Exercise-Associated Oxidative Stress

Christopher M. Deaton and David J. Marlin

During exercise a number of potential sources exist for the production of reactive oxygen species such as superoxide anions, hydrogen peroxide and hydroxyl radicals. Oxidative stress has been dened as a disturbance in the pro-oxidant-antioxidant balance in favour of the former, leading to potential damage (Sies 1991). Oxidative stress does not always result in oxidative damage. However, oxidative stress may result in oxidative damage to lipids, protein and DNA and consequently decrease athletic performance. Here we review the evidence for oxidative stress following exercise and the effects of exercise on the enzymatic and non-enzymatic antioxidant systems in a number of species including the horse. The effects of antioxidant supplementation on oxidative stress and performance during exercise are also evaluated. Key Words: Oxidative stress, exercise, horse, free radicals. Copyright 2004, Elsevier Inc. All rights reserved.

with hydrogen peroxide to produce the hydroxyl radical. The iron-catalyzed reaction with hydrogen peroxide is known as the Haber-Weiss reaction59 (Equations 3, 4, and 5). Hypochlorous acid is formed from hydrogen peroxide and chloride in a reaction catalyzed by neutrophil-derived myeloperoxidase (Equation 6).10 Fe 3 Fe 2 Net equation:
O2 O 2 3 Fe 2

O2 OH Fe 3

(3) (4)

H 2 O 2 3 OH

H 2 O 2 3 OH 2Cl 3 OCl

OH

O2 H 2O

(5) (6)

H 2O 2

Free Radicals and Oxidative Stress

A free radical is a molecule with 1 or more unpaired electrons. The reactivity of free radicals results from their desire to attain an electron of opposing spin direction.6 Nonradical derivatives of oxygen, such as ozone and hydrogen peroxide, which are more reactive than oxygen, are also capable of inducing oxidative damage. Free radicals and nonradical derivatives of oxygen are collectively termed reactive oxygen species (ROS).6 Reduction of oxygen results in the production of superoxide anions (O ) 2 (Equation 1). O2
e 3 O2

Nitric oxide (NO ) can also react with superoxide forming peroxynitrite (ONOO ) (Equation 7), since the rate of reaction is greater than that of superoxide with superoxide dismutase.154 Peroxynitrite has a relatively long half-life and is able to cross membranes.196
O2

NO 3 ONOO

(7)

The signicance of the formation of peroxynitrite, hydroxyl radicals, and hypochlorous acid is that these very reactive oxidants are produced from less reactive ones60,154 resulting in an increased likelihood of oxidative damage.

(1)

Potential Sources of Reactive Oxygen Species Generation During Exercise

Long-term physical exercise of moderate intensity has been demonstrated to increase the longevity of rats75 and reduce the incidence of diseases in humans such as cancer and cardiovascular disease, thereby enhancing life expectancy.18,55,166 However, a considerable number of studies have demonstrated that oxidative stress can occur following a bout of exercise (reviewed by Clarkson and Thompson,29 Kanter,89 Leeuwenburgh and Heinecke,109 and Sen 1995173). A number of potential pathways exist for exercise-related oxidant production. 1. Oxygen consumption can increase several-fold with exercise; for example, at rest the oxygen consumption of a Thoroughbred horse is approximately 5 mL/min/kg of body weight23 compared with a maximum oxygen consumption (VO2max) of between 110 and 220 mL/min/kg during maximal exercise.216 The oxygen consumption of individual muscle bers can be even higher due to increases in both blood ow and arteriovenous difference.116 Electron leak from the mitochondrial electron transfer chain results in the production of superoxide anions. Free radical production measured by electron spin resonance spectroscopy correlates strongly with maximal oxygen consumption.4 Alessio and coworkers,2 however, suggested that oxygen uptake

Superoxide anion has either an oxidizing function, where it is reduced to hydrogen peroxide (H2O2), or a reducing function, in which it is oxidized back to oxygen. On formation, 2 superoxide anions dismutate to form hydrogen peroxide and oxygen (Equation 2).
2O 2

2H 3 O 2

H 2O 2

(2)

The reaction can occur spontaneously, but the rate of reaction under physiological conditions is considerably greater when catalyzed by superoxide dismutase (SOD) (1.9 2.3 109 1 1 6 1 1 22,45,123 mol s versus 1.2 10 mol s ). Hydrogen peroxide can form a number of oxidants; however, 2 of the most reactive are the hydroxyl radical (OH) and hypochlorous acid (HOCl). The hydroxyl radical is formed in the presence of a transition metal, for example ferrous iron (Fe2 ), when superoxide reacts
From the Animal Health Trust, Lanwades Park, Kentford, UK, CB8 7UU. Address reprint requests to Christopher M. Deaton, Animal Health Trust, Lanwades Park, Kenford, UK, CB87UU. E-mail: chris.deaton@ aht.org.uk Copyright 2004, Elsevier Inc. All rights reserved. 1534-7516/04/0203-0006$35.00/0 doi:10.1053/S1534-7516(03)00070-2

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Clinical Techniques in Equine Practice, Vol 2, No 3 (September), 2003: pp 278-291

 Fig 1. Generation of superoxide anions (O2 ) during the oxidation of oxyhemoglobin to methemoglobin.53

with membrane phospholipids.4 Lipid radicals were also measured in blood from human subjects after supramaximal anaerobic exercise for 30 seconds by ESR spectroscopy.57 In addition, exercise of the quadriceps muscle resulted in a signicant increase in oxygen radicals in the femoral vein compared with in the femoral artery, which correlated with the leg oxygen uptake.11 ESR spectroscopy has also been used to measure the superoxide-scavenging ability of equine plasma, thus providing an indication of the antioxidant capacity.79 A horserace (the duration and length of which were not dened) involving only 4 horses increased superoxide scavenging but not signicantly.79

was not the sole determinant of the degree of oxidative stress, as isometric exercise increased the concentration of plasma lipid hydroperoxides without an increase in oxygen consumption, although oxygen uptake of muscle bers was not determined. 2. Xanthine dehydrogenase oxidizes hypoxanthine to xanthine and xanthine to uric acid using NAD as the electron acceptor forming NADH. During intense exercise, bers in active muscles may become hypoxic.143 During ischemia, xanthine is formed via anaerobic metabolism of ATP and xanthine dehydrogenase is converted to xanthine oxidase.87 During reperfusion, with the resulting increase in oxygen load, xanthine oxidase still converts hypoxanthine to uric acid, but utilizes oxygen as the electron acceptor forming superoxide (Equation 8).71 Xanthine H2O 2O2 3 uric acid
O2

