnSOD. A potential limitation of our research relies on the absence of female participants in the experimental protocol. However, the female gender presents dramatic variations in estrogen metabolism related to hormonal contraceptive intake or even to a monthly hormonal oscillation associated to the ovarian cycle that may severely interfere in the obtained results. Previous studies have shown that steroid hormones may influence the modulation of antioxidant enzymes [32], and also affect plasma markers related to O2− production [33,34]. This first study attempted to assess the potential effect of the Ala16Val gene polymorphism on the MnSOD modulation in a less biased in vivo model. Once the results herein obtained pointed out to a differentiate MnSOD modulation across this gene polymorphism after a bout of intense exercise, future studies including female participants are necessary. No differences in MnSOD protein content were found after the bout of intense exercise. Previous research also failed to find differences in MnSOD protein content after a bout of exercise in rats [26] or in humans running on a treadmill at the lactate threshold [35], in which individual baseline values differed markedly and changes in protein synthesis were most likely too small to be detected [35]. In the present study, AA subjects showed higher MnSOD protein content than VV individuals after the intense exercise, which may be related to the higher MnSOD mRNA expression in the former. It has been previously demonstrated that the Val-MnSOD precursor is partly arrested in the inner mitochondrial membrane and that the Ala-precursor generates 30–40% more of the active processed MnSOD homotetramer [36]. In this sense, the VV carriers showed a decrease in MnSOD protein content most likely due to deficient import of the protein into the mitochondria as previously described in in vitro assays [36]. The Val-MnSOD precursor captured in the mitochondrial membrane implies lower O2− dismutation into H2O2, causing cellular damage and possibly inducing apoptosis [37]. The A allele played a key role in the MnSOD activity values reached after exercise, with AA and AV subjects showing higher levels compared
VV
−1
Energy expenditure (kcal·day Fe (mg) Cu (mg) Zn (mg) Mn (mg) Se (μg) Ascorbic acid (mg) Retinol (μg) Calciferol (μg) Tocopherol (mg)
) 1778.7 ± 150.0 1799.1 ± 109.4 1849.0 ± 108.5 16.5 ± 2.2 17.1 ± 1.5 15.9 ± 1.4 0.9 ± 0.2 0.7 ± 0.1 0.7 ± 0.0 9.4 ± 1.0 10.2 ± 0.7 12.0 ± 1.9 3.5 ± 0.6 4.4 ± 2.0 3.6 ± 0.9 65.5 ± 8.0 62.6 ± 8.0 62.7 ± 6.1 128.6 ± 44.6 139.4 ± 29.6 130.0 ± 32.0 593.4 ± 86.0 687.1 ± 83.3 802.3 ± 169.8 7.0 ± 3.2 4.2 ± 1.6 7.3 ± 1.9 9.2 ± 2.4 6.9 ± 0.5 7.5 ± 0.9
Values are the mean±SEM. Kcal: kilocalories; Fe: iron; Cu: cooper; Zn: zinc; Mn: manganese; Se: selenium; mg: milligrams; μg: micrograms.
Table 4 Mean and maximal heart rate during the exercise bout and baseline and post-exercise hematocrit levels. Genotype
AA AV VV
Heart rate (bpm)
Hematocrit (%)
Mean
Maximal
Baseline
Post-exercise
174.7 ± 2.7 179.3 ± 2.7 173.8 ± 3.0
183.8 ± 2.2 186.4 ± 2.5 183.9 ± 3.6
44.3 ± 0.8 45.6 ± 0.8 46.2 ± 0.6
44.1 ± 0.8 45.6 ± 0.8 46.2 ± 0.6
Values are the mean ± SEM.
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Fig. 2. tGSH concentration (A) and −SH groups (B) before (black) and 1 h after (white) the bout of intense exercise. Values are the mean±SEM. *Significantly different from group baseline values. Significant differences set at b0.05. tGSH: total glutathione content. −SH: protein thiols content.
Fig. 1. MnSOD mRNA expression (A), protein content (B), and enzyme activity (C) before (black) and 1 h after (white) the bout of intense exercise. Values are the mean±SEM. *Significantly different from group baseline values; #Significantly different from AA post-exercise. Significant differences set at b0.05. %: percentage from baseline values.
