Aging and acute exercise enhance free radical generation in rat skeletal muscle.

Reactive oxygen species (ROS) are implicated in the mechanism of biological aging and exercise-induced oxidative damage. The present study examined the effect of an acute bout of exercise on intracellular ROS production, lipid and protein peroxidation, and GSH status in the skeletal muscle of young adult (8 mo, n = 24) and old (24 mo, n = 24) female Fischer 344 rats. Young rats ran on a treadmill at 25 m/min and 5% grade until exhaustion (55.4 +/- 2.7 min), whereas old rats ran at 15 m/min and 5% grade until exhaustion (58.0 +/- 2.7 min). Rate of dichlorofluorescin (DCFH) oxidation, an indication of ROS and other intracellular oxidants production in the homogenate of deep vastus lateralis, was 77% (P < 0.01) higher in rested old vs. young rats. Exercise increased DCFH oxidation by 38% (P < 0.09) and 50% (P < 0.01) in the young and old rats, respectively. DCFH oxidation in isolated deep vastus lateralis mitochondria with site 1 substrates was elevated by 57% (P < 0.01) in old vs. young rats but was unaltered with exercise. Significantly higher DCFH oxidation rate was also found in aged-muscle mitochondria (P < 0.01), but not in homogenates, when ADP, NADPH, and Fe(3+) were included in the assay medium without substrates. Lipid peroxidation in muscle measured by malondialdehyde content showed no age effect, but was increased by 20% (P < 0.05) with exercise in both young and old rats. Muscle protein carbonyl formation was unaffected by either age or exercise. Mitochondrial GSH/ GSSG ratio was significantly higher in aged vs. young rats (P < 0.05), whereas exercise increased GSSG content and decreased GSH/GSSG in both age groups (P < 0.05). These data provided direct evidence that oxidant production in skeletal muscle is increased in old age and during prolonged exercise, with both mitochondrial respiratory chain and NADPH oxidase as potential sources. The alterations of muscle lipid peroxidation and mitochondrial GSH status were consistent with these conclusions.     

Glutathione and antioxidant enzymes in skeletal muscle: effects of fiber type and exercise intensity.

Glutathione status and antioxidant enzymes in various types of rat skeletal muscle were studied after an acute bout of exercise (Ex) at different intensities. Glutathione (GSH) and glutathione disulfide (GSSG) concentrations were the highest in soleus (SO) muscle, followed by those in deep (DVL) and then superficial (SVL) portions of vastus lateralis. In DVL, but not in SO or SVL, muscle GSH increased proportionally with Ex intensity and reached 1.8 +/- 0.08 mumol/g wet wt compared with 1.5 +/- 0.03 (P < 0.05) in resting controls (R). GSSG in DVL was increased from 0.10 +/- 0.01 mumol/g wet wt in R to 0.14 +/- 0.01 (P < 0.05) after Ex. Total glutathione (GSH + GSSG) contents in DVL were also significantly elevated with Ex, whereas GSH/GSSG ratio was unchanged. Activities of GSH peroxidase (GPX), GSSG reductase (GR), and catalase (CAT) were significantly higher in SO than in DVL and SVL, but there was no difference in superoxide dismutase activity between the three muscle types. Furthermore, Ex at moderate intensities elicited significant increases in GPX, GR, and CAT activities in DVL muscle. None of the antioxidant enzymes was affected by exercise in SO. It is concluded that rat DVL muscle is particularly vulnerable to exercise-induced free radical damage and that a disturbance of muscle GSH status is indicative of an oxidative stress.     