Detrimental Effects of Reactive Oxygen Species

ROS can have damaging effects on lipids, proteins, and DNA as well as pro-inammatory actions. Damage to proteins may cause structural changes and alter enzyme activity, for example, hydrogen peroxide-induced SOD inactivation28 and HOCl-induced -1 proteinase inhibitor inactivation.32 Oxidants such as hydroxyl radicals may cause DNA strand breakage directly or by activation of a specic metabolic DNA strand-break pathway, for example, a calcium-dependent endonuclease.17,60,122 DNA repair mechanisms exist whereby damaged bases or nucleotides are excised. However misrepair can lead to mutations, modulating the function of the resulting protein. Nearly 10,000 DNA bases per cell are formed each day.112 The degree of DNA damage has been determined predominantly by the measurement of oxidized DNA bases, for example, 8-oxo-7,8-dihydro2 -deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine)183,214 or fragments of damaged DNA by the comet assay.140 Markers of lipid peroxidation have been frequently used as indicators of oxidative damage. Lipid peroxidation is initiated by the abstraction of a hydrogen atom from a bis-allylic site [methylene group [-CH2-] between 2 double bonds of a polyunsaturated fatty acid (PUFA)] giving a carbon-centered lipid radical (L) (Equation 9), which reacts with oxygen forming a peroxyl radical, LOO (Equation 10). The peroxyl radical can react with an adjacent PUFA side chain (L H) forming another lipid radical (L ) and a lipid hydroperoxide [LOOH (Equation 11)] and propagating the chain reaction.46 LH 3 L L LOO H (9) (10) L

2H

(8)

3. Tissue damage resulting from exercise may induce the activation of inammatory cells such as neutrophils, with the subsequent production of free radicals by NADPH oxidase.87 Downhill running by human subjects has been demonstrated to produce a signicant decrease in the plasma concentration of ascorbic acid, which correlated with the concentration of myeloperoxidase in plasma.24 Interestingly, uphill walking at equivalent oxygen consumption (60% VO2max for 35 minutes) did not decrease plasma ascorbic acid or increase myeloperoxidase. 4. Catecholamine concentrations are increased during exercise, and ROS can result from their auto-oxidation.168 5. Muscle mitochondria undergo increased uncoupling and superoxide generation with increasing temperatures. Therefore exercise-induced hyperthermia may cause oxidative stress.165 An increase in temperature and relative humidity from 20C and 40%, respectively, to 30C and 80%, respectively, was associated with a greater increase in the oxidation of glutathione in red blood cell hemolysate and lipid peroxidation in plasma following a submaximal exercise test in horses.130 6. Auto-oxidation of oxyhemoglobin to methemoglobin results in the production of superoxide and the rate of formation of methemoglobin can increase with exercise53 (Fig 1).

O 2 3 LOO L H 3 LOOH

(11)

Lipid hydroperoxides can decompose to a number of aldehydes such as malondialdehyde and 4-hydroxy-2-nonenal, which can diffuse from their site of production and cause further damage by oxidizing protein or DNA.43,202

Denitions of Oxidative Stress and Oxidative Damage Measurement of Free Radical Production During Exercise
Free radical production, measured by electron spin resonance (ESR) spectroscopy (or electron paramagnetic resonance), has been detected in the liver and skeletal muscle of rats33 and in human venous blood samples4 after submaximal exercise to exhaustion. The free radicals were postulated to be secondary alkoxyl radicals formed by primary oxygen radicals reacting
EXERCISE AND OXIDATIVE STRESS

Oxidative stress can occur as a result of increased production of ROS, with a normal antioxidant capacity and function, as a result of normal ROS production but in the presence of decreased antioxidant capacity, a combination of both or due to an imbalance in different antioxidant components (see Figs 2, 3, 4, 5, and 6). Furthermore, the presence of oxidative stress does not automatically imply oxidative damage. The latter can

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Fig 2. Under normal conditions at rest and in healthy individuals there is still background production of ROS, but these are adequately buffered by the nonenzymatic and enzymatic antioxidant systems so that there is a balance between production and removal and no net increase in ROS. This also implies that oxidized antioxidants are regenerated (ie, reduced) to keep pace with ROS production.

Fig 4. Oxidative stress may occur without any increase in normal background production of ROS due to selective or general reduction in components of the enzymatic or nonenzymatic antioxidant defenses.

only be veried by the direct measurement of markers of oxidative damage. Oxidative stress is commonly considered to have occurred if there is a decrease in concentration or activity of nonenzymatic or enzymatic antioxidants or an increase in oxidation of nonenzymatic antioxidants. For example, the ratio of oxidized to total (ie, oxidized reduced) glutathione, which is termed the glutathione redox ratio (GRR), is commonly used. The same expression can be used for reduced and oxidized ascorbic acid. Historically, the GRR has most commonly been expressed as a percentage, with the higher the percentage of the total glutathione in the oxidized form, the greater the inferred oxidative stress. It is now becoming accepted that this is incorrect, as a ratio should not be expressed as a percentage. So no oxidation of glutathione would result in a GRR of 0 while complete oxidation would result in a GRR of 1. However, this is still a misnomer, as a redox ratio implies the ratio of the reduced to the oxidized form. Thus, innitely high values would indicate low oxidative stress (very high reduced and very low oxidized) and a value of 0 would indicate complete oxidation. In this respect some groups are now adopting the latter form of expression.

Exercise, Oxidative Stress, and Oxidative Damage

Lipid peroxidation. Lipid peroxidation is by far the most extensively studied marker of oxidative damage following exercise.143 Both submaximal exercise and short-duration high-

intensity exercise have been demonstrated to induce lipid peroxidation in a number of species.116 In humans, submaximal exercise increased exhaled pentane concentration,39 which is formed from the peroxidation of linoleic acid.38 After a marathon, runners had elevated products of lipid peroxidation and a greater susceptibility to low-density lipoprotein oxidation.86,113 Increases in plasma malondialdehyde (MDA) have been demonstrated after 30 minutes of submaximal exercise in humans.171 In rats, submaximal exercise induced increases in both plasma and muscle MDA.33,173 Endurance exercise in sled dogs increased plasma isoprostanes,72 but not plasma lipid hydroperoxides.15 In the horse, submaximal exercise induced an increase in plasma lipid hydroperoxides within 5 minutes of the end of exercise and had returned to resting levels by 2 hours.130 Increases in plasma lipid hydroperoxides were greater and more prolonged when exercise was performed at 30C and 80% humidity compared with at 20C and 40% humidity.130 Endurance exercise increased the concentration of thiobarbituric acid reactive substances (TBARS) in plasma in horses, and the concentrations of plasma -tocopherol and red blood cell glutathione before exercise were negatively associated with plasma TBARS concentration post exercise.118 In men, exercise at 40% VO2max for 5 minutes decreased plasma MDA, at 70% VO2max plasma MDA was not changed, and at 100% VO2max plasma MDA increased.115 In horses, an

Fig 3. Despite an adequate antioxidant capacity (enzymatic and nonenzymatic), a marked increase in ROS production may overwhelm the antioxidant system, ie, development of oxidative stress.