to baseline values. Moreover, the AA genotype participants presented a higher activity after the bout of exercise than homozygous VV participants, and a genotype dose-response was reached. Previous research demonstrated that MnSOD activity increased in a mouse heart 1 h after a distressing stimulation [38]. We observed an increase of MnSOD activity 1 h after the exercise in A allele carriers; this finding indicates that intense exercise may also enhance MnSOD activation in oxidative stress situations. Investigations with mitochondria extracted from livers of MnSOD+/− and MnSOD+/+ rats found that MnSOD+/− rats have a 50% decrease in enzyme activity and a 30% decrease in GSH concentration [39]. We observed the same tendency in this study, considering that VV participants lacked an increase in MnSOD activity and reached higher levels of thiol group oxidation. The A allele carriers showed an increased MnSOD activity and did not present thiol groups oxidation. The mechanisms related to the decrease of tGSH and thiol groups after muscle contractile activity still need to be clarified; however, a few investigations attribute the mechanisms to thiol group oxidation by exercise-mediated ROS production [40]. It is well known that a few ROS induced a decrease in thiol groups as a response to oxidative stress [25]. The VV genotype participants decreased thiol levels when compared to baseline values, while A allele carriers did not show significant differences. This decrease in thiol content in VV genotype participants is accompanied by decreased protein content and enzyme activity when compared to A allele carriers, which may indicate a deficient antioxidant defense in VV genotype participants. In spite of this, it is also
plausible to argue that different oxidative and inflammatory markers may also present changes after a bout of intense exercise according to the MnSOD Ala16Val gene polymorphism genotype. Oxidant enzymes such as NADPH oxidase or even isoforms of SOD (such as cytosolic SOD) and inflammatory molecules like protein C reactive (PCR) or interleukins (IL-1β, IL-6, TNF-α) may also be influenced by different Ala16Val gene polymorphism genotypes. In this sense, Montano et al. [41] showed in vitro increases of pro-inflammatory cytokines associated with the VV genotype. However, due to the methodological design of the present study, blood sampling was taken before and after exercise and the total amount, sample processing and the number of analysis performed were limiting factors. This first study aimed at the responses of different MnSOD Ala16Val gene polymorphism genotypes to a bout of intense exercise in men. Considering the results found, future studies regarding the impact of the MnSOD Ala16Val gene polymorphism on the oxidative-inflammatory responses related to acute exercise are of interest. The relevance of this study relies on the impact of recent findings concerning disease-related gene associations [5–8,42]. The MnSOD gene polymorphism has been linked to several oxidative stress-related diseases, and the results are still inconclusive to whether the A or V allele may be a determinant on redox status maintenance [4,7]. A step toward understanding the mechanism underlying the involvement of Ala16Val MnSOD gene polymorphism on the mature enzyme synthesis has been taken [36,43]. However, studies with a greater number of participants aiming to assess long term moderate exercise effects on the MnSOD Ala16Val gene polymorphism modulation are also necessary. An increasing number of studies show that moderate exercise training is associated with disease prevention, such as cancer, cardiovascular, and cardiometabolic clinical outcomes. Moreover, a previous study suggests that the disease risk associated with the MnSOD Ala16Val gene polymorphism may be modulated by environmental factors such as diet [9]. Ambrossone et al. [9] showed that women carrying the AA genotype presented a 4-fold increase of breast cancer development;
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nevertheless, the risk was decreased on those women that reported high fruits and vegetables intake. Therefore, an open question is whether moderate exercise training may also prevent disease associated risks in subjects carrying the AA genotype. On the other hand, a consistent number of studies have shown an association between the VV genotype and obesity [7], hypercholesterolemia [5], oxidized-LDL and oxidativeinflammatory markers [6,44], and type 2 diabetes and microvascular complications or coronary heart disease [45,46]. Considering the different response to acute exercise presented by the MnSOD Ala16Val gene polymorphism genotypes in the present research, complementary studies to assess moderate exercise training effects on the modulation of cardiometabolic risk factors, such as obesity, dyslipidemia, and metabolic syndrome are necessary. In conclusion, investigations on the influence of gene variation on MnSOD signaling pathways are still incipient and are needed to better understand the interactions among genetics and redox status maintenance. In this sense, the present study notes the relevance of a gene polymorphism on a specific antioxidant enzymatic pathway to an exerciseinduced oxidative stress model. Nonetheless, matching studies need to be performed to analyze whether other environmental variables, such as antioxidants intake, may also influence the differential response of the MnSOD Ala16Val gene polymorphism genotypes on oxidative metabolic markers submitted to stress stimuli modulation. 5. Conclusions The MnSOD Ala16Val gene polymorphism presents a role on oxidative stress modulation caused by environmental factors, such as intense exercise. This condition could affect clinical and biochemical variables related to chronic disease risks. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments Authors are indebted with Leonardo Barili Brandi for technical support with figure editing. References [1] Li Y, Huang TT, Carlson EJ, Melov S, Ursell PC, Olson JL, et al. Dilated cardiomyopathy and neonatal lethalityin mutant mice lacking manganese superoxide dismutase. Nat Genet 1995;11:76–381. [2] Lebovitz RM, Zhang H, Vogel H, Cartwright Jr J, Dionne L, Lu N, et al. Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc Natl Acad Sci U S A 1996;3:9782–7. [3] Shimoda-Matsubayashi S, Matsumine H, Kobayashi T, Nakagawa-Hattori Y, Shimizu Y, Mizuno Y. Structural dimorphism in the mitochondrial targeting sequence in the human manganese superoxide dismutase gene, A predictive evidence for conformational change to influence mitochondrial transport and a study of allelic association in Parkinson's disease. Biochem Biophys Res Commun 1996;226:561–5. [4] Bica CG, da Silva LL, Toscani NV, Zettler CG, Gottlieb MG, Alexandre CO, et al. Polymorphism (ALA16VAL) correlates with regional lymph node status in breast cancer. Cancer Genet Cytogenet 2010;196:153–8. [5] Duarte MM, Moresco RN, Duarte T, Santi A, Bagatini MD, Da Cruz IB, et al. Oxidative stress in hypercholesterolemia and its association with Ala16Val superoxide dismutase gene polymorphism. Clin Biochem 2010;43:1118–23. [6] Gottlieb MG, Schwanke CH, Santos AF, Jobim PF, Müssel DP, da Cruz IB. Association among oxidized LDL levels, SODMn, apolipoprotein E polymorphisms, and cardiovascular risk factors in a south Brazilian region population. Genet Mol Res 2005;4:691–703. [7] Montano MA, Barrio Lera JP, Gottlieb MG, Schwanke CH, da Rocha MI, Manica-Cattani MF, et al. Association between manganese superoxide dismutase (MnSOD) gene polymorphism and elderly obesity. Mol Cell Biochem 2009;328:33–40. [8] Taufer M, Peres A, de Andrade VM, de Oliveira G, M.E. do Canto G Sá, dos Santos AR, et al. Is the Val16Ala manganese superoxide dismutase polymorphism associated with the aging process? J Gerontol A Biol Sci Med Sci 2005;60(4):432–8. [9] Ambrosone CB, Freudenheim JL, Thompson PA, Bowman E, Vena JE, Marshall JR, et al. Manganese superoxide dismutase (MnSOD) genetic polymorphisms, dietary antioxidants, and risk of breast cancer. Cancer Res 1999;59:602–6.
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
The Ala16Val MnSOD gene polymorphism modulates oxidative response to exercise Guilherme Bresciani a, b,⁎, Javier González-Gallego a, Ivana B. da Cruz b, Jose A. de Paz a, María J. Cuevas a a b
Institute of Biomedicine (IBIOMED), University of León, Spain Programa de Pós-Graduação em Ciências Biológicas: Bioquímica Toxicológica, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria (UFSM), RS, Brazil
a r t i c l e
i n f o
Article history: Received 4 June 2012 Received in revised form 20 November 2012 Accepted 22 November 2012 Available online 5 December 2012 Keywords: MnSOD polymorphism Gene expression Enzyme activity Redox balance Physical exercise
a b s t r a c t Objectives: In humans, the manganese-superoxide dismutase (MnSOD) gene contains a polymorphism (Ala16Val) that has been related to several metabolic dysfunctions and chronic diseases. However, the obtained results suggest that risks related to this polymorphism are directly influenced by environmental factors. Because few studies have analyzed this possible influence, we performed a controlled study to evaluate if the oxidative stress caused by exercise is differentially modulated by the Ala16Val MnSOD polymorphism. Design and methods: Fifty-seven males were previously genotyped and 10 subjects per genotype were selected to perform a bout of controlled intense exercise. MnSOD mRNA expression, protein content, enzyme activity, and total glutathione and thiol content from peripheral blood mononuclear cells were evaluated before and 1 h after a bout of intense exercise. Results: The AA genotype participants showed increased post-exercise MnSOD mRNA expression and enzyme activity compared to baseline values. Conversely, MnSOD mRNA expression did not change but protein thiol content decreased significantly after the bout of exercise in VV carriers. A comparison of the genotypes showed that the AA genotype presented a higher MnSOD protein content than VV volunteers after exercise; while a dose-effect for the A allele was found for enzyme activity. Conclusion: This study supports recent evidence that genotypes of key antioxidant enzymes may be associated with differential oxidative stress modulation and the hypothesis that the risk of disease associated with the MnSOD Ala16Val gene polymorphism may be controlled by environmental factors. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction Cells express a nucleus-encoded, mitochondrial localized manganesesuperoxide dismutase (MnSOD) to protect mitochondria from superoxide (O2−)-mediated damage. Previous studies confirmed that MnSOD is essential for life as illustrated by its neonatal lethality in knockout mice [1,2]. A single nucleotide polymorphism (SNP) has been identified in the MnSOD encoding gene, with genetic variants found to contain a structural mutation of a thiamine (T) substituted for a cytosine (C) in the exon 2. The substitution affects the 16th codon, mutating a valine (GTT) into an alanine (GCT) (Ala16Val). This protein modification produces a β-sheet secondary structure instead of the expected α-helix structure [3]. The Ala16Val polymorphism has been associated with several diseases, such as breast and prostate cancers and cardiovascular dysfunctions, and risk factors, such as hypercholesterolemia [4–8]. However, the obtained results are still contradictory, suggesting environmental influences on risks of disease related to the Ala16Val MnSOD polymorphism [9], and investigations with controlled conditions of environmental variables and this polymorphism are still incipient. Because intense or unaccustomed physical exercise increases oxygen ⁎ Corresponding author at: Laboratório de Bioquímica do Exercício, Centro de Educação Física e Desportos, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail address: [email protected] (G. Bresciani).