萝卜硫素对急性低温暴露鼠骨骼肌Nrf2介导的抗氧化能力的影响

萝卜硫素(sulforaphane,SFN)是一种在十字花科植物中含量丰富,且具有抗氧化效应的天然物质。本文基于核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)介导的抗氧化系统,探究不同时长低温暴露对骨骼肌抗氧化酶的影响及SFN对低温暴露骨骼肌抗氧化能力的作用。首先,30只雄性C57BL/6N小鼠随机分为常温对照组(0 h组)、低温暴露1 h组(1 h组)和低温暴露3 h组(3 h组)。其次,40只雄性C57BL/6N小鼠随机分为PBS常温对照组(PBS+Con),PBS低温暴露3 h组(PBS+Cold),SFN常温对照组(SFN+Con)和SFN低温暴露3 h组(SFN+Cold)。小鼠在急性温度干预前腹腔注射4次SFN或等体积PBS。急性低温暴露后,取小鼠骨骼肌,试剂盒检测活性氧(ROS)水平、总抗氧化能力(T-AOC)、还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量;荧光实时定量PCR检测Nrf2介导的抗氧化酶和参与生成谷胱甘肽相关酶的mRNA转录水平;Western blot检测Nrf2介导的抗氧化酶蛋白表达。结果显示,与0和1 h组相比,3 h组小鼠骨骼肌Nrf 2和抗氧化酶基因(Gpx 1、Hmox1、Cat、Sod 1和Nqo 1)的mRNA转录水平显著降低,ROS水平显著增加。与PBS+Con组相比,PBS+Cold组小鼠骨骼肌Nrf2和抗氧化酶(HMOX1和CAT)蛋白表达、GSH/GSSG比值及T-AOC水平显著降低,而GSSG含量和ROS水平增加。与PBS+Cold组相比,SFN+Cold组小鼠骨骼肌Nrf 2 mRNA及其蛋白表达、抗氧化酶(HMOX1和SOD1)蛋白表达、抗氧化酶基因(Gpx 1、Hmox 1、Cat、Sod 1和Nqo 1)mRNA转录水平、参与GSH生成的酶基因(Gclm和Gss)mRNA转录水平、GSH/GSSG比值以及T-AOC水平显著提高,而GSSG含量和ROS水平显著降低。综上,3 h急性低温暴露降低了Nrf2介导的抗氧化作用。而低温暴露前给予SFN补充,则激活了Nrf2介导的抗氧化酶和谷胱甘肽抗氧化系统,增强了骨骼肌抗氧化能力。     

MRP1/GS‐X pump ATPase expression: is this the explanation for the cytoprotection of the heart against oxidative stress‐induced redox imbalance in comparison to skeletal muscle cells?

Striated muscle activity is always accompanied by oxidative stress (OxStress): the more intense muscle work and/or its duration, the more a redox imbalance may be attained. In spite of cardiac muscle functioning continuously, it is well known that the heart does not suffer from OxStress-induced damage over a broad physiological range. Although the expression of antioxidant enzymes may be of importance in defending heart muscle against OxStress, a series of combined antioxidant therapeutic approaches have proved to be mostly ineffective in avoiding cellular injury. Hence, additional mechanisms may be involved in heart cytoprotection other than antioxidant enzyme activities. The strong cardiotoxic effect of doxorubicin-induced cancer chemotherapy shed light on the possible role for multidrug resistance-associated proteins (MRP) in this context. Muscle activity-induced 'physiological' OxStress enhances the production of glutathione disulfide (GSSG) thus increasing the ratio of GSSG to glutathione (GSH) content inside the cells, which, in turn, leads to redox imbalance. Since MRP1 gene product (a GS-X pump ATPase) is a physiological GSSG transporter, adult Wistar rats were tested for MRP1 expression and activity in the heart and skeletal muscle (gastrocnemius), in as much as the latter is known to be extremely sensitive to muscle activity-induced OxS. MRP1 expression was completely absent in skeletal muscle. In contrast, the heart showed an exercise training-dependent induction of MRP1 protein expression which was further augmented (2.4-fold) as trained rats were challenged with a session of acute exercise. On the other hand, inducible expression of the 70-kDa heat shock protein (HSP70), a universal marker of cellular stress, was completely absent in the heart of sedentary and acutely exercised rats, whereas skeletal muscle showed a conspicuous exercise-dependent HSP70 expression, which decreased by 45% with exercise training. This effect was paralleled by a 58% decrease in GSH content in skeletal muscle which was even higher (an 80%-fall) after training thus leading to a marked redox imbalance ([GSSG]/[GSH] raised up to 38-fold). In the heart, GSH contents and [GSSG]/[GSH] ratio remained virtually unchanged even after exercise challenges, while GS-X pump activity was found to be 20% higher in the heart related to skeletal muscle. These findings suggest that an intrinsic higher capacity to express the MRP1/GS-X pump may dictate the redox status in the heart muscle thus protecting myocardium by preventing GSSG accumulation in cardiomyocytes as compared to skeletal muscle fibres.     