Fig 5. The worst case scenario is both a reduction in antioxidant capacity combined with increases in ROS production. This may happen in certain disease conditions such as human asthma or equine recurrent airway obstruction (RAO). The disease causes chronic reduction in antioxidant defenses and ROS production is also dramatically increased in exacerbations.
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Fig 6. Under certain circumstances, excess intake of dietary nonenzymatic antioxidants can lead to oxidative stress. One example is excess ascorbic acid intake. In the presence of metal ions such as iron and copper, production of potent and damaging ROS can occur via interaction with reduced ascorbic acid.

incremental exercise test to fatigue has been demonstrated to increase both urinary excretion of MDA and plasma lipid hydroperoxide concentration.128,130 Three 2-minute periods at 8, 9, and 10 meter/s increased the concentration of plasma 8-isoprostane96 and 2 gallops at average speeds of 12 and 13 meter/s over 200 meters separated by 10 minutes also resulted in an immediate increase in plasma MDA, which was still elevated after 18 hours.27 Similarly, 2 minutes at 11 meter/s on a 5 gradient increased plasma lipid hydroperoxides, which peaked 1 minute post exercise.131 Interestingly, after administration of allopurinol, a structural analog of hypoxanthine and a potent inhibitor of xanthine dehydrogenase and xanthine oxidase, the exercise-induced increase in plasma lipid hydroperoxides was reduced, suggesting that lipid peroxidation results from anaerobic metabolism.131 However, allopurinol may also act by inhibiting xanthine oxidase-induced activation of circulating neutrophils and scavenging of hydroxyl radicals.73 Oxypurinol, formed from the metabolism of allopurinol, is also a potent inhibitor of hydroxyl radicals.129 It has been postulated that maintaining the availability of glycogen during exercise and therefore preserving the production of NADPH and hence the reducing potential of the cell may decrease the likelihood of lipid peroxidation.36 However, when an exhaustive exercise bout was repeated with lowered glycogen levels, the level of oxidative stress was not different from that of the rst bout.70 Not all studies have demonstrated that exercise induces lipid peroxidation. Submaximal exercise failed to increase blood TBARS in rats.199 No increases in lipid peroxidation products were observed in runners after a half marathon41 or after maximal207 or submaximal208 exercise on a cycle ergometer or following box-stepping exercise.121 The inconsistency of results may be a reection of differences in exercise intensity, duration, type, or training or the method used for assessment of lipid peroxidation.173 No increase was observed in lipid peroxides after maximal bicycle exercise in either trained or untrained human subjects,100 while the concentration of red blood cell TBARS increased in untrained but not trained rats after exhaustive exercise.175 Endurance training of mice reduced the susceptibility of mouse skeletal muscle to lipid peroxidation in vitro.163 At rest, sportsmen had higher plasma concentrations of
EXERCISE AND OXIDATIVE STRESS

TBARS and conjugated dienes, and female gymnasts had greater susceptibility to LDL oxidation than untrained subjects12,58; however, muscle TBARS and expired breath pentane were not different between trained and untrained horses.148 Similarly, plasma TBARS concentrations of adult Thoroughbred horses in training, not in training, and Thoroughbred foals were similar.101 However, all were higher compared with less athletic breeds (crossbred between Percheron and Breton).101 In addition, all Thoroughbred groups had higher superoxide scavenging ability than the other breeds and the scavenging ability of Thoroughbreds in training was higher than in those not in training and foals. The increased superoxide scavenging capacity in untrained Thoroughbreds appears to be due to ceruloplasmin, while the further increase in exercising Thoroughbreds appears to be due to albumin-bound bilirubin.101 Protein oxidation. Metal-catalyzed oxidation converts the side chains of some amino acid residues to carbonyl derivatives.3 Side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation. Exhaustive exercise induced a signicant increase in protein carbonylation in lung tissue157 and skeletal muscle, which may be related to a decrease in lipophilic antioxidants ( -tocopherol, ubiquinone, and ubiquinol).160 An increase in plasma protein carbonyls after acute exercise occurred in untrained but not trained rats188 and protein carbonyl concentration increased in hind limb muscles in response to training in rats.143 Four weeks of training at high altitude also increased protein carbonyls but not TBARS or lipid peroxides in rat skeletal muscle, suggesting that the mechanism of generation or mode of action of ROS, or the antioxidant capacities, differ in lipids and proteins.156 The proteins appeared to be actins, which along with myosin heavy chains are highly carbonylated, indicating that contractile proteins are susceptible to oxidation. The activity of manganese SOD in rat skeletal muscle increased after high-altitude training, but catalase, glutathione peroxidase, and copper and zinc SOD activities were unaltered.156 The authors suggested that the increased activity of mitochondrial SOD but not peroxide scavenging enzymes may have resulted in an elevation in the concentration of hydrogen peroxide.156 Hydrogen peroxide itself is relatively unreactive, but it can be converted to the highly reactive hydroxyl radical, resulting in protein oxidation.64 Exercise increased protein oxidation in heart muscle mitochondria, but not the cytosol, further suggesting that the mitochondrial generated ROS induce protein oxidation.108 DNA damage. Oxidative modications of DNA bases, particularly the 8-hydroxylation of guanine forming 8-hydroxydeoxyguanosine, are mutagenic and have been associated with conditions such as cancer and aging.152 Oxidative DNA damage was induced in human peripheral leukocytes following a 42-km marathon201 and in sled dogs after 3 days of endurance exercise.15 An increase in human DNA strand breaks was also observed 24 hours after a treadmill run to exhaustion.68 However, a number of other studies report conicting results. In voles voluntary exercise did not induce DNA damage,170 and in human subjects 90 minutes at 65% VO2max failed to induce an increase in urinary output of 8-hydroxyguanosine (a marker of oxidative RNA damage).207 Oxidative DNA damage was produced after long-duration intense exercise but not after acute or prolonged moderate-intensity exercise.152 An increase in leukocyte DNA strand breaks was found at various exercise intensities; however, the amount of damage was not related to the intensity of exercise.172 Interestingly, after a single bout of

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downhill exercise in untrained rats, the increase in DNA damage in circulating white blood cells correlated with the plasma creatine kinase concentration,203 and in human subjects following downhill running plasma creatine kinase concentrations were related to the release of superoxide from neutrophils.25 Therefore, following downhill running at least, it appears that oxidative DNA damage is related to muscle damage and neutrophil activation rather than increased oxygen consumption. A number of pathways exist for the production of an array of ROS and a complex antioxidant system therefore exists to prevent or reduce the formation of ROS and the degree of ROSinduced damage.