consumption, enhances reactive oxidative species (ROS) production and thus causes oxidative stress [10,11], exercise may be an environmental variable used as an in vivo model to investigate acute oxidative stress effects on humans [10,12]. The relevance in the analysis of an exercise effect is based on evidence that shows that mitochondria are particularly susceptible to oxidative damage from O2− generated by metabolic pathways activated during exercise [10,13]. Because ROS are reported to induce endogenous antioxidant enzymes [14], exercise-related ROS production is a reliable method to study the molecular pathways related to the MnSOD modulation during oxidative stress. Therefore, we analyzed the influence of the MnSOD Ala16Val gene polymorphism on gene expression, protein content, enzyme activity, and the redox status of biochemical markers after a controlled bout of intense exercise in young men. 2. Methods 2.1. Participants and experimental design Fifty-seven males (20.6 ± 1.8 yr) participated in the study, which was approved by the Ethics Committee of the University of León and carried out according to the Declaration of Helsinki. Written inform consent to enroll in the study was then obtained from participants
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.11.020
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who were not taking inflammatory, hormonal, or antioxidant supplements. After obtaining a blood sample for MnSOD Ala16Val genotyping and full medical screening, the participants were weighed, had their height measured, and had their body fat calculated according to a previously described equation [15]. A VO2max test until exhaustion was carried out on a cycle ergometer with electromagnetic brakes (Ergoline, GmHLindestrable, Bitz, Germany). Participants warmed up for 5 min with 25 W workload. After, the workload was increased by 25 W/min−1 until exhaustion. Achievement of VO2max was considered to be the attainment of at least two of the following criteria: (a) participants' volitional exhaustion, (b) a plateau in oxygen consumption (VO2), or (c) a heart rate (HR) ±10 beats min−1 of age-predicted maximal HR (Polar S810, Polar Electro OY, Kempele, Finland). Ventilatory and gas exchange variables were continuously monitored and collected breath-by-breath using an automated and open-circuit system (CPX plus, Medical Graphics, St. Paul, MN, USA). The data were averaged every 5 s, and the metabolic cart was calibrated with calibration gas mixtures of known O2 and CO2 concentrations (accuracy 0.01%) as provided by the manufacturer (Medical Graphics, St. Paul, MN, USA). The highest VO2 obtained during the incremental exercise test was taken as the VO2max whereas the intensity obtained just before exhaustion is referred to as peak power (Wmax). A bout of intense exercise was performed two weeks after the all-out test. Participants had to maintain a pedaling rate of 70 r·min−1 for 40 min at 75% of the VO2max obtained on the previous test. The Wmax at 75% was adjusted by extrapolation depending on the VO2max obtained during the all-out test. The VO2 was registered along the bout of intense exercise: 1) during the first 5 min; 2) from 10 to 15 min; 3) 20 to 25 min; and 4) 30 min until the end of the test. The HR was continuously monitored along the bout of exercise. Blood samples were obtained immediately before and 1 h after the exercise bout. The participants consumed 500 mL of commercial mineral water between blood sampling to avoid dehydration. A small amount of blood sample was used as a hematocrit measurement (Haemofuge A, Heraeus Sepatech, Germany). The VO2, HR, and W were measured using the same devices along the whole study. 2.2. MnSOD Ala16Val genotyping A peripheral blood sample (5 mL) was drawn from the antecubital vein using EDTA as anticoagulant. Genomic DNA was isolated from leukocytes using a GFX Genomic Blood DNA Purification Kit (Amersham Biosciences Inc., Uppsala, Sweden), and MnSOD genotyping was performed according to Gottlieb et al. [6]. 2.3. Dietary intake A 24 h self-administered dietary recall was used to assess food intake prior to the bout of intense exercise. The food composition present in the records was calculated using the NutriberR software, version 1.1.3 (Spain), and the values are presented as the mean ± standard error of means (SEM). 2.4. Analytical procedures Blood samples (35 mL) were obtained from the antecubital vein and immediately centrifuged at 1500 ×g for 10 min at 4 °C. The peripheral blood mononuclear cells (PBMC) were separated as previously described [17] and used since they are reliable cells to investigate exercise-related oxidative stress and inflammation [16,17]. Total RNA was isolated from PBMCs by using a RiboPure™ Blood Kit (Ambion Inc., Austin, TX, USA) and quantified by the fluorescent method Ribogreen RNA Quantification Kit (Molecular Probes, Leiden, The Netherlands) as described elsewhere [18]. TaqMan primers and probes for MnSOD (Genbank M36693.1 and Hs00167309) and 18S (housekeeping