Age and sex differences in human skeletal muscle: Role of reactive oxygen species

Previous studies, conducted on experimental animals, have indicated that reactive oxygen species (ROS) are involved in the aging process. The objective of this work was to study the relationship between oxidative damage and human skeletal muscle aging, measuring the activity of the main antioxidant enzymes superoxide dismutase (total and MnSOD), glutathione peroxidase (GPx) and catalase in the skeletal muscle of men and women in the age groups: young (17–40 years), adult (41–65 years) and aged (66–91 years). We also measured glutathione and glutathione disulfide (GSH and GSSG) levels and the redox index; lipid peroxidation and protein carbonyl content. Total SOD activity was lower in the 66–91 year-old vs. the 17–40 year-old men; MnSOD activity was significantly greater in 66–91 year-old vs. 17–40 year-old women. GPx activity remained unchanged. The activity of catalase was lower in adults than in young men but higher in the aged. We observed no changes in GSH levels and significantly higher GSSG levels only in aged men vs. adult men, and a significant decrease in aged women vs. aged men. The protein carbonyl content increased significantly in the 41–65 and 66–91 year-old vs. the 17–40 year-old men. Finally, young women have lower lipid peroxidation levels than young men. Significantly higher lipid peroxidation levels were observed in aged men vs. both young and adult men, and the same trend was noticed for women. We conclude that oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging in both sexes; and that men appear to be more subject to oxidative stress than women.     

Age and sex differences in human skeletal muscle: role of reactive oxygen species

Previous studies, conducted on experimental animals, have indicated that reactive oxygen species (ROS) are involved in the aging process. The objective of this work was to study the relationship between oxidative damage and human skeletal muscle aging, measuring the activity of the main antioxidant enzymes superoxide dismutase (total and MnSOD), glutathione peroxidase (GPx) and catalase in the skeletal muscle of men and women in the age groups: young (17-40 years), adult (41-65 years) and aged (66-91 years). We also measured glutathione and glutathione disulfide (GSH and GSSG) levels and the redox index; lipid peroxidation and protein carbonyl content. Total SOD activity was lower in the 66-91 year-old vs. the 17-40 year-old men; MnSOD activity was significantly greater in 66-91 year-old vs. 17-40 year-old women. GPx activity remained unchanged. The activity of catalase was lower in adults than in young men but higher in the aged. We observed no changes in GSH levels and significantly higher GSSG levels only in aged men vs. adult men, and a significant decrease in aged women vs. aged men. The protein carbonyl content increased significantly in the 41-65 and 66-91 year-old vs. the 17-40 year-old men. Finally, young women have lower lipid peroxidation levels than young men. Significantly higher lipid peroxidation levels were observed in aged men vs. both young and adult men, and the same trend was noticed for women. We conclude that oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging in both sexes; and that men appear to be more subject to oxidative stress than women.     

Crucial yet divergent roles of mitochondrial redox state in skeletal muscle vs. brown adipose tissue energetics