2GSH

H 2 O 2 3 GSSG

2H 2 O

(12)

Oxidized glutathione (GSSG) is subsequently reduced by glutathione reductase with the utilization of NADPH (Equation 13).146 The ratio of GSSG to total glutathione (GSH and GSSG), termed the glutathione redox ratio (GRR), has been implicated as a sensitive indicator of oxidative stress.130 GSSG NADPH H 3 NADP GSH (13)

Antioxidant Systems
An antioxidant has been dened as a substance that, when present at low concentrations compared with those of an oxidizable substrate, signicantly delays or prevents oxidation of that substrate.61

Nonenzymatic Antioxidants
Glutathione, ascorbic acid, and uric acid are important hydrophilic antioxidants. Vitamin E is the major lipophilic antioxidant and, as such, is present in membranes and lipoproteins.21,46 Ascorbic acid. Ascorbic acid was identied as the antiscorbutic factor in the treatment of scurvy in the 1920s by SzentGyorgy and Glenn.81 Ascorbic acid acts as a cosubstrate for procollagen, catecholamine, and carnitine biosynthesis.46 In addition, a vast array of ROS (eg, hydroxyl radicals, peroxyl radicals, superoxide anions, hypochlorous acid, and ozone), reactive nitrogen species (eg, peroxynitrite), and antioxidantderived radicals (eg, -tocopheroxyl and urate radicals) are scavenged by ascorbic acid.26 Ascorbic acid is oxidized to dehydroascorbate (DHA) in two 1-electron steps, with the release of a hydrogen atom at each step.63 The 1-electron standard reduction potential of ascorbic acid is low compared with ROS. Therefore, ascorbic acid will be readily oxidized by these highly oxidizing species.63 Furthermore, the intermediate radical produced, semidehydroascorbate (A ), has low reactivity, preventing further oxidation. The concentration of DHA in plasma from healthy human subjects has been reported to range from 0% to 40% of the total ascorbic acid content.37,83,98,116,134,137 This variation is likely to reect artifactual oxidation of ascorbic acid during sample processing, as ascorbic acid is readily oxidized as a function of light and temperature above 4C.37,110 Careful processing techniques are therefore essential to attain reliable measurements of the concentrations of ascorbic acid and DHA. Glutathione. Thiols are strong reducing agents and have negative standard reduction potentials and therefore act as electron acceptors.174 The most abundant nonprotein thiol in mammalian cells is glutathione,209 which has been the focus of extensive research, as it appears to be present in all living cells.174 Glutathione is a tripeptide [L- -glutamyl-L-cysteinylglycine (GSH)]. The active group of glutathione is the sulfhydryl (-SH) group of cysteine.145 Glutathione peroxidase catalyzes the oxidation of glutathione in which 2 glutathione molecules are joined by their sulfhydryl groups in a disulde bridge (Equation 12).

GSH also conjugates with exogenous and endogenous toxic compounds, catalyzed by glutathione sulfur-transferase.145 Uric acid. Uric acid is produced from the breakdown of purine compounds and, in most mammals other than man, is degraded further by urate oxidase.16 The absence of urate oxidase activity in man results in high plasma concentrations of uric acid compared with other species.16 Uric acid functions as an antioxidant by inhibiting iron-catalyzed oxidation of ascorbic acid by binding iron and scavenging ROS such as hypochlorous acid and hydroxyl radicals.176 It is oxidized to the urate radical, which is then either recycled back to uric acid or oxidized further (2-electron transfer), forming predominantly allantoin but also oxonic acid and parabanic acid.16,90 Vitamin E. Vitamin E is a chain-breaking antioxidant derived from the diet. Four tocopherols and 4 tocotrienols exhibiting vitamin E activity have been identied.145 -Tocopherol (ArOH) is the most active form, accounting for approximately 90% of the activity of vitamin E in tissues.117 -Tocopherol reacts with lipid peroxyl radicals forming a lipid hydroperoxide and tocopheroxyl radical ArO, and preventing the propagation of lipid peroxidation (Equation 14).210 In vitro, -tocopherol reacts with peroxyl radicals at a greater rate than peroxyl radicals react with lipid.211 LOO ArOH 3 LOOH ArO (14)

Coenzyme Q. Coenzyme Q also functions as a lipophilic antioxidant as well as playing a fundamental role in the mitochondrial electron transfer chain. The reduced form of coenzyme Q (ubiquinol) acts as an electron donor preventing the initiation and propagation of lipid peroxidation and also reduces the -tocopheroxyl radical back to -tocopherol.42 Ubiquinol, unlike -tocopherol, can be synthesized.42 Carotenoids. Carotenoids such as -carotene and lycopene are plant pigments, which possess antioxidant properties. Carotenoids are prominent scavengers of singlet oxygen and peroxyl radicals.191