gene) rRNA (Genbank X03205.1 and Hs 99999901_s1) were designed from the commercially available TaqMan® Assays-on-Demand Gene (Applied Biosystems, Foster City, CA, USA). Relative changes in gene expression levels were determined using the 2−ΔΔct method as described previously [19]. For Western blot analysis, PBMCs were homogenized with 150 μl of 0.25 mM sucrose, 1 mM EDTA, 10 mM Tris and a protease inhibitor cocktail [20]. Samples containing 50 μg of protein were separated by SDSpolyacrylamide gel electrophoresis (9% acrylamide) and transferred to PVDF membranes. Non-specific binding was blocked by preincubation of the PVDF in PBS containing 5% bovine serum albumin for 1 h. The membranes were then incubated overnight at 4 °C with appropriate antibodies. Bound primary antibody was detected using a peroxidaseconjugated secondary antibody (Amersham Inc., Piscataway, NJ, USA). The blot was stripped in 6.25 mM Tris, pH 6.7, 2% SDS and 100 mM β-mercaptoethanol at 50 °C for 15 min and probed again with antibeta-actin antibodies (Sigma-Aldrich, St. Louis, MO, USA) (42 kDa) to verify equal protein loading in each lane. MnSOD activity was determined in PBMCs by following the MnSOD inhibition of the reaction of O2− with nitroblue tetrazolium (NBT) as previously described [21] and expressed as U/mg protein. The automated glutathione recycling method described elsewhere [22] was used to assess total glutathione content (tGSH) using a microtiter plate reader (Synergy™ HT Multi-Mode Microplate Reader, Bio-Tek Instruments Inc., Winooski, VT, USA). The protein thiol content (−SH) was analyzed with a spectrophotometric method adapted for use on a 96-well plate reader [23]. Total glutathione is expressed as μmol GSH/mg protein and -SH groups are expressed as nmol SH/mg protein. 2.5. Statistical analysis Data were expressed as the mean± SEM. Allele frequencies were estimated using the gene-counting method. Chi square analysis was used to estimate the Hardy–Weinberg equilibrium. The results for RT-PCR and western blotting were presented as percentages from baseline values. Student's t-test was used to determine significant differences between the mean for the individual response to the bout of exercise. A one-way analysis of variance (ANOVA) with the factor genotype was also used. Bonferroni post hoc analysis was applied where appropriate. SPSS 15.0 (Statistical Package for Social Sciences, Chicago, IL, USA) was used, and statistical significance was set at P b 0.05. 3. Results The genotype and allelic frequencies of the initial samples were calculated, and 25% AA (n=14), 33% VV (n=19), and 42% AV (n=24) were observed. Calculated allelic frequencies of the A and V alleles were of 0.456 and 0.544, respectively. The gene frequencies were all in HardyWeinberg equilibrium for the groups investigated (P=0.273), indicating the samples were homogenous, and avoiding possible genetic variations on the measurements performed. Table 1 presents results from the anthropometric and functional parameters, and Table 2 depicts food intake data. No differences between genotypes were observed. VO2 data and respiratory quotient did not significantly differ among genotypes during exercise (Table 3). Mean and maximal HR assessed during the bout of exercise and baseline and post-exercise hematocrit levels were also similar among genotypes (Table 4). Fig. 1 depicts MnSOD mRNA levels, protein content and activity for each genotype. Heterozygous and VV homozygous participants did not show significant changes in mRNA expression from baseline to post-exercise values. The AA homozygous participants registered a significant increase in mRNA gene expression after the bout of intense exercise (P = 0.038). No significant differences were found between genotypes. The AV, AA, and VV genotypes showed no differences between baseline and post-exercise values for MnSOD protein content.
G. Bresciani et al. / Clinical Biochemistry 46 (2013) 335–340 Table 1 Anthropometric and functional assessment of participants.
Age (years) Weight (kg) Height (cm) BMI Body fat (%) VO2max (mL/kg·min−1) Peak power (Wmax)
AA
AV
VV
21.0 ± 0.7 76.4 ± 3.3 176.0 ± 1.8 24.6 ± 0.8 12.0 ± 1.1 58.2 ± 2.8 302.5 ± 9.4
20.0 ± 0.5 73.3 ± 2.2 177.8 ± 1.7 23.2 ± 0.7 10.6 ± 0.6 60.0 ± 1.9 312.5 ± 5.5
20.9 ± 0.4 75.2 ± 1.7 177.3 ± 1.5 23.8 ± 0.2 10.9 ± 0.5 57.8 ± 0.9 317.5 ± 7.5
Values are the mean ± SEM. BMI: body mass index; VO2max: maximal oxygen uptake.