Reduced glutathione (GSH) is the major determinant of redox balance in mitochondria and as such is fundamental in the control of cellular bioenergetics. GSH is also the most important nonprotein antioxidant molecule in cells. Surprisingly, the effect of redox environment has never been examined in skeletal muscle and brown adipose tissue (BAT), two tissues that have exceptional dynamic range and that are relevant to the development of obesity and related diseases. Here, we show that the redox environment plays crucial, yet divergent, roles in modulating mitochondrial bioenergetics in skeletal muscle and BAT. Skeletal muscle mitochondria were found to naturally have a highly reduced environment (GSH/GSSG≈46), and this was associated with fairly high (～40%) rates of state 4 (nonphosphorylating) respiration and decreased reactive oxygen species (ROS) emission. The deglutathionylation of uncoupling protein 3 (UCP3) following an increase in the reductive potential of mitochondria results in a further increase in nonphosphorylating respiration (～20% in situ). BAT mitochondria were found to have a much more oxidized status (GSH/GSSG≈13) and had basal reactive oxygen species emission that was higher (～250% increase in ROS generation) than that in skeletal muscle mitochondria. When redox status was subsequently increased (i.e., more reduced), UCP1-mediated uncoupling was more sensitive to GDP inhibition. Surprisingly, BAT was found to be devoid of glutaredoxin-2 (Grx2) expression, while there was abundant expression in skeletal muscle. Taken together, these findings reveal the importance of redox environment in controlling bioenergetic functions in both tissues, and the highly unique characteristics of BAT in this regard.     

Cellular and biochemical parameters of exercise-induced oxidative stress: relationship with training levels

To better clarify the relationship between physical activity and oxidative stress, we determined the effects of a maximal test in 18 young subjects with different training levels (six professional Athletes and 12 non-agonists (NA)). Redox homeostasis (total antioxidant activity (TAS), vitamin C and glutathione (GSH)), oxidative damage (diene conjugation and hemolysis), lymphocyte cell death and repair systems (apoptosis, micronuclei and Hsp70 expression) were evaluated. We found that agonistic training led to a chronic oxidative insult (high baseline values of oxidized glutathione (GSSG), micronuclei and hemolysis). On the contrary, NA with the lowest level of training frequency showed a well balanced profile at rest, but they were more susceptible to exercise-induced variations (GSSG/GSH and diene increased values), respect to the NA with an higher level of training. As almost all the parameters employed in this study showed inter-individual variations, the GSSG/GSH ratio remains the most sensitive and reliable marker of oxidative stress, accordingly with other data just reported in the literature.     

Antioxidant supplementation enhances the exercise-induced increase in mitochondrial uncoupling protein 3 and endothelial nitric oxide synthase mRNA content in human skeletal muscle

The effects of acute exercise on the mRNA content of selected genes were examined during control conditions and after oral intake of antioxidants. In addition, to provide evidence for formation of reactive oxygen species (ROS) in human skeletal muscle during exercise, cytochrome c reduction was measured in microdialysate from the muscle. For the study on the effects of antioxidants on mRNA content, seven healthy, habitually active, male subjects participated in a double-blinded experimental design in which they, on one occasion, received a placebo and, on another, a mixture of antioxidants containing 1500 mg vitamin C, 120 mg coenzyme Q, and 345 mg alpha-tocopherol every day for 7 days before the experiment. On the experimental day the subjects cycled for 90 min and muscle biopsies were taken preexercise and at 1, 3, and 5 h after exercise. Exercise induced an increase in the eNOS, UCP3, PGC-1alpha, VEGF, Hsp72, and HO-1 mRNA content (p < 0.001), whereas there was no change in the Hsc70 mRNA level. Prior antioxidant treatment further enhanced (p < 0.05) the eNOS and UCP3 mRNA content after exercise. Moreover, the overall level of Hsc70 mRNA tended (p = 0.07) to be higher after antioxidant treatment. In another group of healthy male subjects, cytochrome c reduction was determined in microdialysate from the thigh muscle at rest and during knee extensor exercise to determine ROS formation. There was a significant increase in cytochrome c reduction with exercise both at 14 ( approximately 25%) and at 30 W ( approximately 50%). The data show that ROS are formed within skeletal muscle during exercise and that oral intake of antioxidants can enhance the exercise-induced adaptive mRNA responses of eNOS and UCP3.     