Exercise and Antioxidant Status

Exercise-induced oxidative stress has been evaluated in terms of the effects on antioxidant capacity. Ascorbic acid. A marathon increased plasma ascorbic acid concentrations in human subjects.86 Similarly, an increase in plasma ascorbic acid was observed immediately after a 21-km race, which correlated with the rise in cortisol, suggesting that cortisol may induce the efux of ascorbic acid from the adrenal cortex or cortisol and ascorbic acid may be released simultaneously.51 Twenty-four and 48 hours after exercise, the plasma concentration of ascorbic acid was less than the preexercise concentration.51 Ninety minutes of submaximal exercise increased the plasma concentration of total ascorbic acid in human subjects, and interestingly, the percentage of total ascorbic acid in the reduced state also increased, which may be due to an
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increase in the activity of dehydroascorbate reductase in erythrocytes with exercise.207 Downhill running by human subjects decreased the plasma concentration of ascorbic acid, which appeared to be related to neutrophil activation.24 In contrast, uphill walking at equivalent oxygen consumption did not alter plasma ascorbic acid. Differences between uphill and downhill running may reect the use of different muscle groups and variation in the amount of training between the 2 forms of exercise. In rats, which unlike humans can synthesize ascorbic acid, downhill running was associated with an increase in plasma ascorbic acid.203 In horses, plasma ascorbic acid was not changed after a 1-km race at approximately 16 meter/s; however, the total antioxidant status did increase.212 Following endurance exercise at a mean ambient temperature of 28C, horses demonstrated decreased concentrations of plasma ascorbic acid and red blood cell GSH, which both correlated with the increase in plasma AST.65 No signicant changes in plasma ascorbic acid were observed after endurance exercise at ambient temperatures of either 5C to 11C74 or 15C to 19C.118 However, individual horses demonstrated marked increases or decreases in plasma ascorbic acid.118 In unt ponies 20 minutes of exercise at 4 meter/s on a 7 incline did not alter plasma concentrations of TBARS, ascorbic acid, or -tocopherol or exhaled breath pentane.124 However, horses performing regular exercise had lower concentrations of ascorbic acid and -tocopherol in plasma compared with sedentary controls.148 Glutathione. A large number of studies have measured the concentrations of reduced and oxidized glutathione following exercise to give an indication of the antioxidant-oxidant balance. The concentration of glutathione is negligible in both human and equine plasma.76,186 Therefore, studies have predominantly measured glutathione in whole blood or red blood cells. An increase in blood GSSG was induced by bicycle exercise at a range of intensities (maximal intensity for 14 minutes, 50% VO2max for 30 minutes, and 80% VO2max for 30 minutes), which returned to preexercise concentrations after 24 hours.172 In both rats and humans, an increase in GSSG was observed after graded exercise to exhaustion, but not after running at high speed for a short duration ( 20 s).167 In human subjects, blood GSH was decreased and GSSG increased 15 minutes after submaximal bicycle exercise (90 minutes at 65% VO2peak).53 Exercise to VO2peak did not alter the concentration of GSH or GSSG; however, the concentration of lactate increased considerably.53 No increase in lactate was observed in the submaximal exercise test and the authors speculated that lactic acid might reduce oxidized species by acting as a hydrogen donor preventing the oxidation of GSH.53 Lactate has been demonstrated by ESR spectroscopy to scavenge hydroxyl and superoxide radicals but not lipid radicals.56 Laaksonen and coworkers102 measured an increase in plasma TBARS and blood GSSG and a decrease in GSH after 40 minutes of exercise at 60% VO2max in men. Of interest, higher blood GSH concentrations before exercise were associated with lower exercise-induced increases in TBARS, thereby supporting the contention that red blood cell GSH acts as a sink for circulating ROS.62,102 During the rst 15 minutes of a 90-minute cycle ergometer exercise test at 65% VO2peak, blood GSSG increased and GSH decreased.207 GSH and GSSG did not change further during the remaining 75 minutes of exercise, returning to preexercise levels after 15 minutes of recovery. The increase in GSH oxidation during the onset of exercise may result from
EXERCISE AND OXIDATIVE STRESS

increased rates of loading and unloading of hemoglobin with oxygen and the formation of methemoglobin and superoxide.53,207 In humans decreases in total GSH in red blood cells have been observed after a half marathon41 and 30 and 60 minutes, but not immediately, after exhaustive exercise.167 Decreases in the concentration of total glutathione in red blood cells may be the result of adrenergic stimulation following exercise, decreasing hepatic synthesis of GSH.167 However, adrenergic stimulation also increases glutathione efux from hepatocytes.44 Not all studies have demonstrated an increase in GSH oxidation with exercise. Forty minutes of running exercise in human subjects103 and intermittent submaximal exercise in horses did not signicantly alter the concentrations of GSH or GSSG in red blood cells.97 Similarly, Ji and coworkers84 found that blood GSH and GSSG were not altered following exercise at 70% VO2max to fatigue, but the activity of glutathione reductase was increased. They suggested that GSH in blood may be maintained by hepatic release of GSH and an increase in GSSG may only be observed when glutathione reductase is overwhelmed. Interestingly, the concentrations of GSH are decreased in the liver of rats following exercise to exhaustion111,155 and red blood cell concentrations of GSH are increased after a marathon.86 Ji and Fu (1992)9 observed an increase in rat skeletal muscle GSH as well as GSSG after treadmill running to exhaustion. The activities of glutathione peroxidase, glutathione reductase, SOD, and catalase were also increased, which may have been the result of increased ROS production and therefore increased substrate for these enzymes. In the horse, high intensity, short-duration exercise also did not result in the oxidation of red blood cell GSH.130 Submaximal exercise, however, did increase the GSSG concentration, peaking 5 minutes after exercise and returning to resting levels within 2 hours, while the concentration of GSH was unchanged.130 Total red blood cell GSH was also reduced immediately and 16 hours following a 140-km equine endurance race, with no increase in the oxidation of GSH.118 Red blood cell GSH concentrations did not change immediately after intense exercise, but decreased after 24 hours of rest.13 Uric acid. Endurance exercise in sled dogs,149 box-stepping exercise,121 and a half marathon in humans41 increased plasma uric acid. Exhaustive exercise at 90% VO2max resulted in the accumulation of uric acid in human skeletal muscle.70 Increases in plasma uric acid have been demonstrated in horses after submaximal exercise of between 50 minutes and 9 hours duration, and after intense exercise to fatigue,96,118,130 but not after a 1-km race.212 Administration of allopurinol to horses before galloping at 11 meter/s for 2 minutes prevented the increase in uric acid, indicating that exercise-induced increase in uric acid resulted from the degradation of purine nucleotides.131 There appears to be an exercise intensity threshold where the reamination of IMP cannot keep pace with the deamination of AMP resulting in uric acid accumulation.67,158 Vitamin E. In human runners, an increase in plasma vitamin E has been observed after a 31-km run206 and after cycling at VO2max to exhaustion,150 but not after a marathon.86 Elevations in plasma tocopherol may be due to increased mobilization of tocopherol with free fatty acids from adipose tissue150 or increased ux of fatty acids through the liver stimulating the secretion of very-low-density lipoprotein, which are enriched in tocopherol by tocopherol transfer protein.91 Alternatively,