The AA genotype presented a higher protein content after the bout of intense exercise when compared to the VV group (P=0.044). MnSOD enzyme activity increased significantly for both AA and AV genotypes after the bout of intense exercise (P=0.013; and P=0.018, respectively). A decrease in MnSOD activity was observed for VV subjects, but it did not reach a statistical significance. Moreover, post-exercise values were significantly lower in VV homozygous compared to the AA genotype (P=0.017). The total GSH content showed no differences post-exercise and between MnSOD Ala16Val genotypes. Nevertheless, thiol levels decreased significantly in VV homozygous participants (P=0.042), as observed in Fig. 2. 4. Discussion The results presented herein show differences in MnSOD mRNA expression, protein content, and enzyme activity after a bout of intense exercise according to distinct MnSOD Ala16Val gene polymorphism genotypes. To our knowledge, this is the first study to describe a differential MnSOD total status response related to the Ala16Val gene polymorphism using an in vivo model of increased ROS generation. The research was designed following the recommendations regarding data analysis of genotype prevalence and gene-disease interactions, which can also be applied to gene-exercise interactions [24]. Anthropometric assessment and performance data obtained during the bout of intense exercise showed no significant differences among genotypes. Moreover, data from a 3-day dietary questionnaire were analyzed, and no differences in the intake of antioxidant vitamins (such as C and D) and enzyme cofactors (such as zinc and copper) were found between genotypes [25]. Finally, genotypes and allele distribution were in accordance to the Hardy–Weinberg equilibrium, confirming the homogeneity of the sample to avoid major data bias. Currently, it has become clear that the ROS produced by exercise also act as signaling molecules to enhance the expression of antioxidant enzymes. MnSOD mRNA expression has been reported to increase in skeletal muscle of rats submitted to exercise, partly due to ROS production [26]. MnSOD overexpression is associated with increased levels of H2O2 [27,28], which may reach the cytosol and activate many redox
Table 2 Dietary intake estimation prior to the bout of intense exercise. AA
AV
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Table 3 Oxygen uptake (mL/kg·min−1) and respiratory quotient (arbitrary units) during the bout of intense exercise. Sampling
1 2 3 4
AA
AV
VV
VO2
RQ
VO2
RQ
VO2
RQ
39.7±1.1 43.2±1.7 42.5±2.1 42.6±1.6
1.04±0.01 0.99±0.03 0.95±0.03 0.95±0.02
41.1±1.5 46.6±1.9 45.4±1.6 46.4±2.3
1.02±0.02 1.00±0.02 0.97±0.02 0.97±0.03
41.1±1.5 46.6±1.9 45.4±1.6 46.4±2.3
1.05±0.02 1.00±0.01 0.98±0.02 0.96±0.03
Values are the mean±SEM. 1: first 5 min; 2: from 10 to 15 min; 3: from 20 to 25 min; 4: from 30 min until the end of the test. VO2: oxygen uptake. RQ: respiratory quotient.
signaling events [29,30]. In a study that investigated the differences of H2O2 production between men and women after a bout of exercise, lymphocyte MnSOD mRNA expression and enzyme activity were found to be higher in men; this result was attributed to their higher H2O2 production [31]. H2O2 could be the key molecule implicated in the increased MnSOD mRNA expression observed in the AA participants, in which a higher O2− dismutation would result in greater H2O2 concentrations. Due to its membrane permeability, H2O2 would easily reach the cytosol, where its signaling pathways would activate transcription factors and, in turn, increase the mRNA levels of the gene encoding MnSOD. A potential limitation of our research relies on the absence of female participants in the experimental protocol. However, the female gender presents dramatic variations in estrogen metabolism related to hormonal contraceptive intake or even to a monthly hormonal oscillation associated to the ovarian cycle that may severely interfere in the obtained results. Previous studies have shown that steroid hormones may influence the modulation of antioxidant enzymes [32], and also affect plasma markers related to O2− production [33,34]. This first study attempted to assess the potential effect of the Ala16Val gene polymorphism on the MnSOD modulation in a less biased in vivo model. Once the results herein obtained pointed out to a differentiate MnSOD modulation across this gene polymorphism after a bout of intense exercise, future studies including female participants are necessary. No differences in MnSOD protein content were found after the bout of intense exercise. Previous research also failed to find differences in MnSOD protein content after a bout of exercise in rats [26] or in humans running on a treadmill at the lactate threshold [35], in which individual baseline values differed markedly and changes in protein synthesis were most likely too small to be detected [35]. In the present study, AA subjects showed higher MnSOD protein content than VV individuals after the intense exercise, which may be related to the higher MnSOD mRNA expression in the former. It has been previously demonstrated that the Val-MnSOD precursor is partly arrested in the inner mitochondrial membrane and that the Ala-precursor generates 30–40% more of the active processed MnSOD homotetramer [36]. In this sense, the VV carriers showed a decrease in MnSOD protein content most likely due to deficient import of the protein into the mitochondria as previously described in in vitro assays [36]. The Val-MnSOD precursor captured in the mitochondrial membrane implies lower O2− dismutation into H2O2, causing cellular damage and possibly inducing apoptosis [37]. The A allele played a key role in the MnSOD activity values reached after exercise, with AA and AV subjects showing higher levels compared
VV
−1
Energy expenditure (kcal·day Fe (mg) Cu (mg) Zn (mg) Mn (mg) Se (μg) Ascorbic acid (mg) Retinol (μg) Calciferol (μg) Tocopherol (mg)
) 1778.7 ± 150.0 1799.1 ± 109.4 1849.0 ± 108.5 16.5 ± 2.2 17.1 ± 1.5 15.9 ± 1.4 0.9 ± 0.2 0.7 ± 0.1 0.7 ± 0.0 9.4 ± 1.0 10.2 ± 0.7 12.0 ± 1.9 3.5 ± 0.6 4.4 ± 2.0 3.6 ± 0.9 65.5 ± 8.0 62.6 ± 8.0 62.7 ± 6.1 128.6 ± 44.6 139.4 ± 29.6 130.0 ± 32.0 593.4 ± 86.0 687.1 ± 83.3 802.3 ± 169.8 7.0 ± 3.2 4.2 ± 1.6 7.3 ± 1.9 9.2 ± 2.4 6.9 ± 0.5 7.5 ± 0.9
Values are the mean±SEM. Kcal: kilocalories; Fe: iron; Cu: cooper; Zn: zinc; Mn: manganese; Se: selenium; mg: milligrams; μg: micrograms.