Effect of glutathione depletion on antioxidant enzymes in the epididymis, seminal vesicles, and liver and on spermatozoa motility in the aging brown Norway rat

Reactive oxygen species (ROS) play a role in male infertility, where excessive amounts impair spermatozoal motility. Epididymal antioxidant enzymes protect spermatozoa from oxidative damage in the epididymal lumen. Antioxidant secretions from the seminal vesicle protect spermatozoa after ejaculation. As it is known that with age there is increased generation of ROS, the goals of this study were to determine how aging affects the response of antioxidant enzymes in the epididymis, seminal vesicles, and liver to l-buthionine-S,R-sulfoximine (BSO) mediated glutathione (GSH) depletion, and to examine the impact of GSH depletion on motility parameters of spermatozoa from the cauda epididymidis in young (4-mo-old) and old (21-mo-old) rats. Levels of GSH and glutathione disulfide (GSSG), as well as activities of glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase, were measured in the caput, corpus and cauda epididymidis, seminal vesicles, and liver. Spermatozoal motility was assessed by computer-assisted sperm analysis. Significant age-related changes in antioxidant enzyme activities were found in the liver and cauda epididymidis. Glutathione depletion clearly affected tissues in both young and old. The compounding effect of age was most evident in the cauda epididymidis, seminal vesicles, and liver, where antioxidant enzyme activities changed significantly. Additionally, spermatozoa motility was adversely affected after BSO treatment in both age groups, but significantly more so in older animals. In summary, the male reproductive tissues and liver undergo age-related changes in antioxidant enzyme activities and in their response to GSH depletion.     

Exercise protects against doxorubicin-induced oxidative stress and proteolysis in skeletal muscle

Doxorubicin (Dox) is a potent antitumor agent used in cancer treatment. Unfortunately, Dox is myotoxic and results in significant reductions in skeletal muscle mass and function. Complete knowledge of the mechanism(s) by which Dox induces toxicity in skeletal muscle is incomplete, but it is established that Dox-induced toxicity is associated with increased generation of reactive oxygen species and oxidative damage within muscle fibers. Since muscular exercise promotes the expression of numerous cytoprotective proteins (e.g., antioxidant enzymes, heat shock protein 72), we hypothesized that muscular exercise will attenuate Dox-induced damage in exercise-trained muscle fibers. To test this postulate, Sprague-Dawley rats were randomly assigned to the following groups: sedentary, exercise, sedentary with Dox, or exercise with Dox. Our results show increased oxidative stress and activation of cellular proteases (calpain and caspase-3) in skeletal muscle of animals treated with Dox. Importantly, our findings reveal that exercise can prevent the Dox-induced oxidative damage and protease activation in the trained muscle. This exercise-induced protection against Dox-induced toxicity may be due, at least in part, to an exercise-induced increase in muscle levels of antioxidant enzymes and heat shock protein 72. Together, these novel results demonstrate that muscular exercise is a useful countermeasure that can protect skeletal muscle against Dox treatment-induced oxidative stress and protease activation in skeletal muscles.     

In Situ Microdialysis for Monitoring of Extracellular Glutathione Levels in Normal,Ischemic and Post-Ischemic Skeletal Muscle

Microdialysis probes were inserted into the tibialis anterior muscle and into the femoral vein of anaesthetised Sprague-Dawley rats for monitoring of reduced (GSH) and oxidized (GSSG) extracellular glutathione. The dialysates were analysed using HPLC. The levels of GSH and GSSG were high immediately after implantation in the skeletal muscle and declined to steady state levels after 90 minutes into the same range as that found in the venous dialysate. Total ischemia was induced two hours after implantation of the dialysis probe after steady state levels had been reached. The extracellular levels of GSH increased during total ischemia and had doubled at the end of the ischemic period compared to preischemic values. During the following initial 30 minutes of reperfusion the levels increased further to four-fold the preischemic levels. The levels of GSSG also increased (100%) during the initial 30 minutes of reperfusion. The extracellular GSH levels remained elevated for 1 hour of reperfusion, but the GSSG levels returned to preischemic levels. The results indicate that intermittent hypoxia or anoxia in muscle tissue through hypoperfusion or ischemia decreases intracellular GSH stores by leakage, reducing the intracellular antioxidative capacity and increasing the risk for oxidative reperfusion injury upon final normalization of tissue blood supply.     