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tocopherol may be increased in plasma during its rapid redistribution toward tissues with increased ROS production.150 The plasma concentrations of -tocopherol were not altered following endurance exercise for 80 km (mean speed 8.8 km/h), 140 km (16.5 km/h), or 160 km (8.2 km/h) in horses.65,118 Similarly, the concentrations of plasma and muscle -tocopherol in horses were not altered by submaximal exercise.181 In contrast, exercise to exhaustion by rats104 and a 52-km run by sled dogs resulted in decreases in plasma vitamin E concentration.72 Alterations in plasma antioxidant concentrations following exercise reect the rate of their release from tissue stores and the rate of utilization. An inconsistent effect of exercise on antioxidant status may reect the mode of exercise, time points sampled, the level of training, environmental factors (eg, ambient temperature and altitude), or lack of control for plasma volume.29,127

Antioxidant Manipulation and Oxidative Stress in Association With Exercise

Despite the variation in response to exercise, the number of studies demonstrating evidence of exercise-induced oxidative stress has led to considerable interest into the effects of antioxidant supplementation. Ascorbic acid. Ascorbic acid supplementation has been demonstrated to decrease the concentration of plasma TBARS in human subjects after 30 minutes of submaximal exercise.1 A single 1-g dose of ascorbic acid prevented the increase in systemic free radical production (measured by ESR spectroscopy) and lipid peroxidation after an incremental exercise test to exhaustion5 and prevented the exercise-induced increase in low-density lipoprotein susceptibility to oxidation following a 4-hour race.165 The oxidation of blood glutathione following exhaustive physical exercise in rats was prevented by 0.5 g of ascorbic acid/kg of body weight.167 A single 2-g dose of ascorbic acid reduced the exercise-induced decline in FEV1 in 11 of 20 subjects with exercise-induced asthma.30 Four hundred milligrams of ascorbic acid per day for 3 weeks reduced the strength loss in the triceps surae and increased recovery 24 hours after 60 minutes of box-stepping exercise, suggesting that ascorbic acid prevented exerciseinduced muscle damage.82 In contrast, -tocopherol (400 mg) did not prevent the exercise-induced alterations in contractile function.82 Ascorbic acid supplementation did not however prevent the decrease in neutrophil function (assessed by neutrophil phagocytosis and neutrophil bactericidal ability) after a biathlon.99 Oral ascorbic acid has poor systemic bioavailability in horses.114,188 Dietary supplementation of horses with derivatives of ascorbic acid such as ascorbyl palmitate and calcium ascorbyl monophosphate has been demonstrated to elevate plasma ascorbic acid concentrations.35,189 Intravenous administration of 5 g of ascorbic acid to horses before exercise prevented the slight increase in plasma TBARS concentration after a 1-km race.212 Glutathione. Supplemented GSH has poor tissue uptake in rats.172 There has therefore been considerable interest in the supplementation of glutathione precursors. N-Acetylcysteine (NAC) is a cysteine derivative; therefore, because of its thiol group NAC is able to scavenge hydrogen peroxide, hydroxyl radicals, and hypochlorous acid directly.50 Furthermore, NAC can easily be deacetylated to cysteine, an important precursor

for cellular glutathione synthesis. Exhaustive exercise increased the oxidation of GSH in blood and the lung in rats, which was prevented by intraperitoneal administration of NAC but not GSH.171 NAC infusion also reduced the increase in oxidation of blood glutathione following short-duration highintensity exercise in humans125 and oral NAC supplementation prevented the priming of human circulating neutrophils to produce ROS following short-duration maximal exercise.77 In rats NAC supplementation prevented the oxidation of glutathione in blood after exhaustive exercise.167 Glutathione-decient rats had greater levels of lipid peroxidation after exercise.107,172 Lipoic acid supplementation of rats increased the concentration of GSH in the liver and blood and prevented the increase in TBARS in skeletal muscle, heart, and liver after exhaustive exercise.95 Supplementation of horses with lipoic acid decreased the plasma concentration of lipid hydroperoxides.213 Selenium is a cofactor for glutathione peroxidase. A deciency in selenium results in a reduction in the activity of glutathione peroxidase.215 Selenium deciency increased exhaled pentane in rats, which was reduced by supplementation with -tocopherol.39 Selenium supplementation did not inuence the exercise-induced increase in red blood cell GSSG despite an increase in glutathione peroxidase activity in human subjects.198 In addition, selenium deciency had no effect on endurance capacity in rats and did not alter the degree of oxidation of glutathione despite depleting glutathione peroxidase, suggesting that residual glutathione peroxidase activity or glutathione peroxidase-independent processes are sufcient to detoxify hydroperoxides.104 Vitamin E. Vitamin E has been the antioxidant most extensively studied with respect to reducing exercise-induced lipid peroxidation.89 Supplementation with vitamin E reduced the level of lipid peroxidation in exhaled breath and plasma in human subjects and rats following exercise of varying intensities and duration.40,54,127,193 Vitamin E and selenium supplementation decreased plasma concentrations of MDA in exercising horses.8 A high dose of vitamin E (10,000 IU/kg of diet) decreased the concentration of protein carbonyls in skeletal muscle of rats at rest and following a run to exhaustion.160 The exercise-induced increase in muscle damage after 6 days of successive training in human subjects was also decreased by -tocopherol supplementation.80 Long-term supplementation with -tocopherol decreased the plasma concentrations of MDA and creatine kinase in elite cyclists.162 Exercise-induced DNA damage was reduced by supplementation with vitamin E in human subjects.68 In fact, in 4 of 5 subjects DNA damage was completely prevented by supplementation. During a 90-day training period the concentration of plasma -tocopherol decreased in horses fed 80 IU/kg of diet but not in horses fed 300 IU/kg in addition to a basal diet containing less than 40 IU/ kg.181 Furthermore, in -tocopherol-supplemented ponies, the concentration of ascorbic acid has been shown to increase after submaximal exercise.124 Acute submaximal exercise did not induce lipid peroxidation in vitamin E-decient rats199 and vitamin E supplementation in humans did not reduce the exercise-induced increase in plasma muscle enzymes.69 Antioxidant combinations. The effects of supplementing with a combination of antioxidants have also been investigated as 2 or more antioxidants may have synergistic interactions.78,196 Moreover, oxidized antioxidants can be reduced by other antioxidants; for example, ascorbic acid has been proposed to recycle oxidized -tocopherol and uric acid,16,141
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while glutathione reduces oxidized ascorbic acid.19 Antioxidants also differ in their location of action. For example, -tocopherol is an important lipid antioxidant, scavenging free radicals including superoxide, hydroxyl radical and lipid peroxides,200 whereas ascorbic acid is an important antioxidant in the aqueous phase. Supplementation with -tocopherol, ascorbic acid, and -carotene in humans reduced the increase in expired pentane and serum MDA after submaximal exercise and decreased resting levels.88 The level of DNA damage was decreased in sled dogs fed a supplement of -tocopherol, -carotene, and lutein after 3 days of endurance exercise compared with nonsupplemented animals.15 Supplementation with ascorbic acid and glutathione prevented the increase in GSSG in human blood after exercise to exhaustion.167 Cycling exercise for 45 minutes at 75% VO2max increased plasma tumor necrosis factor- (TNF), interleukin-1 (IL-1 ), and IL-6 concentrations.206 After supplementation with a combination of antioxidants (vitamins E, A, and C for 60 days; allopurinol for 15 days; and N-acetylcysteine for 3 days), plasma IL-1 became undetectable, the exercise-induced increase in TNF- was prevented, and the increase in IL-6 was signicantly reduced, suggesting that exercise-induced cytokine production was stimulated by oxidative stress.206 The exercise regime did not increase plasma concentrations of creatine kinase, suggesting that muscle damage did not occur. In contrast, 90 minutes of downhill running at 75% VO2max induced a marked increase in plasma creatine kinase as well as plasma IL-6 and these were not reduced by supplementation with 500 mg of ascorbic acid and 400 mg of vitamin E.147 In sled dogs -tocopherol, ascorbic acid, and -carotene supplementation did not reduce the exercise-induced increase in creatine kinase activity after endurance exercise.149 Antioxidant mixtures have also been reported to not prevent oxidative stress. A supplement containing selenium, vitamins C, E, and B2, niacin, and -carotene decreased levels of MDA in human plasma at rest, but did not prevent the exercise-induced increase in MDA.85 The authors suggested that the decrease at rest might have mainly been as a result of improved lipid metabolism due to decreased levels of triglycerides and cholesterol rather than inhibition of lipid peroxidation.85 Supplementation with -tocopherol and ascorbic acid and ubiquinone in humans failed to prevent the exercise-induced increase in serum-conjugated dienes.205 A combination of antioxidants including an ascorbic acid derivative, vitamin E, and selenium prevented the exerciseinduced increase in plasma uric acid in horses in horses suffering from recurrent airway obstruction.97 The concentration of plasma ascorbic acid was elevated following submaximal exercise, but only in horses supplemented with the combination of antioxidants.34 The elevation in plasma ascorbic acid may reect the release of stored ascorbic acid from tissues such as the adrenal cortex.51,203