Table 4 Mean and maximal heart rate during the exercise bout and baseline and post-exercise hematocrit levels. Genotype
AA AV VV
Heart rate (bpm)
Hematocrit (%)
Mean
Maximal
Baseline
Post-exercise
174.7 ± 2.7 179.3 ± 2.7 173.8 ± 3.0
183.8 ± 2.2 186.4 ± 2.5 183.9 ± 3.6
44.3 ± 0.8 45.6 ± 0.8 46.2 ± 0.6
44.1 ± 0.8 45.6 ± 0.8 46.2 ± 0.6
Values are the mean ± SEM.
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Fig. 2. tGSH concentration (A) and −SH groups (B) before (black) and 1 h after (white) the bout of intense exercise. Values are the mean±SEM. *Significantly different from group baseline values. Significant differences set at b0.05. tGSH: total glutathione content. −SH: protein thiols content.
Fig. 1. MnSOD mRNA expression (A), protein content (B), and enzyme activity (C) before (black) and 1 h after (white) the bout of intense exercise. Values are the mean±SEM. *Significantly different from group baseline values; #Significantly different from AA post-exercise. Significant differences set at b0.05. %: percentage from baseline values.
to baseline values. Moreover, the AA genotype participants presented a higher activity after the bout of exercise than homozygous VV participants, and a genotype dose-response was reached. Previous research demonstrated that MnSOD activity increased in a mouse heart 1 h after a distressing stimulation [38]. We observed an increase of MnSOD activity 1 h after the exercise in A allele carriers; this finding indicates that intense exercise may also enhance MnSOD activation in oxidative stress situations. Investigations with mitochondria extracted from livers of MnSOD+/− and MnSOD+/+ rats found that MnSOD+/− rats have a 50% decrease in enzyme activity and a 30% decrease in GSH concentration [39]. We observed the same tendency in this study, considering that VV participants lacked an increase in MnSOD activity and reached higher levels of thiol group oxidation. The A allele carriers showed an increased MnSOD activity and did not present thiol groups oxidation. The mechanisms related to the decrease of tGSH and thiol groups after muscle contractile activity still need to be clarified; however, a few investigations attribute the mechanisms to thiol group oxidation by exercise-mediated ROS production [40]. It is well known that a few ROS induced a decrease in thiol groups as a response to oxidative stress [25]. The VV genotype participants decreased thiol levels when compared to baseline values, while A allele carriers did not show significant differences. This decrease in thiol content in VV genotype participants is accompanied by decreased protein content and enzyme activity when compared to A allele carriers, which may indicate a deficient antioxidant defense in VV genotype participants. In spite of this, it is also
plausible to argue that different oxidative and inflammatory markers may also present changes after a bout of intense exercise according to the MnSOD Ala16Val gene polymorphism genotype. Oxidant enzymes such as NADPH oxidase or even isoforms of SOD (such as cytosolic SOD) and inflammatory molecules like protein C reactive (PCR) or interleukins (IL-1β, IL-6, TNF-α) may also be influenced by different Ala16Val gene polymorphism genotypes. In this sense, Montano et al. [41] showed in vitro increases of pro-inflammatory cytokines associated with the VV genotype. However, due to the methodological design of the present study, blood sampling was taken before and after exercise and the total amount, sample processing and the number of analysis performed were limiting factors. This first study aimed at the responses of different MnSOD Ala16Val gene polymorphism genotypes to a bout of intense exercise in men. Considering the results found, future studies regarding the impact of the MnSOD Ala16Val gene polymorphism on the oxidative-inflammatory responses related to acute exercise are of interest. The relevance of this study relies on the impact of recent findings concerning disease-related gene associations [5–8,42]. The MnSOD gene polymorphism has been linked to several oxidative stress-related diseases, and the results are still inconclusive to whether the A or V allele may be a determinant on redox status maintenance [4,7]. A step toward understanding the mechanism underlying the involvement of Ala16Val MnSOD gene polymorphism on the mature enzyme synthesis has been taken [36,43]. However, studies with a greater number of participants aiming to assess long term moderate exercise effects on the MnSOD Ala16Val gene polymorphism modulation are also necessary. An increasing number of studies show that moderate exercise training is associated with disease prevention, such as cancer, cardiovascular, and cardiometabolic clinical outcomes. Moreover, a previous study suggests that the disease risk associated with the MnSOD Ala16Val gene polymorphism may be modulated by environmental factors such as diet [9]. Ambrossone et al. [9] showed that women carrying the AA genotype presented a 4-fold increase of breast cancer development;