In Situ Microdialysis for Monitoring of Extracellular Glutathione Levels in Normal, Ischemic and Post-Ischemic Skeletal Muscle

Microdialysis probes were inserted into the tibialis anterior muscle and into the femoral vein of anaesthetised Sprague-Dawley rats for monitoring of reduced (GSH) and oxidized (GSSG) extracellular glutathione. The dialysates were analysed using HPLC. The levels of GSH and GSSG were high immediately after implantation in the skeletal muscle and declined to steady state levels after 90 minutes into the same range as that found in the venous dialysate. Total ischemia was induced two hours after implantation of the dialysis probe after steady state levels had been reached. The extracellular levels of GSH increased during total ischemia and had doubled at the end of the ischemic period compared to preischemic values. During the following initial 30 minutes of reperfusion the levels increased further to four-fold the preischemic levels. The levels of GSSG also increased (100%) during the initial 30 minutes of reperfusion. The extracellular GSH levels remained elevated for 1 hour of reperfusion, but the GSSG levels returned to preischemic levels. The results indicate that intermittent hypoxia or anoxia in muscle tissue through hypoperfusion or ischemia decreases intracellular GSH stores by leakage, reducing the intracellular antioxidative capacity and increasing the risk for oxidative reperfusion injury upon final normalization of tissue blood supply.     

Thiol/disulfide redox equilibrium between glutathione and glycogen debranching enzyme (amylo-1,6-glucosidase/4-alpha-glucanotransferase) from rabbit muscle

Rabbit skeletal muscle glycogen debranching enzyme is inactivated in a kinetically biphasic manner by GSSG at pH 8.0. The rapid phase results in the loss of 30% activity, while the slower phase leads to total enzyme inactivation. Both the glucosidase and the transferase activities of the enzyme are inhibited by GSSG. The inactivation by disulfides is fully and rapidly reversed in a biphasic manner by reduction with excess reduced dithiothreitol or GSH. After a fast initial recovery of 70% of the initial activity, the remaining 30% of the activity is recovered more slowly. Equilibration of the enzyme with a redox buffer of GSH and GSSG shows a monophasic equilibration of the activity. The ratio of GSH/GSSG where the enzyme is 50% active (R0.5) is 0.06 +/- 0.03. The R0.5 does not vary significantly with the total concentration of glutathione species suggesting formation of protein-SSG mixed disulfides. The ratios of the observed second-order rate constants for GSSG inactivation and GSH reactivation do not lead to a correct value of the observed thiol/disulfide oxidation equilibrium constant. Although the enzyme has sulfhydryl groups, the oxidation of which leads to activity changes, the kinetic and thermodynamic resistance to oxidation suggests that the enzyme is not likely to be subject to regulation by thiol/disulfide exchange in vivo.     

Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae

The reduction potential of a cell is related to its fate. Proliferating cells are more reduced than those that are differentiating, whereas apoptotic cells are generally the most oxidized. Glutathione is considered the most important cellular redox buffer and the average reduction potential (E_h) of a cell or organism can be calculated from the concentrations of glutathione (GSH) and glutathione disulfide (GSSG). In this study, triplicate groups of cod larvae at various stages of development (3 to 63 days post-hatch; dph) were sampled for analyses of GSSG/2GSH concentrations, together with activities of antioxidant enzymes and expression of genes encoding proteins involved in redox metabolism. The concentration of total GSH (GSH+GSSG) increased from 610±100 to 1260±150 μmol/kg between 7 and 14 dph and was then constant until 49 dph, after which it decreased to 810±100 μmol/kg by 63 dph. The 14- to 49-dph period, when total GSH concentrations were stable, coincides with the proposed period of metamorphosis in cod larvae. The concentration of GSSG comprised approximately 1% of the total GSH concentration and was stable throughout the sampling series. This resulted in a decreasing E_h from −239±1 to −262±7 mV between 7 and 14 dph, after which it remained constant until 63 dph. The changes in GSH and E_h were accompanied by changes in the expression of several genes involved in redox balance and signaling, as well as changes in activities of antioxidant enzymes, with the most dynamic responses occurring in the early phase of cod larval development. It is hypothesized that metamorphosis in cod larvae starts with the onset of mosaic hyperplasia in the skeletal muscle at approximately 20 dph (6.8 mm standard length (SL)) and ends with differentiation of the stomach and disappearance of the larval finfold at 40 to 50 dph (10–15 mm SL). Thus, metamorphosis in cod larvae seems to coincide with high and stable total concentrations of GSH.     