mal aerobic capacity. In the horse, supplementation with a variety of antioxidants did not improve lung function during a submaximal exercise test34 and vitamin E supplementation did not increase the time to exhaustion in unt horses during submaximal exercise.124 Supplementation with -tocopherol has been demonstrated to increase anaerobic threshold at high altitude184 and increased swimming time to exhaustion in rats.135 Restriction of thiamin, riboavin, vitamin B6, and ascorbic acid reduced VO2max in healthy human subjects; however, VO2max was not affected when only ascorbic acid was restricted.204 Rats fed a diet decient in vitamin E had a decrease in exercise endurance capacity, which was not prevented with ascorbic acid supplementation (3 g/kg of diet).52 Endurance capacity has also been shown to be lower in ascorbate-decient guinea pigs.142 A high dose of ascorbic acid (approximately 40 mg of ascorbic acid/kg of body weight) has been shown to slow racing Greyhounds by 0.2 s (equivalent to 3 months) in a 500-month race.119 The authors speculated that the increase in run time may have been a result of the dogs supplemented with ascorbic acid being slightly, but not signicantly, heavier than controls. NAC supplementation did not increase the run-time to fatigue in rats or humans.125,172 However, glutathione deciency dramatically reduced run-time to fatigue in rats.171 ROS have been proposed to alter muscle function and increase the rate of muscle fatigue by impairing action potential propagation, altering the sarcoplasmic reticulum calcium pump, oxidizing sulfhydryl groups on contractile proteins, and inactivating enzymes such as succinate dehydrogenase, succinate oxidase, ATPase, NADH oxidase, and NADH dehydrogenase.195 Diaphragm muscle bers have been demonstrated to release superoxide anions and the rate of release is increased with fatiguing muscle contractions.159 Administration of antioxidants reduced the rate of development of limb and respiratory muscle fatigue,14,180,194 while increased ROS production by xanthine oxidase increased the rate of limb muscle fatigue.14 Vitamin E and glutathione deciency increased the susceptibility of muscles to fatigue.33,133 Neither dietary vitamin E nor intravenously administered ascorbic acid prevented exerciseinduced muscle damage in horses.181,212

Enzymatic Antioxidants
Enzymatic antioxidants complement the nonenzymatic antioxidants and either catalyze reactions to remove ROS or to regenerate (reduce) oxidized antioxidants. For example, superoxide dismutase (SOD) acts on superoxide radicals to form oxygen and the lesser reactive nonradical species, hydrogen peroxide, while glutathione reductase can regenerate oxidized glutathione (GSSG, glutathione disulde) to reduced glutathione (GSH). The major antioxidant enzymes include SOD, glutathione peroxidase, glutathione reductase, catalase, and the thioredoxins, glutaredoxins, and peroxiredoxins. Superoxide dismutase (SOD) is primarily located in mitochondria and the cytosol of cells and neutralizes superoxide radicals. Two isoforms of SOD are known to exist in skeletal muscle in mammals; ie, CuZn SOD, which is located primarily in the cytosol, and MnSOD, which is located primarily in mitochondria. SOD activity in muscle is highest in high oxidative muscle bers (ie, types I and IIa). An extracellular form of SOD also exists (ECSOD) that requires Cu and Zn and is found in both plasma and tissues.