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nevertheless, the risk was decreased on those women that reported high fruits and vegetables intake. Therefore, an open question is whether moderate exercise training may also prevent disease associated risks in subjects carrying the AA genotype. On the other hand, a consistent number of studies have shown an association between the VV genotype and obesity [7], hypercholesterolemia [5], oxidized-LDL and oxidativeinflammatory markers [6,44], and type 2 diabetes and microvascular complications or coronary heart disease [45,46]. Considering the different response to acute exercise presented by the MnSOD Ala16Val gene polymorphism genotypes in the present research, complementary studies to assess moderate exercise training effects on the modulation of cardiometabolic risk factors, such as obesity, dyslipidemia, and metabolic syndrome are necessary. In conclusion, investigations on the influence of gene variation on MnSOD signaling pathways are still incipient and are needed to better understand the interactions among genetics and redox status maintenance. In this sense, the present study notes the relevance of a gene polymorphism on a specific antioxidant enzymatic pathway to an exerciseinduced oxidative stress model. Nonetheless, matching studies need to be performed to analyze whether other environmental variables, such as antioxidants intake, may also influence the differential response of the MnSOD Ala16Val gene polymorphism genotypes on oxidative metabolic markers submitted to stress stimuli modulation. 5. Conclusions The MnSOD Ala16Val gene polymorphism presents a role on oxidative stress modulation caused by environmental factors, such as intense exercise. This condition could affect clinical and biochemical variables related to chronic disease risks. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments Authors are indebted with Leonardo Barili Brandi for technical support with figure editing. References [1] Li Y, Huang TT, Carlson EJ, Melov S, Ursell PC, Olson JL, et al. Dilated cardiomyopathy and neonatal lethalityin mutant mice lacking manganese superoxide dismutase. Nat Genet 1995;11:76–381. [2] Lebovitz RM, Zhang H, Vogel H, Cartwright Jr J, Dionne L, Lu N, et al. Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc Natl Acad Sci U S A 1996;3:9782–7. [3] Shimoda-Matsubayashi S, Matsumine H, Kobayashi T, Nakagawa-Hattori Y, Shimizu Y, Mizuno Y. Structural dimorphism in the mitochondrial targeting sequence in the human manganese superoxide dismutase gene, A predictive evidence for conformational change to influence mitochondrial transport and a study of allelic association in Parkinson's disease. Biochem Biophys Res Commun 1996;226:561–5. [4] Bica CG, da Silva LL, Toscani NV, Zettler CG, Gottlieb MG, Alexandre CO, et al. Polymorphism (ALA16VAL) correlates with regional lymph node status in breast cancer. Cancer Genet Cytogenet 2010;196:153–8. [5] Duarte MM, Moresco RN, Duarte T, Santi A, Bagatini MD, Da Cruz IB, et al. Oxidative stress in hypercholesterolemia and its association with Ala16Val superoxide dismutase gene polymorphism. Clin Biochem 2010;43:1118–23. [6] Gottlieb MG, Schwanke CH, Santos AF, Jobim PF, Müssel DP, da Cruz IB. Association among oxidized LDL levels, SODMn, apolipoprotein E polymorphisms, and cardiovascular risk factors in a south Brazilian region population. Genet Mol Res 2005;4:691–703. [7] Montano MA, Barrio Lera JP, Gottlieb MG, Schwanke CH, da Rocha MI, Manica-Cattani MF, et al. Association between manganese superoxide dismutase (MnSOD) gene polymorphism and elderly obesity. Mol Cell Biochem 2009;328:33–40. [8] Taufer M, Peres A, de Andrade VM, de Oliveira G, M.E. do Canto G Sá, dos Santos AR, et al. Is the Val16Ala manganese superoxide dismutase polymorphism associated with the aging process? J Gerontol A Biol Sci Med Sci 2005;60(4):432–8. [9] Ambrosone CB, Freudenheim JL, Thompson PA, Bowman E, Vena JE, Marshall JR, et al. Manganese superoxide dismutase (MnSOD) genetic polymorphisms, dietary antioxidants, and risk of breast cancer. Cancer Res 1999;59:602–6.
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