Prepubertal children with suitable fitness and physical activity present reduced risk of oxidative stress

To assess the impact of fitness status and physical activity on oxidative stress in prepubertal children, we measured selected biomarkers such as protein carbonyls (PC), lipid peroxidation products, and total nitrites, as well as the antioxidant system: total glutathione (TG), oxidized glutathione (GSSG), reduced glutathione (GSH), superoxide dismutase activity, and glutathione peroxidase. A total of 132 healthy children ages 7-12, at prepubertal stage, were classified into two groups according to their fitness level: low fitness (LF) and high fitness (HF). They were observed while engaged in an after-school exercise program, and a questionnaire was created to obtain information on their physical activity or sedentary habits. Plasma and red blood cells were obtained to analyze biomarkers. Regarding oxidative stress markers, the LF group and the sedentary group showed higher levels of TG and GSSG and a lower GSH/GSSG ratio than the HF group and the children engaged in physical activity. A negative association was found between PC and GSSG and TG and between TG and the GSH/GSSG ratio. Moreover, a negative correlation was found between GSSG and fitness, with a positive correlation with the GSH/GSSG ratio. TG, GSSG, and the GSH/GSSG ratio seem to be reliable markers of oxidative stress in healthy prepubertal children with low fitness or sedentary habits. This research contributes to the recognition that an adequate level of fitness and recreational physical activity in childhood leads to better health and oxidative status.     

Haematopoietic stem cells and endothelial progenitor cells in healthy men: effect of aging and training

The number of hematopoietic stem cells (HSC) and endothelial progenitor cells (EPC) is thought to be a marker for neovascularization and vascular repair. Because physical inactivity and aging are risk factors for cardiovascular diseases, these factors may influence the numbers of HSCs and EPCs. Therefore, we examined baseline and exercise-induced levels of HSCs and EPCs in sedentary and trained young and older men. To study the role of aging in eight sedentary young (19-28 years) and eight sedentary older men (67-76 years), baseline and acute exercise-induced numbers of HSCs (CD34+-cells) and EPCs (CD34+/VEGFR-2+-cells) were quantified by fluorescence-activated cell sorter (FACS) analysis. To examine the effect of chronic training, eight age-matched trained young men (18-28 years) were compared with sedentary young men, whereas older men performed an 8-week endurance training. Older men showed significantly lower baseline and exercise-induced levels of HSCs/EPCs than the young men (P < 0.05). In young and older men, acute exercise significantly increased HSCs (P < 0.01), but not EPCs. The absolute increase in numbers of HSCs was attenuated in older men (P = 0.03). Apart from the lower baseline numbers of EPCs after chronic training in older men, training status did not alter baseline or exercise-induced levels of HSCs/EPCs in young and older men. We concluded that advancing age results in lower circulating numbers of HSCs and EPCs and attenuates the acute exercise-induced increase in HSCs. Interestingly, in young as well as in older men chronic endurance training does not affect baseline and exercise-induced numbers of HSCs and EPCs.     

Age-dependent changes of antioxidant activities and markers of free radical damage in human skeletal muscle.