Effect of Manipulation of Antioxidant Status and Performance

The evidence for a positive effect of ascorbic acid or vitamin E supplementation on exercise performance is equivocal.29,47,48 The majority of studies in human subjects have not demonstrated a benecial effect of ascorbic acid49,92-94 or vitamin E39,105,162,177-179,211 on either endurance performance or maxiEXERCISE AND OXIDATIVE STRESS

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Glutathione peroxidase (GSHPx) is found in mitochondria, the cytosol, and the membranes of cells and reduces H2O2 and organic hydroperoxides using reduced glutathione (GSH) as the electron donor. GSHPx activity is selenium dependent and its activity is highest in type I muscle bers (ie, highly oxidative bers). Glutathione reductase is essential for conversion of oxidized glutathione (GSSG) back to the reduced form (GSH) using NADPH as the reductant. Catalase (CAT) is located mainly in peroxisomes and mitochondria and also removes H2O2. CAT requires iron as a cofactor, and similar to GSHPx and SOD, its activity is highest in highly oxidative muscle bers. Thioredoxins (Trx) repair oxidized sulfhydryl groups, remove H2O2, and acts as a radical scavenger. They are also involved in regeneration of oxidized ascorbic acid. The class of thioredoxins consists of thioredoxin, thioredoxin reductase, and thioredoxin peroxidase. Glutaredoxin (Grx) is involved in the protection and repair of proteins and nonprotein thiols such as glutathione, and its function overlaps with that of the Trx family. Peroxiredoxins (Prx) are antioxidant enzymes that protect protein and lipids against oxidant-induced damage. They are also thought to be involved in signaling pathways regulating apoptosis, and cell proliferation, differentiation, and gene expression. They are found in the cytosol, mitochondria, peroxisomes, and plasma. Various different peroxiredoxins exist (Prx1 to Prx6), but they all contain a reactive Cys residue in the N-terminal region and can behave as a peroxidase, with thioredoxin and or glutathione as the electron donor.

for SOD, this is mainly conned to bers with a high oxidative capacity and the majority of the increase occurs within the mitochondria.185 The situation in the systemic circulation, however, is less clear. Overload training of triathletes resulted in an increase in plasma but not red blood cell GSH-Px activity.144 Endurance-trained athletes also had higher red blood cell GSHPx activities than anaerobic-trained athletes (wrestlers).169 In another study GSHPx was higher in sprinttrained compared with endurance-trained athletes.120 Catalase. Catalase has been studied to a lesser extent in relation to exercise and training than either SOD or GSHPx. Endurance training did not result in any change in the activity of catalase in rat heart muscle,197 but conversely a different study by the same authors showed an increase of around 20% with training but also an increase with age.192 Muscle catalase activity has been shown to be increased by endurance training in rat slow-twitch but not fast-twitch muscle groups.151 Resting erythrocyte catalase activity was not increased in human subjects following intense endurance training despite increases in SOD and GSHPx.132 In contrast, erythrocyte catalase activity and GSHPx activity were both positively correlated with weekly training distance in a group of human runners.161 Catalase activity of erythrocytes was reported to be increased in professional cyclists at rest compared with amateur cyclists and sedentary controls, but also decreased after 20 days of racing.126

Enzymatic Antioxidants in Horses

In relation to acute exercise, erythrocyte SOD and GSHPx activities were not changed in pentathlon horses following show jumping.13 While light exercise in Quarterhorses was reported to increase erythrocyte glutathione reductase activity, there was no change in GSHPx activity.20 Ono and colleagues138 reported no change in erythrocyte SOD activity with intense exercise, but erythrocyte GSHPx was signicantly reduced from 69 to 65 IU/g of Hb. In contrast, 3-year-old Maremmana stallions did not show any increase in lymphocyte GSHPx activity with intense exercise.8 Following prolonged exercise (80- or 160-km endurance racing), erythrocyte GSHPx activity was reported to increase.66 Plasma xanthine oxidase activity was increased by exercise in Standardbred trotters, but the increase was reported to be independent of exercise intensity.158 Only a limited number of studies have investigated changes in antioxidant enzymes with training. Maremmana stallions on a diet providing 10 g of selenium and 100 IU of vitamin E per day were maintained in light training for 60 days. Pretraining erythrocyte GSHPx activity was 4.9 U/106 erythrocytes and was decreased by around 40% after 60 days of training.7 The same authors also measured lymphocyte GSHPx activity in 3-yearold Maremmana stallions before and after training and reported a 3-fold increase from around 7 mU/mg of protein to 21 mU/ mg.8 Supplementation with intramuscular vitamin E and selenium had no effect on SOD, GSHPx, or catalase in erythrocytes from Thoroughbred horses,138 and there was no change in GSHPx activity following 4 weeks supplementation with selenium (60 ppm selenium as sodium selenite).20 There appears to be only 1 report in the literature of supplementation of horses with lipoic acid.213 In this study, after 2 weeks supplementation of mature Thoroughbreds with lipoic acid, white blood cells from peripheral blood had a higher GSHPx activity than
DEATON AND MARLIN

Enzymatic Antioxidants and Exercise and Training

SOD. In relation to exercise and training, SOD has been studied to a greater extent than any of the other antioxidant enzymes. Studies in human subjects have shown either no change in muscle total SOD activity following marathon running or red blood cell CuZn SOD activity following short- to moderate-duration cycling.31,126 A tremendous number of studies have looked at SOD activity following exercise in rodents (see Powers and Sen153 for review). Studies have examined SOD activity following short-term and prolonged exercise and tissues investigated have included muscle, heart, lung, liver, bone marrow, diaphragm, brain kidney, plasma, and red blood cells, with the majority of studies showing no increase. Studies in which an increase has been demonstrated have been most often in heart and/or skeletal muscle following exhaustive running. Training has been shown to increase SOD in the muscle and red blood cells of students, swimmers, cyclists, and volleyball players (see review by Powers and Sen153) although there are relatively few studies. In animals the majority of work has been conducted by using rodents, and tissues most commonly studied are muscle, heart and liver. A large number of studies have shown increases following training, but a considerable proportion also show no change, with a small number also showing a decrease. For example, SOD increased in muscle of exercisetrained rats and humans136,139 while GSHPx has shown to be increased in muscle but not plasma.139 In addition, exhaustive exercise causes greater increases in SOD in untrained compared with trained individuals.190 GSHPx. The majority of studies have shown an increase in muscle GSHPx activity with endurance training, eg,106,190 As

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the control group, although both groups showed a decrease over the period of the study.

Conclusion
In summary, there is considerable variation between studies in the degree and severity of systemic oxidative stress induced by exercise, irrespective of species. In horses there is considerably less published work compared with either humans or rodents. However, the ability to manipulate antioxidant status through diet and the possibility of improvements in performance or reduction in exercise-associated damage (eg, to muscles) should ensure that many more studies are forthcoming. Exercise has not been conclusively shown to induce oxidative stress or oxidative damage in all modes. It is likely that factors such as duration, intensity, tness, breed, athletic ability, health, and environmental conditions all have an impact on the occurrence or severity of oxidative stress and damage.

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