This study was conducted in order to provide evidence for the role of reactive oxygen species (ROS) in human skeletal muscle aging. We used human muscle samples obtained from hospitalized patients in an open study with matched pairs of individuals of different ages. The subjects, ranging in age from 17 to 91 years, were grouped as follows: 17-25-, 26-35-, 36-45-, 46-55-, 56-65-, 66-75-, 76-85-, and 86-91-year-old groups. To investigate the relationship between muscle aging and oxidative damage we measured total and Mn-dependent superoxide dismutase (total SOD, MnSOD), glutathione peroxidase (GSHPx), and catalase (CAT) activities; total reduced and oxidized glutathione (GSHtot, GSH, and GSSG) levels; lipid peroxidation (LPO), and protein carbonyl content (PrC). Total SOD activity decreases significantly with age in the 66-75-year-old group, although MnSOD activity increases significantly in the 76-85-year-old group. The activity of the two H2O2 detoxifying enzymes (GSHPx and CAT) did not change with age, as do GSHtot and GSH levels. GSSG levels increased significantly (76-85- and 86-91-year-old groups) with age. We observed a significant increase in LPO levels (66-75- and 76-85-year-old groups), although the PrC content shows a trend of increase without gaining the statistical significance. These results support the idea that ROS play an important role in the human muscle aging process.     

Smaller age-associated reductions in leg venous compliance in endurance exercise-trained men

We determined the independent and interactive influences of aging and habitual endurance exercise on calf venous compliance in humans. We tested the hypotheses that calf venous compliance is 1) reduced with age in sedentary and endurance-trained men, and 2) elevated in young and older endurance-trained compared with age-matched sedentary men. We studied 8 young (28 +/- 1 yr) and 8 older (65 +/- 1) sedentary, and 8 young (27 +/- 1) and 8 older (63 +/- 2) endurance-trained men. Calf venous compliance was measured in supine subjects by inflating a venous collecting cuff, placed above the knee, to 60 mmHg for 8 min and then decreasing cuff pressure at 1 mmHg/s to 0 mmHg. Calf venous compliance was determined using the first derivative of the pressure-volume relation during cuff pressure reduction (compliance = beta(1) + 2. beta(2). cuff pressure). Calf venous compliance was reduced with age in sedentary (approximately 40%) and endurance-trained men (approximately 20%) (both P < 0.01). Furthermore, calf venous compliance was approximately 70-120% greater in endurance-trained compared with age-matched sedentary men and approximately 30% greater in older endurance-trained compared with young sedentary men (both P < 0.01). These data indicate that calf venous compliance is reduced with age in sedentary and endurance-trained men, but compliance is better preserved in endurance-trained men.     

Induced swimming modified the antioxidant status of gilthead seabream (Sparus aurata)

Swimming has relevant physiological changes in farmed fish, although the potential link between swimming and oxidative stress remains poorly studied. We investigated the effects of different medium-term moderate swimming conditions for 6 h on the antioxidant status of gilthead seabream (Sparus aurata), analyzing the activity of enzymes related to oxidative stress in the liver and skeletal red and white muscle. Forty fish were induced to swim individually with the following conditions: steady low (SL, 0.8 body length (BL)·s⁻¹), steady high (SH, 2.3 BL·s⁻¹), oscillating low (OL, 0.2–0.8 BL·s⁻¹) and oscillating high (OH, 0.8–2.3 BL·s⁻¹) velocities, and a non-exercised group with minimal water flow (MF, < 0.1 BL·s⁻¹). All swimming conditions resulted in lower activities of superoxide dismutase (SOD), glutathione reductase (GR), and glutathione-S-transferase (GST) in the liver compared to the MF group, while steady swimming (SL and SH) led to higher reduced glutathione/oxidized glutathione ratio (GSH/GSSG) compared to the MF condition. Swimming also differently modulated the antioxidant enzyme activities in red and white muscles. The OH condition increased lipid peroxidation (LPO), catalase (CAT) and glutathione peroxidase (GPx) activities in the red muscle, decreasing the GSH/GSSG ratio, whereas the SL condition led to increased GSH. Oscillating swimming conditions (OL and OH) led to lower CAT activity in the white muscle, although GPx activity was increased. The GSH/GSSG ratio in white muscle was increased in all swimming conditions. Liver and skeletal muscle antioxidant status was modulated by exercise, highlighting the importance of adequate swimming conditions to minimize oxidative stress in gilthead seabream.